—-—————.— CHANGES mama mums or UNSALTED CHEDDAR CHEESE 'E'hesés far the Degree of M. S. Wéifiéfim S‘E‘AFE fifiiVERSITY MAN? KANT THMUR 1973 llllllllllllllllililI‘lllllllllllllllllllllllillllllllllllllll 3 1293 10444 6632 ABSTRACT CHANGES DURING RIPENING 0F UNSALTED CHEDDAR CHEESE BY Mani Kant Thakur Absence of salt in Cheddar cheese can result in a different pattern of ripening. The prime purpose of this research was to explore the ex- tent of some salient chemical and physical changes occurring in unsalted Cheddar cheese over a ripening period of 12 wk. Results were compared with those in the salted controls made from the same batch of cheese curd. The flavor of unsalted Cheddar cheese was described as flat, fermen- ted, bitter and unnatural. The body and texture was judged to be weak, pasty and gassy. At the end of the 12 wk ripening period, the unsalted cheese had an average flavor score of 36.37-37.20 compared to 39.20-39.50, for the controls, and the body score of 26.87-27.30 compared to 29.00-29.37, for the controls. With the incorporation of 0.5-1.52 salt into the ripened unsalted cheese, the flavor score increased and with 1.02 salt, compared closely to the score of the controls. A precise evaluation of the change in the firmness of the unsalted cheese during ripening was made with a Lee-Kramer Shear Press. The initi- al shear force value for the unsalted cheese was 0.1302 lblg (maximum). At the end of 10 wk the unsalted cheese registered a value of 0.0890 lblg (mi— nimum). The shear force values for the salted controls showed an initial value of 0.2433 lblg, a maximum of 0.3082 at 1 wk and 0.2503 (minimum) at Mani Kant Thakur 10 wk. After decrease for 10 wk there was no significant change observed in the average shear force values. The average pH value for the unsalted cheese increased from 5.16 at 1 wk to a maximum of 5.29 at 7 wk and then variations were observed to 12 wk. During ripening for 12 wk the samples of unsalted cheese showed a lower pH than the corresponding controls. Progress in the ripening of the cheese also was measured by determining nitrogen in water-soluble nitrogen com- pounds, which rose from an initial value of 6.00-8.00% in the unsalted cheese to 36.00-41.102 after 12 wk. The corresponding values for the salt- ed controls varied from 4.60-7.151 to 21.40-29.50% during the same period. The average volatile fatty acids in the unsalted cheese steadily in- creased from 22.90 ml to a maximum of 28.87 ml of N/lO acid per 100 g cheese at 12 wk. Increase in the volatile fatty acids of salted cheese was from 16.70 ml to 22.63 ml of N/lO acid per 100 g cheese at 8 wk. During the subsequent ripening although some variations in the amount of volatile fat- ty acids in the unsalted and salted cheese samples were observed, the un- salted cheese showed higher values than the corresponding controls at each interval of testing. The acetic acid content of unsalted cheese increased from an average value of 427.8 mg/kg cheese to 515.5 mg/kg at 10 wk. Corresponding values for the salted cheese were 285.7 mg/kg and 386.1 mg/kg at 8 wk. At 14 wk ripening, the acetic acid content for unsalted cheese had increased over 10 wk and was much higher than content for the salted cheese. Gas chromatographic analyses of free fatty acids in unsalted and salt- ed cheese samples showed that identical, even-numbered carbon chain fatty acids were liberated during ripening. The quantitation of the individual fatty acids revealed that myristic, palmitic, stearic and oleic acids were Mani Kant Thakur liberated in higher quantities than the rest of the fatty acids in the samples of cheese analyzed during the 12 wk ripening. Also, the overall hydrolysis of fat in the unsalted cheese was found to be more extensive than in the salted controls. CHANGES DURING RIPENING OF UNSALTED CHEDDAR CHEESE BY Mani Kant Thakur A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1973 DEDICATION This dissertation is dedicated to the fond memory of my late elder brother, Mr. Rup Kant Thakur, M.A., Dip-in- Ed., whose energy, cour- age and inspirations will always be symbolic. ACKNOWLEDGEMENTS The author expresses a deep sense of gratitude to his major professors and the academic advisors Dr. Theodore I. Hedrick and Dr. James R. Kirk for valuable advice and constructive criticism of the research and manuscript. The author wishes to thank the other members of the guidance committee: Drs. L. R. Dugan, D. R. Heldman and P. Markakis for their advice and ef- fort in reading this manuscript. Sincere thanks are expressed to Drs. C. L. Bedford, C. M. Stine and other staff members for facilities offered in conducting this investiga- tion. Thanks are also due to Dr. G. M. Trout, Prof. J. M. Jensen and Prof. A. L. Rippen for their able participation in the organoleptic evaluation of cheese samples. Grateful appreciation is extended to the Department of Food Science and Human Nutrition for the financial support that made this project feasible. The author also expresses his deep love and appreciation to his wife, Shail, for her patience and moral support, which helped make the comple- tion of this program possible. 111 TABLE OF CONTENTS Chapter I INTRODUCTION II REVIEW OF LITERATURE Status of Low Sodium Foods Unsalted Cheese Effect of Salt on some Important Physico- Chemical Properties of Cheese Salt in Cheese Physical Changes Influenced by Salting Moisture Retention of Curd Body and Texture of Cheese Chemical Changes Influenced by Salting pH Lactose Fermentation Proteolysis Acetic Acid in Cheddar Cheese Fat Hydrolysis Microbiological Aspects Flavor Development in Cheddar Cheese Salt in the Development of Typical Cheddar Flavor Chemistry of Flavor in Cheddar Cheese Carbonyl Compounds Nitrogenous Compounds iv Page 10 11 12 13 15 15 16 17 18 Fatty Acids Sulfur Compounds Other Flavor Compounds III EXPERIMENTAL Materials and Methods for Cheesemaking Analytical Procedures Measurement of Shear Force Moisture and Total Solids Fat Salt Sodium Nitrogen in Total and Water Soluble Nitrogen Compounds pH Free Fatty Acids Volatile Fatty Acids Acetic Acid Butyric and Higher Acids Organoleptic Judging IV RESULTS AND DISCUSSION Composition of Unsalted and Salted Cheddar Cheese Organoleptic Evaluation of Flavor and Body Firmness Evaluation Using Shear Press Protein Degradation Changes Acid Changes During Ripening pH of Cheese During Ripening Volatile Fatty Acids Acetic Acid Page 19 20 21 22 22 23 23 24 24 24 25 25 26 26 26 26 28 31 32 32 34 39 44 46 46 51 52 Page Butyric and Higher Fatty Acids 55 V SUMMARY AND CONCLUSIONS 63 VI LITERATURE CITED 66 vi LIST OF TABLES Table 1. Comparative analysis of unsalted and salted Cheddar cheese. 2. Comparison of body and flavor scores of unsalted and salted Cheddar cheese. 3. Flavor evaluation of ripened unsalted Cheddar cheese with different amounts of salt added before judging. 4. Comparison of the average shear force in le8 cheese during ripening 5. Comparison of the amounts of nitrogen in the soluble nitro- genous compounds present in the samples of unsalted and salted Cheddar cheese during ripening. 6. Comparison of pH on samples of unsalted and salted Cheddar cheese during ripening. 7. Quantitative changes in volatile fatty acids of Cheddar cheese during ripening. 8. Acetic acid contents of unsalted and salted Cheddar cheese during ripening. 9. Correction factors for calculating the actual amounts of butyl esters of fatty acids in relation to the internal standards (heptanoic and heptadecanoic acids). 10. Analysis of individual free fatty acids in unsalted and salted Cheddar cheese during ripening. vii Page 33 36 39 41 47 49 53 56 S9 60 LIST OF FIGURES Figures 1. Body and texture of Cheddar cheese cured for 4-5 wk. 2. Changes in shear force in the unsalted and salted Cheddar cheese during ripening. 3. Changes in "water soluble" nitrogen of unsalted and salted Cheddar cheese during ripening. 4. Changes in pH of unsalted and salted Cheddar cheese during ripening. 5. Changes in volatile fatty acids in the unsalted and salted Cheddar cheese during ripening. viii Page 38 42 48 50 54 CHAPTER I INTRODUCTION Doctors have been recommending a sodium restricted diet for patients suffering from kidney trouble, hypertension, congestive heart troubles, cirrhosis of liver with fluid retention, or toxemia of pregnancy. Since 1929 salt intake has been believed to be associated with blood pressure. In recent years the theory that excessive sodium intake over an extended period of time might result in hypertension and atherosclerosis in sus- ceptible individuals, is accepted by the medical profession. The harmful effects of excessive use of salt apparently was known for several centu- ries. A documented reference dating back to 1572 indicates that Thomas Tusser, a poet and a specialist in the farming problems mentions, "..much saltness in white meat is ill for the stone." A highly salted 'white-meat' (cheese) was found to harm the patients with 'stone' (a kind of kidney di— sease). To many people cheese is an important source of protein and calcium. ,However, dietary limits of 500-1000 mg of sodium per day is commonly re- commended for many people. A 100 3 slice of Cheddar cheese contributes roughly 600-1500 mg sodium. Sodium restrictions also limit the intake of meats, fish and poultry. Unsalted cheese because of its low sodium con- tent (approx. 25-55 mg/100 3 cheese), high nutritive value and a wide va- riety of uses in foods has potential for many applications in sodium re- stricted diets. Experimental evidence with rats showed that the intake of calcium initiated an interaction between calcium and sodium in a manner that if more calcium was excreted more sodium was excreted (Saunders, 1970). Thus, unsalted cheese should be a valuable adjunct to sodium restricted diets. No comprehensive investigation has been reported on the flavor attri- butes and other physics-chemical ripening aspects of salt-free Cheddar cheese. This study, therefore, was undertaken to investigate some of the salient changes in this type of cheese during a ripening period of up to 12 wk. The primary objective of this study was to determine the effect of the absence of salt in cheese on: 1. production of free fatty acids; 2. degradation of proteins; 3. overall organoleptic evaluation of flavor and body; and 4. body firmness by measuring the peak pound force resistance to shear the cheese sample, by employing a Lee-Kramer Shear Press. CHAPTER II REVIEW OF LITERATURE Status of Low Sodium Foods Salt has been used in cooking and preservation of foods since pre- historic time. The appetite for salt and other additives containing so- dium seems to be an acquired habit rather than a reflection of need, al- though sodium and chloride are necessary for man as constituents of extra- cellular fluids in the body (Gormican, 1972). Functions of sodium and chloride ions have been discussed in greater detail by Davidson (1954): 1. Sodium in the extra-cellular fluids participate in maintaining osmotic equilibrium (water balance) between the solutes of extra—cellular fluids and solutes contained within the cells (fluctuations from osmotic equilibrium may have dire results upon the functional capacity and the viability of the cells); 2. through its function as a buffer base in conjunction with bicar- bonate and phosphate, sodium maintains pH of blood within normal limits; 3. sodium alone or in conjunction with other extra-cellular ions functions to control nervous impulses, muscle contractility and especial- ly the conduction and contractility of heart muscles. Salt utilization varies considerably among individuals and various countries of the world. Saunders (1970) correlated the high salt intake of several populations around the world with the incidence of arterial hypertension. Achard and Leoper (1901) demonstrated that quantity of salt ingested was related to the edema formation in patients with heart disease. Then in 1929 they further postulated that low salt diet seemed to be found fairly frequently with normal blood pressure and the converse was likewise true. While a healthy kidney has the capacity to excrete sodium, there also appears to be growing support for the thesis that excessive sodium intake over a period of time might cause hypertension and atherosclerosis in sus- ceptible individuals (Dahl, 1961). There are a number of clinical condi- tions in which sodium retention occurs, e.g. congestive heart problems, hypertension, kidney disease, cirrhosis of liver with fluid retention, se- vere malnutrition and toxemia of pregnancy (Gormican, 1972). Strict adhe- rence to a sodium restricted diet may become a life or death proposition. As early as 1953, a method was developed by Chaney in cooperation with Los Angeles County Heart Association, which, according to Heap (1968), removed up to 902 of the original sodium content of fresh milk (approxi- mately 120 mg sodium/8 oz milk). Since sodium restriction essentially li— mits the intake of meats and other high-protein foods that normally have high sodium content, the enthusiasm for sodium restriction has to be paral- lelled with an equal zeal for providing the necessary nutrients. White (1957) stressed that the wealth of nutrients furnished by milk especially protein, calcium and riboflavin was no less important in sodium restricted diets. He also suggested that other protective foods with high sodium con- tent could be replaced with low sodium milk. Unsalted Cheese Cheese is an important part of the diet of many Americans. This food has a high caloric value, is easily digested and is rich in protein and fat. However, implication of high sodium intake in certain disease conditions make the use of salted cheese objectionable. Efforts,therefore.were made