dCVW/UU lllllllllllllllllllll[HUIHUI!llllllllllllllllllllllllllllll 31293 00786 4337 l’ l LIBRARY Michigan State L University ‘ L A This is to certify that the dissertation entitled QUICK RIPENING OF BLUE CHEESE CURD USING CONTROLLED ATMOSPHERE PACKAGING presented by Zain E.M. Saad has been accepted towards fulfillment of the requirements for PhD degree in Food Science Major professor Date _.Iannany_l_8,_128_9 MSU is an Affirmative Action/Equal Opportunity Institution 0-12771 PLACE fl RETURN BOX to romavothlo chockom from your noord. TO AVOID FINES return on or baton dd. din. DATE DUE DATE DUE DATE DUE SEP 0 6 200 l__ ; £3 MSU I. An Mandi“ ActloNEan Opportunity Institution cm OUICK-RIPENING OF BLUE CHEESE CURD USING CONTROLLED ATMOSPHERE PACKAGING By Zain E. H. Saad A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1989 w W5 4 ABSTRACT QUICK RIPENING OF BLUE CHEESE CURD USING CONTROLLED ATMOSPHERE PACKAGING By Zain E. H. Saad A Blue cheese of desirable quality was produced by a modification of the quick ripening (QR) procedure of Hedrick g; _L.(1968). The research described herein was designed to investigate the feasibility of quick ripening Blue cheese curd by selecting and maintaining the optimum atmosphere over packaged curd. Variables introduced included salting, temperature of ripening and modified and gas flushed atmosphere packaging. The use of physical methods of preservation such as ionizing radiation and microwave heating to control the accelerated ripening was investigated. To determine package permeability needed, the 03/002 requirement of the curd at various temperatures was investigated. During ripening of the cheese , pH, water—soluble nitrogen, gas headspace concentration, moisture content, and methyl ketone concentrations were determined. Sensory evaluation was also carried out on all batches of cheese. ZAIN E. M. SAAD The rate and magnitude of respiration were directly related to the ripening temperature. The CO, production ranged from 52.45 to 130.70 cc/kg/hr. A mutual dependent relationship existed between respiration and permeation within the Blue cheese packaging system. As the pH values increased there was a corresponding increase in water-soluble nitrogen. The values for pH and water-soluble nitrogen at the end of ripening were 5.46- 6.49 and 32 - 432, respectively, depending on the ripening temperatures and the packaging procedures used. The initial moisture content of Blue cheeses was 50.34% and decreased to 41.21-46.6SZ depending on the temperature, salt content and water vapor transmission of the package materials. The predominant methyl ketone in DR Blue cheese curd was 2-heptanone, 2-pentanone and 2-nonanone were also present in substantial levels. In cheeses irradiated with 1.0 KGy, a visible reduction of mold pigmentation and a slight decrease in methyl ketone concentrations were observed. A slight change of flavor, which was not objectionable, was also noted. Microwave heating the cheese to 53.0“0 and above was effective in reducing mold growth, however these ZAIN E. M. SAAD temperatures were detrimental to the flavor and texture of the cheese. The cheeses ripened in modified atmosphere packaging were comparable or superior, as indicated by sensory evaluation, to commercial Blue cheese when an extended period of two weeks storage at 5°C was applied to the QR cheese. Dedicated To My Wife Safaa My Daughter Sarah and My Son Yassir ii ACKNOWLEDGMENTS In loving memory of my major professor, Dr. Charles M. Stine, the author wishes to express sincere appreciation to him for his support, understanding and guidance which he provided while being a graduate student. Dr. Stine had a profound influence on me and will always be remembered. The author wishes to express sincere appreciation and thanks to Dr. Bruce R. Harte for his helpful suggestions, assistance and effort throughout the course of this study. Appreciation and thanks are extended to members of the guidance committee, Drs. J. Pestka, J. Partridge, P. Markakis, Department of Food Science and Human Nutrition, E. Beneke, Department of Mycology, for their advice and effort in reading this manuscript. The author also wishes to express his sincere appreciation and gratitude to his brother, Dr. Fathi M. Saad and his family for their invaluable encouragement, help, advice and moral support all the way. The author wishes to express his sincere appreciation and special gratitude to his wife, Safaa, for her devotion, patience and encouragement. Her understanding and moral support made it possible to pursue a higher degree of education. 111 TABLE OF CONTENTS Page LIST OF TABLES . ..... ................................ viii LIST OF FIGURES ...... ...................... ......... xi INTRODUCTION ........... ..... ........................ 1 REVIEH OF LITERATURE ................................ 3 Blue Cheese Flavor Components and Their Origin ........................................ 4 Free Fatty Acids .......................... 4 Lipid Metabolism (Lipolysis) .............. 5 Lipases of Other Microorganisms ...... 7 Factors Affecting Lipolysis in Blue Cheese ......................... 8 Temperature ..................... 8 Acidity ......................... 8 Salt Concentration .............. 9 Homogenization .................. 10 The Gas Requirements of Mold ......... 11 Volatile Acidity .......................... 12 Methyl Ketones ............................ l4 Mechanism of Methyl Ketone Formation by Fungi .................. 16 Other Flavor Compounds .................... 20 Protein Metabolism (Proteolysis) .......... 22 Acceleration of Ripening and/or Improving the Flavor of Blue Cheese ..................... 25 Control of Accelerated Ripening ................ 30 Modified and Controlled Atmosphere Packaging ... 32 Effect of Modified and Controlled Atmosphere ............................... 33 Advantages of Modified and Controlled Atmosphere ............................... 35 Cheese Packaging ............................... 37 Packaging Materials Requirements ............... 38 Factors Affecting Permeability ................. 39 iv Page ANALYTICAL PROCEDURES ....... ................... ..... 41 pH ............................................. 41 Moisture Content ............................... 41 Gas Headspace Analysis ......................... 41 Nitrogen (reduced) Determinations .............. 42 Sensory Evaluation ............................. 42 High Performance Liquid Chromatographic (HPLC) Analysis of Methyl Ketones .................... 43 Purification of Solvents .................. 43 Hexane ............................... 43 Benzene .............................. 44 Chloroform ........................... 44 Nitromethane ......................... 44 Acetonitrile ......................... 44 Ethanol .............................. 44 Butanol,l- ........................... 44 Ethylene Chloride .................... 45 Preparation of 2,4-DNPH Derivatives of Standard Methyl Ketones .................. 45 Isolation of Monocarbonyls from Cheese Samples .................................. 45 Extraction of Fat .................... 45 Formation of 2,4—DNP-Hydrazones ...... 46 Removal of Fat from 2,4-DNP— Hydrazones .......................... 46 Fractionation of 2,4-DNP-Hydrazones on Weak Alumina ..................... 46 Determination of Methyl Ketones by High Performance Liquid Chromatography (HPLC).. 47 Chromatography Equipment and Conditions .......................... 47 Recovery of Methyl Ketones ........... 48 Gas Requirements ............................... 48 water Vapor Transmission of LDPE and Laminate (Nylon/PE)..................................... 49 Oxygen Permeation of LDPE and Laminate (Nylon/PE)..................................... 49 Gamma Irradiation .............................. 50 Microwave Heat Treatment ....................... 50 V Page Procedure Used for Manufacture OR Blue Cheese... 51 Statistical Analysis............................ 52 RESULTS AND DISCUSSION .............................. 53 The Gas (Oz/C02) Requirements of E.rongforti... 53 Respiration Rate .......................... 53 Oxygen Depletion .......................... 59 Selection of Product-Package Parameters......... 62 Changes in pH During Ripening .................. 64 Changes in Water-Soluble Nitrogen During Ripening ...................................... 72 Gas Headspace Analysis ......................... 80 A. Modified Atmosphere Packages ......... 80 B. Gas Flushing Atmosphere Packages ..... 83 Change in Curd Moisture Content During Ripening ....................................... 86 Liquid Chromatographic Determination of Methyl Ketones as Their (2,4-DNPH) Derivatives ....... 9O Extraction and Fractionation of Carbonyl Compounds in Cheese Fat .................. 90 High Performance Liquid Chromatographic (HPLC) Separation of Methyl Ketones .................. 91 Mass Spectral Identification of The HPLC Minor Compounds .......................... 97 Ouantitation of Individual Methyl Ketones, C3-C,3 .......................... 104 Recovery Study of C3-C1; Methyl Ketones Extraction .................. 104 Methyl Ketones in Commercial Imported and Domestic Blue Cheese ..................... 106 Methyl Ketones in OR Blue Cheese by Controlled Atmosphere Packaging .......... 109 Sensory Evaluation of OR Blue Cheese by Controlled Atmosphere Packaging ............... 112 Visual Observations............................. 116 Physical Methods of Presevation ................ 117 A. Ionizing Radiation ................... 117 8. Microwave Heating .................... 120 vi Page SUNHARY AND CONCLUSIONS IIIIIIIIIIIIIIIIIIIIIIIIIIIII 1 N N APF.ENDICES II ...... IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII Apper‘dix A I I ...... I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 128 Appendix B I I I I I I I IIIIII I I I I I I I I I I I I I I I I I I I I I I I I 129 Appendix C I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 130 BIBLIOGRAPHY III-IIIIIII IIIII IIIIIIIIIIIIIIIIIII 131 vii Table 1. LIST OF TABLES Page Some Acids Found in Roquefort and Gorgonzola Cheese ................ ..... . ........ 13 Maximum CO, Production, 02 Consumption and Respiration Quotient in Blue Cheese Curd Ripened at Different Temperatures .............. 59 Water Vapor Transmission of LDPE and Laminate (PE/Nylon)Pouches at Various Temperatures....... 63 Moisture Content(Z) of Blue Cheese Curd Salted on Day One (3.5%) and Quick Ripened at 10°C or 15°C over 95ZRH for Two Weeks then Stored at 5°C/95ZRH for Additional Two Weeks........... 87 Moisture Content of Quick Ripened and Commercial Blue Cheeses ........................ 89 The Retention Times of Standard 2,4-DNP- Hydrazones of Methyl Ketones 22- Those in Blue Cheese samples IIIIIIIIIIIIIIIIIIIIIIIIIIII 94 Relative Abundance Comparison of Ca and Cs' for Ion Fragments Higher Than 10% of Base Peak IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 102 Relative Abundance Comparison of C7 and C7. for Ion Fragments Higher than 10% of Base Peak IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 103 viii Table 10. ll. 12. 13. 14. 15. 16. 17. 18. 19. Relative Abundance Comparison of C. and C.‘ for Ion Fragments Higher than 102 of Base Peak IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII Values of Slopes, Correlation Coefficients and Intercepts of Calibration Curves of 2,4-DNPH Derivati V25 0* C3-C3: "EthYl KEtDnES e e e e e e e e e e e Percent Recovery of Standard, C3“Cxaa Methyl Ketones IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII Methyl Ketones Concentration of Imported and Domestic Commercial Blue Cheese ................ Methyl Ketones Concentration of Blue Cheese Quick Ripened in Modified and Gas Flushed Atmosphere Packaging for 2 Weeks at 10°C and 15°C/95%RH.................................. Methyl Ketones Concentration of Blue Cheese Quick Ripened in Modified and Gas Flushed Atmosphere Packaging for 2 Weeks at 10°C and 15°C plus 2 Weeks Stored at 5°C/9SZRH........... Subjective Analyses of QR Blue Cheese Ripened at 10°C and 15°C for Two Weeks ......... Subjective Analyses of QR Blue Cheese ripened at 10°C and 15°C for 2 Weeks and then Stored at 5°C for an Additional 2 Weeks................ Subjective Analyses of Blue Cheese Irradiated and Stored at 5°C/952RH for One Week ........... Methyl Ketones Concentration of QR Blue Cheese Irradiated and Stored at 5°C for One Week ...... Subjective Analyses of Blue Cheese "In-Package", Microwave Heating with Storage at 5°C for One Week ................................... ix Page 103 104 105 107 110 111 114 115 118 119 120 Table Page A. The Molecular Weight of Methyl Ketones and Their 2,4-Dinitrophenylhydrazones .............. 128 8.1 Analysis of Variance for Sensory Evaluation .... 130 8.2 Standard Error (ST‘) for the Main Factors in the Statistical "DdEI IIIIIIIIIIIIIIIIIIIIIIIIII 130 LIST OF FIGURES Figure 1. IOI Respiration Rate in Blue Cheese Curd, containing 3I57I NaCIIIIIIIIIIIIIIIIIIIIIIIIIIIII Respiration Rate in Blue Cheese Curd, containing 3.5% NaCl............................ CO, Production and 0, Consumption in Blue Cheese Curd, containing 3.5% NaCl.-............. C02 Production and 0, Consumption in Blue Cheese Curd, containing 3.5% NaCl............... 0, Depletion in Blue Cheese Curd Ripened at Various Temperatures in 212 0, Atmosphere ...... 02 Depletion in Blue Cheese Curd Ripened at Various Temperatures in 100% 02 Atmosphere ..... Change in pH During Ripening of Quick Ripened Blue Cheese Curd at 10°C in Modified and Gas Flushed Atmosphere Packaging ............... Change in pH During Ripening of Quick Ripened Blue Cheese Curd at 15°C in Modified and Gas Flushed Atmosphere Packaging ........... The Effect of Temperature on pH During Ripening of Quick Ripened Blue Cheese Curd ..... Change in Water-Soluble Nitrogen of Quick Ripened Blue Cheese Curd at 10°C................ xi Page 54 55 57 58 60 61 67 68 71 74 Figure 11. 13. 14. 15. 16. 17. 18. 19. Change in Water-Soluble Nitrogen of Quick Ripened Blue Cheese Curd at 15°C................ The Effect of Salt Concentration on Water Soluble Nitrogen of Quick Ripened Blue Cheese curd at 10°C IIIIIIIIII.IIIIIIIIIIIIIIIIIIIIIIII The Effect of Temperature on Water Soluble Nitrogen of Quick Ripened Blue Cheese Curd ..... Change in Package Headspace Gas Concentration During Ripening of Blue Cheese Curd in Modified Atmosphere Packaging................... Oxygen Depletion During Ripening of Quick Ripened Blue Cheese Curd at 10°C and 15°C in Gas Flushed Atmosphere Packaging ............ HPLC Resolution of 2,4-DNPH Derivatives of Standard Methyl Ketones, C3 - C33, at Different Solvent Ratio of Acetonitrile : Water A) 75:25 (V/V); B) 90:10 (V/V).......... Typical HPLC Separation of 2,4-DNPH Derivatives of Standard Methyl Ketones, C3 - C13, Prepared in Solution Mobile Phase: Acetonitrile/Water (80:20)........ HPLC Separation of 2,4-DNPH Derivatives of Methyl Ketones, C3 - C33, Present in Cbmmercial Blue Cheese Samples ................. HPLC Separation of 2,4-DNPH Derivatives of Standard Methyl Ketones, C; - C‘s, Passed Through Celite/Magnesia and Alumina COlumns IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII xii Page 77 78 82 85 92 93 95 96 Figure Page 20. Typical GC/MS Total Ion Current Chromatogram of 2,4-DNPH Derivatives of Standard Methyl Katones C3-C‘3 eeeeeeeeeeeeeeeeeeeeeeeeeeeeee 98 21. Mass Spectrometry Patterns of 2,4-DNPH Derivatives of Standard 2-Pentanone ............ 99 22. Mass Spectrometry Patterns of 2,4-DNPH Derivatives of Standard 2-Heptanone ............ 100 23. Mass Spectrometry Patterns of 2,4-DNPH Derivatives of Standard 2-Nonanone ............. 101 24. Typical HPLC Separation of 2,4-DNPH Derivatives of Methyl Ketones in Commercial: A) Danish ; B) French Roquefort .............. 108 xiii INTRODUCTION The market for Blue mold cheeses and their related food products, salad dressings, chip dips and snack food has increased tremendously in recent years. Because of the high production costs and labor requirements in manufacturing and curing Blue cheese, attempts have been directed towards more economical and efficient methods of production. Quick Ripened (QR) Blue cheese loose curd offers a superior alternative to the use of Blue cheese flavor. The Blue cheese curd produced by this technique is ready for utilization in approximately two weeks compared to traditional commercial methods which require three months to one year. Although considerable progress has been achieved in modifying, improving, refining and evaluating the quick ripening process, other problems remain to be solved. The labor intensive preparation process, storage space requirement, the risk of microbial contamination prior to packaging, and control of the acceleration of Blue mold growth and metabolism have stimulated the need for a more efficient method to manufacture loose curd Blue cheese. This project was undertaken to investigate the feasibility of: 2 quick ripening of Blue cheese curd by selecting and maintaining the optimum atmosphere over packaged curd. controlling the accelerated ripening of Blue mold cheese curd by means of low temperatures and/or the use of chemical or physical methods of preservation, such as ionizing radiation or microwaving. evaluating the suitability of various flexible films and/or rigid containers for controlling the composition of the headspace gas in packaged curd during quick ripening. reducing handling and post—manufacture processing. Quick ripening of Blue cheese curd by controlled atmosphere packaging could result in less warehouse space needed during the manufacturing and ripening of Blue cheese curd, possible elimination or reduction in growth of both pre-package and post-package contaminant organisms and a reduction in production costs. LITERATURE REVIEW Cheese flavor was described by Harper (1959) as " a complex phenomenon which results from a multiplicity of chemical changes in the cheese system“. The complexity of the system is evidenced by Day and Anderson (1965), where approximately 100 flavor compounds in the volatile fraction of Blue cheese were identified by gas chromatography and mass spectrometry. The major components in the aroma fraction were methyl ketones, secondary alcohols, methyl and ethyl esters of the normal aliphatic acids. Different classes of flavor components in Blue-veined cheese can originate from several sources. The most significant flavor compounds are produced by the metabolism of microorganisms, by the action of natural milk enzymes, and by the concerted action of numerous enzymes of P.roqugforti which are involved in protein and lipid metabolism. Kinsella and Hwang (1976) reviewed the biosynthesis of flavors during Blue cheese ripening. Aspects of the flavor and manufacture of Blue-veined cheese were also reviewed by Bakalor (1962). BLUE CHEESE FLAVOR COMPONENTS AND THEIR ORIGIN FREE FATTY ACIDS The degradation of milk fat forms a major source of flavor compounds in Blue cheese. Currie (1914) stated that the "peppery and spicy" taste of Roquefort cheese was due to the short chain free fatty acids, caproic, caprylic, and capric and their readily hydrolyzable salts. Caproic , valeric and butyric acids in Blue cheese were reported by Thomasaw (1947). Using gas chromatography, Coffman,gtrgl. (1960) analyzed dry Blue cheese and detected butyric, caproic, caprylic, capric, isovaleric and heptanoic acids. Several investigators have obtained quantitative data for free fatty acids in Blue cheese. Morris g; al. (1955) attributed the "pungent. peppery flavor" to free acids and their oxidative products and reported finding an average of 0.77 -mg butyric acid/g of Blue cheese made from pasteurized, homogenized milk. The average caproic and higher acid value (average molecular weight 228) was 24.93 mg/g of cheese. Day and Anderson (1965), utilizing gas chromatography, reported that the major fatty acids in Blue cheese were C2 - 018:3 acids. Similar concentrations of free fatty acids were reported by Blakely (1970) in QR 5 loose curd Blue cheese. The lipid and fatty acid content of Blue cheese was also studied by Fujishima g; a;. (1971). Harte and Stine (1977) reported the same free fatty acid (butyric - linolenic) patterns between QR and commercial Blue cheese. Anderson (1966) showed a difference between the fatty acid content of Roquefort and imported Blue cheese. Roquefort flavor and aroma were more characteristic of caprylic and capric acids due to the difference in composition of the fat in sheep’s milk as compared to bovine milk fat. LIPID METABOLISM (LIPOLYSIS) Lipid hydrolysis during cheese ripening is governed by lipase activity. The lipases in cheese may originate from milk lipases,, microorganisms from the milk, and from the interior or surface of the cheese (Stadhouders and Mulder, 1957). Various authors have demonstrated the presence of more than one natural milk lipase. Two lipase systems existing in raw skim milk with pH optima of 5.4 and 6.3 have been reported by Albrecht and Jaynes (1955). Taressuk and Frankel (1957) observed two lipase systems native to Cow’s milk, a plasma lipase associated with casein and a lipase absorbed irreversibly on the fat globule membrane. Chandan and Shahani (1963) isolated a low molecular weight lipase from milk clarifier slime. This lipase had the ability to 6 hydrolyze milk fat within a pH range of 5.0 - 10.0. Downing and Andrews (1969) reported five lipases in bovine milk. However, those lipases with a pH optimum of 7.0 and above could not continue to be active in Blue-veined cheese manufacture (Bakalor, 1962). Jensen (1964) reported that when mixed triglycerides are hydrolyzed, there is an obvious preference for the fats containing short chain acids. Milk lipase preferentially releases fatty acids from the primary positions (1,3) of a triglyceride. Since butyrate appears to be predominantly esterified at the 3-position, release of butyrate results. In Blue cheese manufacture, spores of Penigilligg rogueforti are used to inoculate the milk or the curd. Currie (1914) stated that a water soluble lipase was produced by E; rogueforti which hydrolyzes milk fat to produce the free fatty acids characteristic of the sharp flavor of Roquefort cheese. Morris and Jezeski (1953) reported two lipase system in E;_ rogugforti, isolated from mycelial extract, one with a pH optimum for milk hydrolysis at 6.5 - 6.8 and one with a pH optimum at 7.0 to 7.2. Niki gt a1. (1966), using butter fat as a medium, found that the optimum lipase activity was obtained at pH 6.0 and 7.5 for both the intra- and extra-cellular lipases. Different strains of E; rogueforti show certain differences in their lipolytic properties. Strains with moderate proteolytic .7 activity and strong lipolytic activity are the most suitable for Blue cheese production (Niki, gt gt., 1966). Three out of eight strains studied by Graham (1958) produced well- veined cheese and good flavor. Soto, gt _t. (1966) found that the amounts of carbonyls and fatty acids differed according to the strain of E; rogueforti used. Therefore, proper strain selection is necessary for the production of a quality Blue cheese. LIPASES OF OTHER MICROORGANISMS Some investigators have suggested that other microorganisms may be involved in the release of fatty acids from triglycerides during cheese ripening. Fryer and Reiter (1967) found a weak lipolytic activity in 56 strains of lactic acid bacteria. Strains of yeast were isolated during the manufacture and ripening of Blue cheese (Devoyod and Sponem, 1970). Of the ones present, Sagchargmxges lactis, Candida ligothica, Torglggsis s hasica, and Torulopsis fragillis have the most important lipolytic properties. Khan, _t gt. (1966) suggested that the microbial lipases released larger amounts of unsaturated fatty acids and smaller amounts of short chain fatty acids compared to milk lipase. 