——_—' '-——— -7 444__<_ A-ngl A; 44‘44. ‘ . ' . . u v p ~.lo- ¢ ' > I.- .‘:"--"-‘" I. . ‘ . A STUDY OF TEE FORMATEGN 0F METHYL KETONES AND FREEFAT'FY ACIBS {N QUICK RIPENEB BLUE CHEESE AND SUBSEQUENT LOSSES WHERE EEHYDRAHON Thesis §or HM Degree 0‘ pk. D. MICHIGAN STATE UNIVERSITY Lee E. Biakely €970 lHriRtg Date 0-7839 WWW?“ 1m 3 1293 10726 This is to certify that the " thesisentitled . ' A STUDY OF THE FORMATION OF METHYL KETONES AND FREE FATTY ACIDS IN QUICK RIPENED’” ' , BLUE CHEESE AND SUBSEQUENT LOSSES DURING DEHYDRATION ' presented by . M Lee E. Blakely .. 5"." has been aocepted towards fulfillment of the requirements for Ph.D. degreein FOOd SCLQQQQ and Human Nutrition W MW professor 11 November 1970 ‘-- ‘u LIBRAR Y1 Michigan S ta te University m-M-"- l I .l- u 0‘.- -"‘~ outw‘sdwOn-p—an'A-MQ" '4' MSU LIBRARIES .—_—. RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. ABSTRACT A STUDY OF THE FORMATION OF METHYL KETONES AND FREE FATTY ACIDS IN QUICK RIPENED BLUE CHEESE AND SUBSEQUENT LOSSES DURING DEHYDRATION BY Lee E. Blakely A study was made of the development of Blue cheese flavor in quick ripened loose curd Blue cheese prepared from cows' milk and coconut oil filled milk. The retention of free fatty acids (FFA) and methyl ketones during spray and freeze drying of 40 per cent solids Blue cheese slurries was also investigated. Free fatty acids were quantitated using gas chromatography and heptanoic and heptadecanoic acids as internal standards. Methyl ketones were converted to their 2,4-dinitrophenylhydrazones, separated from other monocarbonyl classes, and the C3-C13+15 odd carbon number ketones separated and then quantitated spectrophotometrically. Volatile acid development and pH changes in quick ripened Blue cheese dur— ing the 7 days of curing followed a pattern similar to that observed in Blue cheese ripened several months. The volatile acid content of the ripened curd continued to increase during storage at 40 F. Significantly larger quantities of FFA were present in quick ripened Blue cheese than in Danish Blue cheese samples analyzed. Relatively low methyl ketone Lee E. Blakely concentrations were observed in quick ripened Blue cheese compared to the quantity of fatty acid precursor available. Additional ripening at 62 F significantly increased the FFA concentration, but no increase in methyl ketone content resulted. Nonanone-Z was the predominant ketone in all quick ripened Blue cheese samples analyzed. Quick ripened filled Blue cheese samples exhibited extensive fat hydrolysis (67—70 per cent) and contained large quantities of methyl ketone with 2-undecanone predominating. Filled Blue cheese possessed a flavor described as not completely typical of Blue cheese. Larger quantities of FFA were retained during freeze drying of Blue cheese than during spray drying. Over 50 per cent of the butyric acid present in the original cheese was retained during freeze drying while less than 20 per cent of the butyric acid was retained during Spray drying. Greater retention of FFA in both spray and freeze dried cheese was ' observed as compared to methyl ketone retention. Only 0—14 per cent of the original acetone was retained in Spray dried Blue cheese. A STUDY OF THE FORMATION OF METHYL KETONES AND FREE FATTY ACIDS IN QUICK RIPENED BLUE CHEESE AND SUBSEQUENT LOSSES DURING DEHYDRATION By 1‘" 1» Lee EefiBlakely A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1970 \\ \J to ACKNOWLEDGMENTS The author wishes to express sincere appreciation and thanks to Dr. C. M. Stine for his counsel and guidance throughout the course of this graduate program. The author also wishes to thank the members of the guidance committee: Drs. J. R. Brunner and L. G. Harmon, Department of Food Science and Drs. E. J. Benne and H. A. Lillevik, Department of Biochemistry for their advice and effort in reading this manuscript. Appreciation is extended to the Food Science Department for the financial support that made this project possible. ii TABLE OF CONTENTS Page INTRODUCTION. . . . . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE. . . . . . . . . . . . . . . . . 5 Flavor Components of Blue Cheese and Their Origin 5 Free Fatty Acids. . . . . . . . . . . . . . . 5 Glyceride Hydrolysis. . . . . . . . . . . . . S Acids from Sources other than Milk Trigly- cerides . . . . . . . . . . . . . . . . . 14 Methyl Ketones. . . . . . . . . . . . . . . . 15 Secondary Alcohols. . . . . . . . . . . . . . 24 Aldehydes . . . . . . . . . . . . . . . . . . 25 Primary Alcohols. . . . . . . . . . . . . . . 26 , Esters. . . . . . . . . . . . . . . . . . . . 27 Contribution to Blue Cheese Flavor by Microorgan- isms other than 2, roqueforti . . . . . . . . 28 Proteolysis . . . . . . . . . . . . . . . . . 50 Improving the Flavor and/or Shortening the Ripen- ing Time of Blue Cheese . . . . . . . . . . . 52 Filled Cheese 0 O O O O O O O O O C C O O C O O D 58 Dehydrated Cheese . . . . . . . . . . . . 40 Loss of Aroma Compounds in Dehydrated Foods . . . 44 EEERIMENTAL PROCEDIIRES O O O O O O O O O O O O O O O 52 Preparation of Samples for Spray Drying and Freeze Drying . . . . . . . . . . . . . . 52 Freeze Drying of Imported Danish Blue Cheese. . . 52 Spray Drying of Imported Danish Blue Cheese . . . 55 Preparation of Quick Ripened Loose Curd Blue Cheese 0 O O O O O O O O C O O O O O O O O O 0 54 Preparation of Milk for Filled Cheese Manufacture 55 ANALYTICAL PROCEDURES . . . . . . . . . . . . . . . . 56 M01 Sture o O O O O O O O O O O O O I O O O O O O O 56 pH 0 O O O O O O O 0 O O I O O O O O O O O O O 0 O 56 iii TABLE OF CONTENTS--continued Volatile Acidity. . . . . . . . . . . . . . . . . Quantitation of Free Fatty Acids. . . . . . Acetic Acid . . . . . . . . . . . . . . Butyric and Higher Acids. . . . . . . . Quantitation of Methyl Ketones. . . . . . . Purification of Solvents. . . . . . . . . . . Isolation of Methyl Ketones . . . . . . . . . Extraction of Fat . . . . . . . . . . . Reaction of Carbonyls with DNP-hydrazine. Removal of Fat from the DNP—hydrazones. . DNP-hydrazone Class Separation. . . . . . Separation of Ketone DNP-hydrazones into Individual Chain Lengths. . . . . . . Determination of Individual Methyl Ketone Concentration . . . . . . . . . . . . RESULTS AND DISCUSSION. 0 O O O O O O O O O O O O O 0 Flavor Development in Quick Ripened Loose Curd Blue-veined Cheese. . . . . . . . . . . . . . Changes in pH During Ripening . . . . . . . . Changes in Volatile Acidity During Ripening . Quantitation of Free Fatty Acids. . . . . . . . . Factors for Relating the Internal Standards to the Free Fatty Acids . . . . . . . . . Acetic Acid Recovery Trials . . . . . . . . . Free Fatty Acids in Danish Blue Cheese. . . Free Fatty Acids in Quick Ripened Blue Cheese Free Fatty Acids in Quick Ripened Filled Blue Cheese. . . . . . . . . . . . . . . . Quantitation of Methyl Ketones. . . . . . . . . . Recovery of Methyl Ketone DNP—hydrazones from Butteroil . . . . . . . . . . . . . . . Methyl Ketones in Danish Blue Cheese. . . . . Methyl Ketones in Quick Ripened Blue Cheese . Methyl Ketones in Quick Ripened Filled Blue Cheese. . . . . . . . . . . . . . . . . . Retention of Some Aroma Compounds During Spray Drying and Freeze Drying of Blue Cheese . . . Retention of FFA and Methyl Ketones During Spray Drying. . . . . . . . . . . . . . . Retention of FFA During Spray Drying. . . Retention of Methyl Ketones During Spray Drying. . . . . . . . . . . . . . . . Retention of FFA and Methyl Ketones During Freeze Drying . . . . . . . . . . . . . . iv Page 56 57 57 58 64 64 65 65 65 65 66 68 68 71 71 71 75 8O 82 85 85 89 95 104 105 107 .116 124 151 152 155 155 140 TABLE OF CONTENTS--continued Retention of FFA During Freeze Drying . . Retention of Methyl Ketones During Freeze DrYing C O O O C O O O O O O O O O O 0 SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . BIBLIOGMPHY O O O O O O O C C O O C O O O O O O O O 0 APPENDIX 0 O O O O O O O O O C O O O O O O O O O O O 0 Determination of Acetic Acid. . . . . . . . . . . Reagents O O O O O 0 O O O O O O O O O O O 0 Preparation of Adsorbent. . . . . . . . . . . Column Preparation. . . . . . . . . . . . Determination of Butyric and Higher Acids . . . . Reagents. . . . . . . . . . . . . . . . . . . Isolation of FFA from Cheese. . . . . . . . . Removal of FFA from Fat . . . . Materials . . . . . . . . . Procedure . . . . . . . . . Preparation of Butyl Esters Correction Factors. . . . . . . . . . . . Page 145 146 151 154 175 175 175M 175' 175 176 176 1176 177 177 178 179 180 10. 11. LIST OF TABLES Page Some acids found in Gorgonzola and Roquefort cheese. . . . . . . . . . . . . . . . . . . . . 15 Drying conditions used to prepare spray dried Blue cheese powders . . . . . . . . . . . . . . 54 Factors for relating the internal standards to the butyl esters of the other individual fatty aCids O O O O O O O O O O O O O O O O O O O O C 84 Concentration of free fatty acids in Imported Danish Blue cheese (mg acid/kg cheese). . . . . 86 Mole percentages of free fatty acids in Blue- veined type cheese. . . . . . . . . . . . . . . 88 Concentration of free fatty acids in quick ripened loose curd Blue-veined cheese (mg acid/ kg cheese). . . . . . . . . . . . . . . . . . . 91 Concentration of free fatty acids in filled quick ripened Blue—veined cheese (mg acid/kg Cheese) 0 O O O O O O O O O O O O O O C O I O O 97 Percentage recovery of individual methyl ketones at completion of analytical procedure . 108 Concentration of methyl ketones in fat ex- tracted from Blue-veined cheese (micromoles/ .10 g extracted fat) . . . . . . . . . . . . . . 110 Comparison of the mole percentages of the free fatty acids and methyl ketones found in Blue- vein type cheese. . . . . . . . . . . . . . . . 115 Comparison of the mole percentages of the free fatty acids and methyl ketones found in filled Blue-veined cheese. . . . . . . . . . . . . . . 126 vi LIST OF TABLES-~continued TABLE 12. 15. Page Retention of free fatty acids in spray dried and freeze dried Blue cheese. . . . . . . . . . 154 ~Retention of methyl ketones in spray dried and freeze dried Blue cheese. . . . . . . . . . . . 156 vii LIST OF FIGURES FIGURE - Page 1. Changes in pH during ripening of quick ripened Blue cheese curd. . . . . . . . . . . . . . . . 72 2. Changes in volatile acidity during ripening of quick ripened Blue cheese curd. . . . . . . . . 77 5. Separation of homologous series of methyl ketone DNP-hydrazones by thin-layer partition chromatography. . . . . . . . . . . . . . . . . 106 4. Temperature profile of freeze dried Blue cheese; Trial A O O O O O O O O C O O O O O O O O I O O 141 5. Temperature profile of freeze dried Blue cheese; Trial B O O O O O O O O O O O O O O O O O O O O 142 viii INTRODUCTION Blue mold cheeses have been steadily increasing in popularity and concurrently, food products flavored with Blue mold cheeses have been in greater demand. ~The produc- tion costs for Blue cheese have generally been greater than for most other varieties of cured cheese. The high labor requirements in manufacturing and curing Blue cheese have stimulated the develOpment of mechanized procedures for hooping curds and shortened methods for manufacturing and curing. These innovations have included direct acidifica- tion, production of Blue cheese flavor constituents by fer- mentation and the "quick ripening" of loose curd Blue cheese. The present high price for milk fat in relation to vegetable fats and oils has encouraged the formulation and fabrication of dairy products, including cheese, containing vegetable fat. The increasing demand for Blue cheese and for convenience foods by the consumer provides a potentially large market for a dry Blue cheese which could be used for preparation of Blue cheese flavored salad dressing and chip dips. This project was undertaken to evaluate the development of Blue cheese flavor in quick ripened loose curd Blue cheese, to determine if a coconut oil filled Blue cheese possessing a Blue cheese type flavor could be produced and to compare the loss of volatile flavor constituents from Blue cheese during spray drying and freeze drying. REVIEW OF LITERATURE The sharp, peppery flavor of Blue-veined or Blue mold cheeses has intrigued researchers for years. Recently, the origin and development of the characteristic piquant flavor of Blue mold cheeses has been studied quite extensively. The flavor of Blue—veined cheese can originate from several sources. The most significant flavor compounds are produced by the metabolism of microorganisms and by the action of natural milk enzymes during the manufacture and curing of cheese. The intrinsic flavor components of the milk from which the cheese is made and added salt also contribute flavor. ASpects of the flavor and manufacture of Blue-veined cheese were reviewed by Bakalor (1962). Flavor Components 6f Blue Cheese and Their Origin Free Fatty Acids The lipid components of the milk from which Blue cheese is made are probably the single most important source of the flavor associated with Blue cheese. Currie (1914) indicated that caproic, caprylic, and capric acids and their readily hydrolyzable salts were responsible for the "peppery“ taste of Roquefort cheese. Caproic, valeric and butyric acids were found in Blue mold cheese by Thomasow (1947). Using gas chromatography Coffman, Smith, and Andrews (1960) established the presence of butyric, isovaleric, caproic, heptanoic, caprylic and capric acids in a dried Blue cheese flavoring. The flavoring was a commercial product containing Blue cheese dried on a milk product. Succinic, lactic, formic and/or acetic acid were discovered in Roquefort cheese by Simonart and‘nayaudan (1956a). The same workers (1956b) demonstrated the presence of phenylacetic, p-hydroxyphenylacetic and p-hydroxybenzoic acid in Roquefort cheese. The aromatic acids are probably unrelated to fat hydrolysis, but may be important in the flavor of cheese. Utilizing gas chromatography Day and Anderson (1965) identified the major free fatty acids (Cg—C1833) in domestic Blue cheese. No formic, propionic or isovaleric acid was evident in any of the cheese samples analyzed. Morris 2; 3;. (1955) reported finding an average of 0.77 mg butyric acid/g of Blue cheese made from pasteurized homog- enized milk. The average caproic and higher acid value (average molecular weight 228) was 24.95 mg/g of cheese. Sj6str5m and Willart (1959) found 1.22, 0.92 and 26.8 mg/g of acetic, butyric, and caproic and higher (average molecular weight 200) acids respectively in Blue cheese. The C2-C13:3 fatty acids in domestic Blue and imported Roquefort were quantitated by Anderson and Day (1965). Considerable variation in the fatty acid content of the cheeses was noted. A dif- ference was also evident in the flavor and aroma of the cheeses. By calculating the mole percent of the free acids present in the cheeses, Anderson and Day (1965) demonstrated that octanoic and decanoic acids were proportionately more abundant than butyric acid in Roquefort cheese. The entire quantity of fatty acids reported probably did not exist as free acids in the cheese. Cheeses with a pH range of 5.5 to 5.6 may have had approximately 75 to 85 per cent of their free fatty acids in the salt form, assuming an average pKa of 4.8 for the major acids. Salts of long chain fatty acids were soaps and as such possessed definite flavor properties. The odor resulting from the short chain acids would be re- duced if these volatile acids were in the salt form. Glyceride Hydrolysis The enzymes involved in the process of fat hydrolysis in cheese may originate from: 1. true milk lipase (s) 2. microorganisms from the milk, and from the interior or the surface of the cheese (Stadhouders and Mulder, 1957). The presence of more than one natural milk lipase system has been demonstrated by various authors. Albrecht and Jaynes (1955) indicated that two lipase systems existed in raw skimmilk with optima at pH 5.4 and 6.5. According to Tarassuk and Frankel (1957), cows' milk contains at least two lipases, a plasma lipase, associated with casein and a membrane lipase, adsorbed on the fat globule membrane when milk is cooled. Gaffney, Harper and Gould (1966) and Downey and Andrews (1969) obtained results indicating that more than one lipase is present in milk, or that lipase is associated with a variety of other milk proteins of various molecular weights. Milk lipase was observed by Schwartz, Gould and Harper (1956) to have multiple pH optima. Three pH optima at 8.5, 6.5-7.0 and 7.9 were found at high (16.5 per cent) milk fat concentrations. At low fat concentrations (0.92 per cent) a broad optimum between pH 7.0 and 8.0 existed. ‘Chandan and Shahani (1965) isolated a pure milk lipase from milk clarifier slime with a pH optimum of 9.0 and the ability to hydrolyze milk fat within a pH range of 5.0 and 10. Those lipases with a pH optimum of 7 and above could not continue to have a high activity in normal Blue- veined cheese (Bakalor, 1962). When mixed triglycerides are hydrolyzed, there is an obvious preference for the fats containing short chain acids (Jensen, 1964). Milk lipase preferentially releases fatty acids from the primary positions of a triglyceride. Since butyrate appears to be predominantly a primary ester, an ap- parent preferential release of butyrate results. Harwalker and Calbert (1961) reported that lauric and higher acids were the major acids released at all stages of lipolysis, but as lipolysis progressed the ratio of these acids to butyric decreased. Very little change was observed in the mole per- centages of the other acid fractions. In the manufacture of Blue-veined cheese the curd is inoculated with spores of Penicillium roqueforti, although some makers add the spores to the milk before curdling. Penicillium roqueforti produces a water soluble lipase which hydrolyzes milk fat to produce the free fatty acids character- izing the sharp flavor of Roquefort cheese (Currie, 1914). Morris and Jezeski (1955) confirmed the findings of Thibodeau and Macy (1942) that the lipolytic activity of g, roqueforti is due to a lipase rather than an esterase system. Several workers have characterized two lipases associated with 1g. roqueforti. Morris and Jezeski (1955) obtained a lipase from mold mycelia that exhibited a pH optimum for milk fat hydrolysis at 6.5-6.8. Another lipase present in the growth medium had a pH optimum of 7.0-7.2. Reports by Imamura (1960a) and Imamura and Kataoka (1965b, 1966) indicated 2 types of lipase withaxloptimum pH of 6.5 and 7.5 reSpectively. Optimum lipase activity on butterfat was obtained at pH 6.0 and 7.5 on both the intra- and extra-cellular lipase of g, roqueforti examined by Niki, Yoskioka and Ahiko (1966). Using butter oil as a substrate Eitenmiller, Vakil and Shahani (1970) showed the g. roqueforti lipase to be active over a pH range of 7.5-9.0, with a pH optimum at 8. The authors (1970) suggested that differences in the pH optimum observed in their work and in that of other workers may be due to use of differ- ent substrates, different assay conditions and different strains of mold. Penicillium lipase was found by Shipe (1951) to hydrolyze tributyrin, tricaproin, tricaprylin and tripropionin in this order of decreasing rates. Eitenmiller, Vakil and Shahani (1970) demonstrated similar results. Triolein and tripropionin were hydrolyzed at much lower rates than the other trigly- cerides studied. Results obtained by Morris and Jezeski (1955) indicated that there was a decrease in mold lipase activity as the molecular weight of the fatty acid portion of the tri- glyceride increased. The extracellular g, roqueforti lipase studied by Imamura and Kataoka (1965a) liberated caproic, capric and butyric acids selectively from butterfat. Also, WilcOx, Nelson and wood (1955) reported that g, roqueforti lipase preferentially hydrolyzed short chain fatty acids from butter fat. Blue cheese was found by Jensen, Gander and Duthie (1959) to contain large amounts of monoglyceride as compared to whole milk, cream and butter. These monoglycerides may have some influence on the flavor of Blue-veined cheeses. Vujicic and de Man (1967) compared the hydrolysis patterns of the fat in various cheeses with the pattern obtained by Jensen, Sampunga and Gander (1961) when milk fat was hydrolyzed by milk lipase. Hydrolysis of the fat in Danish Blue and Roquefort cheeses left much more of the short chain (4:0, 6:0, 8:0) fatty acids in the partial glycerides than was the case with milk lipase. Monoglycerides in Roquefort cheese had a high medium chain (10:0, 12:0) fatty acid content. In the fat of Danish Blue and‘Roquefort cheese the long chain saturated (14:0, 16:0, 18:0) fatty acids were present in lower concentrations in the monoglyceride fraction than the triglyceride fraction. Short chain acid partial glycerides may contribute to the flavor of cheese (de Man, 1966). Kuzdzal and Kuzdzal-Savoie (1966) concluded that a comparison of free and esterified fatty acids may be of use in studying the agents producing lipolysis. The free fatty acid content of Blue cheese samples was found by Anderson and Day (1965) to be considerably different than that of a Roquefort cheese