to produce unsalted cheese despite the common knowledge that salt renders it more flavorful and is necessary for normal ripening. High calcium content of the unsalted cheese may have an added advan- tage if included in the diet of the patients who need to get rid of excess sodium. This conclusion may be based on the experimental data of Saunders (1970) who found that a greater secretion of calcium was accompanied by a greater secretion of sodium in experimental rats which were fed different doses of calcium and normal saline. Sharara (1956) also reported an in- creasing loss of calcium in the cheese whey as the percentage of sodium chloride in the curd was increased to a certain limit. The effect was dis- cussed in relation to the exchange of calcium in the casein for sodium. In want of an improvement in variety for a palatable, balanced diet with restricted sodium content, unsalted cheese may possibly be used. Details of any comprehensive analysis on physico—chemical aspects of unsalted cheese apparently has not been reported in literature except for the processed "dietary" cheese produced in Holland, which was analyzed and found to contain as high a sodium content as natural salted cheese; possibly due to the use of 'emulsifiers' containing sodium (Mulder and Schouten, 1955). Effect of Salt on Some Important Physico-Chemical Properties of Cheese Salt in Cheese Salting of cheese has been considered an essential step for almost all ripened varieties. Tustin, Jr. (1946) stated that the aim of salting was to get a good quality cheese with maximum yield and minimum shrink— age during ripening. Davies, £5 31. (1937) showed that among a number of externally added chemical substances likely to affect the growth and metabolism of bacteria or the rennet activity, the largest effect on the rate of chemical ripening was exerted by the rennet and salt. Marquardt and Yale (1941) found the salt content of most varieties was between 0.7- 2.02, but the concentration of salt in the aqueous solution was much high- er. Salt concentration affects bacterial growth and thus the ripening of cheese (Tittsler, 1965). Irrespective of the method of salting, the salt concentration in the surface layer may reach 16% or greater. This level of salt is approximately the limit that can be endured by micro— organisms with the exception of a few halophilic types (Davis, 1965). Functions of salt in cheese are to (1) suppress growth of undesirable microorganisms, (2) assist the physico—chemical changes in cheese, (3) retard the growth of lactic acid and other desirable types of microorga— nisms, (4) influence the firmness of cheese and (5) make cheese more tasteful (Marth, 1963). Physical Changes Influenced by Salting. Moisture Retention of Curd. Salt has a marked effect on the moisture content of the curd. In the past, correcting an overmoist curd was accomplished by the addition of salt (Tustin, Jr., 1246). Cheesemakers know that an immediate re— lease of whey from the curd follows the addition of salt. The effect of salt is to cause contraction of the curd and release of moisture. High levels of salt result in lower moisture values, stickiness and retarded ripening (Reynolds, 1946). The omission of salt results in high moist- ure cheese which promotes rapid ripening (Tittsler, 1965, Davis, 1965). Moisture content of the curd at definite stages during cheese making along with the acidity are important factors controlling the quality of the product. Differences in these factors are responsible for the dif- ferent texture and body values and these differences are influenced by moisture content of the cheese. Body and Texture of Cheese. Salting produces an immediate increase in the hardness of the curd (bsiszar, 1949) and salted curd mats more readily. Undersalting and over- salting adversely affect the moisture of cheese. A low salt content re- sults in higher moisture and more likely a pasty and/or weak body and open texture (Riddet, ggugl., 1933 and Irvine, 1955). Studies conducted to assess the effect of salt on the development of 'Dutch'type cheese showed that the characteristic, elastic and plastic properties of the cheese were influenced by the water distribution and by the extent of proteolysis of the paracasein complex; both of which are regulated by the presence of salt (Ramanauskas, 1971). Oversalting causes dry, brit- tle body, cracking of cheese rind which may result in mold growth with— in the cheese (Tustin, Jr., 1246). The presence of salt has also been shown to influence the solubility of nitrogenous compounds in the cheese because salt decreased the hydrolysis of paracasein due to the decrease in the number of the lactic acid bacteria (Sandberg, 35 31., 1930). Failure to salt the curd evenly results in cheese with portions having low salt, which tend to become the regions of off-flavor including putrefaction. The rind may also become soft and the cheese misshapened (Davis, 5351., 1937). Chemical Changes Influenced by Salting. During ripening of cheese, protein, fat and to a less extent lactose undergo complex biochemical reactions, which are responsible for the de- velopment of typical body, texture and flavor of the cheese. The changes during ripening involve fermentation of lactose, partial hydrolysis of proteins, peptides and fat. The mechanism responsible for these diffe- rent biochemical reactions are catalyzed by enzymes from the microorga- nisms produced during the ripening process and to a lesser extent enzymes which are specific in the 'cheese substances' (Schormuller, 1968). The source of the latter are starter microorganisms added in the cheese mak- ing, milk and the rennet (Tittsler, 1965). Important influences on the- se enzymes are the dynamic changes in the composition and pH of the ripen- ing cheese, as well as the decomposition and the end products of these reactions (Schormuller, 1968). p§_ Changes in pH are important to the ripening process. Lactic acid produced in cheese has a physico-chemical action during ripening. It regulates the pH level and ion equilibrium (Schormuller, 1968). The gra— dual increase in pH during storage is caused by destruction of lactic a- cid to nonacidic decomposition products and less highly dissociated acids, which include acetic and carbonic acids, as well as, alkaline products of protein decomposition. pH changes primarily affect the characteristics of the curd as the body is controlled by 'acid-base equilibrium' and enzyme activity (Davis, 1950). Every enzyme has an optimum pH for activity. Therefore, enzyme reactions fluctuate when the pH of the media deviates from its optimum point (Gale, 1951). Sodium chloride is known to affect the activity of the enzyme system or the pH value, which directs the ripening process in a particular di- rection (Schormuller, 1968). Since the omission of salt results in a cheese of higher moisture content, more lactose is retained in the curd which may be available for the production of lactic acid. In an effort to correlate composition, pH and the color of the Cheddar cheese, Irvine (1951) found that 'acid' cheese had a lower pH, high moisture and low fat content. Raadsveld and Mulder (1949) indicated that the hydrolysis of fat was found to take place more quickly in cheese with low pH than in cheese with high pH. In another set of experiments on the reaction of glutamic acid and glycine in the ripening skim milk cheese, using the same species of microorganisms, Schormuller and Leichter (1955, a, b) found that the breakdown of amino acid is dependent not only on the species of microorganisms but also on the pH of cheese. Lactose Fermentation. Lactose in cheese although present in small quantities, may be im- portant in that it serves as a primary energy source of the lactic bac- teria. The changes observed occur first to lactose, then to the decom- position products of the disaccharide, galactose and glucose and finally to calcium lactate (Schormuller, 1968). Alfredsson, g; 31., (1962) found that damaging the homofermentative starter bacteria with high scalding temperature was reflected by higher galactose content and less formation of lactic acid. Lactic acid development repressed the growth of harmful and unde- sirable organisms. The amount and rate of production of this acid in— fluenced the quality of cheese. Its role was different in the develop- ment of various varieties. Harper and Kristoffersen (1956) reported that 10 the glycolysis of lactose to lactic acid occurs with many intermediate stages. Effect of salt on the fermentation of lactose in cheese does not seem to have been investigated. However, the absence of salt obviously would influence the lactose fermentation by the microbial flora. Proteolysis. Proteins in cheese are known to undergo degradation to varying ex- tents depending upon the variety of cheese and degree of curing. Pro- teins provide much of the physical structure, body and textural proper- ties. Casein fractions, the main protein in cheese, are degraded by a mechanism that is common to most varieties. Amino acids are considered to contribute to the flavor of cheese, but their contribution to the characteristic flavor is not certain (Harper and Kristoffersen, 1956). Qualitatively, the amino acids released by proteolysis during the ripen- ing of cheese are the same as those present in the casein (Kosikowski, 1951) and (Kosikowski and Dahlberg, 1954). These amino acids contribute to the substrate for the cheese flora and may produce flavorful compounds. The amino acids of the cheese protein are also of special importance in the formation of a number of fatty acids (Schormuller, 1968). The rate, nature and extent of protein decomposition during cheese ripening are influenced by the kind and concentration of microbial en- zymes, moisture content (Sammie and Germain, 1929), presence of lactic acid (Zaykowski and Slobodska-Zaykowsky, 1925), temperature (Sanders, gtflgl., 1946 and Van Slyke and Hart, 1903), pH, oxidation-reduction po- tential and salts that affect enzyme activity. The reaction is basically the same whether the hydrolysis involves an acid, a base or an enzyme: 11 R' +H20 -—-——-+- R CO NH R COOH + R'NHZ 7 Higher moisture and low salt concentrations increase the rate and extent of proteolysis (Van Slyke and Hart, 1903). Sodium chloride when added in increasing amounts not only decreased the number of streptococci in cheese, but also diminished the amount of 'soluble nitrogen'. Davis (1965) reported that salt affects the mechanism of cheese ripening in the following manner: 1. activates the proteolytic enzymes of rennet, 2. brings the protein fraction into solution for enzyme degradation, 3. changes the acid-base equilibrium in cheese, and 4. alters the proportion of bound and free water. Acetic Acid in Cheddar Cheese. Despite differences in opinion among researchers as to the substrate for acetic acid production, it is generally agreed that this acid is the product of bacterial metabolism. Suzuki, gt_gl,, (1910) reported acetic acid was formed from the lactates present in cheese and reached a maxi- mum at 3 mo. after which a decrease was noted. Honer (1953) noted a de- finite cyclic change in the concentration of acetate in cheese during ripening. It increased, then decreased, again increased and then decreas— ed again. Fruton and Simmonds (1960) have suggested that species of lac- tobacilli, and pseudomonas and E, 221; form acetic acid by beta-oxidation of fatty acids. Kandler (1961) proposed a mechanism by which starter bacteria derived this acid directly from the utilization of carbohydrates. 0hren (1965) stated that acetic acid in Cheddar cheese could be derived from carbohydrate, amino acids or fatty acids. When mixed lactic starter cultures used for Cheddar cheese were grown in milk, lactic acid and small 12 amounts of acetic and propionic acids were formed. Nakae and Elliott (1965, a, b) demonstrated that §, lactis C2 formed acetic acid from ala- nine and serine by deamination and decarboxylation. They also reported that microorganisms will deaminate and decarboxylate other amino acids to form various volatile acids. Fat Hydrolysis. The fact that milk fat is essential for the development of typical flavor of cured cheese is accepted by researchers. The nature of the de- composition of fat in cheese largely depends on the variety and is more extensive in mold inoculated curd. The numerous volatile and nonvolatile carbonyl.compounds present in cheese indicate that fatty acid oxidation might occur in addition to the lipolytic changes in fat due to lipases. Suzuki, 35.31, (1910) isolated a distillate, possessing the flavor of aged cheese, which contained alcohols, esters and fatty acids. The non-nitrogenous products were considered to have been derived from cheese fat during ripening. Lane and Hammer (1939) showed that, as cheese ripen- ed, the acidity of cheese fat increased and was higher in raw milk cheese than in pasteurized milk cheese. Mattick and Hiscox (1939) related levels of acids in cheese to its microflora and concluded that a high volatile acid content was associated with the presence of high numbers of non-lactic bacteria. Bills and Day (1964) observed that fat hydrolysis was a normal chem- ical change taking place in cheese. Dacre (1955) found that ethyl ace— tate and ethyl butyrate, both esters of fatty acids, were present in Cheddar cheese. Day (1967) also reported that lactones, methyl ketones, esters, alcohols and fatty acids were derived from milk fat during cheese ripening. 