8 Factors affecting ligolysis in Blue cheese: Temperature: The optimum temperature of lipase activity in E; roqugfiorti was found to be between 30° to 35°C by Shipe (1951), and 30° - 32°C by Morris and Jezeski (1953). The variation in the optimum temperatures for the lipase activity may be due to the difference in growth condition, fungal strain and analytical method. A combination of temperatures could be used to obtain the optimum balance between the biological and chemical processes involved in Blue cheese ripening (Morris, 1969). Higher temperatures in the early stages of ripening promoted greater production of lipase while lower temperatures in the later stages discouraged mold growth and produced hydrolysis and/or Beta- oxidation of the free fatty acids. Kinsella and Hwang (1977) reported that the lipase system of fit rogueforti has shown to be active at low temperatures of cheese ripening (10°C). Acidity: The acidity of Blue cheese was studied by Coulter, gt gt. (1938b). A rapid reduction to pH 4.7 was observed during the first day of ripening. The pH increased after three months to 6.5, then decreased slowly to 5.7 at nine months. The marked rise in pH from about 2 weeks to 10 weeks was due to continuing proteolysis (Currie, 1914; 9 Morris, et.al , 1951). A tendency for the rate to decrease as the pH approached 6.5 was noted by Morris (1963). The lipase systems in Blue cheese were active in the pH range occurring throughout curing. _glt concgntrgtion: The effect of salt concentration on mold growth and lipolytic activity has been investigated. Gould (1941) demonstrated the inhibitory effect of NaCl on lipase activity in raw homogenized milk and cream. Levels of 5% to 8% NaCl were sufficient to inhibit lipolysis almost completely. Morris and Jezeski (1953) reported that 10- 162 NaCl brine diminished lipase activity by 60%. Most organisms can not survive salt concentrations greater than 6%, and essentially none survive a concentration of 10% (Morris, 1969). Poznanski, gttgt (1967) reported that the increase of salt content from 3.0% to 3.9% caused diminished acid production in Roquefort cheese. Godinho and Fox (1981 a) studied the effect of NaCl on the germination and growth of E; roggeforti. Germination of all strains (excluding "Dairy land" strain) isolated from several Blue cheeses was inhibited by >32 NaCl. Godinho and Fox (1981a) studied the salt diffusion rates and mold growth in ripening of Blue cheese. In the manufacture of Blue cheese, the amount and timing of salt application played an important and critical role in the development of the mold and therefore in the 10 flavor of the cheese (Kondrup and Hedrick, 1963; Godinho and Fox, 1981a). Homoggniggtion: Homogenization of milk prior to Blue cheese manufacture is a critical process in the hydrolysis of milk fat. Homogenization increases the surface area of the milk fat, thus allowing increased lipolysis by milk and mold lipase systems and also alters the composition of the fat globule membrane. Lane and Hammer (1938) reported that homogenized milk yielded cheese which ripened faster, was softer in texture and lighter in color. Morris, et.al (1963) showed the importance of homogenization with progressive accumulation of free fatty acids during Blue cheese ripening. Dozet, gttgt (1972) determined the effect of milk homogenization in manufacture of soft white cheese. The triglycerides in non—homogenized raw milk are not available to lipase due to the intact “membrane" surrounding the fat globule in multi-layers which inhibit attack and hydrolysis. Kinsella and Hwang (1976) reported that homogenization expands the total surface area of milk fat available for lipase attack. The gas reguirements oftgold: The necessity of having exchange of carbon dioxide, produced by the mold, with oxygen from the air during 11 ripening is an important factor involved in maintaining proper mold growth. Golding (1937) stated that the presence of CO: rather than the absence of 02 is the limiting factor in the growth of fit rogueforti in the cheese. The author (1937) reported that the gas requirements of Et rogueforti are functions of both concentration and temperature and varied with different gases. Thom and Currie (1913) reported that fit rogueforti was able to grow at room temperature, with only slight restriction, in an atmosphere of 75% C02 and 5% 02. Brown (1922) found that low temperatures increased the inhibitory power of CO, on the growth of mold, with no mold growth in pure 003. Durrell (1924) showed that CO, in amounts up to 5% stimulated mold growth (Basisgorium gallarum). Karsner and Saphir (1926) found that concentrations of O, of 76% and more had a definite inhibitory effect on certain molds. Golding (1940 II) studied the oxygen requirements of fit rogueforti in an atmosphere diluted with nitrogen and carbon dioxide-free air. The author (1940 II) concluded that only with the greatest O, dilution (2.1% 02) that a significant reduction (between 16% and 54%) of growth was recorded. No significant reduction of growth was observed in an atmosphere of 4.2% O, in nitrogen. There was a tendency for a limiting effect of oxygen on growth to be observed at higher temperatures. Golding (1940 III) concluded that relatively small 12 concentrations of C02 in air increased the growth of some fit roqugforti strains, while large concentrations inhibited growth. The acceleration of growth due to low CO, concentrations took place sooner at lower temperature, while the inhibition of growth, due to high concentrations of C02, is apparent at low temperatures sooner than at higher temperatures. Kinsella and Hwang (1976) summarized the optimum conditions for generation of methyl ketones from fatty acids by spores and mycelium of fit rggueforti. At concentration of more than 5% O: for the spores, more than 4% 02 for the mycelium and more than 0.033 CO; was needed. The authors (1976) stated that carbon dioxide may actually enhance ketone formation by inhibiting the normal oxidation of acetyl-CoA via the Citric acid cycle, thereby causing a “back-up" of the fatty acid oxidation pathway. Volatile acidity: The presence of acids in very low concentrations has been identified in Blue cheese (Anderson, 1966). Table I lists some of the acids found in Roquefort and Gorgonzola cheese (Schormuller, 1968). Bassett and Harper (1958) identified acids such as oxaloacetic, oxalosuccinic and alpha-acetolactic in Blue cheese. 13 Table I Some acids found in Roquefort and Gorgonzola cheese Lactic Succinic Fumaric Acetic Propionic iso-Butyric alpha-Ketoglutaric Oxalic Phenyl propionic Malic p-Hydroxyphenyl pyruvic Formic p-Hydroxyphenyl propionic n-Valeric alpha-Ketobutyric Pyruvic Many microorganisms have the ability to carry out glycolysis, which resulted in the formation of lactic, acetic and propionic acids. Succinic, fumaric, glycoxalic, pyruvic and alpha-ketoglutaric acids are intermediate stages in the acetic acid cycle. Free amino acids are decomposed by trans- deamination to form alpha-hydroxybenzoic and other keto aCids. Keto acids in Blue cheese can also be formed through citric acid fermentation and beta-oxidation. The aroma and taste of these acids differ, but they may contribute to the overall flavor of Blue cheese. METHYL KETONES Many researchers have studied the occurrence of methyl ketones in milk and dairy products. Starkle (1924) reported 14 the presence of 2-heptanone and 2-nonanone in Roquefort cheese. The author (1924) postulated that these methyl ketones were formed by the metabolism of fats by Et roquggorti and that they contributed to the flavor and aroma of this cheese. Patton (1950) isolated acetone, 2- pentanone, 2-heptanone, and 2-nonanone from Blue cheese. It was felt that these methyl ketones, particularly 2- heptanone had an aroma typical of Blue cheese (Hammer and Bryant, 1937). Morgan and Anderson (1956) identified odd- numbered methyl ketones, C3 - ng, in Roquefort, Gorgonzola, Danish and domestic cheese. Harper and Bassett (1959) surveyed the acidic and neutral carbonyl compounds in Blue cheese. Several even-numbered methyl ketones were isolated from Blue cheese such as 2- butanone, 2-hexanone, and 2- octanone (Nawar and Fagerson, 1962). Day and Anderson (1965) confirmed the presence of methyl ketones: C3, C., 0., C7, C., C9, Cgo, Cg; and Cg: in Blue-vein cheeses by means of gas chromatography and mass spectral analysis. Schwartz and Parks (1963) quantitated the odd—numbered methyl ketones from C3 to C,. in domestic Blue cheese using chromatographic lmethods. Schwartz,_t gt. (1963) made similar measurements on Roquefort cheese and 2-heptanone was the major ketone in all samples except one which contained more 2-nonanone. Quantitation of the methyl ketones in Blue-veined cheeses, lay Anderson and Day (1966), indicated a large variation in 15 the quantity of ketones in different cheeses, with 2-heptanone being the most abundant ketone in all samples. Blakley (1970) reported a much lower concentration of methyl ketones, C3 to 0,3, in quick ripened (7 days) Blue cheese. The author (1970) found 2-nonanone to be the major methyl ketone in Danish and quick ripened Blue cheese. In the same study, the author found 2-undecanone to be the most abundant ketone with slightly smaller amounts of 2- nonanone present in quick ripened cheeses made from filled milk containing hydrogenated coconut oil. A much higher concentration of methyl ketones in quick ripened Blue cheese and lower content of methyl ketones in commercial Blue cheese were reported by Albert (1974). Dokhani (1978) quantitated C; to C,; methyl ketones in quick ripened and commercial Blue cheeses utilizing a high performance liquid chromatographic system. The author (1978) concluded that the predominant methyl ketones were C7 and C9. Starkle (1924) investigated the ability of several mold cultures, Penicillium laucum, fit rggueforti, Asgergillus thg5, and at fumigatug, to convert free fatty acids to methyl ketones, C7, C. and C,, from oxidative decomposition products of cocoa fat and butter and compared them with ketones obtained by hydrogen peroxide oxidation of pure fatty acids. A mechanism for the ‘formation of these ketones was advanced by Stokoe (1928). 'The~author (1928) suggested that Penicillium species oxidize 16 a chain compound primarily at the beta-carbon atom with the formation of the beta-keto acid followed by decarboxylation to the methyl ketone and carbon dioxide. Mechagtgm of Mgthyl tho g Fogggtion by Fuggt: Gehrig and Knight (1958, 1961, and 1963) demonstrated that only spores of E; rogueforti were capable of producing methyl ketones from fatty acids. The capacity to form methyl ketones disappeared rapidly and progressively as the spores germinated. In contrast, Lawrence and Hawke (1968 reported the formation of methyl ketones by mycelia of Et rogueforti from low concentrations of fatty acids with less than 14 carbon atoms over a wide range of pH. Dwivedi and Kinsella (1974a and 1974b) carefully obtained mycelia cultures of fit rogueforti and demonstrated the ability of mycelia to oxidize fatty acids and produce methyl ketones. Fan, gt gt. (1976) showed the relative rates of methyl ketone formation from fatty acids by spores of fit rogueforti at progressive stages of germination. They concluded that mycelia were much more active under short incubation conditions: however, the resting spores showed very high activity during a long period of incubation. Lawrence (1966) reported that the addition of Casamino acid or certain amino acids (particularly L-proline or L- alanine) greatly stimulated oxidation of triglycerides by spores of fit roqugtorti. The 17 rate of oxidation was further increased by addition of certain sugars (particularly D-galactose, D-xylose, D- glucose) to the media. The increase in the rate of 2- undecanone formation by the addition of glucose to the resting spores of fit rogueforti was shown by Hwang, gt gt. (1976). Using 1-“C labeled octanoate as substrate, Gehrig and Knight (1963) demonstrated that in small concentration (1 uM), sodium octanoate was oxidized completely by spores of Et,roqugforti; however, in larger concentrations (20 uM), part of the molecule was converted to methyl ketone and part was completely oxidized. The authors (1963) stated that no ketone was produced under anaerobic conditions. Wong, gt gt. (1958) proposed that the methyl ketones obtained from milk fat might be derived from beta-keto acids which are natural constituents of milk fat. Hawke (1966) outlined a schematic pathway of fatty acid oxidation for methyl ketone formation similar to the classical beta-oxidation cycle of fatty acids as shown in scheme I. This mechanism provides for the formation of: 1. only methyl ketones of one less carbon atom than the fatty acids used as substrates. 2. a simultaneous process for B-oxidation of fatty acids. 18 Scheme I E_th§§y of Fatty Acid Oxidation ggd Methyl thonngormation .. ‘2. H R CHzcuzcoscm ...—.3 RCH=CHCOSCoA + 2. H Deacylase CoASH + H20 RCOCH2COOH + CoASH RCOSCoA + CH3COSCoA Decarboxylase C0; + H20 further formation it of cuscoscm R1300“: 4' C02 Kinsella and Hwang (1976, 1977) diagramed the metabolism of fatty acids by at rogggiorti as it occurs in Blue cheese (scheme II). These authors (1976,1977) described the following steps, which are similar to the process outlined by Hawke (1966): 1. Lipolysis of triglycerides to form free fatty acids . 