E "I “‘ Wzfimxaw ‘ ,‘ _ ‘ . “W? _.... ‘ ... ..m...w. :1 L \ L » \.<.‘. 4L d «A... ..\.\ ~_LL 4.”. . A hkvln. Hm I »"=:f‘:,. .' I a ; ~‘ . .4 - 1 .u. ‘ I -. < . 42' x... V ._._ . . a m. aw »..« at.» ...\.'.\-\. .'«.»\.'n:‘ - - :1: “3.1“: . M. { fui'lfi‘l 1'51?! gen. « $3.: ‘2». Al 1 4.. . u. .m. »:1.:... v.1 m. ‘1 " . u-rn .3 _ r: n1 A ‘ . . .m. w E r z » ‘ , . - . . l A u u 2‘.» A ..-m .1 13,15 134 "main,- <' u¢r«:_ ",'. v u .‘. uv n U r w . my a ll“l“llllljllllfiljlllllllllll .. This is to certify that the thesis entitled Melting Properties of Cheddar Cheese as Influenced by Milk Fat Level and Ripening Time presented by Kimie Kawachi has been accepted towards fulfillment Master of the requirements for of Science _ , Food Science degreein <\_) f// /Major professor ’ / 0-7 639 MS U is an Affirmative Action/Equal Opportunity Institution LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MSU Is An Affirmative Action/Equal Opportunity Inuitution Wanna-9.1 ,_.:. __rL‘_.v _ . MELTING PROPERTIES OF CHEDDAR CHEESE AS INFLUENCED BY MILK FAT LEVEL AND RIPENING TIME BY Kimie Kawachi A THESIS submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1993 ABSTRACT MELTING PROPERTIES OF CHEDDAR CHEESE AS INFLUENCED BY MILK FAT LEVEL AND RIPENING TIME BY Kimie Kawachi Cheddar cheeses with six different fat levels (34.3, 31.5, 26.8, 20.5, 12.6 and <0.l%) were manufactured, and allowed to ripen for 4 months at 7°C. Melting properties were studied using the Arnott test and dynamic rheological methods. Water soluble nitrogen measurement and sodium dodecyl sulfate polyacrylamide gel electrophoresis were performed at 1 day, 2 weeks, 1, 2, 3, and 4 month intervals. Meltabilities of the cheeses increased with increase level of fat. Meltabilities of cheeses with 34.3% to 20.5% fat improved in the first two weeks of ripening. Meltabilities of cheeses with 12.6% and <0.l% fat did not improve during the ripening period. a,- caseins were degraded more readily in 34.3% and 20.5% fat cheeses. These cheeses also showed higher water soluble nitrogen. The dynamic rheological testing indicated that the thermal transition of the V complex viscosity (1].) in the cheeses with 12.6% and <0.1% fat, in which 7]. decreased with temperature variations from 25°C to 65°C and then increased from 65 to 85°C. To my husband, Ataru. ACKNOWLEDGEMENTS The author would like to thank her major advisor, Dr. Z. Ustunol, for her inspiration, counsel, and encouragement during the time spent at Michigan State University. Appreciation, is also extended. to Drs. J. Steffe, J. Partridge, and B. Haines for serving as members of the guidance committee and for their very helpful comments and suggestions for the improvement of this thesis. Special thanks are expressed to Dr. T. Nishiya, my section manager at Snow Brand Milk Products Company, Kawagoe, Japan, for his encouragement. iv TABLE OF CONTENTS Page LIST OF TABLES .......................................... Vii LIST OF FIGURES ........................................ viii CHAPTER l.INTRODUCTION .......................................... l 2. REVIEW OF LITERATURE ................................. 3 2.1. Characteristics of Reduced Fat Cheeses .......... 3 2.2. Cheese Ripening ................................. 7 2.3. Cheese Meltability ............................. 11 2.4. Applications of Dynamic Rheological Test Methods for Cheeses ............................ 17 3. MATERIALS AND METHODS ............................... 21 3.1. Preparation of Cheese Milk ..................... 21 3.2. Cheese Manufacture ............................. 21 3.3. Proximate Analysis ............................. 22 3.4. Meltability Test (Arnott Method) ............... 22 3.5. Dynamic Rheological Test ....................... 23 3.6 SDS-PAGE ....................................... 23 3.7 Water Soluble Nitrogen ......................... 26 3.8. Statistical AnalySis ........................... 26 4. RESULTS AND DISCUSSION 4.1. Milk Composition ............................... 28 4.2. Compositon of Cheese and Whey .................. 28 4.3. Meltability Test ............................... 28 V vi CHAPTER Page 4.4. Rheological Dynamic Analysis ................... 37 4.5. SDS-PAGE ....................................... 49 4 . 6 . Water Soluble Nitrogen ......................... 59 5. CONCLUSIONS ......................................... 64 6. FUTURE RESEARCH ..................................... 66 LIST OF REFERENCES ....................................... 67 LIST OF TABLES Table Page 1. Molecular weight standards used for SDS—PAGE ........ 25 2. Composition of milk used for cheese manufacture ..... 