13 Hydrolysis in milk fat occurs in cheese due to action of lipases which may originate from milk and the microorganisms. While lipase ori- ginally present in milk has little effect on fat hydrolysis in cheese, a significant influence of microbial lipase on fat hydrolysis was found (Stadhouders and Mulder, 1953). Gould (1941) reported that sodium chlo- ride had an inhibitory effect on milk lipase in raw homogenized milk and cream, and amounts of S-8Z completely inhibited lipolysis. Factors af- fecting lipolysis were: salt, pH and storage conditions (Stadhouders, 1956). Later, Chandan and Shahani (1964) also noted that various salts, for example, sodium chloride, zinc chloride, magnesium sulfate and mag- nesium chloride were inhibitory to milk lipase. Stadhouders and Mulder (1957) further reported that microorganisms representative of the Alka- ligenes, Achromobacter, Pseudomonas and Serratia groups if added to pas- teurized milk, increased the hydrolysis of fat in cheese made from this milk. Jensen (1964) found that in the hydrolysis of mixed triglycerides enzymes exhibited a distinct preference for the fats containing short chain fatty acids. Milk lipase has a preference for the release of fatty acids from the primary positions of a triglyceride. Butyrate being a primary ester, often was preferentially released. Lauric and higher a- cids were the major acids released at all stages of lipolysis in an ex- periment of Harwalkar and Calbert, (1961) but with the advancement of lipolysis the ratio of these acids to butyric decreased. Microbiological Aspects. Bacterial counts in cheese vary widely but inversely with the salt content (Hoecker and Hammer, 1944). Sodium ions are relatively more toxic than the chloride ions but a concentration of 5% salt in cheese was not 14 sufficient to inhibit growth of certain bacteria. E, ggli_required 12% salt for inhibition, while 3% had a stimulating effect (Davis, 1965). Earlier Hof (1935) found that only a few species could tolerate 6% salt and very few could survive 15%. Starter streptococci were not affected by up to 2% salt but were almost completely inhibited by 5% (Platon, 1942). Sjostrom (1944) observed that some starters may be affected even at 2% salt concentration. Walter, g£_§l,, (1958) reported that §, lactis was generally not inhibited by less than 1.62 and not significantly at 1.6- 2.02 salt. Welter, gt_gl,, (1958) also found that §, cremoris was in- hibited slightly by 1.6% and almost completely by a 2.0% salt, while a mixture of §, lactis and S, cremoris seemed to resist sodium chloride more uniformly than single strains. Malushko (1957) noticed that 0.5- 2.SZ salt had an inhibitory effect on gas producing bacteria in 'Dutch' type cheese made from raw milk; lactic acid bacteria appeared to have been affected less. Davis (1965) noted that a higher concentration of salt at the sur- face than in the interior of the curd exerted an inhibitory effect for several hours. He also indicated that apart from the inherent salty taste of the cheese, quite different flavors would be obtained if cheese was made without salt, because of the different microflora that develop- ed. The production of the common proteolytic and lipolytic bacteria would be far higher and the cheese would almost certainly possess an unpleasant odor and taste. These bacteria normally constitute about 1% of total microflora in good salted Cheddar cheese. Davis and Mattick (1932) suggested that the state of nitrogen in cheese may be a controlling factor. They found that digestion of protein beyond a certain point resulted in an inhibition of lactic streptococci. 15 Studying the effect of salt concentration of the microflora of cheese from the public health view point, Wagenaar and Dack (1953) noted that the salt requirement necessary to inhibit toxin production by S, BREEZ. lgggg decreased with the length of ripening. In order to study the sur- vival of some pathogens in Cheddar cheese, Campbell and Gibbard (1944) experimented with S, typhosa and Yale and Marquardt (1940) with S, pyg;_ 35323. They found that an increase in ripening temperature reduced the survival period of pathogens in both cases. S, typhosa was not affected either by the amount of starter or the acidity of cheese. Mattick, ggugl. (1959) could not recover.S. aureus, added to cheese milk, from any of the Cheddar cheese samples after 14-22 wk. They further related the results with the acidity of cheese during manufacture and reported that S, aureus died out least rapidly in the cheese in which the least amount of acidity was allowed to develop during manufacture. Salt, acidity, temperature and oxygen are factors influencing the growth of microorganisms. Salt content effectively controls the flora distribution (Davis, 1965). Flavor Development in Cheddar Cheese Salt in the Development of Typical Cheddar Flavor. In different lots of Cheddar cheese Irvine (1951, 1955) found a salt content of 0.9-2.92 and reported that low salt cheese more often tended to have unclean flavors and gas holes. The best cheese had about 1.6% salt. Davis (1965) pointed that a cheese made without salt would lose the characteristic flavor and become relatively insipid. Such a cheese would not only lack the inherent salty taste but would acquire quite a different l6 flavor. Salt takes part in the highly complex phenomena of odor and taste formation of individual cheese varieties. According to Harper (1959), cheese aroma can be divided, through distillation of an acqueous cheese suspension, into two large groups: non-volatiles which consisted of lactic acid, amino acids and others, amines, minerals and sodium chlo- ride and the volatiles which consisted of fatty acids, aldehydes, ketones, alcohols, esters, hydrogen sulfide and other sulfides. In the United States salt ranges of 3.3-6.2: for Provolone and 2.9-9.0% for Romano cheese were found in the dry matter (Harper and Gould, 1952). However, there appeared to be no correlation between salt content and the flavor development in these ranges. Chemistry of Flavor in Cheddar Cheese. Kosikowski (1957) stated that the typical flavor of Cheddar cheese is associated with a pleasant, slightly sweet, aromatic, walnutty sen- sation without any outstanding single component. In aged Cheddar cheese a bitty quality which is neither coarse nor unpleasant gives sharpness to the cheese. The list of compounds isolated from the aroma fraction has become extensive, so it has not been possible to attribute Cheddar fla- vor to a single or relatively few compounds. Mulder (1952) was the first to advance a theory on a balance of various flavor components responsible for the production of typical Cheddar flavor. His 'Theory of Balanced Components' explained that the typical aroma was due to the combination of many compounds in proper quantitative balance. Kosikowski and Mocquot (1958) proposed another concept called the 'Component Balance Theory' which states that not only for Cheddar flavor but for a range of food fla- vors, only a small number of compounds are responsible. Thus, for a ty- pical Cheddar flavor the amounts and relative proportions of the flavor 17 compounds were within certain limits in the cheese. Each major variety of cheese is different in flavor, body and texture from other cheese varieties and is characterized by the specific kind of ripening it undergoes. Tittsler (1965) attributed the cheese characte— ristics to the composition, enzyme content, bacterial flora of milk, star- ter, rennet and other added enzymes, amount of added salt and conditions of manufacturing and curing. The compounds considered to be relatively more important in imparting a typical Cheddar flavor included the vola- tile fatty acids (Patton, 1963), aldehydes (Keeney and Day, 1957) methyl ketones (Harvey and Walker, 1960), diacetyl (Calbert and Price, 1948), and sulfur compounds (walker, 1959). Others working in the area of fla- vor chemistry have reported that compounds like amino acids (Mabbitt, 1955), esters (Dacre, 1955), alcohols (Scarpellino, 1961), partial, gly- cerides (deMan, 1966), lactic acid (Mulder, 1952), amines (Silverman and Kosikowski, 1956) and peptides (Harwalkar and Elliott, 1965) also probab- ly affect the typical flavor of cheese. Carbonyl Compounds. Carbonyl compounds identified in Cheddar cheese have been classified as acidic and neutral. Among acidic carbonyls are oxalacetic, oxalsucci- nic, pyruvic, alpha-keto-glutaric, alpha-aceto-lactic and alpha-keto- isocaproic. The neutral carbonyls considered to be the degradation pro— ducts of acidic compounds (Bassett and Harper, 1956) include diacetyl, butyraldehyde, acetaldehyde, actylmethylcarbinol, acetone, methyl ethyl ketone, 2-butanone, 3-hydroxybutanone, 2-pentanone, 2-heptanone, 2-non- anone, 2-undecanone, 2-tridecanone, formaldehyde, 3-methy1thio-propana1 (methional), 3~methy1 butanal and propionaldehyde. Calbert and Price (1949 a) concluded that the presence of diacetyl in concentrations less 18 than 1 ppm was necessary in good cheese. A higher amount was associated with flavor defect. Wolin and Kosikowski (1959) and Wolin (1961) used tritium labelled casein for cheesemaking and found some of the carbonyls in ripened cheese resulted from the degradation of casein. MacLeod and Morgan (1958) report— ed that the bacteria in cheese form aldehydes from amino acids by trans- amination and decarboxylation. Schonberg and Moubasher (1952) suggested that the strecker degradation of amino acids may play a part in the for- mation of Cheddar cheese flavor by production of aldehydes. Day and Keeney (1958) and Walker and Harvey (1959) have reported that carbonyl compounds are only partly responsible for the Cheddar cheese aroma and suggest that 3-methy1thio-propana1, which is produced by strecker degra- dation of methionine, may be a key component of the Cheddar cheese flavor. In general, Kristoffersen and Gould (1959) found no relationship between the presence and relative concentrations of the individual carbonyl com- pounds and Cheddar cheese flavor. Nitrogenous Compounds. An extensive study of proteolysis in cheese has helped identify se- veral reaction products. Despite certain quantitative differences repor- ted by several workers in the occurrence of these nitrogenous compounds, it is generally agreed that amino acids, amines, and peptide fractions do not impart cheese flavor as such, but appear to function by providing a 'brothy' background on which is superimposed the typical Cheddar flavor. While no direct relationship may be established, a higher flavor score of raw milk cheese over the pasteurized milk cheese is ascribed to the higher amino acid content of raw milk (Day, 1967). Day mentioned that further degradation of amino acids served to introduce a variety of compounds 19 which were significant for typical Cheddar cheese flavor. Bitterness in cheese was attributed by Emmons, 3; El. (1960) to the presence of peptides. Schormuller (1968) showed that bitter taste occur— red when certain microorganisms, that participate in cheese ripening and exhibit peptidase activity were missing. Breene, g; 31. (1964) made cheese by direct acidification with hydrocloric acid and found that a bitter flavor which developed was cured by addition of 1% starter. A low salt concentration always accompanied by a higher moisture content tended to favor development of a bitter flavor in Cheddar cheese (Golding, 1947). Tuckey and Ruehe (1940) associated the production of bitter fla- vor in Cheddar cheese with undersalting treatment that only incorporated 1.31 or less salt. The effect was not found when the salt values were 1.72 or more. A very slight bitterness is, however, a part of true Ched- dar flavor. Fatty Acids. Fatty acids originate from fat hydrolysis, metabolism of carbohy- drate or degradation of amino acids. The acid spectrum can differ great- ly with the type of individual cheese. Peterson, g£_gl, (1949) observed that free fatty acids of intermediate chain length were produced during the ripening of cheese and were of importance in the development of cha- racteristic Cheddar flavor. Mabbit and Zielinska (1956) observed that a typical Cheddar flavor did not develop in skim milk cheese, instead a flavor described as, more like a mixture of amino acids was found. Stad- houders (1956) reported that short chain free fatty acids were desirable in the mixture of fatty acids. Kristoffersen and Gould (1960) found that no cheese flavor developed without appreciable fat hydrolysis. 20 Patton (1963) studies the volatile fatty acids (acetic, butyric, caproic, and caprylic) in cheese distillates using sodium bicarbonate to block the functional groups of these acids in cheese and cheese distil- lates. Patton concluded that the volatile fatty acids were the backbone of Cheddar aroma. Acetic acid, a dominant volatile fatty acid was con- sidered to be hportant. Samples of Cheddar cheese made from raw and pas- teurized milks and having a wide range of flavors were analyzed by Bills and Day (1964). They found acetic acid showed the greatest variability in concentration. 0hren (1965) reported that the typical Cheddar flavor was related to the balance of free fatty acids and acetate. The ratio of free fatty acids to acetic acid was calculated to be between 1:0.55 to 1:1.00 in the finest flavored cheese after 180 days of ripening. Sulfur Compounds. Sulfur compounds involved in the formation of cheese flavor are con— sidered to be the degradation products of sulfur containing amino acids of milk, e.g., cystine, methionine, and cystein. Mabbitt (1961) sug- gested that hydrogen sulfide formation may be an indicator of flavor pro- duction and that methyl sulfide may be of more flavor importance. Methyl mercaptan was isolated and identified by Libbey and Day (1963) as a com- ponent of cheese flavor. Kristoffersen (1963) and Kristoffersen, ggugl. (1964) showed that concentrations of hydrogen sulfide and fatty acids are related to the aroma of Cheddar cheese and the concentration of active -SH groups was parallel to the characteristic Cheddar flavor intensity. Sharpe and Franklin (1963) reported that many strains of lactobacilli produced hydrogen sulfide under low pH, anaerobiosis and low sugar con- centrations and they may also cause a similar change in cheese. 