2. Dehydrogenation of the beta-Hydroxy acyl-CoA to form beta-Keto acyl-CoA. 3. beta-Keto acyl—CoA is deacylated to form beta—Keto acid and CoASH by beta-Keto acyl-CoA deacylase or Thiohydrolase. 19 4. Rapid decarboxylation of beta-Keto acid to methyl kehnms (2-Alkanones) and carbon dioxide by beta-keto acid dec arboxyl ase. 5. The IPAlkanones produced are easily reduced to the corresponding 2-Alkanols (secondary alcohols) by a reductase. Scheme II Lipids lipase Egtty Acidg beta-oxidation Egto Acyl-CoA Thiolase 3 Acylf-CoA + ‘*\.‘_ ,” AcetleoA “‘“""~‘"” Thiohydolase KCa Beta-tho Acid CO: 2-Alkgnol Decarboxylase / 2-Alkglong )cyl group may contain from 4 to 16 carbon atoms. ((3 represents the Krebs or Citric acid cycle. 20 'he Thiolase is possibly inhibited by Acyl-CoA (denoted by :he broken line in the scheme), thereby facilitating the jeacylation-decarboxylation step (Kinsella and Hwang,1977). The effect of environmental parameters on the rate of beta- oxidation was demonstrated by Lawrence (1966). The author (1966) observed that the pH optimum for methyl ketone formation varied with the concentration and type of fatty acids. The maximum yield of methyl ketone obtained was 25% from trioctanion and trihexanion at pH 6.0. Hwang, gt; L1. (1975) reported an optimum pH, 6.5 - 7.0, for 2-undecanone formation from beta-ketolaurate. Lawrence (1966) reported an optimum temperature of 27°C for the maximum rate of methyl ketone formation. Gehrig and Knight (1963) suggested an optimum temperature of 25°C for methyl ketone production. Other Flavor Compounds In addition to methyl ketones and free fatty acids, secondary and tertiary products have been found in Blue cheese. Secondary alcohols were identified by Stokoe (1928) as a result of the action of E._ rgueforti on coconut oil. The author (1928) postulated that these alcohols were intermediate products in the oxidation of fatty acids to methyl ketone. He also considered the possibility that they were formed by the reduction of ketones. Jackson and Hussong (1958) isolated 2-pentanol, 2-heptanol, 2-octanol 21 1nd 2—nonanol from Blue cheese. Anderson and Day (1965) identi f i ed 2-propanol , 2-pentanol , 2-heptanol , 2-octanol and 2-nonanol as components of Blue cheese aroma. Nawar and Fagerson (1962) reported the presence of 2-pentanol and 2- heptanol in Roquefort cheese. Anderson and Day (1966) determined the concentration of secondary alcohols in Blue cheese. The alcohols were found in much lower concentrations than the methyl ketones. Kinsella and Hwang (1977) suggested that a musty flavor in Blue cheese may result when 2-al kanols are present in large concentrations. Acetaldehyde, 2-methylpropanal, 3-methylbutanal and furfural were identified by Anderson (1966). The presence of these aldehydes may be due to the action of enzymes involved in the oxidative deamination of amino acids (Kinsella and Hwang, 1977; Anderson, 1966). Ethanol, acetyl methyl carbinol, and diacetyl have been identified in Blue cheese by Bassett and Harper (1958). Methanol, ethanol, n-pentanol, 2- and 3-methylbutanol, and 2-phenylethanol were identified in Blue cheese by Day and Anderson (1965) . The importance of methyl and ethyl esters in the overall flavor profile of Blue cheese was reported by Anderson and Day (1966). Different analogs of methyl, ethyl, and isopropyl esters were identified by Anderson (1966) . Jol l y and Kosi kowski (1975) identif ied and 22 uantitated five different lactones in Blue cheese ontaining added microbial lipase. f'ROTE I N METABOL 1 SH (PROTEOLYS I S) Proteolysis (degradation of protein) is important in cheese ripening for the development of proper texture, for background flavor (peptides and amino acids), for provision of amino acids which act as precursors of flavors (aldehydes, alcohols and esters) and as germination stimulants (Kinsella and Hwang, 1977). The extent of protein degradation varies with the variety of cheese. Primary protein hydrolysis to amino acids occurs to some extent in all cheese varieties. In Blue cheese, proteolysis varies with the strain of E; roqugforti, the temperature of ripening and the pH. Porks gt 1;. (1959) reported that the strains of E rogueforti differed in their lipolytic and proteolytic activities. Niki _t EL. (1966) observed that strains of EA rogueforti possessing low lipolytic activity exhibited high proteolytic activity and vice versa. The authors (1966) suggested that strains with low proteolytic and high lipolytic activity are most suitable for Blue cheese manufacture. Czulak (1959) attributed a marked bitterness in cheese to the accumulation of certain peptides and peptones. Niki g_t_ l. (1966) reported that strains which hydrolyzed over 507. of the protein produced soft fl 23 cheeses with an unpleasant bitter taste. Niki e_t_ §_1_. (1966) detected the presence of an intra and extracellular protease witii an optimum pH of 5.5. The extra-cellular protease contributes to casein degradation during the early stages of ripening, whereas the intra-cellular protease contributes to proteolysis throughout the entire ripening period. rflmdler gt, al. (1974 b ) isolated and examined a pure extra- cellular protease. Hodler e_t_ 1L. (1974 a ) reported that pH Toptima. of extra-cellular proteases were different on different substrates. The authors (1974 a and 1974 b ) demonstrated a wide range of pH’s (3.0 - 6.0) for maximum stability of the extra-cellular protease and showed the optimum temperature to be 45° to 46°C. Using a protease isolated from Penicillium, Gripon and Bergere (1972) reported two pH optima, 3 and b, for casein hydrolysis. The optimum temperature ranged between 35‘ and 43°C. The activity of the protease fell off below 2500. Therefore, ripening temperatures of 10-15°C minimize excessive amino acid and peptide accumulation in Blue cheese. Dominique and Gripon (1981) demonstrated the action of E; rogueforti aspartyl proteinase on purified caseins and identified the main degradation products obtained from beta-casein. The auUuws (1981) demonstrated also the activity in cheese of Una two endopeptidases (aspartyl and metalloproteinase) using commercial Blue cheeses. 24 Sodium chloride added to cheese also affects proteolysis. Gripon and Hermier (1974) demonstrated the iJflwibition of extra-cellular protease by adding sodium chlcu'ide to the medium. Godinho and Fox (1981 b ) studied the diffusion rates of sodium chloride into Blue cheese curd and the influence of salt concentration on the growth of E_,_ roguefcwti in cheese. The authors (1981 b ) reported that the salt concentration in the outer layer of the cheese decreased, presumably as a result of continued inward diffusion of salt. The mold development was affected by the rate of diffusion of salt. A slower rate of diffusion in such cheese permits spore germination. The breakdown of protein is progressive during cheese ripening. Jolly and Kosikowski (1975) reported that soluble nitrogenous compounds comprised about 252 of the total nitrogen in ripened blue cheese and 34% of cheese made with added protease. Ismail and Hansen (1972) reported that the free amino acid levels in samples of Danish blue cheese ranged from 5 to 10% of the total nitrogen. Kuehler (1974) determined the concentration of free amino acids in quick- ripened Blue cheese samples. The amino acid content increased to 28% of the total protein after ripening 39 days compared with 161 after 11 days ripening. Takafuji gt al. (1982 b,c) studied the action of intracellular neutral gnwmeinases from E; camemberti on casein fractions. The 25 authors (1982 b,c ) concluded that initially alphaa-casein is broken down by Chymosin, and after 20 days beta-casein is broken down by fungal proteinase, releasing high molecular weight peptones. The hydrolysis of beta-casein by the metal loproteinase and aspartyl proteinase from EL rogueforti was observed when mold growth was apparent in Bleu d’ Auvergne cheese (Trieu-Cuot and Gripon, 1983). Hewedi and Fox (1984) reported that proteolysis in Blue cheese was rapid between 10 and 16 weeks presumably due to the proteinases/peptidases excreted by E._ r uef ti. Approximately 672 of the total nitrogen in commercial mature Blue cheese was water soluble. ACCELERATION 0F RIPENING AND/DR IMPROVING THE FLAVOR 0F CHEESE Various methods have been described for shortening the period during which desired texture, color and flavor are obtained in Blue cheese. Lane and Hammer (1937) investigated the effect of homogenization of cheese milk on subsequent flavor development. The authors (1937) observed a more uniform flavor development in the cheese made with homogenized milk compared with unhomogenized raw milk. Cheese made from pasteurized-homogenized milk showed a rapid development of volatile acids and a more typical flavor than 26 cheese made from unhomogenized raw milk. Morris gt gt. (1955) reported a 4 - 5 month ripening period for Blue cheese made with raw or pasteurized-homogenized milk compared to a 9 — 11 month ripening period with unhomogenized raw milk. Peters and Nelson (1961 a ) demonstrated that ripened Blue cheese made from homogenized milk, both raw and pasteurized, was higher in quality than ripened cheese made from raw or pasteurized nonhomogenized milk. The same authors (1961 b ) studied the effect of different ripening temperatures on the quality of Blue cheese. A procedure was developed by Kondrup and Hedrick (1963) and patented by Hedrick gt gt. (1968) to shorten the ripening period of Blue cheese from the conventional 90 days to 10 days. The mold was inoculated into milk after setting. Loose curd rather than the normal hooped wheels was ripened at 62°F and 95% relative humidity. Salting was performed in three stages. The curd was agitated daily until a desirable texture, color and flavor was obtained. The cured product was packaged in polyethylene bags with a capacity of 25 pounds and stored at 45°F. Blakely (1970) prepared a quick ripened filled Blue cheese. The procedure used was that of Kondrup gt_ gt, (1963) except that coconut oil was. used to replace milk fat. A high percentage of particular fatty acids and methyl ketones was obtained. The ‘flavor was described as not completely typical of Blue 27 cheeses A modified procedure of Hedrick gt gt. (1968) was used by Kuehler (1974), Harte (1974), Albert (1974) and Harte and Stine (1977) to produce quick ripened Blue cheese with more desirable quality. The authors concluded that homogenization and pasteurization of milk, cooking the cut cheese curd, salting times and temperature of curing were critical for development of a clean, full flavor and control of contamination. Various methods can be used to accelerate cheese ripening. Sood and Kosikowski (1979) reported that the addition of microbial enzymes, fungal protease and fungal lipase to Cheddar cheese curd accelerated the ripening process. Law (1978, 1980) reviewed the possibilities of accelerating cheddar cheese ripening by the use of non- conventional starters and enzymes. The author (1978, 1980) described several methods such as addition of enzymes (microbial proteinases and lipases), increased microbial populations by addition of modified lactic acid bacteria to cheese milk or addition of bacterial concentrates to the curd, and the use of mutant organisms. One notable alternative to increasing enzyme concentrations is to increase the activities of existing enzymes by changing the temperature and moisture levels artificially in slurry systems. The use of lactose to stimulate the growth of normal 28 culture, and the high glucose availability probably encourages the growth of other flavor—promoting microorganisms (g.g. molds in Blue cheese). This principle was applied to shorten the ripening time and speed up the flavor development of several cheese varieties including Cheddar, Mozzarella, Camembert and Blue (Anon, 1977). Law and Higmore (1982) reported that development of Cheddar cheese flavor was accelerated by the addition of bacterial neutral proteinase to the curd. The authors (1982) demonstrated that the addition of fungal acid proteinase resulted in a bitter defect even at low concentrations. Davide and Foley (1981) and El-Soda gt gt. (1981) reported that the use of disintegrated frozen bacterial cell concentrate (DFCC) and cell free extract (CFE) or cell debris (CD) to supplement the normal starter in Cheddar cheese accelerated proteolysis with no effect on glycolysis or