sample analyzed. The three Blue cheese samples contained relatively high percentages of butanoic acid compared to the Roquefort sample analyzed, i.e., 7.2, 9.0, and 9.8 versus 5.9 mole per cent (Anderson, 1966). The Roquefort sample contained more decanoic and octanoic acid than the Blue cheese samples. The Roquefort sample lacked the strong pungent flavor of butyric acid that was evident in the Blue cheese samples. Vujicic and de Man (1967) reported that the tri-, di-, and monoglycerides of Roquefort cheese contained higher amounts of caprylic and capric acids than did the glycerides of Blue cheese. Roquefort cheese is made from sheeps' milk. Sheeps' milk according to Hilditch (1956) con— tains less butyric acid and more capric and caprylic acid than 10 bovine milk. Likewise, Kuzdzal-Savoie and Kuzdzal (1965), Benassi (1965) and Sadini (1965) reported that ewes' milk con- tained greater amounts of capric acid than cows' milk. The differences in fatty acid composition of ewes' milk versus cows“ milk may be partly responsible for the difference in the flavor of a Roquefort versus a Blue cheese. ‘Different strains of g, roqueforti show certain differ- ences in their lipolytic properties. The proper ripening of a Roquefort—type cheese is therefore dependent to some degree upon selection of the appropriate strain of mold (Proks, Dolezalek and Pech, 1959c). Thibodeau and Macy (1942) also found differences in the lipolytic activity of some strains of g, roqueforti. Certain strains observed by Lane and Hammer (1958) combined luxuriant growth with desirable flavor, while other strains yielded cheese having undesirable flavors and poor mold growth. Not one of eight strains of mold examined by Parmelee and Nelson (1947) was found to be suitable for use in making Blue cheese from pasteurized milk. Three out of eight strains studied by Graham (1958) produced well-veined and good-flavored cheese in routine manufacture. The strains used by Parmelee and Nelson (1949b), to prepare a mold-enzyme preparation for use in homogenized milk, varied greatly in their ability to hydrolyze milk fat. The effective use of the mold-enzyme preparation depended upon careful selection of the mold strain to be used. The lipolytic properties of nine strains of g, roqueforti were examined by Proks, Dolegalek 11 and Pech (1956). Chromatographic analyses showed differences in the ability of the nine strains to hydrolyze butyric, caproic and caprylic acids from butterfat.) The results of Niki, YOshioka and Ahiko (1966) demonstrated that high lipolytic strains accumulate lower fatty acids rapidly, subse- quently decomposing them into methyl ketones and other metabolic products. Strains with moderate proteolytic activ— ity and strong lipolytic activity are the most suitable for the purpose of Blue cheese production. Therefore, proper strain selection is necessary for the production of a quality Blue cheese. The pH and temperature of the system are important, because they may influence the activity of the enzyme systems involved in ripening (Morris and Jezeski, 1955). Coulter, Combs and George (1958b) observed a rapid reduction to pH 4.7 during the first day of ripening. After three months, the pH had increased to 6.5; then the pH decreased slowly to 5.7 at nine months. The marked rise in pH from about 2 weeks to 10 weeks was due to continuing proteolysis (Currie, 1914; Morris, Combs and Coulter, 1951). Morris (1965) noted a tendency for the rate of rise of pH to decrease as the pH approached 6.5. The lipase systems in Blue cheese were active in the pH range occurring throughout curing. The Changes in lower free fatty acids during ripening were Studied by Niki, YOshioka and Ahiko (1966). Butyric and caProic acid content increased over a 21 week period as did the 12 total volatile free fatty acid content. According to Morris §£_§l, (1965), the free fatty acids (mainly butyric, caproic, caprylic and capric) increased in concentration during ripen- ing, thus indicating lipase activity throughout the ripening period. The temperature optima for the two mold lipases was found to be between 50-55 C by Shipe (1951) and 50-52 C by Morris and Jezeski (1955). Morris (1969) concluded, in a study of the effect of ripening temperature on the properties of Blue-veined cheese, that those cheeses ripened at 57 F scored better than those ripened at 50 F or 60 F in relation to mold color, body and flavor. Higher temperatures produced more rapid breakdown of fat and protein, but were found to be detrimental to the flavor, body and color of the cheese. Factors other than pH and temperature may also influence the microorganisms and enzymes present in a ripening cheese. Gould (1941) found NaCl inhibited milk lipase in raw homogenized milk*and cream. Levels of 5 to 8 per cent NaCl were sufficient to~inhibit lipolysis almost completely. In cheese of the Swedish "Svecia" type Willart and Sjfistr6m (1959) found that increasing amounts of salt caused decreasing amounts of free fatty acids to be liberated. The lipolytic activity of the intra- and extra-cellular g, roqueforti lipase enzymes, studied by Morris and Jezeski (1955), decreased with increasing salt content. Between 10 and 16 per cent salt, which approximates the salt concentration in the water of Blue cheese, the lipase 15 activity was 40 per cent less than the activity found in the absence of NaCl. Increasing the salt content from 5.0 to 5.9 per cent was found by Poznanski, Jaworski and D'Obyrn (1966) to slow down the increase in fat acidity in Roquefort- type cheeses. Most organisms cannot survive salt concentra- tions greater than about 6 per cent, and essentially none survive a concentration of 10 per cent (Morris, 1969). .2. roqueforti can grow at salt concentrations up to about 16 per cent brine. A 10 per cent brine is the concentration maintained in Blue-veined cheeses. Therefore, about a 4 per cent salt concentration in a cheese of 40-44 per cent moisture will provide the proper brine concentration to discourage contaminating molds, yeast and bacteria from growing in Blue- veined cheeses. Another important factor involved in maintaining proper mold growth is the necessity of having exchange of carbon dioxide, produced by the mold, with oxygen from the air during ripening. Studies by Golding (1940a,b) revealed that mold would grow in a low (4.2 per cent) oxygen concentration, but growth was inhibited by a large concentration of carbon dioxide. Low concentrations of carbon dioxide favored mold growth. Skewering cheese allowed the carbon dioxide to escape, while providing for the admission of oxygen. Other microorganisms, in addition to g, roqueforti, may be involved in the production of fatty acids. In a study of the hydrolysis of milk fat by microbial lipases, Khan §£_§l, 14 (1966) noted that Blue cheese free fatty acid composition suggests that the lipase of g, roqueforti, as well as some other lipase, may be involved in the flavor development of Blue cheese. Microorganisms growing on the surface of cheese were shown by Stadhouders and Mulder (1957) to have a marked influence on fat hydrolysis. Enzymes of milk bacteria re- mained active in cheese long after the bacteria have died and autolyzed. Extracts of the genera Alcaligenes, Achromo- bacter, Pseudomonas and Serratia caused hydrolysis of the fat in cheese. Lipases from the genus Pseudomonas and Achromo- bacter were found to be sufficiently thermoresistant to with- stand pasteurization and remain active in cheese (Stadhouders and Mulder, 1960). Two cultures of Geotrichum candidum were found by Wilcox, Nelson andeood (1955) to release butyric acid from milk fat. »Acids from Sources other than Milk Triglycerides Some of the acids identified in Blue cheese do not occur in milk triglycerides or are present in very low concentrations (Anderson, 1966). Acids such as valeric (Thomasow, 1947). isovaleric and heptanoic (Coffman, Smith and Andrews, 1960), and acetic (Simonart and Mayaudon, 1956a) are in this classi- fication. Table 1 lists some of the acids found in Roquefort and Gorgonzola cheese (Schormfiller, 1968). Acids such as oxaloacetic, oxalosuccinic and alpha-acetolactic have been identified in Blue cheese by Bassett and Harper (1958). 15 Table 1. Some acids found in Gorgonzola and Roquefort cheese # Lactic n-Valeric Succinic Glyoxalic Mblic Pyruvic Fumaric alpha-Ketoglutaric Formic alpha-Ketobutyric Acetic p-Hydroxyphenyl pyruvic Propionic‘ p-Hydroxyphenyl propionic iso-Butyric Phenyl propionic Acetoacetic V/{Many microorganisms have the ability to carry out glycolysis, which provides for the formation of lactic acid and its metab— olites acetic and propionic acid. Intermediate stages of the citric acid cycle provide succinic, pyruvic, fumaric, glyoxalic and alpha-ketoglutaric acid. Free amino acids are decomposed by trans- and deamination to form alpha-keto butyric, p-' hydroxyphenyl pyruvic, p-hydroxybenzoic, and other keto acids. Keto acids in Blue cheese can also be formed through citric acid fermentation and beta-oxidation (Harper, 1959). The value to aroma and taste of all these acids differed, but they may have contributed to the flavor of Blue-veined cheese. Methyl Ketones The contribution of methyl ketones to the flavor and aroma of Roquefort cheese, and the involvement of g, roqueforti in the production of methyl ketones was indicated by Starkle (1924). Roquefort cheese was found to have an odor character- istic of 2-heptanone and 2-nonanone. Patton (1950) isolated 16 2-pentanone, 2-heptanone, and 2-nonanone from Blue cheese and concluded, as did Hammer and Bryant (1957), that these methyl ketones, particularly 2-heptanone, have an aroma typical of Blue cheese. Roquefort, Danish Blue, Domestic Blue and Gorgonzola cheeses were analyzed for their ketone content by Morgan and Anderson (1956). -The C3-C1; odd numbered methyl ketones were present in each of the cheese samples analyzed. One sample contained 2-butanone, which was thought to have originated from valeric acid. The meta- bolic activity of a contaminating organism may have produced the valeric acid. A sample of cheese possessing little, or no, desirable aroma contained no 2-heptanone or 2-nonanone. Proks, DoleEalek and Pech (1959b) found a direct relationship between the amount of methyl ketone present, and the typical flavor of Roquefort—type cheeses. The acidic and neutral carbonyl compounds of Blue cheese were isolated and identified by Bassett and Harper (1958). Acetone and 2-pentanone were among the neutral carbonyl compounds identified. A concentrate of the volatile organic compounds in Blue cheese was analyzed, by Scarpellino and Kosikowski (1961), using gas chromatography. The presence of 2-heptanone and 2-pentanone in the concentrate was noted. Bavisotto, Rock and Lesniewski (1960) identified 2-octanone in Blue cheese, and Nawar and Fagerson (1962) identified 2-butanone, 2—hexanone, and 2-octanone in Roquefort cheese. The C3-C9 odd-numbered methyl ketones, as well as 2-butanone, were found in Niva (Blue-veined cheese) by 17 Doleéalek and Brabcova (1964). Normanna (Norwegian Blue-type) cheese was found to contain the C5, C7 and C9 methyl ketones and 2-nonen (8,9)-one (Svensen and Ottestad, (1969). Gas chromatographic, and mass Spectral identification of the com- ponents of Blue cheese aroma, by Day and Anderson (1965), confirmed the presence of C3, C5, C5, C7, C8, C9, C10, C11 and C13 methyl ketones in Blue cheese volatiles. The ketone content of a well ripened Blue cheese was determined by Nesbitt (1955) to be about 60 to 80 ppm. The C3, C5, C7, C9, C1; and C13+15 methyl ketones in domestic Blue and Roquefort cheese have been quantitated by Schwartz and Parks (1965) and Schwartz, Parks and Boyd (1965) respec- tively. The major ketone in all the cheeses, except one Roquefort sample, was 2-heptanone. The Roquefort sample con- tained more 2-nonanone than 2-heptanone. No definite ratio of ketones was apparent and the quantity of individual ketones in the cheese samples varied considerably. Quantitation of the methyl ketones in Blue-veined cheeses, by Anderson and Day (1966), indicated a large variation in the quantity of ketones in different cheeses, with 2-heptanone being the pre- dominant ketone in all samples. No consistent difference between Blue and Roquefort cheese was found; however no acetone was found in one of the Roquefort samples. Although the cheeses varied in their respective methyl ketone contents all the cheeses were judged as having an acceptable flavor. 18 Starkle (1924) attempted to determine the origin of the methyl ketones, 2-nonanone and 2-heptanone, that had been distilled from Roquefort cheese. Aspergillus niger, A, fumigatus and Penicillium roqueforti when grown for several weeks in pure culture, with individual fatty acids as the carbon source, produced methyl ketones of one less carbon atom than the fatty acids used as the carbon source. A mechanism advanced, for the formation of these ketones, was the oxida- tion of the fatty acid to the beta-keto acid followed by decarboxylation of the beta-keto acid. Penicillium palitans and g, glaucum, but not the nonSporeforming Oidium lactis were found to produce methyl ketones when grown on coconut oil (Stokoe, 1928). Ketone producing molds when grown on the coconut oil were postulated to have been poisoned sufficiently by the fatty acids produced that the normal scheme of oxida- tion was altered. Girolami and Knight (1955) confirmed much of the earlier work by showing that resting cells from surface- grown cultures of g, roqueforti formed a methyl ketone from individual fatty acids. There are conflicting reports on the ability of the vegetative cells of molds to produce methyl ketones. Starkle (1924) and Stokoe (1928) showed that when single fatty acids are used as substrates certain mold mycelia produced methyl ketones. Gehrig and Knight (1958, 1965) demonstrated that the ability to form methyl ketones from fatty acids resided in the mold spore. The capacity to form methyl ketones disappeared rapidly and progressively as the 19 spores germinated. The slow rate of formation of 2-heptanone from octanoic acid by washed Spore suspensions of g, roqueforti was shown, by Lawrence (1965), to be markedly increased by the addition of the same specific amino acids and sugars that have been shown to stimulate mold germination. That only methyl ketones of one fewer carbon atoms are produced from fatty acids, has been confirmed by Gehrig and Knight (1965) with P. roqueforti Spores using {1—14CJ- octanoate as substrate. The 2-heptanone synthesized was un- labelled and 14C02 was produced. Lawrence (1966) found that the 2-heptanone formed from [2-‘4CJ-octanoic acid was radio- active and that some radioactive carbon dioxide was also present. Therefore, ketone formation and beta-oxidation pro- ceed simultaneously. The 2-C moiety (presumably acetate) formed by beta-oxidation is further oxidized by the tricar- boxylic acid cycle. The pathway of fatty acid oxidation and methyl ketone formation may be outlined by the following scheme (Hawke, 1966). 20 Pathway of Fatty Acid Oxidation and Methyl Ketone Formation + RCHgCHaCOSCOA 43-5— RCH=CHCOSCoA -2H J r .i H20 +2H3 RCOCHgCOSCOA RCHOHCHgCOSCoA \\\\ -2H , Deacylase CoASH )1 H20 \ RCOCHZCOOH + CoASH RCOSOOA + CHSCOSCOA Decarboxylase Further formatio:\;f§*C02 + H20 l CH3COSC0A (via TCA cycle) RCOCH3 + C02 This mechanism provides for the formation of only methyl ketones of one less carbon atom than the fatty acids used as substrate and for beta~oxidation to proceed simultaneously. Gehrig and Knight (1965) found that when one micromole of labeled sodium octanoate was used as a g, roqueforti substrate most of the radioactivity was present as carbon dioxide and no ketone was produced. At higher substrate concentrations (20 micromoles), part of the molecule appeared as 2-heptanone and part as carbon dioxide. Neither Gehrig and Knight (1965) nor Lawrence (1966) were able to find any of the intermediate compounds of the beta-oxidation of octanoic acid, such as beta-oxoacyl esters of hexanoic and butanoic acids. Beta- keto acids which are intermediates of fatty acid oxidation 21 have been isolated from Blue cheese by Bassett and Harper (1958). .Van der Ven, Begemann and Schogt (1965) isolated from butterfat six even-numbered carbon (C5 to C13) beta- keto acids. The presence of beta-keto acids supported the contention of Wong, Patton and Forss (1958) that beta-keto esters are the precursors of methyl ketones liberated in butterfat upon heating in the presence of moisture. Lawrence (1965) established that a complete range of methyl ketones with odd numbers of carbon atoms from C3 to C15 were formed during*the heat treatment of butterfat. The beta-keto acid containing glycerides were isolated from butterfat by Parks §t_al, (1964). These glycerides were found to constitute about 0.04 per cent of butterfat. The amount of methyl ketones of intermediate chain length found in the fat phase of cheeses eliminates the natural ketone precursor of milk fat as the direct and only source of ketones in cheeses (Schwartz and Parks, 1965). The small amounts of the C13 and C15 methyl ketones which are found in Blue cheese are probably formed from the natural breakdown of precursors rather than by microbial action. No methyl ketones were liberated from the fat of Blue cheese by heat treatment in the presence of water. Schwartz and Parks (1965) indicated that possibly the methyl ketone precursors in milk are metabolized by micro- organisms and therefore make a small contribution to final methyl ketone levels. 22 Results of work, by Anderson and Day (1966), indicate that the quantity of each methyl ketone produced by the action of _P_. roqueforti in Blue cheese does not depend directly upon the amount of available fatty acid precursor. The average mole per cent of the individual methyl ketones and their fatty acid precursors were compared. The proportion of acetone in fOur cheeses (average 6.2 mole per cent) was rela- tzively low compared to its precursor, butanoic acid (average .50 mole per cent). The amount of C12 acid was high compared tn: the amount of C11 ketone produced. Conversely, the C9, C8 and C10, acids represented a smaller percentage of the fatty acids, while the C5, C7 and C9 ketones constituted a high percentage of the total ketones. The percentage of cieecmanoic acid in a Roquefort cheese sample was almost double 1:1123t: of the Blue cheese samples analyzed, yet the concen- tration of 2-nonanone in the Roquefort was only slightly above tZIIEit: of the Blue cheese samples. Lawrence (1966) determined the oxygen uptake and the formation of methyl ketones by spores of _11. rcmueforti in the presence of fatty acids at various pH values. Methyl ketones ‘Vv‘alrfié not detected when spores were incubated with butyrate or myristate at any pH value, but oxygen uptake was evident. Makimum methyl ketone yield was obtained between pH 5.5 and 7 .‘0- Up to 75 per cent of the octanoate, 45 per cent of the decanoate, 25 per cent of the laurate and 5 per cent of the ekanoate were ox1d1zed to the corresponding ketone. 25 Decanoic acid gave the greatest yield of correSponding methyl ketone at pH 7.5. The optimum pH value for maximum ketone ‘formation depended upon the concentration of the acid being oxidized. Lawrence (1966) postulated that a decrease in the uncoupling of phosphorylation activity from myristic to octanoic acid may be a factor in the progressive increase in the yield of corresponding methyl ketone when these acids were incubated with spores. The optimum temperature for methyl ketone formation was 25-50 C (Gehrig and Knight, 1965; Lawrence, 1966) . The metabolism of triglycerides by spores of g. roqueforti was examined by Lawrence (1967) . Maximum yield of methyl ketone was obtained from trioctanoin and trihexanoin at pH 6.0, with lower yields from tridecanoin, tributyrin and trilaurin. Girolami and Knight (1955) reported that methyl ketones were themselves inhibitory to g. roqueforti QrOWth and suggested that this may prevent excessive mold grOWth in ripening cheese. Different strains of g. roqueforti exhibit certain dif- ferences in their desirability for use in Blue-veined cheese InaIiufacture. Lane and Hammer (1958) observed that the in- tensity of flavor in Blue cheese varied with certain mold S‘tIIE‘ains. A high lipolytic strain and a high proteolytic S‘tI‘ain were studied by Niki, Yoshioka and Ahiko (1966) with r’gspect to their ability to produce methyl ketones. The Q1'1eese made using the high lipolytic strain produced ketones I: . . a~Pldly after seven weeks and reached a max1mum ketone level 24 in twenty-one weeks. A typical Blue cheese flavor was evident after twenty-one weeks. The ketone content was low in cheeses made using high proteolytic strains and the character- istic flavor was weak throughout the ripening period. The high lipolytic strain had a strong ability to produce methyl ketones. The amount of carbonyls produced by certain strains at 1, 2 and 5 months of ripening was determined by Sato 22.2;- (1966). During ripening the quantity of carbonyl compounds increased, but poor mold growth resulted in low amounts of ketone and aldehyde. With strains that provided good mold growth adequate amounts of ketone were produced concomitant with a cheese having a good flavor. Seven strains of g, roqueforti were grown on cream, and their ability to pro- duce ketones determined chromatographically (Dolezalek and Iflaza, 1969). The ability to produce methyl ketones was dependent upon the strain of g. roqueforti used, as well as the temperature of ripening. Secondary Alcohols Secondary alcohols were recognized by Stokoe (1928) as beiJug one of the products that resulted from the action of .3. Inoqueforti on coconut oil. Results appeared to indicate that: secondary alcohols were intermediate products in the OXidation of fatty acids to methyl ketones, but if not inter- :medjiite products they must have been produced by reduction of ketones. The alcohols 2-pentanol, 2-heptanol, and 25 2-nonanol were isolated from Blue cheese by Jackson and Hussong (1958). The secondary alcohols were not observed to appear until considerable amounts of methyl ketone were pro- duced, which suggested that the alcohols were formed as a result of the reduction of the corresponding methyl ketone analog. Indirect support for the reduction of methyl ketones was the presence of beta-ketocaprylic and beta-ketocapric acids in Blue cheese (Bassett and Harper, 1958). Gas chroma- tographic analysis of Roquefort cheese volatiles, by Nawar and Fagerson (1962), confirmed the presence of 2-pentanol and 2-heptanol. The alcohols 2-pentanol and 2-nonanol were found, by Svensen and Ottestad (1969), to be the principal secondary alcohols present in Normanna (Norwegian Blue-type) cheese. Day and Anderson (1965) identified 2-propanol, 2-pentanol, 2-heptanol, 2-octanol and 2-nonanol as components latile flavor entrapped in the dried particle. 