21 Other Flavor Compounds. In addition to those previously mentioned, several other compounds have been isolated and may directly contribute to the flavor or contri- bute to the formation of other compounds, e.g., ethyl and secondary butyl alcohols (Patton, ggugl., 1958, Scarpellino and Kosikowski, 1962). The presence and importance of esters of fatty acids and mono hydroxy com- pounds in cheese were confirmed by Day and Libbey (1964). CHAPTER III EXPERIMENTAL Materials and Methods for Cheesemaking. Cheddar cheese for this investigation was made from whole milk re- ceived from the M.S.U. Dairy farm. A substitute rennet, "Emporase"a, from culture fermentation of Mucor pusillus var. Lindt, was used for the cheesemaking. A freeze-dried lactic cultureb was propagated for the ma- nufacture of Cheddar cheese. The method of manufacturing the unsalted Cheddar cheese was the same as for regular Cheddar cheese except that the salt was not added to a portion of the milled curd. The control cheese contained 1.5% to 1.8% sodium chloride after pressing. A pressure of 60 psig was applied on cheese in 20 1b block hoops and after approximately 2 hr the unsalted lot was removed from the press and kept in cold storage. The hoops of salted cheese were left in the press overnight. The next morning the unsalted cheese blocks were taken out of the hoops, all cheese blocks were then wrapped in a standard cheese aluminum foil coated with waxed paper. Two cheese blocks were sampled, wrapped and coated with wax, in order to pre- vent moisture loss during curing or storage. Waxing of the exposed area after each subsequent sampling was to prevent moisture loss and mold con- tamination during ripening. The cheese samples were cured at 45-50°F and approximately 802 humidity in the curing room. a. Dairyland Food Laboratories, Waukesha, Wis. b. Cultures, Inc., Indianapolis, Ind. 22 23 Analytical Procedures In view of the number of the analyses to be made, a slice of ap- proximately 300-400 g was obtained every 2 wk from the unsalted and con- trol cheese blocks. The samples were packaged in polyethylene bags im- mediately. The samples were analyzed first for the shear force measurements within a few minutes of sampling. The smaller slices resulting from this experiment were then placed in bottles and stored at -10°F and grated with a food chopper the same day. The well mixed samples were then held frozen at -80 to -90°F if not analyzed the same day. Measurement of Shear Force. The Lee-Kramer Recording Shear Press, Model TR-l was used with the 3,000-pound transducer ring consisting of 10 blades, and No. C—lS stan- dard shear compression cell, to measure the firmness of the samples using the following procedure: 1. After removal of the rind portions, a slice of cheese approxi- mately one-half inch thick, weighing 55: 1 g was cut from the cheese samp- 1e and properly sized to fit into the bottom of the test-cell, without undergoing any deformation. 2. The element was fixed on the transducer ring and the cell-box with the weighed sample under cover was placed into position. Since the temperature of the sample was critical, it was constantly held to 55: 2 F. 3. For the salted cheese the range of the Shear Press was set at 20, while for the unsalted cheese the setting was 10 except for l-day old samples, which required a setting of 20. Lower range-settings were made to get amplified peaks. 24 4. Zero adjustment was made to bring the recorder pen to zero. 5. After the upper shear blades assembly and the lower sample cell were in proper position, the shear blades were passed through the sample which was laying perpendicular to the shear grids of the sample cell, and the resistance to shear was recorded on the strip chart. 6. Readings on each sample of cheese were obtained in triplicate. The maximum peaks were read from the recording chart and were subsequent- ly converted to pounds of shear force per gram of product using the fol- lowing formula: Rangiogetting x Peak reading (E830) Pounds force/g sample - Sample weight in grams Moisture and Total Solids. Moisture contents of all Cheddar cheese samples were determined by the modified Mojonnier method for moisture in cheese (Milk Industry Foun- dation, 1952). Moisture content was calculated by the difference in weight of the original sample and the dried solids residue. Determina- tions were made in duplicate. Fat. Fat determination by the Roese-Gottlieb method with Mojonnier modi- fication for cheese was made in triplicate on all samples of cheese (Milk Industry Foundation, 1952). Salt. Total chloride contents in the cheese samples were determined by a method of the Association of Official Agricultural Chemists for cheese (AOAC, 1970) and calculated as sodium chloride. 25 Sodium. Sodium contents of cheese samples were determined using a Perkin- Elmer 303 Atomic Absorption Spectrophotometer. Homogenous cheese samp- les were weighed in paper thimbles in 1.5-2.0 g quantities and dried in a vacuum oven overnight. The dried cheese samples were defatted with approximately 30 m1 petroleum ether (B.P. 30-60 C) in a GoldfisdxFat Ex- tractor for 7-8 hr and dried again. Samples were transferred to Vicor crucibles, weighed and charred before putting into the muffle furnace for the final ashing at 550°C. About 4-5 ml of N/lO hydrochloric acid were added to the ash in each crucible and the contents heated mildly for 10 min. Suitable dilu- tions were made with deionized water to give concentrations within 0.3- 3.0 ug sodium/ml. A standard curve was prepared with sodium chloride solutions containing 1, 2, 3, 4, and 5 ug sodium/ml. The method of ana- lysis was essentially that outlined in the Perkin-Elmer handbook (Anony- mous, 1968). Other conditions were as follows: fuel - acetylene (oxidizing flame) at 10 psig; oxidizer — air at 40 psig, and wave length - 295 on the Spectrophotometer (corresponding to 5890 A). The results were expressed as mg sodium/100 g cheese. Nitrogen in Total and Water-Soluble Nitrogen Compounds. Total nitrogen in cheese samples was determined by the semi-micro Kjeldahl method (AOAC, 1970) and the amount of protein calculated by mul— tiplying the amount of total nitrogen by a factor of 6.38. 26 Using the method of Vakaleris and Price (1959), the amount of nitro- gen in water extracts of cheese containing water soluble nitrogenous com- pounds from protein degradation was determined at weekly intervals. The nitrogen analysis were made singly on duplicate acid-soluble extracts of each cheese sample by the semi-micro Kjeldahl method (AOAC, 1970). PH. The pH of the cheese samples were measured on the filtrate obtained by blending 12.5 g well mixed sample of cheese with 17 ml boiled and cool- ed distilled water and then filtering through a fine muslin cloth. The pH was read to the nearest 0.01 of a pH unit using a glass Calomel elec- trode. The dilution of the sample was kept well within the ratio of 1:2 (cheese:water) to prevent any definite shift of the pH reading (Tittsler, 1965). Free Fatty Acids. Volatile Fatty Acids. The rapid direct distillation method reported by Kosikowski and Dahlberg (1946) to recover 100% of the short chain volatile fatty acids, such as, acetic, butyric and caproic, was used. According to these wor— kers, about 90% of the caprylic and a small percentage of capric and lau- ric acids were recovered by this method. A 10 g sample of cheese drawn every 2 wk during ripening was distilled and the volatile acids in the distillate titrated against N/20 sodium hydroxide solution. Results were expressed in terms of ml N/10 volatile acids in 100 g cheese. Acetic Acid. Development of acetic acid in cheese during ripening was measured. 27 The liquid-liquid partition chromatography procedure of Wiseman and Ir- vine (1957) as modified by Bills and Day (1964) and Blakely (1970) was used. A 20 x 300 mm chromatographic tube was filled with a suspension of 15 g Celite in 150 m1 Skellysolve B and acetone (1:1 v/v), blended with 2.4 ml of indicator solution and 0.1 m1 of N/lO sulfuric acid. Nitrogen pressure (10 psi) was used to pack the adsorbent in the column and expel the liquid until the latter had been expressed to the top of the adsorb- ent . A 1% solution of acetone in Skellysolve B (BAl) was carefully added to the top of the column packing to a depth of about 10 cm. To this was added a slurry of 8 g cap material (sodium sulfate : Celite : ammonium sulfate ::12 : 8 : 1) in 25 ml of BA solution and the cap was compressed 1 using nitrogen pressure (10 psi). Seventy-five ml of BAl solution was passed through the column to wash the acetone-Skellysolve B (1:1 v/v) solution previously used. The sample mixture was prepared by thoroughly grinding and mixing. Then 5 g of this was acidified to pH 1.8-2.0 (using 50% sulfuric acid) with 10 g of silicic acid in a mortar with a pestle. Two grams of this sample mixture were applied to the top of the column for the aceticaeid de— termination. A filter disc was then placed on top of the column. Butyric and higher carbon chain fatty acids were removed from the column with 200 ml of BA1 solution. Acetic acid was eluted with 250 ml of a 15% solution of acetone in Skellysolve B. Titration of the eluted fractions from the column was done in an atmosphere of nitrogen using 0.005 N sodium hydroxide solution. Results were expressed as mg acetic acid per kg of original cheese. 28 Butyric and Higher Acids. For the quantitative analysis of individual free fatty acids from butyric through linolenic, the method of Iyer, _£fl§l,(1967 a) as modi- fied by Blakely (1970) was used. The silicic acid column of McCarthy and Duthie (1962) was used for the isolation of free fatty acids from cheese. A 5 g representative sample of Cheddar cheese was acidified with a 50% solution of sulfuric acid to a pH of 1.8-2.0. Five to 10 mg each of 7:0 and 17:0 fatty acids were added as internal standards, in about 5 m1 of Skellysolve B, to the cheese sample with thorough mixing. Nine grams of silicic acid were also added to the cheese sample and mixed tho- roughly. A 25 x 500 mm chromatographic column was filled with 35 g of pre- viously prepared packing material (Blakely, 1970) slurried in 150 m1 of 1% solution of butanol in Skellysolve B. The column was packed using nitrogen at 5-10 psi. The silicic acid-cheese mixture made previously was added to the top of the column packing. The mortar and pestle were thoroughly washed with a 1% solution of butanol in Skellysolve B, and washings returned to the column. Fat was extracted using 400 ml of 1% solution of butanol in Skellysolve B and the eluate saved for the separa- tion of free fatty acids in another column. A 18 x 200 mm chromatographic column was packed with a mixture of 4 g of specially prepared silicic acid (Blakely, 1970), 8 ml of isopro- panol-KOH solution and 24 m1 ethyl ether. The column was washed with 100 ml of ether and freed of air bubbles with a glass rod. The eluate from the fat extraction column was passed over this column. The column was then washed with 75 ml of ether to remove the lipids. The free fatty a- cids were eluted with 60 m1 of ether containing 2.5% concentrated 29 phosphoric acid (v/v). The eluate from the above column was collected in a 250 m1 centri- fuge bottle and 70-80 ml of ethanol were added to the eluate. This so- lution was titrated to the phenolphthalein end-point with l N methano- lic-KOH solution, under nitrogen. The centrifuge tube was subjected to centrifugation at 1200 rpm for 3-4 min in order to remove the precipi- tated potassium salts of acidic constituents. The clear supernatant containing soluble potassium salts of the fatty acids was transferred to a 500 ml round bottom flask. The contents of the flask were concen- trated to 5-10 ml under vacuum on a rotary evaporator. The 5 m1 concen- trated sample was quantitatively transferred to a 16 x 125 mm screw cap round bottom test tube and evaporated to dryness at 50°C under a stream of nitrogen. Butyl esters were prepared by adding 0.5 ml of n-butanol, 1 drop of 0.03% methyl red indicator in n—butanol and 0.1 ml of concen- trated sulfuric acid into the tube containing the dry salts of the free fatty acids. Butyl esters of free fatty acids were formed by heating the sealed tube in a 100°C water-bath for 1.5 hr. It was removed from the water-bath, cooled and approximately 0.5 g anhydrous sodium sulfate added to the tube. The butyl alcohol solution was quantitatively transferred to a Bab- cock skim milk fat—test bottle by washing the tube with 5-10 m1 of 1% so- dium bicarbonate solution. The esters were brought to the neck of the bottle by adding 20% sodium chloride solution. The butyl esters of the free fatty acids were analyzed by flame ion- ization gas chromatography using a Hewlett Packard Research Gas Chromato- graph; Series 5750 B, fitted with a disc integrator. Columns for the gas chromatograph were prepared in a manner outlined 30 by Dal Nogare and Juvet (1962). A 10 ft by 1/8 in o.d. copper column was packed with 15% diethyl glycol succinate coated on 80/100 mesh acid washed Chromosorb W. Packing was accomplished with the aid of a vibra graver tool. Both columns were matched to insure minimum baseline drift during the temperature programmed analysis of fatty acids esters. After the packing was completed the columns were conditioned in the column oven for 72 hr at the upper limit temperature of 195°C and a nitrogen flow ra- te of 30 ml/min. After