lipolysis. El-Safty and Ismail (1982) demonstrated that addition of free fatty acids to Domiati cheese milk improved cheese.flavor and shortened the ripening period. Kalinowski gt gt, (1982) reported that complex enzyme preparations consisting of E; candidum and St lactis autolysate were used to accelerate ripening of Dutch, Tilsit and lowicki-type cheeses. Grieve and Dulley (1983) used Stregtococcus tggttg Lac“ mutants to accelerate Cheddar cheese ripening. Furtado (1983) reported that Camembert-type cheese was 29 ripened in a loose curd at 13°C and 95% RH for 21 days. The author (1983) reported that the amounts of volatile fatty acids and soluble protein in the experimental cheese were higher than in commercial cheeses. The addition of trace elements has accelerated the ripening rate of Ras cheese (Haghdoub gt gt., 1985). Farahat gt gt. (1985) reported that the ripening period of Ras cheese was reduced to 2 months, compared with 4 months for the control by pre- treatment of cheese milk with beta-D-galactosidase (lactozym). Upadhyay _t gt. (1985) reported that higher ripening temperatures can accelerate Cheddar cheese ripening. The flavor and body/texture scores and total volatile fatty acid levels of cheeses ripened at 12 - 13°C for 4 months were similar to those of cheeses ripened at 5- 6°C for 6 months. Aston _t gt. (1985) reported that the maximum temperature at which Cheddar cheese could be stored for 32 weeks without reduction in quality was 15°C. Bartels EL. gt. (1987) reported that the addition of heat-shocked thermophilic lactobacilli and streptococci to the regular lactic starter accelerated flavor development and proteolysis in Gouda cheese. The acceleration of cheese ripening represents a worthwhile saving in the total cost of cheese manufacture and storage provided that flavor development can be 3O accelerated without impairment of flavor balance (Jameson, 1983). Control of accelerated ripgninq It is critical to control the accelerated cheese ripening since a cheese which matures quickly is also likely ‘to become over—ripe quickly and methods to arrest ripening are not readily available (Law, 1980). In quick ripened Blue cheese, excessive mold growth frequently occurs and this results in the development of a musty, moldy flavor. The possibility of unwanted microbial contaminants may also occur. Jameson (1983) stated that maturation of cheese may be retarded by storage at low temperature, but freezing usually causes breakdown of the cheese structure, resulting in a short, crumbly or mealy body. Stehle (1984) reviewed the possibilities of prolonging storage life of Camembert cheese by heat treatment involving surface pasteurization, microwave heating, autoclaving, irradiation, freezing, and high-frequency heat treatment. Herrmann and Rudolf (1980 a, b) demonstrated in laboratory and full-scale experiments that refrigerated storage of Camembert-type cheese (at 0°- 4°C and 65 - 852 RH, in laminated aluminum foil packaging) from the 10‘" to 39‘" day after manufacture, considerably increased storage life. Slanover (1981) reported that cheeses cooled immediately after salting were 31 unsatisfactory. The author (1981) suggested an initial ripening period before refrigerated storage. Jarmul gt gt. (1985) reported that Camembert cheeses which were frozen and stored at -27°C at 75% RH for 2 - 3 months and then ripened for 1 -3 weeks, after thawing, yielded cheese of satisfactory body and flavor. Tochman gt gt. (1985) studied the effect of microwave heating “in-package“ on the quality of Cottage cheese. The authors (1985) concluded that optimum quality was observed when the packaged cheese was heated to 48.8°C using a 0.5 Kw power source. Kreal _t gt. reported that irradiation of Bryndza cheese at more than 100 krad resulted in substantial reductions in the counts of all microorganisms, but caused flavor defects (burnt, foreign and unclean). Yuceer and Gunduz (1980) reported that preservation by irradiation combined with refrigeration increased the shelf-life of Kashar cheese about five fold. Boyar and Gnanayutham (1981) reported that surface irradiation of cheese using an electron beam facility lead to a delay in mold growth, but the irradiated cheese developed off-flavor at the surface. Ibrahim gt gt. (1987) reported that microbial counts decreased when Ras cheese was exposed to gamma-irradiation of 0.25 M rad at 0.10 M rad/hr. The optimum day to irradiate Ras cheese was day 60 of ripening. 32 Modifigd- and Controlled Atmosghere Packaging The terms ”modified atmosphere" (NA) and "controlled atmosphere" (CA) mean that the atmospheric composition surrounding a perishable product is different from that of normal air (Brecht, 1980). (MA) differs from (CA) only in how precise gas partial pressures are controlled. The initial gas concentrations in (MA) systems are established but not readjusted during storage, while in (CA) systems the selected concentrations of gases are maintained throughout storage (Wolfe, 1980, Ogrydziak and Brown, 1982). The manipulation of the environment can take place in an (MA) system, where an actively respiring and metabolizing product reduces the oxygen and increases the carbon dioxide in the ambient air within a chamber in which various barriers and restrictions to gas exchange exist (Smock, 1979). Three barriers exist: the product itself, the package and the storage room. Lee (1987) stated that fresh produce respires at a given rate under constant temperature. If the package allows the influx of oxygen and efflux of carbon dioxide at the same rate as that of the fresh produce, a constant concentration of oxygen and carbon dioxide can be maintained inside the package. The author (1987) reported that the influx of oxygen and efflux of carbon dioxide are determined by the permeability of the package, thickness and surface area of the film, and the partial pressure gradients 33 inside and outside the package. The respiration rates vary with type of product and storage conditions. A dynamic equilibrium exists between the endogenous gases produced by the product and the exogenous gases surrounding the product (Burton, 1978). The balance between exogenous and endogenous gases influences the rate of diffusion into and out of the product. Dynamic changes due to gas permeation, absorption, and/or bio-chemical conversion via respiratory activity must be also considered (Wolfe, 1980). Eftgct oftgodifigd- gnd controlled gtmogghere A number of studies have been conducted to determine the effects of modified- and controlled atmosphere on metabolism and microorganisms growth. Smock (1979) and Burton (1978) reviewed the influence of (CA) on respiration rate and other basic catabolic processes of stored fruits and vegetables. They concluded that the effects of (CA) systems on respiration were dependent on the plant material and on the concentration gradient that develops between the centers of metabolic action and the outer integument of the plant material. Oxygen levels ranging from 3 to 21% have been shown to influence the Krebs cycle, whereas levels below 3% also inhibit the glycolytic system (Brecht, 1980). An oxygen level of 2.1% was shown to reduce the growth of fit 34 rogueforti significantly (Golding, 1940 II). The respiration rates of a number of different organisms as a function of the carbon dioxide tension in the storage atmosphere have been studied (Gill and Tan, 1980). The organisms which were studied included common spoilage organisms such as Pseudomonas, Acinetobacter, Enterobacter and Hicrobacterium thermosghactum. The authors (1980) concluded that the level of carbon dioxide that gave maximum inhibition for the common spoilage organisms was approximately 200 mm-Hg, equivalent to 26% CO, in air at 30°C. Silliker and Holfe (1980) reported that lactic acid bacteria, such as streptococci and lactobacilli, are less affected by elevated levels of CO, and comprise the pre— dominating flora on meats stored in modified atmospheres. Goldings (1940 III) concluded that carbon dioxide concentrations up to 10% increased the growth of strains of at rogueforti in seven days at various temperatures. The effect of temperature on growth of microorganisms in relation to CO, concentrations has been studied. The acceleration of growth of E; rogueforti due to low concentrations of CO2 and the inhibitory effect due to high concentrations of CO, occur sooner at lower temperatures of. growth than at higher temperatures (Golding, 1940 III). The rate of fit fluorescens growth on complex medium in 20% CO, in air was inhibited by 802 at 5°C compared to 20% at 30°C 35 (Gill and Tan, 1979). The increased inhibitory efficiency of carbon dioxide at lower temperatures is related to the dissolution of CO, into the medium (Finne, 1982). Since the solubility of gases is much higher at low temperature, the CO, concentration in the medium will increase as the temperature is lowered. Harned and Davies (1943) reported that the solubility of C02 decreases as the salt concentration of a solution increases. The authors (1943) reported that the solubility of CO, at 0°C in 0.2, 0.5 and 1.0 M sodium chloride solutions is 94.2, 86.7 and 76.4%, respectively, of the solubility in water. The CO, molecule is more soluble in water than are most other gases. More than 99% of CO, exists as the dissolved gas in water (Knoche, 1980). Less than 1% exists as H2CO3, which partly dissociates to give H‘, HC03- , and 003°. Advantages of ggdifigg_gnd controlled atmogghere Various beneficial effects of modified and controlled (atmosphere have been reported. Examples of such effects area retarding senescence (ripening and aging), reducing product sensitivity to ethylene and reduction in the incidence and severity of decay of fruits and vegetables (Brecht, 1980; Smock, 1979), extending the shelf life of Fumaltry,meat and seafoods (Holfe, 1980; Ogrydziak and Brown, 1982), and inhibiting the growth of Gram-negative bacteria 36 and other related psychrotrophs which produce off odors and flavors (Silliker and wolfe, 1980). Extension of product shelf life by Controlled Atmosphere Packaging (CAP) has also great economical advantage to retailers (Hansen and Duckworth, 1982). Controlled Atmosphere Packaging allows a full range of products to be kept on display for a long time which allows products to be packed in a central location and in turn releases the floor space needed for preparation and packaging and increases the availability of the sales area. An advantage which might be derived from modified or controlled atmosphere package is the enhancement of product properties. This may be accomplished by selecting or maintaining the optimum atmosphere over the packaged product. Once the optimum gas mixture required by the product is established, a package material with optimum barrier properties (gases and water vapor transmission) for the product can be selected. In the process of selecting the optimum package material, parameters such as the respiration rate and/or the resgiration guotient of the product as defined by the ratio of carbon dioxide evolved to (oxygen consumed (Karel, 1974), composition of the product, and storage conditions (temperature, relative humidity, light,etc.) become very critical. These parameters affect tuoth the selection of package material and the metabolic activity of the product (Karel, 1974). 