48 The effect of processing variables on the loss of diacetyl and acetoin in acidified, freeze-dried cream was studied by Radanovics (1969). Increases in platen temperature resulted in increased losses of both diacetyl and acetoin. A 45.5 per cent loss of diacetyl was observed at a 75 F platen temperature, while a 79.1 per cent loss occurred at a platen temperature of 225 F. Overall losses of acetoin appeared to be higher than diacetyl, which is contrary to what would be expected, if the losses were a function of boiling point alone. Increased platen temperature, and reduction of product layer thickness reduced the drying time. Varying the absolute pressure in the vacuum chamber within a range of 20-550 microns did not influence the relative loss of diacetyl or acetoin. When aroma compounds are contained in a complex food structure their loss during dehydration is reduced due to various physical and chemical interactions with the food com— Ponents. Using a glucose—glycine-acetone system in freeze- drying experiments, Rey and Bastien (1962) demonstrated that 'theeretention of acetone increased with increasing glucose chncentration. Whether acetone retention was increased by sOrption on the glucose, or by reduction in the rate of diffu— siran through the dry surface layer with higher levels of glu- c308a was not clear. In the spray-drying of coffee, aroma retention was dependent upon the concentration‘of dissolved Sc>lids in the solution to be dried (Sivetz and Foote, 1965) . Retention of acetone during the air drying of aqueous 49 malto-dextrin droplets was studied by Menting and Hoogstad (1967). Larger amounts of acetone were retained as the initial concentration of malto~dextrin in the droplets was increased. Acetone retention was believed to have been caused by the formation of a film that is permeable to water, but impermeable to acetone. The film is formed on the surface of the droplet, and the higher the initial malto-dextrin concentration the faster the film forms. For the membrane around a drying drop- let to act selectively it must have a moisture content of 9 per cent or lower. Flink and Karel (1970) also observed a critical moisture level in a freeze-dried model system contain- ing maltose. Below a certain critical moisture level volatile loss ceased. The authors (1970) postulated that as the water content reaches a critical level association between carbo- hydrate molecules increases as hydrogen bonds between carbo- hydrate hydroxyls and water are replaced by carbohydrate- carbohydrate hydrogen bonds. The small size of the water mole- cule allows it to pass through the sealed microregions, while tile volatile is sealed or trapped in the carbohydrate sealed midcroregion. Radanovics (1969) found the critical moisture cCnatent of freeze-dried sour cream to be somewhere above 1.9 per cent for diacetyl retention. Increasing the moisture to 4-€3 per cent resulted in a diacetyl loss of 82.6 per cent. .Thfe diacetyl was believed to be adsorbed more strongly than th€e water in the freeze-dried system. 50 The per cent retention of diacetyl during spray-drying of skimmilk was shown by Reineccius and Coulter (1969) to increase as the total solids of the infeed increased. Virtually all the added diacetyl was retained when 50 per cent skimmilk was used as a carrier. During the vacuum drying of fruit juices Saravacos and Moyer (1968a) observed that the retention of volatile compounds was improved, if concentrated juices were used. A 15 degree Brix grape juice when dried, retained less aroma compounds than a 50 degree Brix juice after drying. The presence of large amounts of sugars in solution appeared to trap significant amounts of aroma compounds. Dimick, Schultz and Makower (1957) prepared "locked-in" fruit flavors by trapping aroma compounds with carbohydrates. In the early stages of drying, water evaporation is at a constant rate and the loss of aroma compounds is determined by their relative volatility as compared to water (Malkki and ‘Veldstra, 1967). Saravacos and Moyer (1968b) made some ob- servations on the relative volatility of various compounds in Enqueous solutions. Acetic acid in aqueous solution was less VCDlatile than water, while ethyl butyrate which has about the Same vapor pressure as acetic acid was extremely volatile. A luigh boiling ester, methyl anthranilate, found in grape jtliJce was more volatile than water in aqueous solutions. The esters being only partially soluble in water are more volatile, ‘Wfiille acetic acid is less volatile than water, because the maici is completely soluble in water. Retention of 95 per cent 51 of methyl anthranilate (500 ppm) in a freeze-dried cellulose gel was observed even though the volatility of anthranilate was greater than the water. Issenberg, Greenstein and Boskovic (1968) used frontal analysis gas chromatography to study the adsorption of volatile organic compounds on microcrystalline cellulose powder, and to obtain adsorption isotherms for hexane and acetone. The resulting isotherms indicated the possible existence of an interaction between adsorbent and adsorbate. Frontal analysis gas chromatography was employed by Issenberg, Boskovié and Hwang (1969) to determine sorption and'desorption isotherms of some aliphatic straight chain alcohols and acetates of these alcohols on microcrystalline cellulose. The use of a flame ionization detector in frontal analysis gas chromatography permitted study of concentration ranges (ppm) encountered in real dehydrated foods. EXPERIMENTAL PROCEDURE Preparation of Samples for Spray Dpyinq and Freeze Drying Thirty-nine pound lots of Imported Danish Blue cheese were comminuted and slurried with distilled water to 40 per cent total solids. Eight pounds of slurry were poured into a 18% x 25 x 1 inch stainless tray resulting in a layer of cheese-é inch thick. Prior to freezing the slurry to -50 F in the freeze dryer, 5 thermocouples (iron-constantan) were inserted at various locations in the slurry to permit tempera- ture measurement throughout the drying cycle. Two pounds of slurry were placed in a beaker and stored at 40 F for subse- quent chemical analyses. Prior to Spray drying the remainder 0f the slurry was heated to 120 F, and homogenized at 2000 PSig--two stage with a Manton-Gaulin homogenizer. Freeze Drying of Imported Danish Blue Cheese The Danish Blue cheese slurries were freeze dried in a Vilftis RePP #FFD 42 WS freeze dryer capable of removing 50 IKNJITds of water per drying trial. Condenser and platen Ibmn£>erature and vacuum adjustment were automatically controlled 52 55 during the drying cycle. The temperature of the product was constantly recorded throughout the drying cycle by means of thermocouples placed at the bottom, center, and at the sur- face of the product layer, and connected to a Honeywell Electronic 15 Multipoint Strip Chart Recorder. -The product was freeze dried at a platen temperature of 100 F and a vacuum of 10'3 Torr. The freeze drying process was terminated when the temperature indicated by the thermo- couple located on the bottom of the product layer was within 10 F of the platen temperature. The freeze dried cheese was removed from the tray, sized to pass through a U. S. Standard #8 sieve (2.58 mm openings) and stored at 40 F in polyethylene bags. Spray Drying of Imported Danish Blue Cheese Heated, homogenized slurries were fed to a Manton-Gaulin liigh pressure pump equipped with a fluid drive variable speed Chantrol. The automatic speed control permitted control of tlie spray chamber temperature at the desired preset point, by Cllanges in the pump speed to suit variation in inlet tempera- tlxre, temperature of the fluid feed, and per cent of solids 111 'the fluid feed. The slurries were atomized through a SPraying Systems nozzle (Table 2) into an experimental vertical do“rm-draft Spray dryer designed and manufactured by the Malflriott Walker Corporation, Birmingham, Michigan. The drying ccunditions used to prepare the spray dried Blue cheese powders 54 are shown in Table 2. The dried product was collected in a high-efficiency cyclone dust collector and continuously dis— charged through an air lock valve into a collection vessel. The powder was stored at 40 F in polyethylene bags. Table 2. Drying conditions used to prepare spray dried Blue cheese powders. Conditions Variable Trial 1 _ Trial 2 Inlet air temperature, F 285 294 Exit air temperature, F 178 17? Ambient air temperature, F 64 80 Nozzle SBC SBC Core standard standard Orifice diameter 0.058 0.058 Atomization pressure, psig 400 450 Preparation of Quick Ripened Loose Curd glue-Cheese Blue cheese curd was manufactured by the method of HeBdrick, Kondrup and Williamson (1968) using pilot laboratory allxipment described by Kondrup and Hedrick (1965). To facili- 'tate whey drainage the manufacturing procedure was modified to Zinclude a bagging step. Subsequent to draining, the curd was placed in coarse muslin bags and the bags turned frequently 55 for 10 minutes. The curd was removed from the bags and placed on a fine mesh stainless steel screen at a depth of 2-5 inches. The product was moved to a curing room maintained at 62 F and 95 per cent relative humidity. A ripening period of 7 days produced a curd possessing sufficient flavor. The ripened curd was packaged in polyethylene bags and stored at 40 F until analyzed. Preparation of Milk for Filled Cheese Manufacture Filled milk (4 per cent fat), for use in making filled cheese, was formulated from skimmilk and vegetable fat. A hydrogenated coconut oil with an iodine value of 4 (Modler, 1969) was used as the fat source. Fat and skimmilk mixture heated to 150 F were homogenized in a two stage Manton-Gaulin homogenizer at pressures of 1700 and 400 psig on the first and second stages respectively, Continuous agitation on the intake :iide of the homogenizer, by means of a model F Lightnin stirrer, “Has necessary to insure that the oil portion was uniformly distributed in the aqueous phase during homogenization. The Clleese manufacturing procedure followed was the same as that fcxr quick ripened loose curd Blue cheese (Hedrick, Kondrup and Wj-ngiamson, 1968), except that filled milk was used in place .QE’ standardized (4 per cent) cows‘ milk. ANALYTICAL PROCEDURES Moisture Moisture contents of cheese samples, slurries, and reconstituted cheese powders were determined by a method of the Association of Official Agricultural Chemists for cheese (AOAC, 1965). The percentage moisture in spray dried and freeze dried Blue cheese powders was determined by the Karl Fischer method (American Dry Milk Institute, Inc., 1954) employing a Beckman Model KF-2 Aquameter equipped with a duo- platinum electrode. 23 The pH measurements were made with a Beckman Expandomatic Ffii meter using a calomel half cell and a glass electrode. The Pfi‘was determined to the nearest one-tenth of a pH unit. Volatile Acidity Volatile acidity was determined by the rapid direct- distillation method of Kosikowski and Dahlberg (1946). A 10 g sarnmple of quick-ripened loose curd Blue cheese was analyzed 56 57 each day throughout the ripening period and the results re- ported as ml of volatile acid per 100 9 sample. gpantitation of Free Fatty Acids Acetic Acid The liquid-liquid partition column of Wiseman and Irvin (1957), as adapted by Bills and Day (1964) for Cheddar cheese analysis, was used with some modifications for acetic acid Modification analyses. See Appendix for complete procedure. involved the following: 1. A 20 x 500 mm chromatographic tube with a 500 ml liquid reservoir attached at the top, and equipped with a fritted- glass filter and 2 mm Teflon delivery stopcock, was used. 2. Adsorbent was prepared in a quantity'sufficient for the preparation of one column. Two and four tenths ml of alphamine red-R (5-[4-anilino-1-naphthylazo]2,7-naphtha1ene UQ 93024.5 |||| 0.0 mug—E .34 §m>< inn Hd Ow 75 coincided with the first visible signs of mold growth and the initial salting of the curd. After initial salting the pH decreased gradually to about 5.8 at the end of the seventh day. During subsequent storage at 40 F the pH dropped to about 5.7. Wide variations in pH on the third, fourth and fifth days respectively were evident. Fresh curd prior to being placed in the curing room exhibited a pH of approxi- mately 5.5. The maximum acidity in the quick ripened Blue cheese batches analyzed was not reached until approximately 48 hr after manufacture. The Short curing method of Kondrup and Hedrick (1965) used to prepare-the quick ripened Blue cheese utilized 0.5 per cent starter, which may account for the Slow decrease in pH during the initial stages of ripening. Maximum acidity is generally reaChed within 24 hours in Blue cheese manufacture (Foster g£_§l,, 1961). Most procedures used for the manufacture of Blue cheese utilize 2 to 4 per cent starter. Coulter, Combs and George (1958b) and Thibodeau and Macy (1942), using 2 to 5 per cent starter, found pH values reached a minimum about 24 hours after the manufacturing process had been initiated. The early increase in acidity was attributed to the production of lactic acid by Streptococcus lactis-type starters. Average pH values of 4.7 to 4.9 were observed on the first day of manufacture by Bakalor (1965) when 4 per cent starter was used to prepare Blue cheese from pasteurized homogenized milk. Slower acid development was 74 noted by Morris (1964) when 0.75 per cent starter was used with a maximum acidity being attained on the seventh day of ripening. The pH of quick ripened Blue cheese curd increased from its initial minimum to a maximum of 6.2-6.6 at 4 days. The increase in pH between the second and fourth days of ripening may have resulted from proteolysis by the starter organisms and Penicillium roqueforti. The initial gradual decrease in acidity between the second and third day in the quick ripened Blue cheese samples analyzed may have been the result of lactic acid destruction by g, roqueforti. -Foster g£_§l, (1961) suggested lactic acid destruction as a reason for initial gradual decrease in acidity in normally cured Blue cheese. Lane and Hammer (1958), Morris, Combs and Coulter (1951) and Currie (1914) noted that there was continuing proteolysis and an increase in pH as Blue cheese ripened. Cheese made from heat-treated homogenized milk was observed by Morris QEIQL. (1965) to reach a maximum pH of 6.0 at about 24 weeks. Data presented by these authors (1965) revealed a progressive increase in tyrosine during ripening indicating proteolysis may have been responsible for the rapid rise in pH after the fourth week. Amino nitrogen values were shown by Coulter, Combs and George (1958a) to increase with time in essentially a straight line relationship during ripening. Increased levels of free fatty acids from lipolysis by the mold lipase system may have been responsible for the 75 decrease in pH to 5.7 at seven days in the quick ripened Blue cheese. Morris (1964), Coulter, Combs and George (1958b) and Morris 2; El- (1965) noted a slow decrease in pH to about 5.7 at 4 to 9 months in Blue cheese and attributed this decrease in pH to an accumulation of free fatty acids in the cheese. Changes in Volatile Acidity During Ripening Lane and Hammer (1958) concluded that a general relation- ship existed between volatile acidities and flavor scores. A highly significant correlation was found between volatile acidity and flavor scores of Blue cheese made from pasteurized homogenized milk to which lipases or organisms producing lipases had been added (Parmelee and Nelson, 1949c). In general, cheese with the highest flavor scores had volatile acidities in the range of 50-55 ml of 0.1 N acid per 200 g of cheese. Since there is a relationship between volatile acidity and the flavor scores of Blue cheese, batches of ripened Blue cheese were analyzed for their volatile free fatty acid content. A sample of cheese, as prepared for pH determinations, was analyzed each day of the ripening period for volatile acidity. The rapid direct-distillation method used to deter- mine volatile acidity was reported by Kosikowski and Dahlberg (1946) to recover nearly 100 per cent of the short chain volatile fatty acids, such as acetic, butyric and caproic. 76 The authors (1946) found that about 90 per cent of the caprylic and a smaller percentage of capric and lauric acid was re- covered. The distillate collected contained the water soluble volatile acids; the condenser was rinsed with 25 ml neutral ethanol to recover the water insoluble volatile acids. The mean volatile acidities for 16 batches of quick ripened Blue cheese, determined at 24 hour intervals, are shown in Figure 2. The standard deviations of volatile acidities for the cheese in all trials are also Shown. From the first to the Second day of ripening the average volatile acidity of quick ripened Blue cheese decreased from an average of 21.1 to 15.5 ml of 0.1 N acid per 100 g of cheese. Between the second and fourth day there is a Slow, but definite, increase in volatile acidity. Mold growth was first visible and initial salting took place on the fourth day, after which the average volatile acidity increased very rapidly such that by the seventh day there was an average of 77.2 ml of 0.1 N acid per 100 g of cheese. A wide variation h.- in volatile acidity was apparent on the sixth and seventh days as the standard deviation (9.4) for those days illustrates. 5. Quick ripened Blue cheese stored for a month or more at 40 F commonly had volatile acidities in excess of 150 ml of 0.1 N ~e acid per 100 g of cheese. The gradual decrease in pH follow- ing the fourth day of ripening as shown in Figure 1 corres- ponds, in point of time, with the rapid increase in volatile acidity illustrated in Figure 2. The decrease in pH after the MATILE ACIDS (0: ml of ”In oddpa'chhoue) 77 % H i t—+—-# AGE 0Foucu< RIPEIED ELLE CHEESEJJAYS Figure 2. Changes in volatile acidity during ripening of quick ripened Blue cheese curd. 78 fourth day of ripening may have been due to the great increase in volatile acidity which occurred after day four. The decrease in volatile acidity, between day one and two during curing of quick ripened Blue cheese, may have been- duemtggmetabolism of the free fatty acids by g, roqueforti. Hammer and Bryant (1957) found that the volatile acid content of a sterile milk system, to which E, roquefprti spores and free fatty acids had been added, decreased during early stages of ripening. Since the mold could utilize the lower fatty acids, the authors (1957) suggested that during the early stages of ripening the fatty acids were destroyed as rapidly as they were formed. ‘the liberation of volatile acids by action of milk lipase, prior to pasteurization, may account for the presence of volatile acidity 24 hr after curing of the quick ripened Blue cheese was initiated. Raw milk prepared for quick ripened Blue cheese manufacture was preheated to 150 F, homogenized and then batch pasteurized. The time that elapsed between homogenization and pasteurization may have been suf- ficient for the occurrence of some lipolysis in the milk that was subSequently used for the manufacture of quick ripened Blue cheese. Trout, Halloran and Gould (1955) reported that raw milk homogenized at temperatures between 100 and 150 F became rancid within a very short period of time, in some cases only a few minutes. 