conditioning the columns were attached to the detector system and gas leaks eliminated. The instrument was then ready for the analysis of the fatty acid esters. The following operating con— ditions were established for gas liquid chromatographic analysis: injection port temperature, C 270, detector block temperature, C 280, hydrogen flow, ml/min. 65, air flow, ml/min. 500, nitrogen carrier gas flow, ml/min. 30, range setting 103, attenuation, lower limit 1, column temperature - programmed 4 C/min from 40°C until first appearance of butanol (solvent), then pro- grammed at the rate of 8 C/min. to 195°C. Identification of esters was accomplished by their retention times compared to those of the standard butyl esters (obtained from Applied Science Laboratories, Inc., State College, Pa.). Both symetric and asy- metric peaks were quantitated using Disc trace according to the method out- lined in the 'Series 200, Disc Integrator Manual' (Disc Instruments, Inc., 1969). Individual fatty acids including the internal standards (heptanoic and heptadecanoic), each in about 10 mg quantity were dissolved in ether, applied to the silicic acid-KOH column and then treated according to the method outlined earlier. The standard fatty acids passed through six different columns were butylated separately and analyzed in triplicate to 31 obtain the correction factors. These correction factors were obtained for C4:0, C6:0 and C8:0 compared to C7:0 and for C10:0, C12:0, 014:0, Cl6:0, C18:0, C18:1, 018:2 and C18:3 compared to Cl7:0 according to the formulas outlined by Bills, g£_gl,, (1963). Use of these factors elimi- nate the need for precise measurement of the quantity of the sample- injection and permit injection-levels that had desired peak-heights. Organoleptic Judging. A small judging panel of 3-5 members of experienced cheese judges scored the cheese. The flavor evaluation was made in a room free from extraneous background odors. Flavor was scored according to the ADSA method. A flavor score of 40 required no criticism. The same score card was used to score the body and texture. The normal range for this score was between 25-30. A score of 30 required no criticism on body and tex- ture. Samples of the salted and unsalted cheese were scored by the jud- ges within an hour after they were drawn from the cheese blocks. Each trial was judged for flavor development every 4 wk. CHAPTER IV RESULTS AND DISCUSSION Composition of Unsalted and Salted Cheddar Cheese. The summary of chemical analysis of unsalted and salted Cheddar cheese made at the end of the 12 wk curing period appears in Table 1. It shows the average moisture, fat, total solids, total nitrogen, sol- uble nitrogen, salt and sodium contents of three batches of unsalted and salted Cheddar cheese. Higher moisture content, lower sodium and higher soluble nitrogen were associated with unsalted cheese. The to- tal solids, fat and total nitrogen contents of all salted cheese samples were higher than the unsalted samples. A higher percent of moisture in the unsalted cheese could be partly attributed to the absence of salt, which causes contraction of the curd releasing moisture from protein, (Davis, 1965). The second cause could be the reduced pressing time for the unsalted cheese. The estimated nitrogen in the soluble nitrogenous compounds hydro- lyzed from the insoluble proteins during ripening was found to be higher in the unsalted cheese samples. Again, a higher moisture content and pos- sibly the lack of salt inhibition may account for the higher degree of proteolysis in the unsalted cheese samples. The addition of salt is primarily important in controlling the growth of undesirable microorganisms, such as, proteolytic bacteria which are par- ticularly sensitive to sodium chloride in the concentrations found in the 32 33 .33 NH um masons w ooa use monsooaoo moocowouuwc oHpsfiom :« nowouuwa we ate .omooao w OOH use cowouuas we A « om.oee oo.mm o~.~mn cm.as oH.s0e oo.e~ m ooa\ma .asasom .e we.e mo.o we.a eo.o oa.a Ho.o N “Ham .0 oo.msa oo.osme oo.~maa oo.oase oo.mom oo.~wme Aee caucuses «Henson .m oo.mmsm oo.HHmm oo.oewm oo.mamm oo.seam oo.~amm A«aowouuea sauce .s om.em s~.oe so.em Hm.Hs oa.em se.os n «nausea: .m om.me ea.am Hm.~e a~.mm os.me em.mm N season House .N wo.mm Na.~m Na.mm 5H.Nm ~o.em ha.em N use .H souamm assesses soueam souammas museum woodman: 0 you n you < uoa mucosuuuusoo .omoono espouse houses was wouaomas mo mamhasam o>qumusnaou .H manna 34 cheese (Foster, g£_§l,, 1957). The unsalted samples from lots A, B, and C contained very low levels of chlorides expressed as sodium chloride. However, these estimations are based on the chloride contents which may come from compounds other than salt. The salts of milk were considered to be chlorides, phosphates and citrates of potassium, sodium, calcium and magnesium (Corbin and Whittier, 1965). Organoleptic Evaluation of Flavor and Body. The absence of salt on the development of flavor and body of Cheddar cheese was assessed by an organoleptic evaluation pannel. Evaluations were conducted at 4 wk intervals as the ripening progressed. The data presented in Table 2 reveal that unsalted dheese samples were usually characterized as having off-flavors, such as, bitter, acid, fruity/fer- mented, flat and unnatural from the first through the third month of ripening. The corresponding salted controls ripened without any serious flavor criticism. A Cheddar flavor developed with advancement of the ripening period. As the ripening of unsalted cheese proceeded, the fla— vor score definitely improved at 8 wk over the score at 4 wk. However, an overall decline was observed in the flavor score of unsalted cheese at 12 wk, while the controls showed an overall increase in flavor score as ripening advanced. The undesirable flavor of the unsalted cheese could not be related to any one chemical change occurring in the cheese during ripening, except for the bitterness which could be singled out as a do- minant flavor. Salt appears to be a controlling factor in the flavor development of cheese by affecting the kind, extent and direction of physico-chemical changes which occur during ripening. Van Slyke, g£_§;, (1903) 35 investigated the rate of proteolysis in Cheddar cheese as a measure of ripening and found it increased with a higher moisture content and less salt. Latter, Czulak (1959) ascribed the bitterness in cheese to the high proteolytic activity of rennet at low pH, the accumulation of poly- peptides including bitter peptones and the inability of bacterial pro- teinases at low pH to convert the polypeptides to amino acids. Compounds responsible for the production of fruity flavor in Ched- dar cheese were identified by Bills, ggflgl. (1965), who attributed this defect to the excessive alcohol production and to the subsequent forma- tion of ethyl esters of free fatty acids. In general, the presence of compounds imparting unnatural and undesirable flavors in cheese could be due to the new balance of microflora and the changed environment with- in the cheese, such as lower pH, higher moisture content, lower osmotic pressure, and other related physico-chemical changes associated with low salt concentration. Salt by itself also has a strong masking effect on the off flavors. Harper (1959) divided cheese flavor into the non-volatile and volatile components. He reported that the non-volatile constituents, which in- clude lactic acid, amino acids, amines, minerals and common salt influ- enced the flavor. Salt masked some of the undesirable flavors of cheese and improved the overall flavor scores. This was demonstrated by a se- parate set of experiments during this investigation. The data in Table 3 are in agreement with that published by Harper (1959). A panel of four cheese judges was served with cheese after 6, 12 and 24 wk of cu- ring. The unsalted cheese was blended with 0.5%, 1.0% and 1.5% sodium chloride. The control with 1.48% salt was cured for the same periods. Blended cheese flavor ranged from 35.75 to 37.75. As the percentage of 36 Table 2. Comparison of body and flavor scores of unsalted and salted Cheddar cheese. Lot Average flavor Remarks Average body Remarks 39:. score score 4 Weeks A. Unsalted 35.50 Bitter, flat, unna- 26.50 Pasty, weak, mealy. tural, acid, whey taint. Salted 39.00 None. 28.00 Curdy, corky, short, mealy. B. Unsalted 35.33 Bitter, acid, unna- 26.00 Pasty, weak, mealy. tural, flat. Salted 38.16 Flat, heated, whey 27.50 Corky, crumbly, short, taint. mealy. C. Unsalted 37.62 Acid, flat, whey, 27.12 Gassy, open, pasty, taint. weak, mealy. ‘Salted 39.12 Falt. 28.62 Corky, curdy, short, mealy. 8 weeks A. Unsalted 37.33 Flat, unnatural, acid, 26.33 Gassy, pasty, weak, sl. bitter. open. Salted 39.00 81. flat, heated. 28.83 Curdy. B. Unsalted 36.50 Flat, unnatural, acid, 26.33 Gassy, open, weak. bitter. Salted 38.83 Acid, flat. 28.66 Open, short. C. Unsalted 37.00 Acid, fermented, flat, 26.87 Pasty, gassy, open, bitter. weak. Salted 39.50 Flat. 29.25 Corky. 12 weeks A. Unsalted 36.37 Flat, fermented, bitter. 26.87 Pasty, open, gassy, weak. Salted 39.50 Flat. 29.37 Corky, open, weak. B. Unsalted 36.37 Flat, fermented, bitter, 27.12 Pasty, open, weak. Salted 39.25 Acid, flat. 29.37 Open. C. Unsalted 37.20 Acid, bitter, whey taint, 27.30 Pasty, open, weak. sl. flat, fruity. Salted 39.20 Fermented, flat. 29.00 Curdy, open, sl. curdy s The flavor and body scores represent the average of products judges. 3 to 5 trained dairy 37 psalt increased from 0.0 to 1.0, usually the overall flavor score increased. However, the addition of 1.5% salt did not necessarily improve the fla- vor over 1.0%. At 6 wk curing the flavor score of cheese ripened with- out salt but scored after blending with 1.0-1.5% salt surpassed the fla— vor score of the corresponding salted controls. This may be explained by the more rapid curing of the unsalted cheese, the presence of flavor compounds expected in salted cheese after a longer curing period and the masking of undesirable flavors by the incorporation of salt. The development of body and texture characteristics is comparative- ly a quicker change in unsalted cheese than that of the flavor. In all organoleptic evaluation conducted at the end of 4, 8 or 12 wk, the un- salted cheese was criticized as having a weak, pasty, open, mealy or gas— sy body (Table 2). The increase in the protein hydrolysis and higher moisture may explain the weak and pasty body of unsalted cheese. The photograph (Fig. 1) taken of a cross-section of two lots of 4-5 wk old salted and unsalted cheese illustrates the conspicuous body and textu- ral differences. While some of the openings in the unsalted cheese may be gas holes, most of them appear to be mechanical openings resulting from incomplete fusion of the curd during pressing. Another explanation is that the unsalted curd chunks after milling do not undergo the physi- cal shrinkage from the addition of salt, so when placed in the hoop and pressed, the outer mass of the curd in the hoop immediately yields to the pressure applied in the press. As a result of this, a firm outer layer is formed providing a barrier against the elimination of moisture and air during pressing. In an anaerobic environment and acidic medium, characteristic of ripening Cheddar cheese, Platt and Foster (1958) showed that homofermen- tative streptococci produced significant amounts of acetic acid, carbon 633m @5306 633mm: 36.5 .x: m4 you wanna 38:0 .83er mo oasuxou one hoom .H 95me 39 Table 3. Flavor evaluation of ripened unsalted Cheddar cheese with different amounts of salt added before judging. % salt added Average flavor scores 6 wk 12 wk 24 wk 0.00 37.00 34.62 36.00 0.50 36.88 35.75 36.50 1.00 37.75 36.10 37.25 1.50 37.12 36.87 37.00 Control: 1.48 37.12 37.12 37.37 The flavor scores are the average given by a panel of four trained dairy products judges. dioxide and ethanol, in addition to lactic acid. Bang (1949) also noted that degradation of milk sugar by S, lactis can take place both homo- and heterofermentatively, the latter occurring at lower temperatures (S9-60'F). In the initial stages of ripening, bacterial population chiefly consists of lactic streptococci, outgrowing other microbial flo- ra. Therefore, production of gas in unsalted cheese, would not be sur- prising because of the demonstrated ability of the starter bacteria to produce carbon dioxide in the existing environment. Irvine (1951, 1955) also reported after an extensive survey of cheese that low-salt cheese had a tendency to have unclean flavors and gas holes. Firmness Evaluation Using Shear Press. To obtain a more precise evaluation of the body of the unsalted and salted Cheddar cheese, the Lee-Kramer Recording Shear Press, Model TR-l 40 was used with the 3,000 lb test-ring and No. C-lS standard shear com- pression cell. The firmness was evaluated by measuring the extent of deformation of the proving ring from the force required to compress and shear the sample in the test cell. The compression-shearing action of the standard cell thus simulates the action of a tooth in the chewing of food (Anonymous, 1967). Table 4 presents comparative data on the shear force expressed as le8 sample, for each pair of unsalted and salted Cheddar cheese obtained over the 12 wk ripening period. The re- sults indicate that the shear force values of salted cheese appreciably increased in the 1,2 and 4 wk of ripening and then declined to 10 wk. No increase was found in the unsalted cheese as the ripening advanced, with one exception. Generally, unsalted cheese samples decreased very slightly in the shear force as the ripening advanced after 2 wk (Fig. 2). The curves have been shown with standard deviations, which are compara- tively smaller for the unsalted samples than for the salted controls. However, the shear force measurement for unsalted cheese at all stages of ripening were found to be lower than those for the controls. In all trials of salted and unsalted cheese samples, the ratio of shear force (salted/unsalted) was minimum for the 1-day old cheese and increased to the 4 wk. At 6 wk the value declined until 10 wk. The increase in the ratio was attributed to the increase in the shear force values of salted cheese and decrease in those of unsalted cheese. The ratio had a wide range of variation, the minimum being 1:1.8686 on the first day and maximum 1:3.0425, at the end of 4 wk. Foster, ggugl., (1957) reported that solubilization of protein and the high moisture content of the cheese are the important factors responsible for the softness of the cheese. This finding supports the results obtained in this investigation 41 .muoou oueowaeauu mo owmuo>m use one sumo any a mmew.mua cmm~.o ammo.o ceo~.o Nowo.o w-~.o smmo.o wnnw.o ono.o #3 NH mNHw.