37 Cheese Packaging Three methods currently predominate the film packaging of cheese at the retail level: tight wrapping, vacuum-gas flushing, and vacuum packaging. Pearson and Scott (1978) provided an excellent comparative discussion of the three methods. Tight wrapping involves either machine or hand wrapping of plastic films or foils around cheese portions followed by heat sealing (Pearson and Scott, 1978). Tight wrapping utilizes less expensive materials and is applicable to many different cheese shapes, sizes and textures. Packaged cheeses will also stack more easily and thus optimize available space. The disadvantages associated with tight wrapping are that it is time consuming and the materials utilized are more easily punctured than more complex laminated structures (Pearson and Scott, 1978). Vacuum packaging involves the removal of air from the surface of perishable food stuffs, such as bacon, cooked meats, continental sausages, cheeses,etc. and the pack sealed under a vacuum (Ross, 1982). This method is widely recognized in the cheese industry and is often employed at one stage or another in the handling and storage process (Anonymous, 1977). A variation of vacuum packaging is called gas flushing. In this process, air is removed from the package and an inert gas is then introduced. A single gas such as nitrogen 38 may be employed, or a mixture of gases such as carbon dioxide and nitrogen, or for some applications, oxygen, to preserve or enhance the properties of the product (Anonymous, 1977; Ross, 1982). This process is generally used for small portion size packs of the product at point of sale, although on occasion, three hundred pound drums or barrels of product are gas packed. A gas flushing operation might be a possibility in facilitating cheese ripening provided that the gas mixture required for optimum ripening is established and the package material chosen meets these requirements. Packaging Naterial Reguirements Shaw (1977) reported that packaging films should be suitable for printing, contribute no off flavors or odors to the cheese and of course must not contain toxic substances capable of migration into the food. The author (1977) concluded that toxicological safety data must be provided by the manufacturer to the Food and Drug Administration (FDA) before approval of the materials for use in food packaging. Ionson (1979) stated that an extremely important requirement of packaging films is that they adequately control the headspace environment of the packaged cheese. Seal integrity, barrier properties of the film, extent of air entrapment and gas production by the cheese will influence 39 the extent of mold development and can be controlled by selection of suitable films. If the atmosphere within a package is oxygen deficient and/or carbon dioxide rich, the metabolism of Blue cheese will be suppressed. Metabolism can be depressed by decreasing oxygen concentration, decreasing temperature, increasing carbon dioxide concentration, or any combination of the three (Kidd and West, 1974). Gas flush systems require complex laminates. The components of laminated films are chosen so that each component contributes a property or properties desirable to the total system (Ionson, 1979). For example, PVC/nylon or PE/nylon are often used as components of laminates and provide excellent barrier and flexural properties. When selecting a film, it is vital to know the water vapor and gas.permeability of the film. Factors affecting germeabi l i ty Film, gas and water vapor permeabilities are affected byr a ‘number of physical factors. Some of these factors iru:1ude the area of the film, the time of exposure and the partial pressure exerted on the film. Other factors such as temperature, relative humidity and film thickness affect permeability in a less direct manner. Karel (1974) reported that storage at high relative humidity or direct ccurtact between hydrophilic material and cheese can 40 adversely affect the water vapor and gas barriers. Decreasing temperature will improve the barrier characteristics of films, the extent of change being dependent upon the particular material (Karel, 1974; Hanlon, 1983). Polyethylene is more strongly hydrophobic than the other organic films so water vapor penetrates it to a limited degree. This particular property along with its high rate of gas permeability are of considerable value in the packaging of items which require a low moisture loss, while maintaining the ability to transmit significant amounts of oxygen or carbon dioxide. ANALYTICAL PROCEDURES The pH measurements were determined using an Orion digital pH/mv meter equipped with a combination electrode (Standard Methods for Examination of Dairy Products, 1985). Measurements were taken initially and continued on a daily basis. The pH was read to the nearest one-hundredth of a pH unit on the meter scale. Motgturg Contggt The moisture content of the cheese curd was determined by the vacuum oven method (AOAC, 1085). Determinations were made in triplicate and performed initially and on a weekly basis. Gas Headsgace Analysis Package headspace analysis was accomplished using an oxygen‘ analyzer (Model S-3A/11, Applied Electrochemistry) coupled with a Carbon Dioxide analyzer (Model CD-3A, Applied Electrochemistry). Dow Corning silicon rubber was deposited on each pouch to serve as a septum. A syringe needle was inserted through the pouch material to withdraw a 1cc. of headspace gas and then injected into the analyzer. 41 42 Samples were taken initially in duplicate, then daily every 6 - 12 hours. Seven pouches were used for each temperature and determinations were made in duplicate. Nitrgggn (rgducgd) determination Total and soluble nitrogen were determined using a micro-Kjeldahl unit consisting of a Buchi 342 control unit, Metrohm 655 Dosimat, Metrohm 614 impulsomat and Metrohm 632 pH-meter (Brinkmann). Determinations were made in duplicate and performed initially and on a daily basis. Sensory evaluation An experienced panel of cheese Judges scored and rated the cheese curd. Flavor, color (mold appearance) and texture were evaluated. A crumbled, commercial Blue cheese, aged at least 6 months, was used as the control. It was felt that flavor was the most important criterion because OR Blue cheese curd is ideal for direct incorporation into Salad dressings, cheese dips and similar products. Color was rated second in importance and texture last. On this basis, flavor was assigned a score of 20 points, color 15 points and texture 10 points. A cheese with a score of approximately 30 out of the total of 45 was considered acceptable. Ideal mold growth is referred to in this study as a well pronounced, even distribution of mold, and blue 43 coloring which appears on the cheese surface and/or throughout cheese curd. High Performance Liquid Chroggtoqraphic (HPLC) Analysis of Dethvl Ketones Purificgtton of golvggtg Hexane: Carbonyl-free hexane was prepared from redistilled hexane by the methods of Hornstein and Crow (1962), and Schwartz and Parks (1961). A column was prepared using 45 ml of concentrated sulfuric acid blended with 75 g of Celite 545 (John Manville). A small amount of anhydrous sodium sulfate was placed at each end of the column to serve as a desiccant. The eluate from this column was passed over a 2,4-DNPH-reaction column (Shwartz and Parks, 1961). Five- tenths of a gram of recrystallized 2,4- Dinitrophenylhydrazine (2,4-DNPH) was dissolved in 6.0 ml of 85% phosphoric acid by grinding. Deionized distilled water (4 m1)-was added and the precipitated DNPH was redissolved by grinding. Ten grams of Celite 545 was ground with the solution until a homogeneous bright yellow blend was obtained. The mixture was packed in a chromatographic column containing 25 ml carbonyl-free hexane. The column impurities were flushed away with 50 ml carbonyl—free benzene. The eluate from the reaction column was mixed with 44 Sea Sorb 43 (Adsorptive magnesia, Iodine No. 80, Johns Manville) as suggested by Blakely (1970) and filtered to remove the carbonyl derivatives from hexane. Benzene: Carbonyl-free benzene was prepared by refluxing reagent grade benzene (Fisher Scientific Co.) with 1 g of 2,4-DNPH per 500 ml for 2 hrs. and then redistilled. Chloroforgt Analytical grade chloroform (Fisher Scientific Co.) was refluxed over potassium hydroxide for 30 min. and redistilled. Nitromethane: Analytical grade nitromethane (Aldrich Chemical Co.) was redistilled over boric acid and stored. eggtonitrilgt High purity acetonitrile (b.p. 81 - 82° C) was obtained from Burdick and Jackson Lab, Inc. Ethanol: Carbonyl-free ethanol was prepared by distilling 95% ethanol (Fisher Scientific Co.) over 2,4-DNPH, using 1 g 2,4-DNPH per 100 ml ethanol. Butgnol.1-: Distilled in glass Butanol-1 was obtained from Fisher Scientific Co. 45 Ethylene Chloride: Ethylene chloride (Mallinckrodt,lnc.) was redistilled over anhydrous potassium carbonate. All the purified solvents were stored in brown bottles. Preparation of 2.4-DNPH Dgrivgtiygg, of Standard Methyl Ketones: Standard methyl ketones, 99% purity, were obtained from Aldrich Chemical Co., Inc. and were used to form hydrazone derivatives according to Shriner gt gt. (1964). The recrystallized 2,4-DNP—hydrazones were filtered, dried at room temperature and then stored in the freezer. Isolation of Mono-cgrbonylg frogyCheese sagples: Extrgction of tht Ten grams of a representative cheese sample was mixed with 15 grams Celite 545, dried for 24 hrs. at 150° C, and ground to a homogeneous blend. The mixture was packed into chromatographic column. The fat was extracted with 200 ml carbonyl-free hexane. A similar column was packed and extracted with 200 ml carbonyl-free hexane into pre-weighed soxhlet flasks to determine the fat recovery. 46 Formation of 2,4-DNP-hydrazone derivatives: The hexane extract of the cheese sample was passed through a 2,4-DNPH reaction column to convert the carbonyl compounds in the fat extract to their corresponding 2,4-DNP- hydrazones (Schwartz and Parks, 1961). The column was washed with 200 - 300 ml carbonyl-free hexane and the solvent was removed using a rotary evaporator (Precision Scientific, Inc.). Egmovgl of fgt frog 2.4-DNP-hydrgzonggt The fat containing the hydrazones was dissolved in 5- 10 ml carbonyl-free hexane and the 2,4-DNP-hydrazones were isolated from the fat as described by Schwartz gt gt. (1963) with slight modification by Dokhani (1978). The fat was washed off the column by subsequent addition of 200 ml hexane, 100 ml hexane: benzene (1:1 v/v) and 200 ml benzene. The hydrazones, which were adsorbed on the column as a deep brown band, were eluted by adding 250 ml chloroform- nitromethane (3:1 v/v). The eluate was evaporated using a rotary evaporator. [figggtiongtion of 2.4-DNP-hydcgzongg on Weak Alumina: The mono-carbonyl derivatives were separated from Keto- qglycerides as described by Schwartz gt al (1963) on a weak alumina column (Schwartz and Parks, 1961). The DNP- 47 hydrazones were dissolved in 5 ml hexane and applied to a weak alumina column. The mono-carbonyl derivatives were eluted by adding 50 ml hexane-benzene (1:1 v/v). The eluate was evaporated using a rotary evaporated. Determination of Mgthyl thopggt,by Hiqh Pgrformance Liguig Chropgtoqrgphy (HPLC): A high performance liquid chromatographic system was used to isolate and quantitate the methyl ketones present in the mono-carbonyl fraction of the cheese samples (Dokhani, 1978). Chromatography Eguipment gnd Conditiongt The system consisted of a Waters Associates Model 730 Data Module, M-45 Solvent Delivery System, U6K septumless injector and a Model 441 Absorbance Detector (Pharmacia Fine Chemicals). A Bransonic 221 sonicator (Branson Cleaning Equipment Co., Shelton, CT.) was used to degas and uniformly mix the mobil phase. A reversed phase u Bondapak C18 stainless steel column (3.9 x 300 mm; Waters Associates) was employed to separate components for analysis. A Hamilton 100 ul syringe was used to inject 50 ul sample volumes. The following chromatographic conditions were used: Temperature : ambient Detector : U.V. monitor; 254 nm 48 Mobile phase : acetonitrile : water (80 : 20 v/v) Flow rate : 1.5 ml/min Chart speed : 0.65 cm/min Egggvgry of mgthyl kgtonggt The percent recovery of individual methyl ketones was determined in order to confirm the reliability of the extraction and chromatographic procedure. A mixture of accurately weighed standard methyl ketones: C3, C5, C7, C9, C11 and C13 (99.5% purity) were spiked into a 250 9 cheese sample and subjected to the extraction and chromatographic procedure, described in the experimental section. An identical cheese sample was subjected to the same preparation and aliquots of the resulting samples were injected into the HPLC. The values were compared with the untreated C3 — C13 standard methyl ketones. The percent recovery of each standard ketone, as determined by triplicate analyses, was used to calculate ketone concentrations in the cheese samples. Peak area was used for quantitation of methyl ketones. Gas reguirements: The respiration rate was determined experimentally as suggested by Lee (1987). The cheese was kept in airtight Mason jars (500 cc) with two ports. The air was fed into 49 the in-port and allowed to flow over the cheese curd and exit from the out-port. The oxygen consumption and carbon dioxide production were monitored over time. Samples were taken initially in duplicate and then daily every 6 - 12 hours. Seven Mason jars were used for each temperature and determinations were made in duplicate. Water Vgpor Transmission of LDPE and Laminate (Nylon/PE): The water vapor transmission of the test packages was determined as described in ASTM E-96 (1980). A test specimen was placed over the mouth of standard aluminum dishes which contained desiccant and sealed with molten wax. The assembly was placed in an atmosphere of constant temperature (5°, 10°, and 15°C) and humidity (95%). The weight loss or gain of the assembly was used to calculate the rate of water vapor transmission through the materials. Oxygen Permeation of LDPEtgnd Laminate (Nylon/PE): The oxygen permeation rate was determined according to ASTM D-3585 (1981) applied to plastic film and sheet using a coulometric sensor. A Mocon Oxtran 100 (Modern Controls, Inc., Minneapolis,MN.) was used to measure the oxygen permeation rate. A standard polyester film was used to calibrate a chart scale prior to testing the film samples (LDPE and Laminate). 