79 Mold growth usually appeared in the quick ripened Blue cheese on the fourth day of ripening and subsequently large increases in volatile acidity were evident (Figure 2). Thibodeau and Macy (1942) demonstrated that lipase enzymes were released from g, roquefoppl after Sporulation. In quick ripened Blue cheese the appearance of mold growth was ob- served when sporulation occurred and subsequent to sporula- tion an increase in volatile acidity was noted. Morris g£_§l, (1965) detected no appreciable quantities of FFA in Blue cheese made from pasteurized homogenized milk until mold sporulation was evident in the cheeses. The volatile acidities of the quick ripened Blue cheese samples analyzed were Slightly higher than those acidities (69.0and 75.5 ml 0.1 N acid per 100 g of cheese) observed in the research reported herein, for two Danish Blue cheese samples described as possessing a "sharp" Blue cheese flavor. Quick ripened Blue cheese samples with volatile acidities above 80 ml of 0.1 N acid per 100 g of cheese were usually judged as excessively sharp by competent judges. The large quantity of volatile acids found in some quick ripened Blue cheese samples may have been resPonsible for the observed excessively "sharp flavor”. Previous work by Coulter, Combs and George (1958a) indicated that a "good" flavored Blue cheese aged 9 months had a volatile acidity of 50 ml of 0.1 N acid per 100 g of cheese. A Blue cheese prepared by Coulter and Combs (1959) from milk containing added steapsin, and 80 considered "strong" by consumers, had a volatile acidity of 68.4 ml of 0.1 N acid per 100 g of cheese. As illustrated in Figure 2 the average volatile acid content of the quick ripened Blue cheese batches increased rapidly after the fourth day of ripening. The volatile acidity was observed to increase even when the cheese was stored at 40 F in polyethylene bags. Apparently lipolysis continued during storage. Quick ripened Blue cheese prepared in the research reported herein, and stored at 40 F for 1 to 2 months had volatile acidities of 150 to 180 ml of 0.1 N acid per 100 g of cheese. Samples held 2 months at 40 F were described as bitter and soapy. Kondrup e£_§l, (1964) reported that a consumer panel described the flavor of quick ripened Blue, stored 2 months at 45 F, as “too strong". Also, Coulter and Combs (1959) found that Blue-type cheeses made with steapsin, but which were not inoculated with mold, exhibited volatile acidities of 75.5 to 186.8 ml of 0.1 N acid per 100 g of cheese and were termed rancid. Therefore, a means of inhibiting lipolysis during storage of quick ripened Blue cheese would seem to be a prerequiste for the commercial production of such a cheese with desirable flavor attributes. Quantitation of Free Fatty Acids The method described by Iyer gp_al, (1967a, b) for isolat- ing and quantifying the free fatty acids (FFA) from butyrate 81 through linolenate in Provolone and Gouda cheese was evaluated to determine its application to Blue cheese. Blue cheese has substantially larger amounts of FFA (Anderson and Day, 1965) than does Provolone or Gouda cheese. The capacity of a 4 g silicic acid-KOH column for the quantity of FFA observed in Blue cheese was therefore investigated. Fatty acids, approximating the total quantity of FFA expected in a Blue cheese were dissolved in ethyl ether, as described in the experimental procedure, and added to a silicic acid-KOH column (column I). Columns prepared in this manner were washed with ethyl ether, as described by McCarthy and Duthie (1962), to remove tri-, di- and mono-glycerides from the column. To determine whether column I retained all of the added fatty acids the ether eluates from column (I) were passed over a second silicic acid—KOH column (II). The FFA retained on column I and any FFA added to column II via the column I eluate were eluted from their respective columns with 60 ml of ether containing phosphoric acid (2.5 per cent, v/v). Both columns were washed twice with 40 ml portions of ethyl ether. The eluates from each column were then analyzed as described for FFA analysis of Blue cheese. Gas chroma- tograms of the butyl esters prepared from the eluates of both columns and a blank column were compared. The gas chrom- tograms of column II eluates were identical to chromatograms of the butyl esters prepared from blank column eluates. Thin- layer chromatograms, prepared and developed using the method 82 previously described for determining completeness of esteri— fication, exhibited no Spots with Rf values comparable to known fatty acidva values when Spotted with a sample of the initial ethyl ether wash from column I. Therefore, the capacity of a 4 g silicic acid-KOH column was considered to be sufficient for the quantity of FFA normally present. .Factors for Relating the InternallStandards to the Free FattyiAcids Since recorder response was not linear with respect to weight for different esters, and recoveries for different esters may vary, correction factors were established to relate peak areas of the esters of internal standards to the peak areas of the esters of FFA. Heptanoic (7:0) and heptadecanoic (17:0) acids were used by Bills, Khatri and Day (1965) and Iyer §p_§l, (1967a) as internal standards, because of their very low concentration in the triglycerides of milk fat (Magidman §E_al,, 1962). Weighed amounts of about 10mg of each fatty acid, includ- ing the internal standards were dissolved in ether, applied to silicic acid-KOH columns and treated as described for the FFA analysis of Blue cheese to obtain the butyl esters. Standard fatty acids from nine separate columns were analyzed in quadruplicate to obtain the correction factors. ~ Table 5 lists the correction factors used to quantify the FFA in Blue-veined type cheese samples. The standard devia- tions of the correction factors were small and appeared to 85 fall within the bounds of accuracy to be expected for gas chromatographic analyses (Table 5). The correction factor for butyrate was 1.058, indicating quite good recovery of this acid. The confidence limits at the 95 per cent level are also listed in-Table 5. The correction factors obtained were similar to those reported by Bills, Khatri and Day (1965) and Iyer §£_§l, (1967a). Standard deviations were smaller than those reported by Bills, Khatri and Day (1965), but Slightly larger than those reported by Iyer §§_al, (1967a). Use of correction factors circumvented the necessity of attempting to inject precisely measured quantities and per— mitted injection levels that gave desired peak heights. Acetic Acid Recovepy Trials Known amounts of butyric, propionic, acetic and succinic acid dissolved in water were chromatographed using the liquid- liquid partition column of Wiseman and Irvin (1957) with the modifications outlined under experimental procedure. The eluates were titrated with 0.005 N sodium hydroxide to the cresol red end point under a stream of nitrogen. ApprOpriate. blank conditions were obtained from titers of equal volumes of eluents passed through blank columns under similar condi- tions. An average recovery of 99.8 per cent for acetic acid was obtained when composite samples were passed over five separate columns. The standard deviation for the acetic acid recovery trials was 0.55. The percentage recovery was used 84 . mamamm p . fig .mumuwamsupmsv CH can Hmwuu nommm emo.ofiemm.a oomo.o smm.a w mama mao.ofim¢d.a mwoo.o mea.d e mums oeo.ofimeo.a mmoo.o meo.a e Hume moo.oflmao.a mmoo.o meo.e m oume ........... ...... nooo.a m ones eao.ofim~m.o msdo.o msm.o m 0.86 mmo.oflmmm.o oamo.o mmm.o m ones mmo.ofimmm.o mmeo.o mam.o m onus mao.ofissm.o memo.o sem.o m ouoa mao.ofiwao.a mmeo.o meo.a m oum ........... ...... nooo.a m ous mao.OHmao.d emao.o mao.d m one meo.osmmo.a momo.o mmo.a m one mUHEHH sowumw>mp uouumm mmamauu wand mocmpflmcou &mm pnmpcmum mmmumiw mo HmQEDz .mesom muumm Hmsefl>uvcu Hmnpo may no mumumm prsn mnu ou mpumpcmum Hmcumucfl may mswumamu How mHOuUMh .m magma 85 for calculating the amount of acetic acid in Blue cheese samples. Free Fatpy Acids in Danish Blue Cheese Imported Danish Blue cheese was purchased in 5 pound wheels, the surface Slime removed, the cheese comminuted and slurried with distilled water to 40 per cent total solids. Cheese samples were slurried to facilitate subsequent Spray drying of the cheese.‘ Results obtained by Anderson and Day (1965) indicated that samples taken from different areas of cheese wheels varied considerably in their fatty acid content. To obtain reproducible results cheese samples for analysis were taken from thoroughly mixed 40 per cent solids slurries. The data in Table 4 illustrate the quantities of free fatty acids found in the cheese samples analyzed. Samples A and B were made by the same manufacturer, but had different lot numbers and were obtained 6 months apart. Results are given in mg acid/kg cheese and represent the average of dupli- cate analyses. The average per cent deviation of the dupli- cates from their mean for all fatty acid determinations made was: 2:0, 5.7; 4:0, 5.1; 6:0, 5.9; 8:0, 5.0; 10:0, 5.1; 12:0, 5.6; 14:0, 5.4; 16:0, 5.4; 18:0, 4.2; 18:1, 4.7; 18:2, ' 4.1; 18:5, 4.5. As shown in Table 4 there was significant variation in the free fatty acid content of the two cheese samples. ‘Sample B had higher amounts of all the free fatty acids, except linoleic, linolenic and butyric. Sample B was 86 @m 4 . Concentration of free fatty acids in Imported Danish Blue Cheese (mg acid/kg cheese). Cheese sample1 Acid A2 B3 2:0 700 910 4:0 980 900 6:0 600 660 8:0 570 740 10:0 2,160 2,850 12:0 2,900 5,600 14:0 8,470 11,710 16:0 20,580 25,960 18:0 6,670 8,690 18:1 21,500 22,150 18:2 1,900 1,710 18:5 2,260 2,050 lAverage of duplicate analyses. 2Total solids, 51.07%. 3Total solids, 55.58%. 87 judged as lacking the ”sharpness” that is associated with Blue cheese flavor. The lack of ”sharpness" may be attributed to the low amounts of butyric, caproic and caprylic acids present in samples A and B. Calculations of the relative mole percentages of each acid helps illustrate this observation. The relative mole percentages of the FFA found in imported Danish Blue cheese samples are shown in Table 5. The rela- tive mole percentages of 5.1-5.9, 1.8-1.7 and 1.4-1.5 for butyric, caproic and caprylic acids in the Danish Blue cheese samples analyzed are low. The mole percentages, for all the other FFA in the Danish Blue cheese samples analyzed, agree quite closely with those values reported by Anderson (1966) for domestic Blue cheese. The butyric, caproic and caprylic acid content of domestic Blue cheese samples analyzed by Anderson and Day (1965) was slightly higher than the amounts of the same acids found in the imported Danish Blue cheese samples (Tables 4 and 5). V/Without knowledge of the manufacturing history, possible reasons for the low mole percentages of butyric, caproic and caprylic acid in the Danish Blue cheese samples analyzed in this thesis might be use of a cheese milk containing low amounts of these acids and/or use of a mold strain possessing limited ability to hydrolyze these acids from milkfat. . Roquefort cheese was reported to lack the pungent flavor and aroma of butyric acid commonly found in domestic Blue cheese (Anderson and~Day, 1965). Roquefort cheese is made 88 II II m.d o.N N.H m.m m.m mama II I: m.a N.N m.d m.« ¢.N Nuwd o.H m.o m.mm o.¢m o.mm m.wm m.>m dumd o.w m.m w.m >.m m.m m.m e.m oumd m.m m.> «.mm o.mm m.mm o.mm m.mN oumfi N.>a «.ma o.dd m.¢a m.md m.md m.mfi owed m.mm m.mm m.¢ m.w d.m w.m N.m oumd ¢.m N.m m.m o.¢ >.m N.m m.¢ ouOd o.m >.m ¢.d ¢.H m.d m.d ¢.a oum m.o O.H m.N m.d H.N 5.6 m.d one II II m.m e.m m.m a.m m.m one Mmmmflo pmHHHM U Umcwmwm xUflad 4 mosam amassed pave mmmmnu .mmmmno mmhu cwm>lmsam ca mpflom huumm mmum mo mmmmusmoumm macs rm magma 89 from ewes' milk which is low in butyric acid (Sadini, 1965; Kuzdzal-Savoie and Kuzdzal, 1965; and Benassi, 1965). Danish Blue cheese is made from cows' milk which contains larger mole percentages of butyric acid than does ewes' milk (Hilditch, 1956): Therefore, while low levels of butyric acid may account for the lack of "sharpness" in the Danish Blue cheese samples analyzed in this thesis, the low levels of butyric acid probably were not the result of using a milk low in butyric acid for the manufacture of the cheese. The low levels of butyric, caproic and caprylic acid in the Danish Blue cheese samples may have resulted from use of a mold strain possessing limited ability to hydrolyze these acids from milkfat. ProkS, Dole§51ek and Pech (1956, 1959c) found that the lipolytic properties of various strains of g, roqueforti varied. All strains examined produced acetic acid, but varied greatly in their ability to hydrolyze butyric, caproic and caprylic acids from butterfat. Those strains that produced small quantities of volatile acids were capable of producing considerable non-volatile acidity as evidenced by the substantial amounts of acid found in cheese samples containing low quantities of butyric, caproic and caprylic acid. Free Fatty Acids ip_Quick Ripened Blue Cheese Sensory comparisons of Blue cheese with quick ripened Blue cheese, suggested that the flavor of the latter was dif- ferent than commercial Blue cheese samples evaluated. 90 The flavor of quick ripened Blue cheese was variously described as very strong, bitter, atypical and/or as possessing a very pronounced fatty acid flavor. To more completely ascertain the reasons for these flavor judgments, samples of quick ripened Blue cheese were analyzed for their FFA content. Samples of quick ripened Blue cheese manufactured by the method of Hedrick, Kondrup and Williamson (1968) were comminuted and slurried as described for the preparation of Danish Blue cheese for FFA analysis. To provide a basis for comparison among samples the cheese was uniformly ripened 7 days at 62 F.. The FFA, reported as mg acid/kg cheese, for three samples of quick ripened Blue cheese are given in Table 6. Samples A and C were taken from cheese ripened 7 days. Sample B was a portion of the curd from A that was permitted to ripen 8 days. Samples A and C differed considerably in their FFA content. Sample C contains considerably more of all the FFA, except linoleic and linolenic. Significantly larger quantities of FFA were present in the sample ripened 8 days, when compared to the same sample ripened 7 days. The most typical Blue cheese flavor was possessed by sample A. A strong free fatty acid, slightly bitter and soapy flavor was detected in Sample C, which was described as atypical of good Blue cheese. The relative mole percentages of the FFA in the quick ripened Blue-veined cheeses are presented in Table 5. The total quantity of FFA in each of the three samples of quick ripened Blue cheese was greater than the total quantity 91 Table 6. -Concentration.of free fatty acids in quick ripened loose curd Blue-veined cheese (mg acid/kg cheese). Chggse sample1 3 Acid .A2 B c4 2:0 420 780 540 4:0 1,820 1,950 2”610 6:0 770 790 1,080 8:0 610 670 840 10:0 2,010 2,560 2,540 12:0 5,190 5,580 2,890 14:0 9,950 11,280 .10,550 16:0 24,180 25,550 34,270 18:0 8,550 9,420 11,450 18:1 22,280 25,040 28,650 18:2 1,680 2,080 .1,540 18:5 1,040 1,860 1,180 1Average of duplicate analyses. 2Total solids, 55.15%. 3Total solids, 55.10%. 4Total solids, 56.56%. 92 of FFA reported in any of the Blue cheese samples analyzed by ~Anderson and-Day (1965). However, the relative mole per- centages of the FFA in the quick ripened Blue cheese batches were about the same as those calculated by Anderson (1966) for domestic Blue cheese. Sample C had about 50,000 mg/kg cheese more FFA than any sample analyzed by Anderson and Day (1965). The method of Kondrup and Hedrick (1965) used to prepare the quick ripened Blue cheese provides for ripening of Blue cheese as a granular curd at an elevated ripening temperature (62 F). As evidenced by the large quantities of FFA produced (Table 6) within 7 days, the rate of FFA production has been stimulated. During a 24 hr period of ripening there was a significant increase in the quantity of FFA as shown by the greater quantities of FFA in sample B as compared to sample A. The ripening temperature of 62 F (17 C), for quick ripened Blue cheese, is closer to the optimum temperature for mold lipase activity than are the temperatures normally employed in ripening domestic Blue cheese. While FFA development in the quick ripened Blue cheese batches was accelerated the flavor of these cheeses was described as atypical. Ripening temperatures used to manufacture domestic varieties of Blue cheese include 50 F (Peters and Nelson, 1948), 48-52 F (Parmelee and Nelson, 1949a) and 50-55 F (Babel, 1955). The optimum temperature for g; roqueforti lipase activity has been reported as 50-55 C (Shipe, 1951), 95 50-52 C (Morris and Jezeski, 1955), 7 C (Imamura and Kataoka, 1965a, b) and 21—27 C (Eitenmiller, Vakil and Shahani, 1970). Higher temperatures of ripening produced more rapid break- down of fat and higher volatile acidities than did lower temperatures, but mean flavor scores were significantly higher for cheeses held at 57 F than for cheeses held at 50 or 60 F (Morris, 1969). Large concentrations of carbon dioxide inhibit the growth of g, roquejprti (Golding 1940a, b). Skewering of cheese was thought to be important in promoting carbon dioxide escape from the cheese while admitting oxygen. Ripening of Blue cheese as a granular curd provides access of oxygen, necessary for mold growth, to the mold and prevents any accumulation of carbon dioxide which might inhibit or Slow mold growth. The hydrolysis of fatty acids from milk triglycerides in quick ripened Blue cheese is accelerated, but probably follows the same pattern as that in conventional Blue cheese curing. The mole percentages of FFA found in quick ripened Blue cheese (Table 5) approximate those found in domestic Blue cheese by Anderson (1966). Hydrolysis patterns, such as those produced by milk lipase alone, would yield different mole percentages of FFA than those percentages found in the quick ripened Blue cheese samples analyzed. Vujicic and de Man (1967) noted that the composition of the partial glycerides in Danish Blue cheese indicated a pattern of lipolysis dissimilar to that usually observed with milk or pancreatic lipase. Blue cheese FFA 94 composition suggested that the lipase of g, roqueforti as well as some other lipase may be involved in its flavor develop- ment (Khan §£.§l,, 1966). Anderson and Day (1965) suggested that the entire quantity of fatty acids does not exist as free acids in cheese. Between 75-85 per cent of the acids may exist as salts. The salts of long chain fatty acids are soaps and as such they possess definite flavor properties which may contribute to the back- ground flavor of Blue cheese. Sample C contained relatively large quantities of the long chain fatty acids (14:0, 16:0, 18:0), which may explain why sample C was judged as having a slightly soapy flavor. The data presented in Table 6 indicate that there was considerable variation in the fatty acid content of the cheeses. Anderson and Day (1965) demonstrated that variation in the fatty acid content of cheese samples was reflected in the flavor and aroma of the cheeses. Samples A and C were made from milk produced by the same herd, ripened 7 days, inoculated with the same mold strain and manufactured in the same manner yet they exhibited considerable variation in their FFA content and flavor. The volatile acidity present after 7 days of ripening (Figure 2) shows wide variations. Variations in the quantity of short chain fatty acids was reflected in the flavor of the quick ripened Blue cheese. Duplication of the flavor from batch to batch appears to be difficult. 