~"H mom~.c oomo.o wq-.o mmao.o umc~.o ammo.o Hmn~.o mmmo.o #3 CH omom.~u~ mwsm.o mmoo.o emmm.o onmo.o oqmu.o Hmao.o omwm.o wHao.o #3 m meom.~”a Hmm~.o oHcH.o Hue~.o mooa.o maw~.o naoa.o oomm.o como.o #3 o quo.mHH oHNm.o “moa.o aHaN.o NQHH.o ooam.o aooa.o «mam.o mfloa.o :3 e Hnmn.~”H «wom.o oNHH.o mmq~.o omOH.o seam.o neon.o momm.o nmua.o :3 N omow.aua mneu.o NomH.o menu.o ~mma.o «Hmu.c oooH.o N¢ . a: . meanness magnum «moose AH we wou0m Moons omeuo>e oau mo confluemaoo c capes SHEAR FORCE, Ibf/g SAMPLE 18 16 14 12 10 45 40 35 30 25 20 Figure 2. 42 UNSALTED _ Mean value Standard deviation J l 10 12 _L l 4 6 i l o 2 Changes in shear force in unsalted and salted Cheddar cheese during ripening. 8 WEEKS OF RI PENING 43 and explains why the unsalted cheese had a lower shear force than the salted controls, According to Van Slyke (1928), the form in which the protein exists affects the elasticity of the curd and therefore, affects the characte- ristics of the ripened cheese. A green cheese is spongy and curdy. As it ripens it becomes soft and mellow. Van Slyke (1928) theorized that casein in a rennet curd cheese exists as dicalcium paracaseinate and when lactic acid is formed by the starter organisms, it reacts with the paracaseinate as follows: 1. dicalcium paracaseinate + lactic acid-——+.monocalcium paraca- ceinate + calsium lactate; 2. monocalcium paracaseinate + lactic acid.——+.free paracasein + calcium lactate. Free paracasein and its monocalcium salt have widely different physical properties. As lactic acid is produced in appreciable amounts, cheese curd begins to show elasticity. Van Slyke (1928) further attributed the elastic and ductile properties of cheese curd to the presence of monocalcium paracaseinate (reaction 1). He demonstrated that this com- pound comprises over 90% of the nitrogenous materials in the cheese a few hours after the curd is put into the press. As the acid produc- tion continues, the curd loses much of its elasticity, becoming hard and brittle, probably due to the formation of free paracasein (reaction 2). Paracasein does not show the elastic properties of its monocalcium salt. The formation of free paracasein may account for the increase in the shear force values of salted cheese at the start of the ripening. In the case of unsalted cheese, this effect is probably counteracted by the increased moisture of the cheese causing the lower shear force va- lues in these samples. A report from Sazabo (1966) supports this inference 44 by stating that the viscosity and elasticity of the product are lower- ed if its moisture content or fat content is raised. Sohngen, g; 1;. (1937) attributed the physical changes in the curd to viscosity and the degree of hydration of casein. While studying the rheological changes occurring in the small brick Dutch-type cheese during the first 3 wk of ripening, Kunakhov (1967) reported that the maximum shearing stress increased immediately after the pressing and the relative residual de- formation decreased to 1/5 of fresh cheese by the 10th day. Thus, comparatively lower shear force values for all samples of un- salted Cheddar cheese at various stages of ripening may be ascribed to a high moisture content, extensive solubilization of protein and possib- ly to the mechanical openings and the gas holes present in the cheese. Protein Degradation Changes. During cheese ripening, protein is one of the major milk consti- tuents undergoing physical and chemical changes. The extent of pro- teolytic action and specific compounds resulting there from help to de- termine the characteristics of the final cheese. Foster, ggugl. (1957) stated that some of the products of protein decomposition contribute to the flavor. Peptones in general, are bitter and they may account in part, for the background flavor. Certain of the amino acids and simple peptides have considerable influence on taste. The enzymes responsibfie for the proteolytic decomposition in cheese primarily originate from rennet or other added natural enzyme prepara- tion and microorganisms. Rennet, which is used in the manufacture of most types of cheese, has a primary function to coagulate casein, but its proteolytic action also continues during the ripening period (Harper 45 and Kristoffersen, 1956). Practically all the nitrogenous constituents of uncured cheese exist as water insoluble protein, but as the ripening progresses, part or all of the protein is hydrolyzed enzymatically to simpler components that are water soluble. The general course of these changes may be il- lustrated as: Protein —>Proteoses -—>-Peptones -—>— Peptides -—>—Amino acids. In the above scheme, all but the protein are water soluble. Among its several functions in cheese, salt helps control moisture and acidity development. Of primary importance is the function of salt in controlling growth of undesirable microorganisms. Strongly proteo- lytic bacteria, for example, are sensitive to sodium chloride in concen- trations found in most cheeses (Foster, 25.51,, 1957). Davis, g£_g;, (1937) indicate that, of a number of chemical substances added to Ched- dar cheese, salt exerted the largest effect. Omission of salt resulted in a 50% increase in ripening rate as measured by protein breakdown. If moisture is mechanically retained in a normal salted cheese (by waxing), an increased rate of protein breakdown is obtained (Van Slyke and Price, 1949). It is, therefore, difficult to calculate the specific effects of salt concentration and moisture separately. The decomposition of protein in the cheese can be followed in any of several ways, the most common method being the determination of the amount of nitrogen in the water extracts of cheese at different ages. As protein is hydrolyzed to water soluble compounds, the value increases, showing the progress of ripening. In this experiment, all nitrogen ana- lyses were made on duplicate acid-soluble extracts of each cheese by 46 the procedures essentially described by Vakaleris and Price (1959). The experimental data presented in Table 5 indicate a progressive solu- bilization of milk proteins both in unsalted and salted Cheddar cheese samples throughout the 12 wk ripening. In cheese curd held at 50°F the 'water-soluble' nitrogen expressed as per cent of total nitrogen in cheese increased from the range of about 4.60-7.15% to a maximum of 21.4- 29.5% in the salted cheese, compared to 6.0-8.0% to a maximum of 36.0~ 41.1% in the unsalted cheese during 12 wk ripening. The curves in Figure 3 present the mean 'water-soluble' nitrogen values and standard devia- tions of three different batches of unsalted,and salted Cheddar cheese. Acid Changes During Ripening pH of Cheese During Ripening. Since pH influences the activity of enzyme systems involved in the ripening (Morris and Jezeski, 1953), the change of pH in the cheese made with salt and without salt was determined. Baribo and Foster (1951) stated that the apparent influence of pH in the cheese might be explain- ed by the effect on the proteolytic activity of rennin.The lower the pH the greater the activity that occurs. Foster, g£_gl, (1957) reported that the rate of acid formation increased rapidly, because the majority of organisms were concentrated in the curd rather than lost with the whey. Unsalted cheese because of its relatively higher retention of lactose provides more substrate for the formation of acid, which in turn, lowers the pH. The starter organisms continue to grow and produce acid using lactose as their substrate. This may account for the initial de- cline in the pH of cheese when the ripening starts. The unsalted cheese had a lower pH during ripening than the salted cheese. The data on pH 47 0.00o w.nqu O.m¢m w.ONMH N.HMHH 0.0BQH o.mow m.NwMH #3 NH w.w¢m o.mmMH m.mma O.MQMH 0.0NHH m.wm€H m.mah w.OBMH #3 HH N.HNm o.qBMH m.w¢m N.¢MMH m.NOHH m.MN oaxz.mmimm mo 0&4 .wowaoaqu magnum snooze umovonu mo mvwom huuom oawuoao> aw newsman o>aumuwuamsc .n canoe 54 32 30 UNSALTED Mean value Standard deviation SALTED 30 VOLATILE FATTY ACIDS, ml N/lO PER lOOg CHEESE 4 0 - l I J L l I l 0 2 4 6 8 10 12 14 WEEKS OF RIPENING Figure 5. Changes in volatile fatty acids in the unsalted and salted Cheddar cheese during ripening. 55 the modifications of Blakely (1970). Concentrations of acetic, propi- onic and butyric acid in water were used for the recovery mixture using the method described in the experimental section. The percentage re- covery of the acetic acid was found to be 98.7 and was used as the cor- rection factor for the results obtained on the samples. Average acetic acid content in the samples of unsalted cheese in- creased steadily during 10 wk of ripening. At the end of 12 wk a slight decrease was observed. Salted controls generally showed a gradual in- crease in the acetic acid content up to 8 wk. Variations were observed for the 10, 12 and 14 wk analysis. The acetic acid in the unsalted cheese was higher than in the salted controls during ripening.- These data on acetic acid in Cheddar cheese during ripening do not agree with the results of Peterson, 35 El, (1949). They showed butyric acid to be much higher than acetic acid even on the zero day and observed no ap- preciable change in the acetic acid content throughout a ripening period of 420 days. Throughout this investigation, acetic acid was the pre- dominant acid in unsalted as well as the salted cheese. Similar fin- dings were observed by Berridge, g£_§l, (1953), Windlan (1955) and 0hren (1965). Butyric and Higher Fatty Acids. The method described by Iyer, g£_§l, (1957, a, b) as modified by Blakely (1970) was used for isolation and quantitation of free fatty acids from butyrate through linolenate in cheese samples. Recovery factors were determined and are shown in Table 9. As appears in Table 10, there was a marked difference in the free fatty acid contents of the unsalted and salted cheese samples, although qualitative results showed that identical fatty acids were liberated 5.6 w.~mm o.HNm m.mmm H.¢Hn m.omq N.©Hm «.moq o.~mm #3 «H $.0mm n.0Hm m.~m~ m.nom c.0He n.m~m n.5He m.w¢< #3 NH 5.3m n63 n.mm~ moon H.-.~ m.mom o.~mm no: :3 3 down 0.93 noun 563 563. H63 m6?» m.o~m #3 w H63 wow.» aému H53 méom $53 mémw odmm #3 w N.cmm w.Nm¢ o.m¢H H.mnq N.wnm w.¢~¢ m.m~¢ «.mmc #3 e 0.0.3 «.03 TNHN ~43 amen méac H.3m 1me x3 N h.nmN w.n~e n.a©H m.th «.mmu a.uoc N.No< «.wmc haw H vouaam vouammsb oouaem wouamuab wouamm meadows: $333 @3323 u 63 m as a see $33.: one: coo: .0893 mm M mafia. a a .kuo caused wanna—om .waacoawu weaned snooze unvoono vouamm use vouammso mo mucouaoo wave caused .w manna 57 in all lots of cheese. AIleven-numbered carbon fatty acids from butyric to linolenic were found in each lot of cheese throughout the 12 wk ripen- ing period. The concentrations of specific fatty acids varied among the three batches but, in all cases, myristic, palmitic, stearic and oleic acids were present in relatively greater concentrations than the other fatty acids. This was true throughout the ripening period for both un- salted and salted cheese. During this investigation, it was noted that some of the fatty acids were at maximum concentration at 4 wk of ripen- ing and then decreased, while others increased to the 8 wk of ripening before a decrease was observed. Some fatty acids showed variations in concentration as the ripening progressed. In most cases the fatty acids higher than capric had a tendency to increase for the first 8 wk of ri- pening and then to decrease toward the end of 12 wk. Linolenic acid was relatively low in salted as well as unsalted cheese. 0hren (1965) found a similar trend in the development of free fatty acids in Ched- dar cheese. He observed an increase in these acids toward the end of a 6 mo. ripening period, although a decrease in several free fatty a- cids at 8 wk and 12 wk ripening period was also observed. An initial increase in fatty acids followed by a decrease, may be due to the uti- 1ization of these fatty acids by some organisms in the cheese (Foster, 55 51. , 1957). Peterson, 35 21, (1948) demonstrated that the reason for an in- crease in fatty acids in Cheddar cheese was because lipases became more active in young Cheddar cheese after 5-10 days of ripening. These li- pases may represent intracellular enzymes of lactic acid bacteria, li- berated by bacterial autolysis. They also reported that part of free n-butyric acid and all of free caproic, caprylic and capric acids 58 present in 420 day old cheese were the result of the action of these liberated intracellular bacterial lipases on the milk fat in the cheese. 