29 3. Composition of cheeses manufactured with different fat levels ................................ 3O 4. Whey composition and pH at draining ................. 31 5. Meltability of Cheddar cheese as influenced by ripening period and fat level as determined by the Arnott method ................................ 32 6. Regression coefficients obtained from the linear equations relating meltability to the chemical composition of Cheddar cheese at 4 months of ripening ............................. 36 7. Regression coefficients obtained from the linear equations relating the minimum value of complex modulus (G*) to the chemical composition of Cheddar cheese at 4 months of ripening ........... 50 8. Water soluble nitrogen of Cheddar cheese as influenced by ripening period and fat level ......... 61 LIST OF FIGURES Figure Page 1. 10. Meltability of Cheddar cheese as influenced by ripening period and fat level .................... 33 Effect of fat levels of cheeses on storage modulus (G’) and loss modulus (G") 1 day after cheese manufacturing .......................... 38 Effect of fat levels of cheeses on storage modulus (G') and loss modulus (G") 2 weeks after cheese manufacturing .......................... 39 Effect of fat levels of cheeses on storage modulus (G') and loss modulus (G") 1 month after cheese manufacturing .......................... 40 Effect of fat levels of cheeses on storage modulus (G’) and loss modulus (G") 2 months after cheese manufacturing .......................... 41 Effect of fat levels of cheeses on storage modulus (G’) and loss modulus (G") 3 months after cheese manufacturing .......................... 42 Effect of fat levels of cheeses on storage modulus (G’) and loss modulus (G") 4 months after cheese manufacturing .......................... 43 Effect of temperatures on loss tangent (tan 8) of Cheddar cheeses 4 months after cheese manufacturing ....................................... 45 Effect of temperatures on complex viscosity (n') of Cheddar cheeses 4 months after cheese manufacturing ....................................... 47 Natural logarithm of complex viscos_ity (n ) of Cheddar cheeses with 1/T (° Kelvin) 4 months after cheese manufacturing ................. 48 viii ix Figure Page 11. Relationship between the minimum complex modulus (G*) and the meltability of Cheddar cheese during ripening from 1 day to 4 months ....... 50 12. The SDS-PAGE of the Cheddar cheeses with various fat levels 1 day after cheese manufacturing ......... 53 13. The SDS—PAGE of the Cheddar cheeses with various fat levels 2 weeks after cheese manufacturing ....... 54 14. The SDS-PAGE of the Cheddar cheeses with various fat levels 1 month after cheese manufacturing ....... 55 15. The SDS—PAGE of the Cheddar cheeses with various fat levels 2 months after cheese manufacturing ...... 56 16. The SDS—PAGE of the Cheddar cheeses with various fat levels 3 months after cheese manufacturing ...... 57 17. The SDS—PAGE of the Cheddar cheeses with various fat levels 4 months after cheese manufacturing ...... 58 18. Water soluble nitrogen of Cheddar cheese as influenced by ripening period and fat level ......... 62 INTRODUCTION Diet is a factor in the etiology of diseases such as heart disease, stroke, and cancer (Grundy, 1991). The American Heart Association.has promoted lowering the intake of dietary fat and cholesterol as a means of preventing cardiovascular disease (AHA, 1986). The recommended diet is one in which no more than 30% of calories are supplied from fats, and saturated fat should provide no more than 10% of total calories. Milk fat has been suspected of being injurious to health, because of its high saturated fatty acid and high cholesterol content. Milk fat is characterized by a high proportion of saturated.fatty acids (approximately 60%), appreciable amounts of monounsaturated fatty acids (approximately 32%), and small amounts of polyunsaturated fatty acids (Campbell and Marshall, 1975). During cheese manufacturing, when curd is separated from whey, most milk fat is retained in the curd. A Cheddar-type cheese retains 94% fat of milk (Bassette and Acosta, 1988). Semi—hard type cheeses widely consumed in the United States mostly have fat contents in the range of 20% to 30% in total weight (Bassette and Acosta, 1988). Cheese continues to be a 1 2 valuable source of protein and calcium (Best, 1991) and is a major ingredient in many of our favorite foods. Therefore, the demand for reduced-fat cheese is steadily growing. In many cheeses, especially Mozzarella, Cheddar, and process American, melting characteristics are the prime factors in determining the quality for particular product applications. Reduced-fat