95 Free Fatty Acids inéguick Ripened Filled Blue Cheese The flavor of Blue cheese is due in part to the presence of butyric, caproic, caprylic and capric acids or their hydrolyzable salts. Hydrolysis of milk triglycerides must occur to provide a high concentration of free fatty acids in Blue cheese. A fat containing Short chain fatty acids was desired for a filled Blue cheese, because lipase activity is required for flavor development. The lauric acid oils such as coconut oil contain the six-, eight- and ten-carbon chain fatty acids, but there is no known vegetable oil source for butyric acid. A hydrogenated coconut oil was selected for this study, because the melting point (94 F) of the hydro- genated oil was close to that of butteroil (approximately 95 F). A commercial modified lauric fat with added lipolized butteroil has been suggested by Ryberg (1968) for use in a cheese dependent on lipase activity for flavor. The cheese manufacturing procedure followed was the same as that for quick ripened loose curd Blue cheese (Hedrick, Kondrup and Williamson, 1968), except that filled milk con- taining hydrogenated coconut oil was used in place of standardized cows' milk. Quick ripened filled Blue cheese rapidly develOped a flavor similar to a Blue cheese within 5 days. At the end of seven days of ripening the cheese possessed a "strong“ flavor which was not completely typical of Blue cheese. A soapy, slightly bitter, high FFA and ketone flavor was evident in 96 the cheese samples. The concentrations of the individual free fatty acids in samples of quick ripened filled Blue cheese are given in Table 7. Large quantities of caprylic, capric, lauric, myristic, Stearic and small amounts of palmitic and oleic acid were present in the filled cheese as compared to the amounts of the same FFA in Danish Blue (Table 4) and quick ripened Blue cheese (Table 6). Lauric acid was the predominant FFA present in filled Blue cheese followed by myristic acid. The mole percentages of the FFA in the filled cheese samples are presented in Table 5. .As can be seen from Table 5 lauric acid comprises over 50 per cent of the total moles of existing FFA. Higher mole percentages of caprylic, capric and lauric acid were observed in filled cheese samples than in either the Danish or quick ripened Blue cheese samples analyzed and higher than those percentages reported for domestic Blue or Roquefort cheese (Anderson, 1966). Smaller mole percentages of caproic, palmitic, stearic and oleic acids were found in the filled Blue cheese than in Blue cheese made from either cows' or Sheeps' milk. A trace of butyric acid was observed in the coconut oil filled Blue cheese. Since coconut oil contains no butyric acid (Bailey, 1964), the butyric acid may have originated from the small amount of residual milkfat that invariably remains in skimmilk following centrifugal separation. The skimmilk used for the manufacture of quick ripened filled Blue cheese contained between 0.05-0.1 per cent milkfat. 97 Table 7. Concentration of free fatty acids in filled quick ripened Blue-veined cheese (mg acid/kg cheese). Cheese samplel 2 Acid A 133 2:0 550 260 4:0 trace trace 6:0 1,000 920 8:0 7,250 6,150 10:0 12,490 12,210 12:0 95,120 89,800 14:0 52,240 55,460 16:0 17,150 18,660 .18:0 16,620 14,580 18:1 2,060 2,490 18:2 -- -- 18:5 -- -- 1Average of duplicate analyses. 2Total solids, 49.90%. 3Total solids, 48.20%. 98 The relative percentages of the 6:0, 8:0, 10:0, 12:0, 14:0 and 16:0 free fatty acids found in quick ripened filled Blue cheese were 0.5, 4.0, 6.8, 51.7, 17.5 and 9.5 reSpec- tively. The fatty acid composition reported by Bailey (1964) for coconut oil of 0-0.8, 5-9, 6-10, 44-62, 15-19 and 8-11 for the 6:0, 8:0, 10:0, 12:0, 14:0 and 16:0 fatty acids respectively approximates those percentages found in the filled Blue cheese samples. A higher concentration of stearic (9.0 per cent) and a lower amount of oleic acid (1.1 per cent) were observed in the filled Blue cheese as compared to the 1-5 and 5-8 per cent reported by Bailey (1964) for stearic and oleic acid reSpectively in coconut oil. Oleic and linoleic acid are converted to stearic acid when coconut oil is fully hydrogenated (Bailey, 1964), which probably accounts for the higher levels of stearic and lower levels of oleic acid found as FFA in the filled Blue cheese samples analyzed. The percentages of FFA found in the filled Blue cheese samples were similar to those percentages of FFA released by the lipolytic activity of g, roqueforti in a coconut oil emul- sion (Alford and Pierce, 1961). Alford and Pierce (1961) observed that different percentages of fatty acids were hydro- lyzed from coconut oil triglycerides at various temperatures. The filled Blue cheese was ripened at 62 F and the mold lipase activities on emulsified coconut oil were determined at 95 F, which may account for the slight difference in the percentages of FFA found in the filled cheese as compared to those 99 observed in coconut oil. About 90-94 per cent of the fatty acids of coconut oil are’saturated; they consist primarily of lauric, myristic and palmitic acids, with lauric predominating (Bailey, 1964). The primary FFA in filled Blue cheese were lauric, myristic and palmitic, with lauric acid predominating (Table 5). By comparing the relative mole percentages of FFA in Danish, quick ripened (Table 5) and domestic Blue cheese (Anderson, 1966) with those calculated for coconut filled Blue cheese a significant difference in the percentages of butyric, capric, lauric, palmitic, oleic, linoleic and linolenic acid can be ! observed. The fatty acid concentrations are different for Blue and filled Blue cheese which may account for the fact that the filled Blue cheese samples were described by judges as not possessing a typical Blue cheese flavor. The total quantity of FFA in filled Blue cheese was greater than the amounts observed in quick ripened Blue cheese, which in turn contained larger total quantities of FFA than domestic or Danish Blue. 'The large quantities of \ FFA may account for the "strong" flavor, and the slightly } bitter and soapy flavor of the filled Blue Cheese. Anderson? 4 and Day (1965) reported that the salts of long chain fatty acids in cheese are soaps and as such possess definite flavor proPerties. Since between 75-85 per cent of the FFA in Blue cheese may exist as salts depending upon the pH of the cheese, a large number of the long chain FFA in filled Blue cheese 100 probably exist as soaps. The total quantity of FFA in quick ripened filled Blue cheese, as determined by gas chromatography, suggested that about 70 per dent of the fatty acids had been released from the coconut oil triglycerides. To substantiate these find- ings several 5 9 samples of cheese were acidified to pH 1.9 with 50 per cent sulfuric acid and each sample then mixed in a mortar and pestle with 9 g of silicic acid. The mixture was applied to the silicic acid-ethylene glycol partition column described by Keeney (1956). The FFA were extracted by passing 600 ml of 1 per cent butanol in hexane over the column. The eluate was titrated with 0.05 N alcoholic KOH under a stream of nitrogen to the phenolphthalein end point. A mean molecular weight of 211.76, calculated from.saponifi- cation values (Bailey, 1964) was used to determine the total mg FFA/5 9 cheese. To determine the total quantity of fat extracted identical extractions were performed, the eluate evaporated to dryness and the residue weighed. Results indi- cated that 67 per cent of the fatty acids had been hydrolyzed from the coconut oil and serve to corroborate the seemingly high levels of FFA measured by gas-liquid chromatography. A partial explanation, for the fact that the lipase of E, roqueforti was apparently more active in coconut filled Blue cheese than in quick ripened Blue cheese, may be that the glycerides in coconut oil are not as complex as those of milkfat. The fatty acid distribution in the glycerides of 101 butterfat is regarded as highly complex (Webb and Johnson, 1965). Coconut oil consists of about 84 per cent fully saturated glycerides (G63), 12 per cent disaturated-mono- unsaturated glycerides (652U) and 4 per cent monosaturated- diunsaturated (GSUa) glycerides (Hilditch, 1956). Fractional crystallization of the fully saturated components did not yield any simple trilaurin, but dilauromyristins as well as other triglycerides containing two like fatty acids were present in considerable quantities. 'Coconut oil was hydrolyzed by the lipase of Pseudomonas fragi more rapidly than butterfat, corn, cottonseed, olive or soybean oil (Nashif and Nelson, 1955). A mycelial preparation of g, rogpeforti was shown by Morris and Jezeski (1955) to exhibit decreased activity as the molecular weight of the fatty acid portion of the trigly- ceride increased. The arrangement of fatty acids in the triglyceride molecule apparently influenced lipase activity, because greater activity was observed on."synthetic butterfat" composed of simple triglycerides than on natural butterfat which contains a complex mixture of mixed triglycerides. The authors (1955) suggested that some of the lower molecular weight fatty acids in mixed triglycerides may be more difficult to hydrolyze than when they occur in a simple triglyceride due to steric hindrance by longer carbon chain fatty acids. The fat in the filled Blue cheese was hydrolyzed ex- tensively, which may account for the fact that the percentages of FFA approximated the percentages of fatty acids in the 102 original coconut fat. In a study to determine if the rela- tive amounts of the various fatty acids liberated changed with time, Alford pp.§l, (1961) observed that 60 per cent of the triglyceride fatty acids had been released by Pg, fgggl. after 6 days. There was a difference in the percentages of fatty acids in the original fat and the percentages of some of the FFA liberated in the early stages of lipolysis. After 6 days the percentages of fatty acids released approxi- mated the percentages of fatty acids in the original fat. Goldman and Rayman (1952) found that the degree of hydrolysis of coconut fat by Pseudomonas fluorescens exceeded 80 per cent after 42 days. The authors (1952) demonstrated the importance of having finely-divided globules available in order to provide a highly effective surface for bacterial lipase action. -Microbial hydrolysis appeared dependent upon the particle Size of the emulsified fats and not on the molecular composition of the component triglycerides. The same degree of cleavage was achieved with fats possessing widely varying fatty acid compositions as butter, lard, tallow, coconut, soybean and peanut oils. Emulsified fats, subjected to attack by Pg, fluorescens, underwent hydrolysis to a degree which was practically complete (90-95 per cent) at fat con- centrations below 10 g per 100 ml of substrate. The extent of hydrolysis decreased at higher fat levels. In the filled milk system used to manufacture filled Blue cheese, the fat globules may have been more susceptible 105 to mold lipase action than the "natural" milk fat globules in the quick ripened Blue cheese. Coconut oil was emulsified into skimmilk prior to the cheese manufacturing process using a homogenizer. Whole milk, which contains fat in a naturally emulsified form, was standardized and subsequently homogen- ized prior to its use for the manufacture of quick ripened Blue cheese. Emulsions containing appreciable amounts of dispersed phase are stabilized by adsorption of a third phase at the interface (Patton and Jenness, 1959). The membrane in a natural milk system was postulated to consist of a group of materials adsorbed and oriented in the interface between the fat globules and the plasma. Coconut oil as added to skim- milk would possess no such natural membrane. Tarassuk‘ and Richardson (1941) suggested that the natural membrane material around milk fat globules as it exists prior to activation protects the fat from lipolysis. Changes in the natural adsorption layer around the fat globules as accomplished by activation treatments were necessary for adsorption of the lipase of normal milk on the surface of fat globules.‘ Milk fat globules did not require "resurfacing" or "denuding" for steapsin or naturally active lipase action on the fat as it exists in milk. Brunner 23.3l, (1955a, b, c) have reported that the proteins in the fat globule membrane in homogenized milk and nonhomogenized milk are different in both number and nature. 104 Proteins as adsorbed at the coconut oil globule surface after homogenization may not interfere with mold lipase action as much as the natural milk fat globule membrane and material adsorbed at the milk fat globule surface following homogenization. If the milk lipase were less restricted in its action on coconut oil,this may help account for the ex- tensive lipolysis observed in the filled Blue cheese. Quantitation of Methyl Ketones Initial determinations for methyl ketones in Blue cheese revealed that acetone and 2-pentanone were difficultly separated from the saturated aldehyde, 2-enal and 2,4-dienal classes. To obtain cleaner class separations on the 10 g Celite 545-SeaSorb 45 (1:1 w/w) column the monocarbonyls, after passing over the weak alumina column to remove ketoglyceride DNP-hydrozones, were subjected to partition chromatography. Schwartz, Haller, and Keeney (1965) found that butteroil con- tained a class of naturally occurring compounds, other than the four common classes, which elute from alumina with the monocarbonyl fraction. This class was relatively nonpolar, moving with the front on the partition column, and was easily removed from the more polar hydrazones. The first peak from .the partition chromatogram.was collected and subjected to class separation. The remaining bands on the partition column were collected, pooled, the solvent evaporated and then class 105 separated. Any ketones found in the first band removed from the partition column were placed with the ketones separated from the remaining bands. The combined ketones were then subjected to the column partition chromatography method of Corbin, Schwartz and Keeney (1960) to separate the methyl ketone hydrazone class into its individual members. Identity of individual methyl ketone was confirmed using the thin-layer chromatography method of Edwards (1966). This method was modified using Eastman Chromagram sheets and Dow Corning 551 silicone oil and enabled the resolution of the homologous series of the methyl ketones (Figure 5). The first 15 members of the methyl ketone series separated sufficiently although the longer chain members 2—tridecanone and 2-penta- decanone (not shown) were somewhat crowded. Recovery of Methyl Ketone DNP-pydrazones from Butteroil The percentage recovery of the individual methyl ketones was determined by passing a standard mixture of C3, C5, C7, C11 and C13 methyl ketones over the DNP-hydrazine reaction column (Schwartz and Parks, 1961) and subsequently adding butteroil. The mixture was analyzed as described for Blue cheese. Average percentage recovery of each ketone for five recovery trails was used to calculate ketone concentrations in samples of Blue cheese. Average recoveries of the indi- vidual methyl ketones at the completion of analysis are Shown in Table 8. The recovery rates were lowered with 106 SOLVE NT FRONV FiQure 5. ORIGIN Separation of homologous series of methyl ketone DNP-hydrazones by thin-layer partition chroma- tography. Support: Silica gel: stationary phase: Silicone oil: mobile phase: 75 per cent methanol—25 per cent water. Diagonally from top to bottom: free hydrazine, acetone, 2-pentanone, 2-heptanone, 2-nonanone, 2-undecanone and 2-tridecanone. Column on right is a mixture of all of the compounds. 107 decreasing chain length as was earlier observed by Anderson (1966), Allen and Parks (1969) and Arnold and Lindsay (1969). Standard deviations of the recoveries and the confidence limits at the 95 per cent level are presented in Table 8. The deviations indicated considerable variation among samples, which might be expected, since the sample is passed over 5 separate columns before Spectrophotometrically determining the quantity recovered. Anderson (1966) using a similar procedure for methyl ketone quantitation obtained somewhat higher recoveries for the C5, C7, C9 and Cl; ketones. The average recoveries reported by Allen and Parks (1969) and Arnold and Lindsay (1969) for the methyl ketones as determined using a similar procedure, but modified for the determination of ketones in sterile concentrated milk, were similar to those recorded in Table 8. Methyl Ketones in Danish Blue Cheese Imported Danish Blue cheese samples for subsequent ketone analysis were obtained from 40 per cent solids cheese slurries prepared as described for FFA analyses. Initial separations of ketone homologs on the Corbin, Schwartz and Keeney (1960) partition column revealed that the C13 derivative could not be separated from an unidentified forepeak on the partition column, therefore C13 could not be quantitated. By passing the monocarbonyl fraction from the weak alumina column over a partition column prior to class separation the concentration 108 Table 8. Percentage recovery of individual methyl ketones at completion of analytical procedure. Methyl ketone Average percentage Standard 95% Confidence chain length recoverya Deviation limits 5 65.8 1.9 65.8i2.5 5 64.8 2.6 64.8:4.1 7 65.7 2.7 65.71A.4 9 67.7 5.5 67.7:6.5 11 67.5 5.1 67.516.1 15 70.6 2.9 70.612.6 aAverage of 5 trials. 109 of C13 + C15 methyl ketone could be determined. Tridecanone- 2 and 2-pentadecanone were very difficult to separate, therefore both were determined as the C13 methyl ketone and were reported as the C13 + C15 ketone. Results for the quan- titative analysis of the C3, C5, C7, C3, C11 and C13 + C15 methyl ketones in Danish Blue cheese samples are presented in Table 9. The A and B samples used for free fatty acid analy- sis of Danish Blue cheese were also used for quantitation of methyl ketones. Results are reported as micromoles/10 g of extracted fat and represent the average of duplicate analyses The average per cent deviation of the duplicates from their mean for all methyl ketone determinations made was: acetone, 6.1; 2-pentanone, 2.9; 2-heptanone, 5.8; 2-nonanone, 1.9: 2-undecanone, 1.5; 2-tridecanone + 2-pentadecanone, 2.5. ReSults obtained from the Danish Blue cheese analyses are comparable to the findings of Morgan and Anderson (1956). Schwartz and Parks (1965) and Anderson and Day (1966) that the individual ketones in Blue cheese vary considerably. Sample B was judged as possessing the most flavor. This was confirmed by the fact that B contained the largest quantity of methyl ketones and as illustrated by its FFA content ex- hibited the most extensive fat breakdown (Table 4). Both samples were described as being high in ketone. Nonanone-2 was the most abundant ketone in both the Danish Blue cheese samples. Schwartz and Parks (1965) reported that 2-heptanone was the most abundant ketone found in three 110 .mwm>amcm wumuwamsp mo wmmum>¢a momnu mommy v.0 m.o N.H m.m m.d ma N.>¢H m.mmd ¢.m m.m m.m «.md w.ad dd m.ada m.m0a m.m >.¢H N.md m.om «.dn m «.mm m.mm m.¢ o.e m.¢ m.nm m.mm e N.N N.m m.o m.o m.o d.m m.m m momuu momma «.0 mummy oomuu m.d m.o m m < o m e m e numsma semen mmmmsu pwaawm Umcmmen xowsm, wSHm amazon wsOpmx Hmnumz Hmmamfimm mmmmno .Aumm pmpomnuxm mo m Ofi\mmHoEouoflEv mmmono pmcww>lmsam Eoum pmuumuuxm umm ca mmcoumx Hwnumfi mo cowumuucmocoo pm manna 111 domestic Blue cheese samples. For five domestic Blue and two Roquefort cheese samples analyzed Anderson and Day (1966) found that 2-heptanone was the predominant ketone. Two of the domestic Blue cheese samples contained almost equal molar concentrations of 2-nonanone and 2-heptanone. A Roquefort sample analyzed by Schwartz, Parks and Boyd (1965) contained more 2-nonanone than 2—heptanone. A high lipolytic and a high proteolytic strain of g, roqpeforti was used by Niki, Yoshioka and Ahiko (1966) to make two experimental batches of Blue cheese. The high lipolytic strain produced markedly higher quantities of ketone in Blue cheese than did the high proteo- lytic strain. The cheese manufactured using the lipolytic strain contained large amounts of 2-heptanone and 2-nonanone after 21 weeks with 2-nonanone predominating. Sato 2; El. (1966) observed the quantity of numerous carbonyl compounds produced at various stages of ripening by different strains of mold. Strains of mold that grew well produced larger amounts of 2-heptanone and 2-nonanone than strains that grew poorly. Five of the samples contained more 2-nonanone than 2-heptanone after a 5 month ripening period. A small peak which moved ahead of 2-tridecanone on the partition column used to separate the individual ketone homo- logs found in the Danish Blue cheese samples analyzed had an absorption maxima in chloroform of 565 nma Class authenticity was verified using thin-layer chromatography (Schwartz §l_§l,, 1968) and by fading studies of the unknowns spectra in alkaline 112 ethanolic solution (Jones, Holmes and Seligman, 1956). Chain length was assigned using reverse phase thin-layer chromatog- raphy (Edwards, 1966) and the compound tentatively identified as 2-pentadecanone. The existence of the C13 and C15 methyl ketones in the Danish Blue cheese was probably due to their formation from the natural breakdown of the bound beta-keto acids of milk (Schwartz and Parks, 1965). No methyl ketones were liberated from the fat of Blue cheese by heating in the presence of water. Schwartz and Parks (1964) suggested that the methyl ketone precursors in milk were metabolized by the mold and thus make a small contribution to the final methyl ketone levels. Lawrence (1966) working with washed Spores of ,2. roqueforti noted that no methyl ketone was detected when spores were incubated with myristate at any pH value although there was oxygen uptake. A comparison of the mole percentages of the FFA and methyl ketones in Danish Blue cheese is given in Table 10. Both samples of Danish Blue cheese contained a greater mole per cent of 2-nonanone than 2-heptanone. The methyl ketones have been shOwn to be produced from fatty acids by g. roqueforti spores (Gehrig and Knight, 1958, 1965). Lawrence (1966) ob- served that washed spores of g, roqueforti oxidized fatty acids to methyl ketones. The proportion of acetone in the Danish Blue cheese was relatively low compared to its precursor, butyric acid (Table 10). Conversely, the concentration of 115 2-heptanone was