0hren (1965) found that the microflora in the milk and cheese had more influence in promoting lipolysis in Cheddar cheese than normal milk li- pases. He observed that increasing fat hydrolysis was accompanied by an increase in acetic acid production. Although it is well known that lipolysis takes place in Cheddar cheese, the source of lipase enzyme has been a matter of controversy. The mechanism proposed for the lipase reaction on milk fat results in the following mixture of compounds: triglyceride + lipase-—+-trig1yceride + diglycerides + monoglycerides + glycerol + fatty acids. In order to further investigate the role of various factors in the lipo- lysis of fat in the Cheddar cheese, 0hren (1965) studied the effects of lactic acid, rennet, starter organisms and microorganisms isolated from Cheddar cheese. He found that lactic acid and rennet did not effect an increase in the fat hydrolysis over a period of 49 days. However, star- ter organisms, as well as, microorganisms isolated from cheese exhibited lipolytic activity when grown in sterile milk. He also discovered that a micrococcus organism isolated from cheese was lipolytic in sterile milk. This evidence, in part, may explain the increased level of fatty acids in the samples of unsalted cheese. Salt has an inhibitory effect on the bacterial population in the medium. Therefore, unsalted cheese is expected to have a much higher population of ripening organisms, which may liberate lipase through autolysis. Gaffney and Harper (1965) empha- sized that the lipolysis in cheese may not be due to one type of orga- nism, but the total lipolysis may be due to an additive effect of 59 Table 9. Correction factors for calculating the actual amounts of butyl esters of fatty acids in relation to the internal standards (heptanoic and heptadecanoic acids). Acids Number of Mean correction Standard trials factor deviation 4:0 (Butyric) 6 0.88843 10.0137 6:0 (Caproic) 6 0.88133 10.0273 7:0 (Heptanoic) 6 1.00000 --- 8:0 (Caprylic) 6 0.86085 10.0146 10:0 (Capric) 6 0.72803 10.0468 12:0 (Lauric) 6 0.77188 10.0197 14:0 (Myristic) 8 1.03510 10.0409 16:0 (Palmitic) 6 0.87546 10.0255 17:0 (Heptadecanoic) 6 1.00000 --— 18:0 (Stearic) 6 1.15115 10.0342 18:1 (Oleic) 5 0.93360 10.0602 18:2 (Linoleic) 5 1.98840 10.0868 18:3 (Linolenic) 5 1.11180 10.0308 different microbial lipases as well as an other source, such as, the lipase of leucocytes. 6O moonwucou omoono wx\wfium we « m.mm w.~nq m.mmm «.mmm o.wmm «.mom «.mHH q.ecH m.mm “.mq m.oo x3 NH 0.0m m.omm m.qmm ¢.ooq H.wwm ¢.Hmm H.5HH H.¢NH m.~m o.mo m.Hc #3 w m.mm m.mmm 0.8mm ~.m~q H.mqa m.omm o.moH w.mm o.HN o.mq m.oo #3 q m.mm n.nom H.m~q m.qmq o.mqe ~.nmm m.moa o.o~ m.n~ «.mq m.mq hop H a m.q~ o.mm q.enw «.mcq m.mom H.mme c.5NH m.mm o.mm m.on n.5o #3 NH m.mm o.omH w.amw H.Hem m.mmma o.mao o.~na o.mm m.mq m.~o m.Hm :3 m N.¢N o.mmH ~.mon H.~mn «.mmm n.mme m.HmH n.HHH o.mm p.00 q.co x3 a m.oN 0.0mm H.~Hm m.amm o.~Hn o.o¢m m.em o.co w.H~ c.Hq m.nm moo H « wouaumce lludauoou ofiaoaoafia uaoaocaa cacao oaucoum owewfiamm owumwuhz cannon saunas oHHmummo uwounmo uwuhuam mo Hm>uouaH < uog .waaaoefiu weaken masons neocono voufimm was mouammas cw mowom huuom menu stva>woca mo mwmhamq< .OH wanna 61 omacHucoo m.qH o.mo m.mnq H.~em e.anm o.omm e.o0H n.mo m.Hm o.mm m.mq #3 NH w.NH N.mHH ~.qqm N.oom o.H¢~H o.qu H.mmH «.mNH n.mq o.qn n.ow x3 w o.om n.m~m H.0wq m.m~q H.cow o.qmm m.mm o.Hw m.mm m.qm «.mq #3 q q.HH H.wm~ m.-m m.n¢~ w.qmm n.m¢~ m.mo H.mm o.~H ~.mH H.0N amp H wmmflwm. m.HNH ¢.m0H H.mon o.mmm c.¢u~ n.8Hm m.mOH «.mmH q.w~ n.mm n.0m #3 NH m.oq m.mo m.~o~H H.mno m.~mQH H.wqw o.omH H.omH «.05 o.mw H.¢~H #3 m m.mH ~.wn H.oqm c.HHm H.5mn m.¢Hm w.wm o.oo e.qm m.mq w.mo #3 q e.m m.oo o.-< m.wN~ m.nem m.me <.Ho c.5m m.oH m.m~ m.mm how H wouHmmcD 1mmHumou UHcoHoaHH UHoHoaHH UHoHo UHumoum oHuHEHmm oHumHuhz 0HuomH oauamu oHHhuamo 0Hounco uuuhuom mo Hu>uouaH m uOH .AoosaHuaoov .OH oanH 62 .mumwu ouHUHHaso mo owmuo>m onu one some m.mN m.mom N.mH< N.qu w.eHo N.on w.Nm N.HN H.NH H.NN 33 NH m.mH N.mNN o.mom o.mwe N.moN o.oom o.Nm N.ww m.0N q.wm #3 w n.0N o.mNH e.nooH «.mmm H.meH m.won N.qu m.qQH «.mm H.ow #3 q m.No H.qu c.0mm m.Noq m.oow n.0mN N.ow m.mm N.wH N.Nq moo H a o.oN «.mQN m.mMMH H.NNw m.owNH H.u0ucH W464 .AoooaHucouV .OH oHan CHAPTER V SUMMARY AND CONCLUSION Three lots of unsalted Cheddar cheese were made with salted con- trols and examined during a 12 wk ripening period. The following were the findings: 1. The flavor of unsalted cheese was flat, fermented, bitter and unnatural, and the body, weak, pasty and gassy during the first 12 wk of ripening. At the end of this period the flavor score for the un- salted cheese was 36.37-37.20 compared to 39.20-39.50 for the controls (salted) and the body score for the unsalted cheese was 26.87-27.30 compared to 29.0-29.37 for the controls. When 0.5-1.5% salt was incor- porated into the ripened unsalted cheese, the flavor score was higher and at 1% salt closely compared with controls. 2. The shear force values for unsalted cheese were lower than tho- se for the salted cheese. The ratio of shear force (salted/unsalted) varied from 1.8686 on the first day to 3.0425 at the end of 4 wk. The mean shear force value of 0.1302 1bf/8 sample on the first day for the unsalted cheese steadily declined to 0.0890 lbf/B sample at 10 wk com- pared to the values 0.2433 le8 sample on the first day increasing to 0.3082 1bf/8 sample at 1 wk and then steadily declined to 0.2503 lbf/g sample at 10 wk for the salted controls. A slight increase occurred at 12 wk in the shear force values of unsalted and salted cheese. 3. Average pH values for the unsalted cheese was 5.16 at 1 wk and increased steadily to a maximum of 5.29 at 7 wk. Then variations were observed. In all samples the pH of unsalted cheese remained lower than the controls, which had an average pH of 5.46 at 1 wk and 5.62 at 2 wk. 63 64 4. Soluble nitrogen expressed as the percentage of total nitrogen in the unsalted cheese samples was found to be between 35.96-41.11 com- pared to 21.39—29.49 in the controls at the end of 12 wk ripening. 5. The average volatile fatty acids in the unsalted cheese steadi- ly increased from 22.90 ml to a maximum of 28.87 m1 of N/10 acid per 100 g cheese during 1 wk. Increase in salted cheese was from 16.70 ml to 22.63 m1 of N/10 acid per 100 g sample during 8 wk. During subse- quent ripening the volatile fatty acids showed a variation in both salt- ed and unsalted cheese, but remained higher in unsalted cheese. 6. Acetic acid was more in unsalted cheese during ripening despite the variations within the samples at different intervals. Mean values for acetic acid were 427.8 mg/kg at 1 day increasing to 515.5 mg/kg cheese at 10 wk for the unsalted and 285.7 mg/kg cheese at 1 day in- creasing to 386.1 mg/kg cheese at 8 wk for the salted controls. Acetic acid content increased in unsalted as well as the salted cheese at 14 wk ripening, after small decreases between 8-12 wk. 7. The salted and unsalted cheese samples had identical free fat- ty acids liberated in all trials. These were the even-numbered carbon fatty acids from butyric through linolenic. Based on the amounts of free fatty acids liberated during cheese-ripening, the extent of the fat hy- drolysis in unsalted cheese was greater than in salted cheese. In most trials the free fatty acids increased initially and then decreased to- ward the end of the ripening period. Myristic, palmitic, stearic and oleic acids were present in higher amounts during the ripening of both kinds of cheese. In conclusion, unsalted cheese ripened at a faster rate than the salted cheese. But, due to its less desirable flavor and the weak and 6S pasty body it has a lower acceptability. The manufacture of unsalted cheese with a better body and texture and flavor continues to be a chal- lenge. LITERATURE CITED Achard, C. and Leoper, M., 1901. Sur la retention des chlorures dans les tissus au cours de certains etats morbides. Compt. rend Soc. biol. 53:346. Alfredsson, St., Nilsson, R., and Sjostrom, G., 1962. The course of lactose and citric acid breakdown in cheese and the influence of high cooking temperature on these processes. XVI Int. Dairy Congr. B, 4:747. Anonymous, 1952. Lab Manual. Methods of analysis of milk and its pro- ducts. Milk Industry Foundation, Washington, 629 pp. Anonymous, 1967. Bulletin C. Allo Precision Metal Engineering, Inc., Rockville, Md. Anonymous, 1968. Analytical Methods for Atomic Absorption, Spectro- photometry, 1968. Perkin-Elmer, Norwalk, Conn., p. Nal. Anonymous, 1969. Instruction Manual, Series 200, Disc Integrator. Disc Instruments, Inc., Calif. 25 pp. Anonymous, 1970. Association of Official Agricultural Chemists. Offi- cial Methods of Analysis. Eleventh Edition. AOAC, Washington, D.C. 1015 pp. Bang, F., 1949. Substrate breakdown by betacoccus cremoris and S, lac- tis. Rept. Proc. 4th Int. Congr. Microbiol. 1947, 515. Chem. Abstr. __4_:9083, 1950. Baribo, L. E. and Foster, E. M., 1951. The proteinases of Streptococcus lactis and lactobacillus casei and their relationship to cheese ripening. J. Dairy Sci., 345475. Bassett, E. W. and Harper, W. J., 1956. Acidic and neutral carbonyl compounds in cheese. J. Dairy Sci., 323918. Berridge, N. J., Hiscox, E. R. and Zielinska, M., 1953. Some flavor constituents of English cheese. XIV Int. Dairy Congr., 23587. Bills, D. D., Khatri, L. L. and Day, E. A., 1963. Method for the deter- mination of free fatty acids of milk fat. J. Dairy Sci., 46:1342. Bills, D. D., and Day, E. A., 1964. Determination of the major free fat- ty acids of Cheddar cheese. J. Dairy Sci., 415733. Bills, D. D., Morgan, M. E., Libbey, L. M., and Day, E. A., 1965. Iden- tification of compounds responsible for fruity flavor defect of experimental cheese. J. Dairy Sci. 4841116. 66 67 Blakely, L. E., 1970. A study of the formation of methyl ketones and free fatty acids in quick ripened Blue cheese and subsequent losses during dehydration. Ph. D. Thesis., Michigan State University. Breene, W. M., Price, W. V. and Ernstrom, C. A., 1964. Changes in com- position of Cheddar curd during manufacture as a guide to cheese- making by direct acidification. J. Dairy Sci., 41:840. Brown, L. W. and Price, W. V., 1934. A study of the relationship be- tween hydrogen ion concentration, titratable acidity and quality in Cheddar cheese. J. Dairy Sci., 11333. Calbert, H. E. and Price, W. V., 1948. Studies on sources of the ty- pical flavor in Cheddar cheese. J. Dairy Sci., 31:713. Calbert, H. E. and Price, W. V., 1949 a. Study of the diacetyl in cheese. I Diacetyl content and flavor of Cheddar cheese. J. Dai- ry Sci., 323515. Calbert, H. E. and Price, W. V., 1949 b. Study of the diacetyl in cheese. 11 The changes in diacetyl content of Cheddar cheese du- ring manufacturing and curing. J. Dairy Sci., 325521. Campbell, A. G. and Gibbard, J., 1944. The survival of E. typhosa in Cheddar cheese manufactured from infected raw milk. J. Canad. F. Publ. H1th., 22:158. Chandan, R. C. and Shahani, K. M., 1964. Milk lipase: A review. J. Dairy Sci., 41:471. Corbin, E. A. and Whittier, E. 0.. 1965. The composition of milk. Fun- damentals of Dairy Chemistry. The AVI Publishing Company, Inc., Westport, Conn. 827 pp. Csiszar, J., 1949. Hardness of Trappist cheese during ripening. XIII Int. Dairy Congr., 2566. Czulak, J., 1959. Bitter flavor in cheese. Austr. J. Dairy. Technol., 14:177. Dacre, J. C., 1955. A chemical investigation of the volatile flavor principal of Cheddar cheese. J. Dairy Res., 22:219. Dahl, L. K., 1961. Possible role of chronic excess salt consumption in the pathogenesis of essential hypertension. Amer. J. Cardiol., 8:571. Dahlberg, A. C. and Kosikowski, F. V., 1947. The flavor volatile acidi- ty and soluble protein of Cheddar and other cheeses. J. Dairy Sci., 30:165. Dal Nogare, S. and Juvet, R. 5., Jr., 1962. Gas-Liquid Chromatography Inter Science Publishers, N.Y., 258 pp. 68 Davies, W. L., Davis, J. C., Dearden, D. V. and Mattick, A. T., 1937. Studies in Cheddar cheese. V The effect of chemical substances on the ripening process. J. Dairy Res., 8:92. Davis, J. C., 1950. A Dictionary of Dairying. Leonard Hill Limited, London, 856 pp. Davis, J. G., 1965. Cheese, Vol. I, Basic Technology. American Else- vier Publishing Company, Inc., New York, N.Y., 463 pp. Davis, J. G. and Mattick, A. T. R., 1932. Studies in the metabolism of the lactic acid bacteria. I Nitrogen metabolism. J. Dairy Res. 4:81. Day, E. A., 1967. Cheese flavor. Chemistry and Physiology of Flavors. The AVI Publishing Co., Inc., Westport, Conn., 331 pp. Day, E. A., and Keeney, M., 1958. Identification of volatile carbonyls from Cheddar cheese. J. Dairy Sci., 41:718. Day, E. A. and Libbey, L. M., 1964. Cheddar cheese flavor. Gas chro- matographic and mass spectral analysis of the neutral carbonyl com- ponents of the aroma fraction. J. Food Sci., 22:583. Davidson, C. S. (Chairman), 1954. Sodium restricted diets; the ratio- nale, complications and practical aspects of their use. Pub. no. 325. Committee of Food and Nutrition Board, Washington, Division of Biology and Agriculture, National Research Council, 71 pp. deMan, J. M., 1966. Partial glycerides in the fat of Cheddar cheese. J. Dairy. Sci. 49:343. Emmons, D. B., Mcguan, W. A. and Elliott, J. A., 1960. Nitrogen dis- tribution in Cheddar cheese as related to bitterness and strain of starter culture. J. Dairy Sci., 43:862. Foster, E. M., Nelson, F. E., Speck, M. L., Doetsch, R. N. and Olson, J. C., 1957. Dairy Microbiology. Prentice-Hall, Inc., Englewood Cliffs, N.J. 492 pp. Fruton, F. S. and Simmonds, S., 1960. General Biochemistry. 2nd Ed., John Wiley and Sons, Inc., New York, N.Y., 608 pp. Gaffney, P. J. and Harper, W. J., 1965. Lipase activity in somatic cells from separator slime. J. Dairy Sci., 48:613. Gale, E. F., 1951. The Chemical Activities of Bacteria. University Tutorial Press, London, 199 pp. Golding, N. S., 1947. Bitter American Cheddar from pasteurized milk. Proc. Inst. Dairying, Wash., 16:146. Gormican, A., 1972. Sodium in foods and beverages.. J. Milk Fd. Technol. 