cheese tends not to melt when heated, which is a major problem for cooking applications or processing. The objective of this study was to investigate how milk fat levels and ripening parameters affected the melting properties of reduced—fat Cheddar cheese. This will be investigated by the Arnott Test, a commonly used method for measuring meltability, and the dynamic rheological methods, which have been recently applied to cheese studies (Shoemaker et al., 1992). mfi’m‘r'bi. .:< r". ' REVIEW OF LITERATURE 2.1. Characteristics of Reduced-Fat Cheeses The characteristics of reduced fat cheeses were reviewed by Olson and Johnson (1990), Jameson (1990), and Johnson and Chen (1991). Some common defects of reduced—fat cheeses are: 1)body' defects; too firm. or too soft, weak, or' pasty, 2)reduced meltability, 3)lack of flavor, 4)development of off- flavors: bitter, meaty-brothy, or unclean, and 5)a shorter shelf life. Reduced-fat cheeses are usually firmer and.more elastic (often. described. as "rubbery“) than their full-fat counterparts (Lawrence et al., 1983). Masi and Addeo (1986) showed that the moduli of elasticity of Mozzarella cheese increased linearly as the fat content of the cheese solids was reduced. The body of reduced-fat cheese remains tough and rubbery even after ripening (El-Neshawy et al., 1986). Lowering the fat content in cheese results in an increase in its water and protein content. The necessity of increasing the moisture content of reduced—fat cheeses makes them more susceptible to growth of contaminating bacteria, resulting in a shorter self life and unclean and meaty-brothy off flavors (Olson, 1991). 4 Reduced—fat cheeses tend to melt poorly when heated. A film is formed on the surface of the cheese which maintains the shape of the cheese and prevents the cheese from flowing. When it cools down, reduced fat cheese takes on a plastic consistency (Jones, 1992). The effect of fat and moisture content of Mozzarella cheese on its rheological properties was studied by Tunick at al. (1991) using a texture profile analysis. Reduced fat content resulted in higher values for hardness, springiness, and cohesiveness, and it also resulted in reduced meltability. Many approaches have been attempted to produce acceptable reduced-fat cheeses. The general strategies to improve the properties of reduced-fat cheese are: 1)control of pH, 2)control of moisture content, 3)decreased curd cooking temperature, 4)washing the curd, 5)homogenization of thernilk, 6)accelerated ripening, 7)fat substitutes, and 8)starter selection. Chen et al. (1992a) studied manufacturing parameters (milk pasteurization temperatures, starter culture levels, rennet levels, drain pH, and mill pH) of 33% reduced-fat Cheddar cheese. A curd pH of 6.37 versus 6.13 at draining produced cheeses with higher moisture, higher Cheddar flavor intensity and increased body breakdown, and they concluded that pH control was the most critical factor in producing quality 33% reduced—fat Cheddar. It was realized early on that the moisture content in the ’ ~ My...” fl: "ti“: 4 5 final cheese must be increased to levels substantially greater than those of typical full—fat Cheddar (Bassette and Acosta, 1988) to improve the textural properties of the cheese (Simard, 1991). Tunick et al. (1991) studied the effects due to moisture content on texture and meltability of lower-fat Mozzarella cheeses (9.5% and 11.1% fat) and higher-fat Mozzarella cheeses (21.0% and 25.1%); and they discovered that reduced moisture levels resulted in greater values for hardness, and that higher moisture (57.4% compared to 51.8%) was desired to improve the meltability of reduced—fat cheese. Simard (1991) described that decreased curd cooking temperature and increased.curd washing improved the texture of reduced-fat Cheddar cheese because these modifications reduced the lactose and the lactic acid, and increased the water content. Banks et a1. (1989) also used a lower cooking temperature (35°C) and less cheddaring time to produce a high- moisture low-fat cheese. At 25% fat content, a mild Cheddar flavor was produced, and the texture improved. However, Johnson and Chen (1991) reported that higher moisture cheeses (more than 47%) develop undesirable flavors more readily than lower moisture cheeses (43% to 44%), and. body qualities deteriorate rapidly with age. The lower moisture cheeses require more ripening to develop desirable body characteristics, but have a longer shelf life. Emmons et al. (1980) repbrted that homogenization of the milk resulted in a slightly softer, less elastic reduced-fat . .z" erg-4‘4 a ‘14-‘44 6. cheese of a slightly higher moisture