relatively high compared to its precursor, caprylic acid. The 6:0, 10:0, and 12:0 acids appeared to be converted to their corresponding methyl ketones less readily. Results obtained by Anderson and Day (1966) and Lawrence (1966) indicated that the quantity of each methyl ketone produced does not depend directly upon the amount of available fatty acid precursor. Anderson and Day (1966) concluded that during curing the mold spores appeared to convert the 8:0 acid to the C7 methyl ketone readily, while the 4:0, 6:0, 10:0 and 12:0 acids were converted to their corresponding ketones less readily. Girolami and Knight (1955) found that except for the short chain acids, as the carbon chain lengthened up to 9 and 10 carbon atoms the amount of oxygen consumed by g, roqueforti increased. Pelargonic and capric acids exhibited the greatest oxygen uptake. Spores were observed by Gehrig and Knight (1965) to use fatty acids as a source of carbon when the sub- strate was added in small quantities. However, when larger amounts of substrate were added the spore converted at least part of the acid to ketone. The amount of fatty acid avail- able per spore was reported by Lawrence (1966) to determine the rate of oxidation. Maximum methyl ketone formation occurred between pH 5.5 and 7.0 with caprylic acid being the acid most readily oxidized. Capric acid gave the greatest yield of corresponding methyl ketone at pH 7.5. Between pH 5.5 and 6.5 up to 75 per cent of the caprylic, 45 per cent of 114 the capric, 25 per cent of the lauric and 5 per cent of the caproic acid salts were ultimately oxidized to the corres- ponding methyl ketone. By comparing the average mole per cents of the individual methyl ketones with their fatty acid precursors (Table 10) an indication that the quantity of ketone produced does not di— rectly depend on the amount of available fatty acid precursor can be ascertained. That the mole per cent of C9 ketone is greater than the mole per cent of C7 ketone may be related to the relatively high mole per cent (26-50) of capric acid. Anderson (1966) reported finding 16-18 mole per cent capric acid in domestic Blue cheese. A Roquefort sample analyzed by Anderson (1966) containing 52 mole per cent capric acid had the highest amount of 2-nonanone. Larger amounts of C11 ketone were observed in the Danish Blue than has been reported for domestic Blue cheese (Schwartz and Parks, 1965: Anderson and Day, 1966). Danish Blue had a considerably greater mole percentage of lauric acid (51-52 per cent) than that percentage reported for domestic Blue (16-19 per cent). If 75 per cent of the caprylic, 45 per cent of the capric, 25 per cent of the lauric and 5 per cent of the caproic oxidized to their corresponding methyl ketones as determined by Lawrence (1966), then the variations in the mole per cents of the individual ketones between the Danish Blue analyzed in this thesis and domestic Blue cheese (Anderson, 1966) may have resulted from significant differences in the 115 Table 10. Comparison of the mole percentages of the free fatty acids and methyl ketones found in Blue- vein type cheese. Acid Mole %1 Mole % Ketone Mole % chain acid in acid in chain ketone in Cheese length milk fat cheese length cheese Danish 4 44.5 25.4 5 1.2 Blue A 6 12.2 10.9 5 5.5 8 9.0 8.2 7 54.8 10 15.2 26.5 9 45.6 12 21.4 50.8 11 15.1 Danish 4 44.5 18.2 5 1.5 Blue B 6 12.2 10.2 5 5.0 8 9.0 9.1 7 51.9 10 15.2 50.2 9 48.2 12 21.4 52.5 11 15.4 Quick 4 44.5 55.0 5 -- Ripened A 6 12.2 11.2 5 1.9 8 9.0 7.1 7 16.6 10 15.2 19.6 9 58.7 12 21.4 27.0 11 22.7 Quick 4 44.5 54.4 5 -- Ripened B 6 12.2 10.2 5 2.4 8 9.0 7.4 7 16.1 10 15.2 21.5 9 59.5 12 21.4 26.5 11 22.2 Quick 4 44.5 59.8 5 0.6 Ripened C 6 12.2 15.0 5 2.9 8 9.0 8.1 7 26.5 10 15.2 .18.9 9 50.0 12 21.4 20.1 11 20.0 1Calculated from data compiled by Hilditch (1956). 116 amount of fatty acid precursor present. Methyl Ketones in Quick Ripened Blue Cheese As described previously, sensory evaluation of quick ripened Blue cheese suggested that the flavor of such cheese was somewhat different than commercial samples evaluated. Quick ripened Blue cheese, while having a very pronounced fatty acid flavor often was described as possessing little typical methyl ketone flavor. Sterkle (1924) theorized that the typical flavor of Roquefort cheese was due mainly to the formation of methyl ketones from certain fatty acids. Hammer and Bryant (1957) confirmed that methyl ketones, particularly 2-heptanone contributed to Blue cheese flavor. To ascertain if quick ripened Blue cheese qualitatively and quantitatively contained the methyl ketones associated with Blue cheese (Schwartz and Parks, 1965: Anderson and Day, 1966) the methyl ketone content of quick ripened Blue cheese was determined. Samples of quick ripened Blue cheese for subsequent ketone analysis were obtained from slurries prepared as described for FFA analysis of Danish Blue cheese. The methyl ketones, reported as micromoles/10 g of extracted fat, for three samples of quick ripened Blue cheese are presented in Table 9. The A, B and C samples are identical to those used for FFA analysis of quick ripened Blue cheese. Samples A and C were ripened 7 days. Sample B was a portion of sample A ripened 8 days. The C3 and C11 methyl ketone 117 content of Samples A and C were significantly different and the latter sample contained more acetone than sample A. The quantity of methyl ketone in sample A was not signifi- cantly different from.that in sample B. Sample A possessed the most typical Blue cheese flavor. Sample C was described as atypical and lacking in ketone. A comparison of the mole percentages of the FFA and methyl ketones in quick ripened Blue cheese is given in Table 10. Two small anomalous peaks were observed during develop- ment of the partition column chromatogram when separating the homologous series of methyl ketones found in quick ripened Blue cheese. One peak was eluted from the column between the C9 and C7 peak, While the other peak was removed ahead of the C5 peak, but subsequent to the C7 peak. Both peaks had absorption maxima in chloroform of 565 nm. Class authenticity was verified using thin-layer chromatography and by determin- ing the stability of each fraction in alcoholic base as previously described for the tentative identification of 2—pentadecanone. Chain length was assigned using the elution volume from the partition column and reverse phase thin-layer chromatography. The fractions were tentatively identified as 2-hexanone and 2-octanone. The results show a variation in the total quantity of ketone in the cheeses as described previously for Danish Blue cheese (Table 9). Samples A and B contained only a trace of acetone, while sample C contained 0.1 micromole of acetone/10 118 of extracted fat. Schwartz and Parks (1965) reported that two out of three Blue cheese samples analyzed contained only a trace of acetone. No acetone was reported in a Roquefort sample analyzed by Anderson and Day (1966). The quantities of C5, C7 and C11 ketone in the quick ripened Blue cheese were less than those found in the Danish Blue cheese samples analyzed, but were equivalent to those quantities reported by Schwartz and Parks (1965) and Anderson and Day (1966) for domestic Blue cheese. As was the case in Danish Blue cheese 2-nonanone was the predominant ketone. Nonanone-2 was the predominant ketone in a number of samples analyzed by Schwartz, Parks and Boyd (1965), Niki, Yoshioka and Ahiko (1966) and Sato g£_pl. (1966). A strong lipolytic strain of g. roqueforti used by Niki, Yoshioka and Ahiko (1966) to manufacture Blue cheese produced a cheese containing more 2—nonanone than 2-heptanone. A high lipolytic strain produced a cheese con- taining predominantly 2-heptanone. The mold powder used for the manufacture of quick ripened Blue cheese may have contained a strain of g, roqueforti that oxidized capric acid more read— ily than caprylic thereby producing relatively more C9 ketone. In quick ripened Blue cheese as in the Danish Blue cheese the amount of ketone formed by g, roqueforti spores was not directly related to the amount of fatty acid precursor avail- able. As illustrated in Table 10 the proportion of acetone is relatively low compared to its precursor butyric acid. The concentration of 2-heptanone is high relative to its precursor, C2 k: 119 caprylic acid. A larger mole per cent of the C11 methyl ketone was present in the quick ripened than in the Danish Blue cheese analyzed. The larger amount of 2-undecanone may have resulted from the strain of g, roqueforti used in ripen- ing and/or as discussed for Danish Blue cheeses, the larger 'amounts of fatty acid precursor available. The relative mole per cents of the C10 and C12 fatty acids were lower in quick ripened Blue cheese than in Danish Blue, which may indicate that the C10 and C12 acids were more readily con- verted to their corresponding methyl ketones by the particular strain of g, roqueforti used in the manufacture of the quick ripened Blue cheese. Sample A and B of quick ripened cheese contained the same amount of methyl ketone. No significant increase in methyl ketone content resulted from ripening sample A an extra day (Table 9). There were significantly larger quanti— ties of FFA present in the sample ripened 8 days, when com- pared to the same sample ripened 7 days (Table 6). Additional ripening of quick ripened Blue cheese apparently did not in— crease the methyl ketone content, but did significantly increase the FFA levels. The increase in FFA with the extra day of ripening did not change the relative mole percentages of the FFA and ketones (Table 10). Sato §£_3l. (1966) noted that in cheese made using a strain of g, roquefprplnthat grew well the amount of 2-heptanone and 2-nonanone were highest after a ripening period of one month and thereafter decreased 120 rapidly with advancing age. A high proteolytic strain ex- amined by Niki, Yoshioka and Ahiko (1966) produced maximum quantities of ketone after 15 weeks of ripening. At 21 weeks the C3, C5, C7 and C9 methyl ketone content had decreased. The short chain fatty acid content increased throughout the 21 week ripening period. Of the three quick ripened Blue cheese samples analyzed sample C contained the greatest FFA levels, yet sample C exhibited a lower methyl ketone content than either sample A or B. Generally, those Blue cheese samples analyzed by Anderson (1966) that contained large amounts of FFA also con- tained the greatest quantities of methyl ketone. The Danish Blue cheeSe sample containing the highest level of FFA also contained the largest amount of methyl ketone (Table 4 and 9). The quantity of methyl ketone in the quick ripened Blue cheese samples analyzed was not proportionate to their FFA content. The relatively low quantities of methyl ketone may be related to mold growth. In the ripening of Blue cheese g, roqueforti mycelial lipases provide lipolytic enzymes (Thibodeau and Macy, 1942) which supply fatty acid substrate fior conversion through beta-oxidation to ketones (Girolami and Knight, 1955). Meyers and Knight (1958) noted that when propagating mold cells in a synthetic medium where no sporula- tion occurred cells lacked the ability to form methyl ketones from fatty acids. Gehrig and Knight (1958, 1965) found that sporulation was a requisite for ketone formation and that the 121 rate of ketone formation was directly proportional to the de- gree of sporulation. The Spores, other than mycelia, were forming the ketones. Active vegetative inocula provided enzymes for hydrolyzing the fat, and then as spores were formed by the inoculum the FFA were converted to methyl ketones (Gehrig and Knight, 1965). The absence of quantities of methyl ketone in proportion to the amount of FFA in the quick ripened Blue cheese may have resulted from poor sporulation or from poor mold growth which in turn resulted in a low number of spores. Quick ripened Blue cheese A possessed more visible mold growth than did sample C. Therefore, the large quantities of FFA liberated by mycelial lipase may not have been converted as readily to methyl ketones due to an insufficient number of spores. Nelson (1970) reported on the production of a Blue cheese flavor using submerged culture procedures. A spore-rich inocula was added to a sterilized substrate which contained lipolyzed milk fat and the mixture allowed to ferment. Lipolyzed milk fat was added at intervals to simulate the pro- gressive release of FFA that occurs in Blue cheese manufacture. A product was obtained within 24-72 hr which possessed a flavor strength three to six times that of Blue cheese on an equal weight basis. The flavor of quick ripened Blue cheese was described as atypical, lacking ketone and as having a pronounced fatty acid flavor. That the samples were low in ketone and high in fatty 122 acid was substantiated by the FFA and methyl ketone determina- tions which revealed large amounts of FFA in proportion to the amount of ketone. The large quantities of FFA in propor- tion to the methyl ketone content may also have contributed to the resulting atypical flavor. Anderson and Day (1966) discussed the formulation of a synthetic Blue cheese flavor with reSpect to the importance of the relative amounts of flavor compounds in the flavor, rather than the total amounts. Anderson and Day (1966) quantified the secondary alcohols in Blue cheese. The alcohols were present in approximately the same ratios as the ketones, but in lower concentrations. Secondary alcohols were recognized by Stokoe (1928) as result- ing from the action of g, roqueforti on coconut oil. Jackson and Hussong (1958) suggested that the secondary alcohols were formed by reduction of the corresponding methyl ketone. The low levels of methyl ketone in quick ripened Blue cheese may have in turn resulted in low amounts of secondary alcohols which might contribute to an atypical flavor. A mixture containing the FFA, ketones and alcohols found in Blue cheese produced a flavor similar to Blue cheese, but ‘was not considered typical (Anderson and Day, 1966). Large quantities of methyl and ethyl esters as well as lesser quan- tities of primary alcohols have been identified in Blue cheese (Anderson and Day, 1966). Addition of certain esters and alcohols to the FFA, methyl ketone and secondary alcohol mix— ture resulted in an improved flavor, but not a completely 125 typical flavor. The addition of amino acids and sulfur com- pounds originating from proteolysis was suggested as a means of improving the mixtures flavor. Amino acids were suggested by Harper and Kristoffersen (1956) as contributing to the background flavor of cheese. Further investigation of quick ripened Blue cheese might reveal that the methyl and ethyl esters, primary alcohols, amino acids and sulfur compounds necessary for a typical Blue cheese flavor are either absent or present in insufficient quantities to obtain a "balanced" Blue cheese flavor. The role organisms other than 2, roqueforti play in flavor development may influence cheese flavor. The absence of organisms other than P, roqueforti or the presence of such organisms may have influenced the flavor of the quick ripened Blue cheese. Morris, Combs and Coulter (1951) noted that cheeses with normal surface slime had a slightly finer flavor than did cheeses with no slime. Five groups of microorganisms were isolated from the slime of Blue cheese (Hartley and Jezeski, 1954). A yeast isolated from Roquefortcheese by Maxa and. Jidinsky (1956) produced ethanol which was believed to be important for ester formation. Yeasts associated with Blue cheese were found by Anderson and Day (1966) to be capable of ,reducing methyl ketones to secondary alcohols. The authors (1966) speculated that yeasts may play a role in Blue cheese flavor by producing ethanol, 2-methyl- and 2-methyl butanol, and other alcohols, and certain esters. 124 The presence of C3 and C3 methyl ketones.in the quick ripened Blue cheese was not unexpected. Bavissoto, Rock and Lesniewski (1960) identified 2-octanone, and Day and.Anderson (1965) 2-hexanone, 2-octanone and 2-decanone. Butanone—2 was isolated from Blue cheese by Morgan and Anderson (1956). The even numbered ketones identified in Blue cheese probably originated from trace amounts of odd numbered fatty acids in milk fat (Anderson, 1966). Pentanoic, heptanoic and nonanoic acid were reported by Herb §£_§l, (1962) to comprise 0.02, 0.05, and 0.05 per cent respectively of the total saturated fatty acids in milk fat. Methyl Ketones in Quick Ripeneleilled Blue Cheese The homologous series of methyl ketones makes an im- portant contribution to the flavor and aroma of cheeses in which 3. gguefgrti is the ripening agent. Stfirkle (1924) found that P, roqueforti and 2 Species of aspergilli produced methyl ketones when grown for several weeks on fatty acids of intermediate chain length or on neutral fats such as cacao oil. Hydrogenated coconut oil contains relatively large amounts of the intermediate chain length fatty acids. Stokoe (1928) noted that methyl-n-amyl, methylheptyl and methylnonyl ketone were produced when Penicillium_palitans was grown on coconut oil. Grown on coconut oil, P, roqueforti could pro- duce the methyl ketones necessary for the aroma and flavor of Blue cheese. 125 Quick ripened filled Blue cheese made from filled milk containing hydrogenated coconut oil possessed a "strong", but not completely typical Blue cheese flavor. Judges noted a very pronounced ketone flavor in the samples of filled Blue cheese evaluated. Concentrations of the individual methyl ketones in two samples of quick ripened filled Blue cheese are presented in Table 9. Batches of filled Blue cheese had noticeably more abundant mold growth than Similar batches of quick ripened Blue cheese. Both filled Blue cheese samples contained substantially more methyl ketone than any of the previous Danish Blue or quick ripened Blue cheese samples analyzed (Table 9). Table 11 provides a comparison of the mole percentages of the fatty acids in coconut oil with the percentages of FFA found in quick ripened filled Blue cheese. The mole per- centages of the individual fatty acids in coconut oil and the FFA in filled Blue cheese are very similar. Extensive hydroly— sis of the coconut oil in filled Blue cheese may have resulted in the marked similarity between the mole percentages of the FFA liberated and the fatty acids in coconut oil. The per- centages of fatty acids released from lard by Pseudomonas fragi after 7 days approximated the percentages of fatty acids in the original lard (Alford £3.3l3, 1961). In early stages of hydrolysis variations between the percentages present in the lard and the percentages liberated were noted. Stokoe (1928) studying the action of g, palitans on coconut oil reported that 126 Table 11. Comparison of the mole percentages of the free fatty acids and methyl ketones found in filled Blue-vein type cheese. Acid Mole%l Mole% Ketone Mole % chain acid in acid in chain ketone in Cheese length coconut oil cheese Length cheese 4 ---- ---- 5 ---- Ripened A 6 1.2 1.4 5 0.7 8 11.7 8.5 7 20.9 10 15.2 12.0 9 51.9 12 71.9 78.5 11 46.4 4 ---- ---- 5 ---- 6 1.2 1.4 5 0.6 8 11.7 7.5 7 25.9 10 15.2 12.4 9 52.5 12 71.9 78.7 11 42.8 1Calculated from data reported by Bailey (1964). 