50 Gamma irradiation: The effect of gamma irradiation dose and time on Blue cheese curd was investigated using a variable flux gamma irradiator (Department of Chemistry, Michigan State University) with a Co°° source. Cheese samples were placed in LDPE pouches and subjected to 0.5, 1.0, 2.0 and 2.5 KGy. Cooling was accomplished by flowing compressed air into the sample cavity to maintain temperature below 27°C. The samples were stored for one week at 5°C and then analyzed for methyl ketones and sensory properties. Microwave heat treatment: Blue cheese curd was packed in LDPE pouches and subjected to heat treatments using a 2.8 Kw microwave source (Kenmore, model #721). Two weeks after processing and packaging, the cheese was heated "in-package“ to various temperatures. The physical appearance and sensory properties were evaluated. 51 Procedure used for manufacturing Ouick Ripened (QR) Blue Cheese Blue cheese curd was produced according to the method of Kondrup and Hedrick (1963) and Hedrick _t gt. (1968). The modification of Kuehler (1974), Albert (1974), Harte and Stine (1977) was used with some additional modifications. Raw milk, obtained from Michigan Milk Producers Association, was preheated to 130 - 135° F and homogenized at 1500 psi on the first stage and 500 psi on the second stage. The milk was pasteurized at 145° F for 30 minutes and cooled to 86°F. A frozen concentrated cheese starter culture (Hansen’s Lab.,Inc., Milwaukee, WI) was then added to the cheese milk. When the acidity of the milk had increased 0.025 to 0.030, 116 g of Blue mold powder (E. rogugforti), obtained from Dairyland Food Lab, Inc., Waukosha, WI, and 60 ml of rennet per 1000 pounds of milk were added. After setting for approximately one hour, the curd was cut into 3/8 inch cubes. The curd was stirred gently 30 minutes after cutting and heated to 95° F for approximately one hour. The drained curd was placed on a fine mesh stainless steel screen in the ripening rooms which were maintained at a relative humidity of 90%. As the purpose of this project was to study the feasibility of 52 quick ripening Blue cheese by modified and gas flushing atmosphere packaging, many adaptations to the original method were attempted. These modifications dealt with salting times and amounts, temperature of curing, modified and gas flushing atmosphere packaging, type of packaging materials and physical treatment of Blue cheese curd such as gamma irradiation and microwave heating. Statisticgl Analysis Results of sensory evaluation were analyzed using a five-way analysis of variance (ANOVA) with interactions among random factors and fixed factors (Gill, 1978). The data were analyzed by the following statistical mixed model: Ythlnn = u + A. + BJ + AB,J + (3n + D, + Ac,k + A0,; + BC,“ + 8D,; + CD“; + Fn + AF," + BFJH +CFkn +DF1n +CDFI¢1H +E Where Y,Jk1n- = sampling variables u = true mean of the Y distribution A, = replicates B, = panelists C. = packaging treatments D; = temperatures Fn = storage times E = random experimental error Differences between means were analyzed for significance by Tukey’s test (Gill, 1978). RESULTS AND DISCUSSION The Gas (Oz/C02) Reguirements of P. roqugforti The main objective of this research was to accelerate the ripening of Blue cheese curd by maintaining the optimum atmosphere over the packaged curd. Once the gas requirements of the mold and the optimum storage conditions are established, a package material with optimum barrier properties (gases and water vapor transmission) for the cheese can be selected. Eggpirgtion Rgtg Figures 1 and 2 represent the respiration rate in Blue cheese curd, shown as CO, production (cc/kg/hr), salted with 3.5% salt concentration and stored at temperatures of 5°- 20°C. The pattern of these curves appears to be similar to classical growth curve. Cheeses ripened at 20°C, coincided with first visible signs of mold growth, were faster than cheeses ripened at the other temperatures. The amount of CO, produced and the magnitude of the increase were directly related to the temperature employed in ripening. The maximum mold growth and CO, production were observed earlier at higher temperatures. 53 54 160 { V--V C02 L. 4 _ i 1 O. V 20% 3‘ 120 \ r 8 > \J 100» \\ 7- ’ v v 0 V 80- / F- / v V \ o v v’ \ V v 8 50. ‘\V/ v/ \v a 1 \v o. 40 p \v E? . 0 20 [/V OV_L l .L A 1 i 1 . 1 - 1 - i - n O 2 4 6 8 1O 12 14 16 18 TIME ( DAYS) 160~ f: 140’ U—D C02 { T 1500 3’ 120 - g . D V 100 *- \ g ’ C] r: 80 ’ o \ 8 ’ /D\ 8 60 .. D‘U/ \ /D U m ’ D\ /U \, a. 40 . O D\ 8' ' [:1 , D/ OLE/{i . i k L 1 1 + l L L l 2 J O 2 4 6 8 1O 12 14 16 18 TIME ( DAYS) Figure 1. Respiration rate in Blue cheese Curd, containing 3.5% NaCl. 160p 140- 120 v T—v 100 v 1'— V I v ' coz PRODUCTlON ( cc/kg/hr) (I) O 55 A-—-A coz 100C ‘\ / A /A \ /A A A\ A’ A\ /A \X’A/ A\A /A 2 4 6 8 10 12 14 16 18 TIME(DAYS) o—o c 02 soc (J-O g A m-m-Q’Q-Q/ O 160- E 140- \\ . E" 120 - 8 _ r V 100 "' g b 80+ ‘5 1 8 50 - 8 P m 40- 3" 1 o 20- 1 OC 0 Figure 2. 2 4 6 8 1O 12 14 16 18 TIME ( DAYS ) Respiration rate in Blue cheese curd, containing 3.5% NaCl. 56 The decrease in CD: production, presumably due to reduction of growth of E.ro ueforti, was followed by an increase in CO; production for cheeses ripened at 20°C. This increase was also marked with signs of slime formation which was followed by development of orange and reddish color appeared on the cheese surface. The surface micro—flora most likely consisted predominantly of yeasts, several types of micrococci and Brevibacterium linens (Hartley and Jezeski, 1954). The excessive slime formation and discoloration for cheeses ripened at 20°C was detrimental to the flavor and color of the cheese. The discoloration was absent in cheeses ripened at 15°C and 10°C, however, slime formation was still slightly present. Figures 3 and 4 represent the CO, production and 02 consumption in Blue cheese curd containing 3.5% NaCL and ripened at different temperatures. The respiration quotient was almost constant throughout the ripening period. The amount of O, consumed was slightly higher than the amount of CO2 produced (Table 2). Subjective analyses on Blue cheese ripened at different temperatures were performed by the evaluators. Cheeses ripened at 20°C were heavily criticized in all areas. At this ripening temperature, excessive mold growth occurred which resulted in a sticky, gummy texture, and moldy flavor. Cheeses ripened at 5°C were criticized for lacking in mold 160 140 120 100 C02 PRODUCNON ( cc/ kg/ hr ) m o ' I w I f I ' I V I 57 OF- 1 s 1 - l 1 l 1 l 1‘ 1 A L A 1 - 1 O 2 4 6 8 1O 12 14 16 18 TIME ( DAYS) 160 v 1: 140’ D-—-D C02 g , -—- 02 1501: < 120 - l 8 » U V 100 - / Z . D ,I 8 * .’ o /a§fi 8 1 U D [Cl/Cr \-\I O O 20 1- / 1 O’ Oél 1 i A it 1 A l i A 1 Figure 3. TIME ( DAYS ) CO: production cheese curd, and 02 consumption in Blue containing 3.5% NaCl. 58 160v A ' A—A C02 100C E “4)b A-—-A 02 \ r 3‘ 120 ~ \ F U i: 100 ~ ’\ z ’ l/ A E): 80 ' A \ \ ,A Q ' A ‘ A \A 8 50 " /A \ \ / \A O A ,A\ E 40 - \‘ (A A 3 ’ A/u A, A\A 0 20 " / \ A-A/A A O réA’A L 4_ i _A i 1 x - L e l - l -———l O 2 4 6 8 1O 12 14 16 18 TIME ( DAYS) 160v é “*0 f 0—2 802 SOC 32‘ 120 ~ ' 2 \ . O ‘3 100- 8 80 ~ g r 8 50. o a 8 ’ ,3?e\8" 5‘ ’ /e_8»0 \d\ o 20 h ’ ’8/ o 0:4,»0-0:8:8 8’0- 14* #yl 0 2 4 5 8 1O 12 14 16 18 TIME ( DAYS) Figure 4. C02 production and 02 consumption in Blue cheese curd, containing 3.5% NaCl. 59 growth and having very mild flavor. Cheeses ripened at 10°C and 15°C were considered good but the major fault was a mild flavor. Table 2. Maximum CO: production, 02 consumption and Respiration Quotient in Blue cheese curd ripened at different temperatures. Temperature Time‘ cc/kg/hr Respiration (°C) (Days) Quotient 002 02 ( C02 3 03 ) 5 13 52.45 55.68 1 : 1.06 10 8 91.39 96.20 1 : 1.05 15 6 110.39 122.66 1 : 1.11 20 5 130.70 148.00 1 : 1.13 ‘ Time required for ideal mold growth. Oxygen Depletion: Oxygen concentrations of 76% and more have been shown to demonstrate a definite inhibitory effect on certain molds (Karsner and Saphir, 1926). In the present study, the effect of oxygen concentration (21% and 100% 02) on the growth of E.rogueforti was investigated using a closed system with a COz-free atmosphere. Figures 5 and 6 represent the oxygen depletion at initial oxygen concentrations of 21% and 100%, respectively, in Blue cheese curd ripened at various temperatures. In an oxygen atmosphere of 21%, complete depletion of oxygen was Oxygen Depletion ( 96 ) Figure 5. 60 0 20 I 40 60 80 100 120 ‘10 PERIOD (HOURS) 02 Depletion in Blue cheese curd ripened at various temperatures in 21% 0: atmosphere. 100 90 80 7O 60 50 4O 30 20 10 Oxygen Depletion ( 96 ) Figure 6. 61 .. -\ - \ \ 0—0 5% I .\o\ A—A 10% I' V A\ o I—I150C j A \ v—v 2000 f I \1 '\. ~ \ \ \ 1 v a ‘ o\ . \ \ \1 o I l\ \ \, L V I ‘ \ \ , \ \_ . . b v\ \ \ \o c l L 4 A 144 £>+__l:_.——l 2 4 6 8 1O 12 14 TIME ( DAYS ) 0: Depletion in Blue cheese curd ripened at various temperatures in 100% O3 atmosphere. 62 reached between 1-5 days of ripening. No indication of mold growth was observed for any of the temperatures used. In an oxygen atmosphere of 100%, good mold growth was observed on days 4, 6, 8, and 11 of ripening at 20°, 15°, 10°, and 5°C, respectively. Theses data are in agreement with Golding (1940 II) and supported the contention that the absence of oxygen was a limiting factor in the growth of E.rogueforti in the Blue cheese. It also appeared that an initial oxygen concentration of 100% did not inhibit the growth of E.roqugforti. Selection of Product-Package Parameters: Based on the data obtained, the following parameters were selected to investigate the possibility of ripening Blue cheese curd in modified and gas flushed atmosphere packaging: 1. Temperatures of 10° and 15°C were shown by the sensory analysis to be ideal for cheese ripening. The cheese improved in flavor as the temperature of ripening was lowered. Lowering the ripening temperature probably retarded contaminating organisms more than retarding the desired fermentation caused by E.rogueforti (Kuehler, 1974). 2. Flexible pouches of material, Low Density Polyethylene (LDPE), were selected for the modified atmosphere 63 packaging. The low water vapor transmission rate (Table 3) and the high gas permeability rate of LDPE (as was determined experimentally) were considered critical to allow the cheese to ripen. Other important functional characteristics of LDPE, such as flexibility tasteless, odorless, and chemically inert (Hanlon, 1983) were also considered. The transparency of LDPE permits the ripening process to be monitored without the need to open the packages. The oxygen permeability rate of 1 mil LDPE (Dow USA, Midland,MI) was 1920 cc/m2/24hrs., which was comparable to the oxygen requirement of Blue cheese curd as determined by the respiration rate. Table 3. Water Vapor Transmission of LDPE and Laminate (PE/Nylon) pouches at various temperatures. gram/100 in2/24 hr Temperature (°C) LDPE PE/Nylon 5 0.61 0.41 10 0.88 0.66 15 1.19 0.87 Pre-formed pouches made from a good barrier film laminate (Nylon/PE), obtained from Koch Co., Kansas 64 City, MO., were used for the gas flushed atmosphere packaging. The oxygen permeability rate of the laminate (3.0 mil) was 0.041 cc/m2/24hrs. The water vapor transmission rates are shown in Table 3. The possibility of ripening Blue cheese curd by flushing the pouches with 100% initial oxygen concentration was then explored. 