35:1. 69 Could, I. A., 1941. Effect of certain factors upon lipolysis in homo- genized raw milk and cream. J. Dairy Sci., 23:779. Harper, W. J., 1959. Chemistry of cheese flavors. J. Dairy Sci. 42:207. Harper, W. J. and Could, I. A., 1952. Italian cheese ripening: com- position of commercial Provolone and Romano cheese. Butt. Cheese Milk Prod. J.,_fl3:24. Dairy Sci. Abstr. 23:801, 1952. Harper, W. J. and Kristoffersen, T., 1956. Biochemical aspects of cheese ripening. J. Dairy Sci., 32:1773. Harvey, R. J. and Walker, J. R. L., 1960. Volatile compounds in New Zealand Cheddar cheese and their possible significance in flavor formation. III Time of first appearance of volatile carbonyl compounds during ripening. J. Dairy Res. 22:335. Harwalkar, V. R. and Elliott, J. A., 1965. Isolation and partial puri- fication of bitter compounds from Cheddar cheese. J. Dairy Sci., 48:784. Harwalkar, V. R. and Calbert, H. E., 1961. Specificity of milk lipases. Dairy Sci. Abstr., 23:2673, 1961. Heap, Beth. 1968. Low sodium milk, current status. J. Amer. Dietetic Assoc., 33:43. Hoecker, W. H. and Hammer, B. W., 1944. Salt migration in Cheddar cheese curd and its effect on moisture content, pH and bacterial content. Food Res., 2:278. Hof, T., 1935. Investigation concerning bacterial life in strong brines. Rec. Trav. Bot. Neerland, 32:92. Biol. Abstr. 29:8823, 1936. Honer, C. J., 1953. The isolation of intermediates in the Kreb citric acid cycle in cheese and fermented milk. Ph. D. Thesis. Univer- sity of Illinois. Irvine, O. R., 1951. Composition, pH and color of Ontario Cheddar cheese. Canadian Dairy Ice Cr. J., 32:27. Irvine, 0. R., 1955. Salting Cheddar cheese. Canadian Dairy Ice Cr. J., 34:38. Iyer, Meena, Richardson, T., Amundson, C. H., and Boudreau, A., 1967 a. Improved techniques for analysis of free acids in butter oil and Provolone cheese. J. Dairy Sci., 39:285. Iyer, Meena, Richardson, T., Amundson, C. H., and Tripp, R. C., 1967 b. Major free fatty acids in Gouda cheese. J. Dairy Sci., 39:385. Jensen, R. G., 1964. Lipolysis. J. Dairy Sci., 32:210. 7O Kandler, 0., 1961. Metabolism of starter organisms. Milchwissen— schaft, 29:523. Dairy Sci. Abstr., 29:166, 1962. Keeney, M., and Day, E. A., 1957. Probable role of strecker degra- dation of amino acids in development of cheese flavor. J. Dai- ry Sci., 99:874. Kosikowski, F. V., 1951. The liberation of free amino acids in raw and pasteurized milk Cheddar cheese during ripening. J. Dairy Sci., 39:235. Kosikowski, F. V., 1957. Cheese Flavor. Chemistry of Natural Food Flavors: A symposium. Quartermaster Research and Engineering Center, Natick, Mass. 200 pp. Kosikowski, F. V. and Dahlberg, A. C., 1946. A rapid direct distil- lation method for determining the volatile fatty acids of cheese, J. Dairy Sci., 29:861. Kosikowski, F. V. and Dahlberg, A. C., 1954. Free amino acids in for- eign type cheese. J. Dairy Sci., 32:167. Kosikowski, F. V. and Mocquot, C., 1958. Advances in cheese technolo- gy. F. A. 0. Agr. Stud., 38 pp. Kristoffersen, T., 1963. Measuring thiamine disulfide reducing sub- stances in Cheddar cheese. J. Dairy Sci., 93:1135. Kristoffersen, T. and Gould, I. A., 1959. Carbonyl compounds in Ched- dar cheese and their possible relationship to flavor. XV Int. Dairy Congr., London, 2:720. Kristoffersen, T. and Could, I. A., 1960. Cheddar cheese flavor. II Changes in flavor, quality and the ripening products of commercial Cheddar cheese during controlled curing. J. Dairy Sci., 93:1202. Kristoffersen, T., Gould, I. A. and Purvis, G. A., 1964. Cheddar cheese flavor. III Active sulfhydryl group production during ripening. J. Dairy Sci., 92:599. Kunakhov, I. M., 1967. Changes in rheological properties of Dutch cheese during first 3 weeks of ripening. Trudy vologod. moloch. Inst., 33:70. Dairy Sci. Abstr., 33:162, 1969. Lane, C. B. and Hammer, B. W., 1939. Relationship of fat hydrolysis to flavor development in Cheddar cheese. J. Dairy Sci., 22:432. Libbey, L. M. and Day, E. A., 1963. Thin layer chromatography of some 2,4-dinitrophenylhydrazene derivatives of Cheddar cheese volatiles. J. Dairy Sci., 93:598. Mabbitt, L. A., 1955. Quantitative estimation of the amino acids in Cheddar cheese and their importance in flavor. J. Dairy Res., 22: 224. 71 Mabbitt, L. A., 1961. Reviews of the progress of dairy science. Section B. Bacteriology. The flacor of Cheddar cheese. J. Dairy Res., 28:303. Mabbitt, L. A., and Zielinska, M., 1956. Flavor production in Cheddar cheese. XIV Int. Dairy Congr., 2:323. MacLeod, P. and Morgan, M. E., 1958. Differences in the ability of lac- tic streptococci to form aldehydes from certain amino acids. J. Dairy Sci., 92:908. Malushko, V., 1957. Effects of salting on the development of gas pro- ducing bacteria in cheese. Mol. Prom., 29:30. Dairy Sci. Abstr., 29:717. 1957. Marquardt, J. C. and Yale, M. W., 1941. Observations on the composi- tion of Cheddar cheeses. Nat. Butter Cheese J., 22:16. March, E. H., 1963. Microbiological and chemical aspects of Cheddar cheese ripening. A review. J. Dairy Sci., 99:869. Mattick, A. T. R. and Hiscox, E. R., 1939. The volatile acidity in relation to the flora of Cheddar cheese. Proc. Soc. Appl. Bac- teriol., 139:39. Mattick, A. T. R., Neave, F. K. and Helen, R. C., 1959. Staphylococcus aureus in Cheddar cheese. XV Int. Dairy Congr., 2:1914. McCarthy, R. D. and Duthie, A. H., 1962. A rapid quantitative method for the separation of free fatty acids from other lipids. J. Li- pid. Res., 2:117. Morris, H. A. and Jezeski, J. J., 1953. The action of microorganisms on fats. II. Some characteristics of lipase systems of 2: rogue- forti, J. Dairy Sci., 29:1285. Mulder, H., 1952. Taste and flavor forming substances in cheese. Nether- lands Milk and Dairy J., 9:157. Mulder, H. and Schouten, G., 1955. The salt content of some commercial samples of dietary cheese. Voeding, 29:936. Agri. Call. Wagenin- gen, Netherland. Nakae, T. and Elliott, J. A., 1965 a. Volatile fatty acids produced by some lactic acid bacteria. I Factors influencing production of volatile acids from casein hydrolysate. J. Dairy Sci., 99:287. Nakae, T. and Elliott, J. A., 1965 b. Production of volatile fatty acids by some lactic acid bacteria. II Selective formation of volatile fatty acids by degradation of amino acids. J. Dairy Sci., 99:293. 0hren, J. A., 1965. The relation of flavor development in Cheddar cheese to chemical changes in the fat of the cheese. Ph. D. Thesis. Uni- versity of Illinois. 72 Patton, S., 1963. Volatile acids and the aroma of Cheddar cheese. J. Dairy Sci., 99:856. Patton, S., Wong, N. P. and Fross, D. A., 1958. Some volatile compo- nents of Cheddar cheese. J. Dairy Sci., 92:857. Peterson, M. H., Johnson, M. J. and Price, W. V., 1948. Lipase acti- vity during making and ripening of Cheddar cheese. J. Dairy Sci., 31:39. Peterson, M. H., Johnson, M. J. and Price W. V., 1949. Liberation of fatty acids during making and ripening of Cheddar cheese. J. Dai- ry Sci., 92:862. Platon, B., 1942. The influence of salt on acid production by lactic acid bacteria. Svenska Mejeritidin.,‘99:427. Biolo. Abstr., 29: 20653, 1946. Platt, T. B. and Foster, E. M., 1958. Products of glucose metabolism by homofermentative streptococci under anaerobic conditions. J. Bact., 29:453. Raadsveld, C. W. and Mulder, H., 1949. Influence of the temperature on the ripening of cheese. Neth. Milk & Dairy J., 9:117. Ramanauskas, R., 1971. Effect of salt on the development of cheese con- sistency. In proceedings of Inter University Dairy Conference. Food Sci. Technol. Abstr., 9:807. Reynolds, J. N., 1946. Salt in American Cheddar cheese. Proc. St. Coll. wash. Inst. Dairying, 9 pp. Riddet, W., McDowell, F. H. and Valentine, G. M., 1933. Factors affect- ing the rate of ripening of New Zealand export cheese. N. 2. Dept. Sci. Ind. Res. Bull. No. 38, 40 pp. Sammie, J. L. end Germain, L., 1929. Experiments on cheese composition and quality. Butter cheese, J., 29:13. Sandberg, E., Haglund, E. and Barthel, C., 1930. The analysis of the juice expressed from cheese as a means of determining the degree of maturity. Lait, 29:1. Chem. Abstr., 29:1679, 1930. Sanders, P., Walter, H. E. and Tittsler, R. P., 1946. The influence of the quality of milk and curing temperature on the ripening of pasteurized milk Cheddar cheese. J. Dairy Sci., 29:497. Saunders, Elizah., 1970. Dietary salt (sodium chloride) intake and arterial hypertension: Part 1. Maryland State Medical J., 29:103. Sazabo, C., 1966. Factors affecting the rheological properties of pro- cessed cheese. XVII Int. Dairy Congr., 9:251. 73 Scarpellino, R. J., 1961. The application of gas chromatographic tech- niques to the study of volatile components in Cheddar cheese. Diss. Abstr.,‘22:421. "Scarpellino, R. and Kosikowski, F. V., 1962. Evolution of volatile com- pounds in ripening raw and pasteurized milk Cheddar cheese gbser- ved by gas chromatography. J. Dairy Sci., 99:343. Schonberg, A. and Moubasher, R., 1952. The strecker degradation of al- pha-amino acids. Chem. Revs., 99:261. Chem.Abstr., 92:1596, 1953. Schormuller, J., 1968. The chemistryiand biochemistry of cheese-ripening. Advances in Food Research., 29:231. Schormuller, J. and Leichter, L., 1955.a. Glutamic acid decarboxylase in ripening sour milk cheese. 2. Lebensmitt Untersuch., 102:13. Dairy Sci. Abstr., 29:173. 1956. Schormuller, J. and Leichter, L., 1955 b. Lysine decarboxylase in ri- pening sour milk cheese. Z. Lebensmitt Untersuch., 102:13. Dairy Sci. Abstr., 29:174, 1956. Sharara, H. A., 1956. Retention of calcium and phosphorus in Egyptian Domiati cheese. Indian J. Dairy Sci., 9:17. Sharpe, M. E.,and Franklin, J. G., 1963. The incidence of bacteria in cheese milk and Cheddar cheese and'their association with flavor. J. Dairy. Res., 99:87. Silverman, G. J. and Kosikowski, F. V., 1956. Amines in Cheddar cheese. J. Dairy Sci., 99:1137. Sjostrom, G., 1944. Experiments on the effect of rapid heating, cooling, addition of salt and of lactic acid at different times on the de- velopment of starter culture. Svenska Mejeritidn., 99:419. Dairy Sci. Abstr., 2:260. Sohngen, N. L., Wieringa, K. T. and Pasveer, A., 1937. Curdling of milk-study of the curdling enzyme. Rec. trav. Chem. Pays-Bas., 99:280. Chem. Abstr., 92:4004, 1937. Stadhouders, J., 1956. The hydrolysis of the cheese fat in relation to the flavor of cheese. Medelingen Van de Landbouhoge School, Nether- land, 56(11). Dairy Sci. Abstr., 29:592, 1957. Stadhouders, J. and Mulder, H., 1953. The ripening of Dutch cheese. Int. Dairy Congr., 2:681. Stadhouders, J. and Mulder, H., 1957. Fat hydrolysis and Cheddar cheese flavor. I. The enzyme responsible for the hydrolysis of fat in cheese. Neth. Milk & Dairy J., 22:164. Suzuki, S. R., Hastings, E. G. and Hart, E. B.,l910. Volatile acids and esters in Cheddar cheese J. Biol. Chem., 2:431. 74 Tittsler, R. P., 1965. Cheese Chemistry Part II. Fundamentals of Dairy Chemistry. The AVI Publishing Company, Inc., Westport, Conn. 827 PP- Tuckey, S. L. and Ruehe, H. A., 1940. Relation of salt content to bitter flavor development in Cheddar cheese. J. Dairy Sci., 29: 517. Rustin, Jr., E. B., 1946. Effect of varying amounts of salt on the quality of Cheddar cheese. Nat. Butter & Cheese J., 92:44. Vakaleris, D. G. and W. V. Price, 1959. A rapid spectrophotometric method for measuring cheese ripening. J. Dairy Sci., 42:264. Van Slyke, L. L., 1928. A summary of studies relating to casein and some of their applications. Geneva Agr. Exp. Station Tech. Bull No. 139, N.Y. Van Slyke, L. L. and Price, W. V., 1949. Cheese. Orange Judd Publishing Co., New York, N.Y., 522 pp. Van Slyke, L. L. and Hart, E. B., 1903. Conditions affecting the chemi- cal changes in cheese. Geneva Agr. Expt. Station Bull. No. 236. N.Y' Van Slyke, L. L., Smith, G. A. and Hart, E. B., 1903. Experiments in curing cheese at different temperatures. Geneva Agr. Expt. Station Bu11. No. 234. N.YO wagenaar, R. 0. and Dack, G. M., 1953. Some factors influencing the growth and toxin production of 9: botulinum experimentally ino- culated into surface-ripened cheese. J. Dairy Sci., 99:563. walker, J. R. L., 1959. Some volatile compounds in New Zealand Ched- dar cheese and their possible significance in flavor formation. II. Volatile compounds of sulfur. J. Dairy Res., 29:273. walker, J. R. L. and Harvey, R. J., 1959. Some volatile compounds in New Zealand Cheddar cheese and their possible significance in fla- vor formation. I. Identification of the volatile carbonyl frac- tion. J. Dairy Res., 29:265. walter, H. E., Sadler, A. M., Mitchell, C. D. and Hargrove, R. E., 1958. Effect of salt on acid development in Cheddar cheese. J. Dairy Sci., 92:718. White, F. L., 1957. Low Sodium Milk. Council of Foods and Nutrition. J. Amer. Med. Assoc., 167:739. Windlan, H., 1955. The influence of milk coagulating enzyme upon some of the constituents and flavor of Cheddar cheese during ripening. Ph. D. Thesis. Cornell University. 75 Wiseman, H. G. and Irvine, A. M., 1957. Determination of organic acids in silage. Agr. Fd. Chem., 9:213. Wolin, A. G., 1961. The application of tritium to the study of casein degradation and its relationship to cheese ripening. Diss. Abstr., 21:2243. WOlin, A. G. and Kosikowski, F. V., 1959. The use of tritium labelled casein in the study of cheese ripening. XV. Int. Dairy Congr., 2:685. Yale, M. W. and Marquardt, J. C., 1940. Factors affecting the survi- val of 9: pyogenes in cheese. J. Milk Technol., 9:326. Zaykowski, I. J. and Slobodska-Zaykowsky, N., 1925. Chemical and bac- teriological factors in the ripening of cheese. Chem. Abstr., 29:951, 1926. HICHIGQN STQTE UNIV. LIBRRRIES llHlllllllHHIIIIVIIINIIIIIIlllWWIHIIIIIIHIIIHIHII 31293104446632