127 the fatty acid composition of the original coconut oil and the FFA separated from the rancid fat were similar. Far greater concentrations of 2-nonanone and 2-undecanone were present in quick ripened filled Blue cheese than had been previously reported for the C9 and C11 ketones in Blue cheese (Schwartz and Parks, 1965; Anderson and Day, 1966). The large amounts of the C9 and C11 methyl ketones may be a reflection of the amount of fatty acid precursor available. The quantity of 2-heptanone formed is high relative to the amount of its precursor, caprylic acid when compared to the amount of 2-undecanone formed from its precursor, lauric acid. However, the amount of ketone formed by g, roqueforti spores during the ripening of filled Blue cheese may be more closely related to the amount of fatty acid precursor than was the case in Danish Blue (Table 5 and 9) or domestic Blue cheese (Anderson, 1966). Filled Blue cheese contained 9 times more free lauric than caprylic acid; two times more 2-undecanone than 2-heptanone. Danish Blue samples analyzed contained 4 times more free lauric than caprylic acid; more 2-heptanone than 2-undecanone was formed. During fatty acid oxidation the amount of oxygen consumed by g, roqueforti decreased as the fatty acid chain length increased beyond 10 carbons (Girolami and Knight, 1965). Between pH 5.5 and 6.5 Lawrence (1966) found that 25 per cent of the laurate present was oxidized to 2-undecanone, but greater amounts of caprylate and caprate were converted to 128 their corresponding methyl ketones. Lawrence (1967) reported that at pH 6.8 approximately 2 per cent of the trilaurin was metabolized by spores of g, roqueforti to the corresponding ketone with one less carbon atom. The decreased ability of the mold spores to convert lauric acid to 2—undecanone may have been somewhat masked in the filled Blue cheese by the very large amounts of lauric acid precursor available. No even numbered methyl ketones were found in the quick ripened filled Blue cheese samples. Trace amounts of C3 and C13 methyl ketone were observed. Stokoe (1928) did not find methylundecyl ketone in distillates of a g, pplitans growth medium.which contained coconut oil. Steam-distilled coconut oil, which like milk fat is characterized by a high propor- tion of C3-C13 saturated fatty acids, contained no methyl ketones, except acetone (Lawrence and Hawks, 1966). The authors (1966) Speculated that the occurrence of beta-keto acids in triglycerides was possibly unique to milk fat. If the C3-C13 beta-keto acid were incorporated into the trigly- cerides of coconut oil they were thought to have been reduced in their original position in the triglyceride rather than as the free beta—keto acid. Small quantities of residual milk fat in the skimmilk used for preparation of quick ripened filled Blue cheese may account for the C3 and C13 methyl ketones observed in filled Blue cheese. Trace amounts of butyric acid were found in filled Blue cheese (Table 7). Tridecanone-2 may have originated from trace amounts of the C14 129 beta-keto acid ester found in milk fat (Boldingh and Taylor, 1962; Parks 35 gl,, 1964). As evidenced by the appearance of large numbers of blue— green g, roqueforti conidiospores mold growth was abundant throughout the ripening of quick ripened filled Blue cheese. The larger total quantity of methyl ketone in quick ripened filled Blue cheese than in quick ripened Blue cheese may have resulted from good mold growth and sporulation or from the availability of greater amounts of fatty acid precursor. The FFA are converted to methyl ketones by P, roqueforti spores (Gehrig and Knight, 1958). Studies by Lawrence (1966) re- vealed that the rate of fatty acid oxidation depends upon the amount of fatty acid available per spore. Quick ripened Blue cheese was often characterized by poor mold growth. Anderson and Day (1966) emphasized the importance of the relative amounts of flavor compounds in a food, rather than the total amounts. The amounts of 2-heptanone, 2-nonanone and 2-undecanone were proportionally higher in filled Blue cheese than in domestic Blue (Schwartz and Parks, 1965; Anderson and Day, 1966). The amount of 2-pentanone in filled Blue cheese was approximately equal to that quantity of the other ketones found in the filled Blue cheese samples. Filled Blue cheese contained only trace amounts of butyric acid, therefore acetone levels were relatively low. Butyric acid contributes to the peppery flavor of Blue cheese and low concentrations of this acid may have contributed to the 150 atypical flavor of the filled Blue cheese. Flavor variations between filled Blue and domestic Blue cheese may have resulted from an improper "flavor balance". Stokoe (1928) observed the action of P, palitans on coconut oil distillates of a g, palitans ferment yielded a mixture of FFA, methyl ketones and the secondary alcohols-- methylheptyl, methylamyl and methylnonyl carbinol. Esters of caprylic acid were also identified. An acceptable filled Blue cheese would seem possible, since mold action on coconut oil produces many of the flavor compounds associated with Blue cheese such as FFA (Currie, 1914), methyl ketones (Stdrkle, 1924), secondary alcohols (Jackson and Hussong, 1958) and methyl and ethyl esters of fatty acids (Day and Anderson, 1965). By abbreviating the ripening period for quick ripened filled Blue cheese a milder, more desirable flavor may have been obtained. Use of a modified fat system, such as that suggested by Ryberg (1968) for cheeses requiring lipase activ- ity for flavor development, may have resulted in a better "flavor balance." A modified lauric with added lipolized butteroil could provide a flavor profile similar to butterfat for a cheese dependent on lipase activity for flavor (Ryberg, 1968). Anderson and Day (1966) described the development of a synthetic Blue cheese flavor which contained FFA, methyl ketones, secondary alcohols and fatty acid esters. Addition of a selected synthetic flavor to quick ripened Blue cheese 151 curd to "balance" the flavor might produce a product which more closely resembles Blue cheese organoleptically. Compounds such as methyl and ethyl esters, which are formed during the long aging period of normal cheese, might be of value in preparing a quick ripened cheese with‘the flavor characteristics of a normally cured Blue cheese (Anderson, 1966). Further study on the flavor chemistry of quick ripened Blue cheese is needed before such additions can be satis- factorily carried out. Control of mold growth and/or mold Sporulation is necessary to produce a quick ripened Blue cheese which would consistently possess the same flavor characteristics from batch to batch. Retention of Some Aroma Compounds During Spray Drying and Freeze Drying 9f Blue Cheese The relative amounts of flavor compounds retained during the Spray and freeze drying of Blue cheese were compared by drying Blue cheese slurries containing 40 per cent solids. Portions of the same slurry were used for both the spray and freeze drying trials to provide identical quantities of aroma compounds in the slurries prior to dehydration. The dried Blue cheese powders were diluted to 40 per cent total solids with distilled water and mixed to a homogenous slurry in a Waring blendor prior to analysis. The FFA and methyl ketone content of the reconstituted cheese powders was determined 152 in duplicate as previously described for the analysis of Danish Blue cheese. Retention of FFA and Methyl Ketones During_Spranyrying Spray dried Blue cheese powders were prepared using the drying conditions outlined in Table 2. Trial 1 and 2 cor- respond to the conditions used to dry Danish Blue cheese samples A and B respectively. Efforts were made to achieve drying conditions that produced the least heat damage to the cheese powder commensurate with the desired moisture content. Exit air temperatures above 176 F were found by Bradley and Stine (1964) to produce a stale flavor in Cheddar cheese powders. Sivetz and Foote (1965) have stated that a low inlet air temperature (less than 599 F) favors retention of volatile flavors. Inlet and exit air temperatures were found by Reineccius and Coulter (1969) to have an effect upon the re— tention of added flavors. They found that retention of flavor volatiles varied inversely with increase in inlet air tempera- ture. The lowest inlet air temperature (520 F) used by Reineccius and Coulter (1969) resulted in optimum retention of added flavors. High exit air temperatures (194-221 F) favored retention. Improved retention at higher exit air temperatures may have been caused by the lower relative humid- ity of the drying air which resulted in an increased rate of water removal from the atomized particles. A selective mem— brane may have formed rapidly around the partiCles and thereby improved retention of volatile flavor. 155 Retention of FFA During Spray Drying: Retention of FFA in spray dried Blue cheese is reported in the data of Table 12. Table 4 contains information concerning the FFA concen- tration for cheese samples A and B prior to drying. There appeared to be complete retention of the FFA containing 16 or more carbon atoms. A very small amount of the C14 fatty acid was lost. Over 80 per cent of the butyric acid was lost during the preheating and spray drying of Blue cheese. The retention of the C4-C14 FFA increased as the chain length of the acid increased. Retention of these acids appeared to be related to their volatility. The boiling point of the flavor compound was in direct relation to the losses occurring 9(Bradley and Stine, 1968; Saravacos and Moyer, 1968a). Butyric acid with a boiling point of 165.5 C at 757 mm Hg is much more volatile than myristic acid with a boiling point of 250.5 C at 100 mm Hg (Merck Index, 1960). Boudreau, Richardson and Amundson (1966) noted a similar trend in FFA losses when Spray drying butter. More than two- thirds of the butyric and caproic, and about one-half of the caprylic and capric acids were lost during the drying of butter. The retention of butyric and capric acid in Spray dried butter was greater than the retention of the same acids in the Spray dried Blue cheese samples. Aroma retention was shown.by Sivetz and Foote (1965), Rey and Bastien (1962), Menting and Hoogstad (1967), Reineccius and Coulter (1969) and Saravacos and Moyer (1968a) to increase as the concentration 154 Table 12. Retention of free fatty acids in spray dried and freeze dried Blue cheese. Aroma retention1 (%) Fatty Spray-dried2 Freeze-dried3 acid A B A B 4:0 14.5 19.0 54.1 58.5 6:0 28.4 50.7 68.2 71.6 8:0 59.6 65.1 85.0 86.1 10:0 74.8 76.0 90.0 91.5 12:0 82.2 85.5 98.4 99.5 14:0 92.7 94.6 97.5 100.5 16:0 99.6 100.4 99.5 100.2 18:0 96.9 97.7 99.8 97.4 18:1 100.0 101.8 97.6 100.5 18:2 99.7 102.9 99.7 102.9 18:5 98.8 100.8 96.5 101.1 1Initial concentrations of the fatty acids are given in Table 4. 2Drying conditions are shown in Table 2. 3Drying conditions are illustrated in Figures 4 and 5. 155 of the dissolved solids in the solution to be dried increase. The dissolved solids concentration of the butter, FFA, non- fat dry milk, water mixture dried by Boudreau, Richardson and Amundson (1966) was higher than that in the Blue cheese slurries dried. These authors (1966) found that losses of butyric acid were greater from powder in the cyclone collector than from that in the base of the dryer. Per cent recoveries of added fatty acids from spray dried butter were determined on powders removed from the base of the dryer, while Blue cheese powders were removed from the cyclone collector. The Blue cheese slurries that were Spray dried had pH values of pH 5.4-5.6. The pH of the butter mixture spray dried by Boudreau, Richardson and Amundson (1966) was reported as 6.9. At these pH values some of the FFA will be in the salt form and therefore less volatile. Conversion of fatty acids to their sodium salts was shown by Boudreau, Richardson and Amundson (1966) to significantly reduce their volatility. At a pH of 5.4-5.6 more of the FFA will be in the acid form than at pH 6.9. Therefore, greater losses might occur from a Blue cheese slurry at pH 5.4-5.6 than from butter at pH 6.9. Reineccius and Coulter (1969) observed that the concentration of diacetyl added to skimmilk prior to drying influenced the proportion of diacetyl retained during drying. Retention of Methyl Ketones During Spray Drying: The data illustrating retention of methyl ketones during the spray dry- ing of Blue cheese are presented in Table 15. Samples A and B 156 Table 15. Retention of methyl ketones in spray dried and freeze dried Blue cheese. Aroma retention1 (%) Methyl Ketone Spray-dried2 Freeze-dpied3 chain length A B A B 5 0.0 14.4 0.0 2.1 5 15.1 25.9 6.0 9.0 7 12.9 22.5 5.0 5.5 9 19.5 29.1 4.4 4.5 11 51.2 59.5 27.2 20.8 15 + 15 58.1 65.9 69.9 70.7 1Initial concentrations of methyl ketones are given in Table 9. 2Drying conditions are shown in Table 2. 8Drying conditions are illustrated in Figures 4 and 5. 157 prior to dehydration contained the quantities of the methyl ketones reported for Danish Blue cheese samples A and B respectively (Table 9). Both samples of Danish Blue cheese exhibited a marked reduction in methyl ketone content upon spray drying. The 10ss of 2-heptanone, 2-nonanone, 2-unde- canone and 2-tridecanone plus 2—pentadecanone during Spray drying appeared to be related to the boiling points of these compounds. Retention of 2-heptanone was about 15-22 per cent, while 58-64 per cent of the 2-tridecanone plus 2-pentadecanone was retained under the same drying conditions. There may have been a slight tendency for greater reten- tion of 2-pentanone than 2-heptanone in the samples analyzed. The concentrations of acetone and 2-pentanone were very low initially in Danish Blue cheese compared to the other ketones. Samples A and B contained prior to spray drying 4 and 5 ppm 2-pentanone reSpectively. The concentration of 2-heptanone was approximately 62 and 79 ppm for samples A and B reSpective— ly. Reineccius and Coulter (1969) noted that the concentra- tion of diacetyl added to skimmilk prior to spray drying in- fluenced the proportion of diacetyl retained during drying. A greater percentage of diacetyl was retained when the lower concentration was added than when the higher concentration was dried. There may have been a slightly greater retention of 2-pentanone than 2-heptanone in Blue cheese powder, because of the concentration differences between the C5 and C7 methyl ketones. 158 The relative solubility of 2-pentanone in water or the fat phase compared to 2-heptanone may also have resulted in greater retention of 2—pentanone. Saravacos and Moyer (1968a) reported that the volatility of aroma compounds in aqueous solutions depends upon the vapor pressure and the solubility in water of that compound. Those compounds partially soluble in water have relatively high volatilities. Lipid compounds found in foods may have an effect on the retention of flavor compounds since most of these compounds are fat soluble (Saravacos and Moyer, 1968b). The solubility of 2—pentanone in the water and fat of Blue cheese may be such that its relative volatility compared to 2-heptanone is low. Larger amounts of methyl ketone were retained in sample B than in A. The Spray drying conditions were standardized as close as possible, but when drying a small quantity of cheese (17 lbs of solids) in a large commercial size dryer some variations are to be expected. Some of the powder pro- duced from sample A Blue cheese may have remained in the dryer longer at dryer temperature by virtue of the fact that prob- lems were encountered with particles adhering to the walls of the dryer during the initial stages of drying. These cheese powders may not be representative of the magnitude of aroma retention that one might have obtained during a longer drying run. However even in commercial spray drying of Blue cheese there is a great tendency for powder to "hang up" or accumu- late on the walls of the dryer. 159 The moisture contents of samples A and B were 1.54 and 2.55 per cent respectively. The variation in moisture con- tent between the powder samples would not appear to have influenced the relative proportion of aroma retained in light of the findings of Menting and Hoogstad (1967), Flink and Karel (1969) and Radanovics (1969). A critical moisture con- tent of 9 per cent was reported by Menting and Hoogstad (1967) for a malto-dextrin model system. At moisture contents below 9 per cent no adsorption of acetone occurred, while at higher moisture levels the uptake of acetone increased with the in- crease in moisture. Rehumidification studies on dried mal- tose systems showed that retained volatiles were not removable until a critical moisture content (5.24 per cent) was reached (Flink and Karel, 1970). During freeze drying of a maltose model system volatiles were lost only when moisture contents in excess of 4.8 per cent were reached. A critical moisture level for freeze dried sour cream somewhere above 1.9 per cent was reported by Radanovics (1969). Methyl ketones were lost in greater proportions during spray drying than were the FFA. The C3-C9 methyl ketones have lower boiling points than the 205 C reported for caproic acid (Merck Index, 1960). Possibly due to differences in their volatilities losses of the C3-C3 ketones were more extensive than those losses found for caproic and higher fatty acids. Bradley and Stine (1968), using gas chromatography, noted that the majority of low boiling compounds were lost during the Spray drying of cheese slurries. 140 Retention of FFA and Methyl Ketones During Freeze Drying The temperature profile of the Blue cheese slurries and the temperature of the platens during freeze drying were measured with the aid of thermocouples (Figures 4 and 5). The temperature profiles for Blue cheese show close similarity with freeze drying curves obtained when dehydrating sour cream (Radanovics, 1969), except for the 26 hr drying time required for Blue cheese slurries. A platen temperature of 100 F was chosen to keep the product surface temperature below 100 F during dehydration. A higher temperature may have been detrimental to final product quality (Harper and Tappel, 1957). The temperature of the platen upon which the samples were placed was 20 F lower than the control platen. The lower temperature, measured at the interface of the surface of the platen and sample tray was attributed to several factors (Radanovics, 1969): (1) The constant BTU requirement for the latent heat of sublimation, (2) The cooling effect of the sample which had a temperature of about -20 F, (5) Poor heat transfer from the heating fluid to the surface of the platen. The temperature of the product was constantly monitored and freeze drying terminated when the temperature at the bottom of the sample was within 10 F of the sample platen temperature. Samples A and B at the termination of the drying trial had 1.25 and 1.79 per cent moisture respectively. 141 .¢ HmHHB “mmmmnu msam Umfiup museum mo waflmoum musumumewB 25: . us: 62:5 mWIIe NW LIMWIII1IILWIII4IILflI _ w _ W Mg “.0 SOP—.00 a umahguazmhlm witsmwmo mESbShEZEh flflmmamlk O\III 95.5; zmb; mcfibfimtfimh ZNDHI .xxfl2bu .e musmfim 4. :1 ‘ 3801V838W31 142 .m amass “mmmmso msam Umwup mummum mo mammoum musumummfima mane: . m2: oon AW _ cw _ oWIIIdIILfl 4 IIwI s wfl ,4 «I s udifimruo Zbkkxuha mfisbflmizwrlm mg no me—Kmmazw... many—3m IF m¢953m£2wh ZHEUE mfliiim 13% z .dXKZQu .m musmflm o :I ‘ BHDIVEIBdWBJ. é 145 The surface temperature of the product increased above the temperature at the bottom of the sample after 5 hours of. drying and continued to increase until drying was complete. Apparently as long as there were subliming vapors passing through the dried upper layer the temperature of the surface remained cooler than the platen supplying the heat. Retention of FFA DuringiFreeze Drying: The loss of butyric, caproic, caprylic and capric acids during freeze drying of Blue cheese appeared to be related to the boiling points of these compounds (Table 12). Butyric acid (b.p. 165.5 C at 757 mm Hg) losses were 42-46 per cent while only 10-11 per cent capric acid (b.p. 268-270 C) was lost under identical drying conditions. The higher boiling Ola-€13 saturated fatty acids and oleic, linoleic and linolenic acids were completely retained during the drying process. The boil- ing point of flavor compounds was reported by Boudreau, Richardson and Amundson (1966), Bradley and Stine (1968) and Saravacos and Moyer (1968a) to be in direct relation to the losses occurring during dehydration. Freeze dried Blue cheese retained more FFA than did spray dried Blue cheese. Reconstituted freeze dried Blue cheese contained substantially larger quantities of the short chain volatile fatty acids. Over 50 per cent of the butyric acid present in the original cheese was retained during freeze drying, while less than 20 per cent of the butyric acid was retained in spray dried Blue cheese. Reineccius and Coulter 144 (1969) compared the retention of diacetyl added to Skimmilk during roller—, spray- and freeze-drying. Double-drum drying resulted in retention of a negligible amount of diacetyl. Freeze drying resulted in retention of 59-67 per cent of added diacetyl, while the Spray dried powder retained about 61 per cent of the added aroma compound. Losses of volatile acidity from foam spray dried sour cream were greater than the acidity losses observed in freeze dried sour cream (Desai, 1966). A 17.5 per cent fat sour cream that was freeze dried retained over 70 per cent of the volatile acidity present in the con- trol, while a foam spray dried sample retained about 50 per cent of its original volatile acidity. Greater volatile acid loss during Spray drying was attributed to subjection of the sour cream to high temperatures. During freeze drying losses in the volatile acids content is considerably minimized and restricted to a decrease in amounts of those water soluble, low molecular weight compounds which were easily removed dur- ing sublimation of ice. The drying procedure for Blue cheese was studied by Sj6str6m and Willart (1959) with regard to the loss of FFA and their effect on errors in the determination of water and dry matter. At a temperature of 212 F for 4-5 hr no acetic, butyric, caproic or caprylic acid was retained. About 12 per cent of the capric, 40 per cent of the lauric and 85 per cent of the palmitic and myristic acids were retained. Spray drying does not subject the drying product to such severe 145 heat treatments, therefore greater retention of volatiles would be expected. The temperature of the drying particle during spray drying would be close to the wet-bulb tempera- ture early in drying due to the cooling effect of evaporation (Copley and Van Arsdel, 1964). As more water is removed and the particle approaches dryness the particle temperature would approach that of the exit air temperature. Heating the cheese slurry to 120 F prior to spray drying may have also contributed to volatile fatty