4. The salt concentrations (3.5% and 4.5%) were added to the drained Blue cheese curd on the first day. The following day, the Blue cheese curd was then packaged (100 g/pkg) into the selected LDPE and Laminate pouches (6" x 8.5"). The pouches were then sealed with an impulse heat sealer. A portion of the cheese was maintained as a control (unpackaged). All initial analytical data were performed as described in the Experimental Section. Chanqeg_in pH During Ripgning The pH of Cheese is considered important because it could influence the activity of enzyme systems during the ripening period. Therefore, the changes in pH during ripening of Quick Ripened (OR) Blue cheese were determined. A comminuted, representative sample of cheese was analyzed each day of the ripening period. Fresh curd exhibited a pH value of approximately 5.64 prior to being placed in the , ‘1 65 curing room. The hydrogen ion concentrations reached a maximum at about pH 5.19 - 5.38 on the second day of ripening. ’This was followed by a rapid increase to 6.03 - 6:98) which generally coincided with the first visible signs of mold growth and occurred on the fourth, fifth and/or sixth days of ripening. At the end of ripening, the pH stabilized or in some cases decreased slightly to approximately 5.46 - 6.:9‘for the remainder of ripening. Varying treatments such as ripening temperature and packaging procedure were responsible for the wide differences found in pH value during ripening. Figures 7 and 8 represent the change in pH during ripening of OR Blue cheese curd at 10°C and 15°C, respectively. These cheeses showed similar trends in pH during ripening. The initial increase in acidity is due to the production of lactic acid by Strgptococcug tgctig starter culture (Morris, 1963). The maximum acidity was not reached until 48 hours after manufacture. The low production and slow acid development might be attributable to the relatively low amount of starter used (1%), and the early salting time which may retard the starter activity. Kuehler (1974) and Harte and Stine (1977) reported that the hydrogen ion concentration reached a maximum at about 4.66 and 4.91, respectively, for OR Blue cheese ripened at 17°C and salted on days 4, 5 and 6. The authors attributed the 66 slow acid development during the initial stages of ripening to the low amount of starter used (1%) as opposed to the generally accepted commercial amount of 2 - 4% of starter. Slower acid development was noted also by Morris and Jezeski (1953) when 0.75 % starter was used. A gradual increase in pH occurred 48 to 96 hours, after manufacture before a rapid increase in pH was noted. Foster gt gt.(1961) and Harte (1974) suggested lactic acid degradation by E.rogueforti as a reason for the initial rise in pH. The maximum increase in pH occurred on the 6tg and 7th days of ripening and may be attributed to proteolysis by the protease enzymes associated with E.roqugforti. Lane and Hammer (1938), Morris gt gt. (1963), Kuehler (1974) and Harte and Stine (1977) noted that pH increased as proteolysis increased in the ripening cheese. The final stabilization or the slight decrease in pH values near the end of ripening period may be explained by the increasing levels of free fatty acids liberated by the mold lipase system. Coulter gt 1. (1938) and Morris gt gt. (1963) noted a slow decrease in pH to 5.7 at 4 to 9 months in Blue cheese and attributed this decrease in pH to an accumulation of free fatty acids in the cheese. A decrease in proteolysis could also have contributed to the stabilizing effect. Modler gt gt. (1974) noted that the activity of the protease(s), isolated from E.rogueforti, dropped off rapidly below 30°C. 67 7.5-1 e—e CONTROL 10°C a—a LDPE e—e LAMINATE (PE/NYLON) 7.0- 6.5- I a. .. 6.0- 5.5 \s 5.0 .~ . r r r . , I - o i 4 ('5 8 10 12 14 TIME (DAYS) Figure 7. Change in pH during ripening of quick ripened Blue cheese curd at 10°C in modified and gas flushed atmosphere packaging. I’LI 1191.1.- 68 7350- 0—0 CONTROL 15°C H LDPE e—e LAMINATE (PE/NYLON) 7.00d ELSO- I O. 6.00-1 ‘ 5.50-1 \ / 51M) r 1 r I . I r 1* r T ' I 1““ I O 2 4 6 8 1O 12 14 TIME (DAYS) Figure 8. Change in pH during ripening of quick ripened Blue cheese curd at 15°C in modified and gas flushed atmosphere packaging. 69 The most obvious effect of temperature on cheese ripening was that as temperature was increased, there was a decrease in the time required for ripening (Kuehler, 1974). The 22°C ripened cheese reached a minimum pH on the first day and 6 days were required for the 9°C cheese to obtain a comparable hydrogen ion concentration (Kuehler, 1974). Figure 9 reveals the effect of temperature on changes in pH during ripening. The rapidity of change in pH and magnitude of these changes were directly related to the temperature employed in ripening. A significant increase (P < 0.05) was observed between 3-6 days of ripening. This was possibly due to increased activity of the starter cultures at the elevated temperatures used. The rapid increase in pH, which was attributed to proteolysis, at 15°C was delayed and became more gradual as the temperature was lowered to 10°C. The control cheese at 15°C reached a maximum pH of 6.98 on the 6th day, while a pH value of 6.84 was attained on the 7tp_day of ripening at 10°C. There was no significant differences (P < 0.05) between cheeses ripened at 10°C and 15°C for the remainder of the ripening period. Figure 7 demonstrates the effect of modified and gas flushed atmosphere packaging on the change in pH during ripening as opposed to the control cheese. These cheeses showed varying pH values. A gradual increase in pH was 70 noted for modified atmosphere packaged cheese as opposed to the rapid increase in pH for the control cheese. Cheeses in modified and gas flushed atmosphere packaging had a maximum pH value of 6.03 and 6.19, respectively, as opposed to a value of 6.84 for the control cheese at the same temperature (10°C). The considerable variation in pH values between the control and the cheeses in modified and gas flushed atmosphere packaging is likely due to the reduced 02 level and increased CO: concentration in the package headspace. The low 0, content inside the pouches can lead to a decrease in mold growth and activity (Golding, 1940 II). The adsorption of CO, on the cheese surface and partial dissociation of carbonic acid may lead to increase in hydrogen ion concentration. The solubility of CO: increases as the temperature decreases (Ogrydziak and Brown, 1982). This may explain the slight increase in hydrogen ion concentration at 10°C as compared to 15°C. Another factor that could be considered and may have had an effect on the pH of the cheese is the treatment given to milk before, during and after its manufacture into Blue cheese. m 71 : - -‘ yrs-.1 - ' .y‘ .u a , - I v u r a I H I -I:‘\i TP'-I1 . : ‘1‘, I Q - 1" -‘ ‘ ~ "\ , /' 1x- ' 7\-Hk\ I / x I _l ! ./ ,I I , I o; A .‘ ‘1 d // /I/ EN. - ' - - l I f / * | g / L. i , ,9 . I 1 / - I“ - I c , ..., “ I - I/ i 2" ”5""? . I 1 . I 1 I I : f \ 'l l ’ --. '- f9 2 .c'.‘ - ’4. .I' x . \T‘f ./ ‘I i 1X '/ I §# . "6 t: ’4’ 7 fl . | i . T Y I V ‘1 u 2 4 D 8 ID 12 I4 Figure 9. :1 In [:1 I; r) The effect of temperature on pH during ripening of quick ripened Blue cheese curd. 72 Changes in Water Soluble Nitrogen Durinq Ripgninq: Water soluble nitrogen values were used as an indicator of proteolysis occurring during the ripening of OR Blue cheese. Morris gt _t.(1963) used tyrosine liberation, while Kuehler (1974) used NPN values as an indicator for protein degradation. Water soluble nitrogen was defined in this study as the percent of total protein soluble after precipitation of casein at pH 4.6 using the sodium citrate- hydrochloric acid procedure (Vakaleris and Price, 1959). Figures 10 and 11 represent the changes in water soluble nitrogen during ripening of OR Blue cheese curd at 10°C and 15°C, respectively. The curves obtained were similar in all preparations of Blue cheese curd. The gradual increase in water soluble nitrogen was observed in the early part of ripening. This was followed by a rapid increase in the amount of soluble protein liberated from the cheese. A slight increase in soluble protein was the characteristic of the last days of ripening in all preparations of Blue cheese curd. Generally, 32 - 43% of the total protein was present in the soluble form at the end of ripening. Keuhler (1974) reported that 45 - 60% of the total protein in OR Blue cheese was present in the soluble form following precipitation with 12% TCA. Jolly and Kosikowski (1975) reported that soluble nitrogenous compounds comprised about 25% of the total nitrogen in 73 ripened Blue cheese and 34% of cheese made with added lipase. Godinho and Fox (1982) reported that concentration of NPN reached a maximum of 25 -35% of the total nitrogen in Blue cheese. The increase in soluble protein early in the ripening period was likely due to proteolysis resulting from the starter culture and rennet (Kuehler, 1974). The rapid increase in soluble protein occurred 48 - 72 hours after manufacture and probably was due almost exclusively to the protease system of E.rogueforti. The rapid increase in soluble protein also coincided with the rapid increase in pH and the first appearance of mold growth. These data are in agreement with Morris gt gt.(1951), Kuehler (1974), Trien- Cuot and Gripon (1983) and supported the contention that the rapid increase in pH was due to proteolysis. Toward the end of ripening there were slight increases in soluble protein and this level was maintained during an extended ripening period was used. Several factors may have attributed to the slight increase. Imamura and Kataoka (1966) demonstrated that volatile fatty acids formed by action of E.rogueforti lipase were inhibitory to the mold protease. Variation in treatments such as salting time and concentration, ripening temperature and packaging procedure were responsible for the wide differences found in soluble protein values. 74 A 44.5 o—o CONTROL 10°C 1 H LDPE 5 . e—e LAMINATE o) __. 0* L. :2: Z .2 13 -—1 2 (D U) L. a) 4.1 C) 3 0) C3 z-f OJ F- 0 II. D. 2’ p. O p. L\° 405 V l I I— U h T r U I f *Ifi I 7" 0 2 4 6 8 10 12 14 TIME (DAYS) Figure 10. Change in water-soluble nitrogen of quick ripened Blue cheese curd at 10°c. 75 J e—e CONTROL 15°C .1 H LDPE 5 44 5_ H LAMINATE Ch . o 1 a L. :2: z ‘1 0 d B . H .3 '3 34.54 c: W J L. m 4 4—1 0 -I 3: .1 U) 0 24.5- 2: . OJ . p. (D . D: a. d ...I 14.5- S . O p. . N 4 ‘L5 1 T ' I’ r I 4 T ”T’T'T 0 2 4 6 8 10 12 14 TIME (DAYS) Figure 11. Change in water-soluble nitrogen of quick ripened Blue cheese curd at 15°C. 76 Figure 12 represents the effect of salt concentration on water soluble nitrogen of OR Blue cheese curd at 10°C. The concentration of soluble protein reached a maximum of about 30.8%, at a salt concentration of 4.5%, on the 9th day of ripening while at 3.5% salt concentration the maximum increase was attained on the 7gp day and reached 38.5%. A significant decrease (P < 0.05) in soluble protein resulted as the salt concentration increased from 3.5 to 4.5%. The early application of salt and the increasing salt concentration appeared to retard proteolysis. Kuehler (1974) reported that proteolysis was retarded when at least 50% of the salt was added prior to the fourth day of ripening. Gripon and Hermier (1974) demonstrated the inhibitory effect of sodium chloride on the extracellular protease. Kinsella and Hwang (1976) and Godinho and Fox (1982) noted the same effect. The effect of temperature on the changes in soluble protein is shown in Figure 13. The amount of soluble protein increased rapidly at 15°C and reached a maximum of about 42.1% on the 6th day of ripening while at 10°C the maximum increase was attained on the 7th day and was 38.5%. A significant increase (P < 0.05) was obtained when the temperature was increased to 15°C. This level of increase was maintained throughout the ripening period. Kuehler (1974) noted the same effect of temperature on the changes in NPN of OR Blue cheese. 77 50.0- H CONTROL (3.57; SOIt Cone.) c H CONTROL (4.5% Salt Conc.) m '1 CD IO :5: 400‘ s 2: . _ .93. JD 4 2 (3 U) #4. L. 30.0" a) 4.1 C! ; (I) < _2: 20.0- OJ p. O 0: O. _J g 10.0~ O #- I.\° 01) I l 6 i 4 E 8— 1'0 12 14 TIME (DAYS) Figure 12. The effect of salt concentration on water- soluble nitrogen of quick ripened Blue cheese curd at 10°C. r (.'..1|">1111)1¢;f 1"~li|I