acid losses. Bradley and Stine (1968) showed that the preheat treatment effected the losses of natural flavor compounds in Cheddar cheese slurries. Cheese powders dried from slurries heated only to 120 F showed better retention than powders prepared from slurries heated to 180 F. Approximately 55 per cent of added diacetyl was retained in skimmilk if the diacetyl was added to fluid skimmilk concentrate and then Spray dried (Reineccius and Coulter, 1969). When skimmilk with added diacetyl was condensed in a vacuum pan and then spray dried, only 15 per cent of the added diacetyl was retained in the resultant skimmilk powder. A large portion of the volatile flavor constituent originally present had distilled from the food product with the water. Caproic, caprylic and capric acids were suggested by Currie (1914) as being responsible for the characteristic "peppery" flavor of Roquefort cheese. Parmelee and Nelson (1949c) found a significant correlation between volatile 146 acidity and the flavor scores of Blue cheese made from pas- teurized homogenized milk. Losses of the volatile acids acetic, butyric, caproic and caprylic would result in a sig- nificant change in the flavor of the reconstituted dried Blue cheese powder, particularly since the volatile FFA are important to the flavor and aroma of Blue cheese. Freeze drying produces a powder retaining substantially higher quantities of the volatile FFA than does spray drying. Retention of Methyl Ketones During Freeze Dryipg: Relatively small quantities of methyl ketone were retained during the freeze drying of Blue cheese (Table 15). Less than 10 per cent of the acetone, 2-pentanone, 2-heptanone and 2-nonanone were retained in freeze dried Blue cheese. Larger quantities of 2-undecanone and 2-tridecanone plus 2-penta- decanone were retained. Sterkle (1924) found that distillates of Roquefort cheese had an odor characteristic of 2-heptanone and 2-nonanone, and concluded that the flavor and aroma of the cheese was due in part to these ketones. Substantial losses of these ketones would greatly influence the flavor of the reconstituted dried cheese. Residual quantities of less than 1 ppm acetone and 2—pentanone were in the freeze dried Blue cheese samples analyzed. Greater deviations between duplicates resulted when such small quantities of methyl ketones were determined. For 2-pentanone, samples A and B had deviations of 4.5 and 5.6 per cent respectively. A deviation of 12.2 per cent between 147 duplicates was observed for acetone in sample A. The large deviations for these ketones may have contributed to the slightly greater quantities of 2-pentanone than 2-heptanone and 2—nonanone or the small amounts of 2-pentanone in the original cheese (Table 9) may have been retained to a greater extent than the longer chain length ketones. As discussed previously, lower concentrations of diacetyl were retained to a greater degree than were higher concentrations (Reineccius and Coulter, 1969). The retention of the C3, C5, C7 and C9 methyl ketones does not appear to be related to the boiling points of these compounds. Because such small quantities of ketone were retained the effect of increased boiling points may not have been detectable. Factors other than boiling point may have influenced the retention of methyl ketones. Lipid compounds found in food may have an effect on the retention of flavor compounds, since most of these compounds are fat soluble (Saravacos and Moyer, 1968b). However, addition of monoglycerides to gels and apple did not improve retention of flavor compounds in the freeze dried product. Saravacos and Moyer (1968a) reported that the volatility of aroma compounds in aqueous solutions depends on the vapor pressure and the solubilities in water. Acetic acid which had about the same vapor pressure ras ethyl butyrate was less volatile than the butyrate because it was completely soluble in water. 148 The slightly higher retention of methyl ketones in Spray dried Blue cheese as compared to freeze dried Blue cheese was unexpected and contrary to the results obtained for re- tention of FFA in Spray- and freeze-dried Blue cheese (Table 12). As previously mentioned, boiling point and relative volatility may effect the retention of volatile aroma com- pounds. Issenberg, BoSkovié'and Hwang (1969) and Issenberg, Greenstein and BoSkovid (1968) noted that rate of loss of volatile organic compounds is partly determined by resistance to diffusion through dried material and rate of water removal. When solids were present adsorption of flavor components may have been significant. The affinity of the volatile compound for the system in which the compound was present determined its ability to adsorb. In drying methods such as spray drying, the formation of a membrane is observable soon after initiation of drying (Menting and Hoogstad, 1967). The membrane selectively re- tained volatiles, but not water in model systems. Thijssen (1965) explained the high aroma retention in the spray drying of concentrated solutions by assuming that during drying a film was very rapidly formed around the droplet. The film would act as a concentrated layer of solids, where adsorption could take place for both water and flavor volatiles. The twater molecules desorbed as the drying continued and the forces of bonding between the solids and flavor volatile being stronger helped diminish losses of flavor volatiles. 149 During freeze drying there is no experimental evidence for the theory of membrane formation. The water and other volatile compounds pass through various layers by hydro- dynamic flow through the porous media. The driving force is a total pressure gradient between the ice interface and the surface (Harper and Tappel, 1957). Radanovics (1969), Issenberg, Boékovié and Hwang (1969) and Issenberg, Greenstein and BoSkovié (1968) concluded that adsorption rather than mem- brane formation is responsible for the retention of flavor volatiles during freeze drying. Flink and Karel (1970) in- terpreted results of their studies on organic volatile loss from freeze dried maltose solutions as being consistent with entrappment of volatile material with amorphous microregions of hydrogen bonded carbohydrate molecules rather than reten- tion~by adsorption. The retention of methyl ketones in spray versus freeze dried Blue cheese may also have been influenced by the mechanism involved in volatile retention during the respective drying processes. Interpretation of volatile losses from a complex food system such as Blue cheese is very difficult due to the many factors-influencing volatile retention. Nawar (1966) demon- strated the relationship between the concentration of a specific compound in the vapor phase at a given temperature -and the interplay of the following variables: vapor pressure of the compound; type of medium in which flavor is distributed: degree of its solubility in the medium; concentration of the 150 compound in the liquid phase; its miscibility with other organic compounds in the mixture; and the presence of salts and sugars. Nawar (1966) found that the addition of an equal amount of acetone to 2-heptanone reduced the headspace response of the latter by about one-half. Dilution of the acetone, 2-heptanone mixture with water, which is very miscible with acetone, doubles the concentration of 2-heptanone in the headSpace. In Blue cheese the individual methyl ketones are not present in the same concentration and losses may be influenced by such concentration differences as well as inter- play of other variables. These variables make the interpreta- tion of losses of volatile materials in Blue cheese a formid— able task. SUMMARY AND CONCLUSIONS Batches of quick ripened loose curd Blue cheese were prepared and changes in pH and volatile acidity for 16 batches were determined at 24 hr intervals during the 7 day ripening period. Mean pH values for the batches reached a minimum of 4.9 after 48 hr of ripening and a maximum of 6.4 at 4 days. Following the fourth day of ripening the pH declined gradually. Average volatile acidities reached a minimum after 48 hr of ripening and steadily increased to a maximum of 77.2 ml of 0.1 N acid per 100 g of cheese at the seventh day. The vola- tile acids content of the cheese continued to increase during subsequent storage at 40 F. Changes in pH and-volatile acid- ity during curing of quick ripened Blue cheese followed the same patterns previously observed for normal Blue cheese hav— ing a curing time of several months. Imported Danish Blue cheese samples analyzed exhibited wide variations in their FFA and methyl ketone content. Relatively low amounts of butyric, caproic and caprylic acid in the cheese samples resulted in the lack of "sharp" flavor normally attributed to such cheese. Nonanone-2 was the most ”abundant ketone in both Danish Blue cheese samples. The quan- tity of methyl ketone produced did not depend directly upon the amount of available fatty acid precursor. 151 152 To ascertain why the flavor of some quick ripened Blue cheese samples was described as strong and atypical samples were analyzed for their FFA and methyl ketone content. The total quantity of FFA in each of three samples was far greater than the total quantity previously reported for nor- mally cured Blue cheese. Compared to the quantity of fatty acid precursor available quick ripened Blue cheese contained relatively low quantities of methyl ketone. Ripening a sample of cheese an additional day significantly increased the FFA content of the cheese but did not produce a concomi- tant increase in the methyl ketone content. Quick ripened filled Blue cheese developed a flavor similar to Blue cheese within 5 days. A high FFA and methyl ketone flavor was evident in the cheese samples. Filled Blue cheese samples contained substantially more methyl ketone than that reported for Blue cheese made from.bovine milk. The most abundant ketone was 2-undecanone, with slightly smaller amounts of 2-nonanone present. Extensive fat break- down (67-70 per cent hydrolysis) was evidenced by the large quantities of FFA present, with lauric acid predominating. The high mole percentages of lauric, caprylic and capric acid plus high 2-undecanone and 2-nonanone content resulted in cheeses possessing a flavor described as not completely typical ‘of Blue cheese. The relative amounts of flavor compounds retained during spray and freeze drying of Blue cheese were compared. 155 The retention of the C4-C14 even number carbon fatty acids during spray drying increased as the boiling point of the acid increased as did the retention of the C4-C1. fatty acids during freeze drying. Freeze dried Blue cheese re- tained substantially larger quantities of the short chain volatile acids than were retained during spray drying. Over 50 per cent of the butyric acid present in the original cheese ‘was retained during freeze drying, while less than 20 per cent of the butyric acid was retained in spray dried Blue cheese. The loss of methyl ketones during spray drying appeared to be related to the boiling point of the C3-C15 odd numbered carbon methyl ketones. Contrary to the findings with reSpect to FFA losses from Blue cheese during dehydration more methyl ketones were retained in spray than in freeze dried Blue cheese. 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Cresol red: One and three tenths ml of 0.1 N sodium hydroxide added to 50 mg o-cresolsulfonphthalein in 20 ml of 95 per cent ethanol, and made to 50 ml with distilled water. Alphamine red-R: Four tenths gram in 100 ml of water. Preparation of Adsorbent (1) Two and four-tenths ml of Alphamine red-R indicator were mixed in a 50 ml beaker with 6 ml of a sugar-water solu- tion (2:1) and 0.1 ml of 0.1 N sulfuric acid. (2) The indicator mixture was added Slowly to a swirling suspension of 15 g Celite and 150 ml of Skellysolve B and acetone (1:1) in a Waring Blendor. The adsorbent was mixed vigorously for 5 min. Column Preparation (1) The adsorbent slurry was poured into a 20 x 500 mm chromatographic tube and the column repeatedly inverted to 175 174 allow air bubbles to escape to the surface. (2) The adsorbent was packed using nitrogen pressure (10 psi). The pressure was released when the solvent had been expressed to the top of the adsorbent. (5) A.1 per cent acetone in Skellysolve B (BAl) solution was carefully added to the surface of the packing to a depth of about 10 cm. (4) Eight grams of cap material (sodium sulfate, Celite, ammonium sulfate in the proportions 12:8:1) were added to the column as a slurry in 25 mls of BA;. The cap was compressed using nitrogen pressure 10 psi. (5) Seventy—five ml BA; were forced through the column to remove all the acetone-Skellysolve B (1:1) present. (6) Five grams of cheese slurry or reconstituted dried cheese were acidified to pH 1.9 with 50 per cent sulfuric acid. Ten grams of silicic acid, Mallinckrodt 2847, dried at 175 C for 72 hr., were ground with the cheese sample in a mortar with a pestle. Two grams of prepared sample were added to the top of the column by sifting it into a layer of BA;. A filter paper disk was then placed on top of the column. (7) Elution of butyric and higher fractions was achieved using 200 ml of BA;. (8) After the BA; had just entered the cap material, approximately 250 ml of 15 per cent acetone in.Skellysolve B were added to elute the acetic acid. The elution of acetic acid was observed visually as a light blue band moving down the column packing. 175 (9) Twenty milliliter fractions were collected until the blue acetic acid band was within 5 cm of the bottom of the column packing, at which time 100 ml fractions were collected until the acetic acid band was completely removed. (10) Titration of fractions was accomplished using a 5—ml buret graduated to 0.01 ml. A stream of nitrogen over the sample prevented fading of the end points. The sample was agitated using a magnetic stirrer. One drop of cresol red indicator per 20 ml of solution to be titrated served as the indicator. A 0.005 N sodium hydroxide was used as the standard base. Recovery of acetic acid was determined, by adding 2 ml aliquots of a composite test solution containing known amounts of butyric, propionic, acetic and succinic acid to prepared liquid-liquid partition columns. The procedure used was as outlined above, except that the composite sample was added to the sodium sulfate-Celite-ammonium sulfate mixture in the column. The sample was mixed with the sulfate-Celite slurry utilizing a stainless steel rod 1.5 x 500 mm with a loop 10 mm in diameter formed at the end. The rod was suspended ver- tically from the chuck of a drill motor. The motor was started and the rod moved up and down so that the rotating loop homog- enized the sample and cap material. The rod was rinsed with BA; before removal from the column. 176 Determination of Butyric and Higher Acids Reagents Silicic acid: Mallinckrodt 2847, 100 mesh, activated 175 C for 18 hr. Stored in a desiccator. Glycol reggent: Dissolved 700 mg bromocresol green, sodium salt, in 700 ml ethylene glycol by warming on a steam bath. Cooled. Added 200 ml water to the 700 ml ethylene glycol-bromocresol green solution; then added 40 ml 0.1 N ammonium hydroxide and enough water to make one liter. Column packing: Mixed 100 g of silicic acid/95 ml glycol solution in a mortar with a pestle to form a homogeneous powder. Stored in tightly stoppered bottle. Isopropanol-KOH: Twenty—five grams of KOH pellets (85 per cent) was dissolved in 400 ml of isopropanol by warm- ing on a steam bath and swirling. The supernatant isopropanol— KOH solution was decanted from aqueous KOH clinging to the bottom of the flask. Solution was cooled and stored at 40 F. Isolation of FFA from.Cheese (1) Five grams of a representative sample of cheese were acidified to pH 1.9, as determined by acidifying a separate sample with 50 per cent sulfuric acid. (2) Five to ten milligrams each of 7:0 and 17:0 fatty acids were added in about 5 ml of Skellysolve B to the cheese with thorough mixing. 177 (5) Nine grams of silicic acid were added to the cheese with thorough mixing. (4) The chromatographic column, described by Keeney (1956), was prepared by mixing 55 g of packing with 150 ml of-1 per cent butanol in hexane to form a slurry. The slurry was added to a 25 x 500 mm column, equipped with a fritted—glass filter, 2 mm Teflon stopcock and a detachable 500 ml separatory funnel. The column was packed using 5-10 psi nitrogen pressure so as to obtain a flow rate of approximately 5.5 ml/min. The silicic acid, cheese mixture was added as a cap to the column packing. (5) Fat was extracted with 400 ml of Skellysolve B con- taining 1 per cent butanol. The eluate was saved to be passed over a silicic acid-KOH column. Removal of FFA from Fat Materials Silicic acid. Coarser particles of silicic acid were selected by suspending 100 g in 400 ml methanol and decanting and discarding the Silicic acid that did not settle within 5 min. This procedure was repeated once with methanol and once with 400 ml of acetone. The remaining silicic acid was rinsed with ethyl ether and air dried. (1) Fifty grams of the coarse particle preparation were washed 5 times with 150 ml ether containing 2.5 per cent phosphoric acid and then washed 2 times with 150 ml ether. 178 (2) The silicic acid was air dried, after which it was exhaustively washed with distilled water and redried. (5) Silicic acid was activated at 175 C for 18 hr and stored in a desiccator. Procedure (1) Four grams of prepared silicic acid were weighed into a 50 ml beaker. Eight ml of isopropanol-KOH and 24 ml of ethyl ether were added to the silicic acid with mixing. After standing 5 min, the silicic acid was slurried into a 18 x 180 mm chromatographic column, equipped with a fritted glass filter, 2 mm Teflon stopcock and a detachable 250 ml liquid reservoir. The column was washed with 100 ml of ether and air bubbles removed with a glass rod. (2) The eluate from the fat extraction column was passed over the silicic acid-KOH column at a rate of 5 ml/min. (5) The column was washed with 75 ml of ether to remove the lipids. **(2) The FFA were eluted with 60 ml of ether containing concentrated phOSphoric acid (2.5 per cent, v/y) at a flow rate of 10 ml/min. The eluate was collected in a 250 ml centrifuge bottle. (5) Column was washed 2 times with 40 ml portions of ethyl ether and eluate collected in centrifuge bottle. (6) Methanol (70-80 ml) was added to the combined eluates and the solution titrated to the phenolphthalein end point with 1 N methanolic-KOH under nitrogen. / 179 (7) The precipitated potassium salts of acidic constitu- ents other than fatty acids were removed by centrifugation (1200 rpm) and the clear supernatant containing soluble salts of the fatty acids transferred to a 500 ml round bottom flask. The supernatant was evaporated at 50 C, to a volume of 5-10 ml, using a Rinco rotary evaporator. (8) The 5 ml sample was transferred to a 16 x 125 mm screw-cap round bottomed test tube and evaporated to dryness at 50 C under a stream of nitrogen. Preparation of Butyl Esters (1) One-half ml of n-butanol along with 1 drop of 0.05 per cent methyl red indicator in n-butanol was placed in a 5 ml beaker. (2) One-tenth ml of concentrated sulfuric acid was added to the n-butanol, methyl red mixture and the mixture added to the test tube containing the dried salts. The tube was sealed and placed in a boiling water bath until the dried salts had dissolved. The tube was removed and cooled. (5) Additional concentrated sulfuric acid was added until the methyl red end point was reached. A 0.025 ml excess sulfuric acid was added and the tube resealed. (4) Butyl esters of FFA were formed by heating the sealed tube in a 100 C water bath for 1.5 hr. (5) Tube was removed from the water bath and cooled. Anhydrous sodium sulfate was added and the mixture allowed to stand at room temperature for 45 min. 180 (6) The butyl alcohol solution was transferred quanti- tatively to a Babcock skimmilk bottle, by washing the tube with 5-10 ml of 1 per cent sodium bicarbonate. The skimmilk bottle was shaken vigorously. (7) The esters were brought up to the neck of the bottle by adding, without agitation, 20 per cent sodium chloride brine. (8) The bottle was spun briefly in a Babcock centrifuge and the esters brought into the neck of the bottles with brine. Appropriate aliquots of the butyl alcohol solutions were withdrawn directly from the necks of the Babcock bottles with a Hamilton 5 microliter syringe and injected into the gas—liquid chromatograph. Correction Factors Correction factors were obtained for 4:0, 6:0 and 8:0 compared to 7:0, and for 10:0, 12:0, 14:0, 16:0, 18:0, 18:1, 18:2 and 18:5 compared to 17:0. A mixture of standard fatty acids consisting of 5-10 mg of each standard acid was dissolved in ether and applied to the silicic acid-KOH column and treated as outlined above to obtain the butyl esters. The appropriate factor for each acid was calculated from the resulting peak areas of the butyl esters (Bills, Khatri and Day, 1965). The factors were used in solving for the weight of the fatty acids present in the cheese sample.