content. Tunick et al. (1992) reported that at 10300 kPa homogenized. pressure, meltability and textural quality of low—fat and full-fat (22% 'and 46% fat-in-dry-matter) Mozzarella cheeses were improved. Tunick et al. suggested that these general effects, compared with non—homogenized controls, were attributed to the formation of lipid-protein complexes during homogenization. Fat substitutes are filling materials which replace some of the fat, but should not produce undesirable flavors or affect the structure of the product. El-Neshawy et al. (1986) tried to produce reduced fat Cehalotyre (Ras) cheese using 1% to 2% fat milk and gums: carboxymethylcellulose (CMC), and carrageenan. The addition of the gums to the milk enhanced the softness and smoothness of low-fat cheese. The starters which are used for full—fat cheese often produce meaty—brothy, off-flavors in aged reduced- fat cheeses . The slower acid producing starters are more desirable for reduced—fat cheese production (Johnson and Chen, 1991). Chen et al. (1992b) used four different starter cultures, Lactococcus lactis subsp. lactis and. Lactococcus lactis subsp. cremoris, and two Streptococcus salivarius subsp. thermophilus, for 33% reduced-fat Cheddar cheese, and found that the two Lactococcus strains produced a: more typical Cheddar type cheese. Olson (1991) suggested that strains that are less proteolytic and are slower acid producers are preferred for reduced-fat cheeses. 2.2. cheese Ripening The ripening, curing, or maturation of a cheese means placing a green or young cheese in a temperature and relative humidity controlled area for a specific period. During this ripening, the cheese curd brakes down into a smooth body and a characteristic flavor (Kosikowski, 1982). In cheese making, coagulation of milk for rennet cheeses is accomplished by the specific cleavage of casein micelle- stabilizing protein, K—casein, at the phenylalanine (105)— methionine(106) bond by a milk-clotting enzyme (Fox, 1989). The hydrophobic 1—105 fragment of K—casein remains in the casein micelle, and the casein micelles aggregate to form chains and then a network in which the fat globules are entrapped. As more linkages form between caseins, the structure shrinks and the whey is squeezed out. The fat globules become distorted and their membranes are ruptured. The final cheese consists of fat entrapped in a protein matrix (Green et al., 1981). The casein micelles consist of an—, afl-, B-, and K- casein, accounting for 35.6%, 9.9%, 33.6%, and 11.9% of casein micelles, respectively (Bringe and Kinsella, 1985). Cheese ripening involves the conversion of the casein fractions to lower molecular weight products. Cheese undergoes a series of complex, sequential changes during ripening that are caused.by proteinases from milk, milk—clotting enzymes, lactic starter cultures, and other microorganisms (Grappin et al., 1985). 8 There are two distinct proteolysis phases during ripening (Lawrence, 1987) . The initial phase occurs in the first 7 to 14 days. The primary target for proteolysis in cheese made with rennet is the (Isl-casein, with the initial cleavage yielding oral-I . The network is greatly weakened when about 20% of the (tn-casein single bonds are hydrolyzed. The second phase involves a more gradual change over the following weeks. In ten week old cheeses, 90% of the “.1'1 peptide was hydrolyzed, yielding (In-2, and “51'3/4 peptide. DiMatteo et a1. (1982) found that almost all of the Gin-casein was broken down into (In—I and (1.1-2. However 95% of the B-casein was still intact after ten weeks of ripening. The (In-casein is degraded more rapidly than {S-casein (Bertola et al., 1992) . 0f the two major casein fractions, (In-casein is hydrolyzed first, and is extensively degraded (Grappin et a1. , 1985) . B- Casein may be expected to undergo two types of hydrolysis during the first stage of ripening: one by the action of rennet to form 13-1, 8—2, and 8-3 peptides; and one by plasmin, indigenous milk proteinase, giving y-caseins. Enzymes from starter bacteria together with the other proteinases will further degrade these peptides (Grappin et al., 1985). The proteins of cheeses are cleaved at various sites during ripening and the protein network loses part of its original structure, which alters the rheological properties of cheese (Grappin et al., 1985) . Bertola et a1. (1992) determined the proteolysis indices, the water-soluble and 12% 9 TCA—soluble nitrogen, and the rheological parametersof Tybo Argentino (a semi-hard cheese) by force-deformation curves and relaxation curves at 20°C. During aging, both proteolysis indices and adhesiveness increased, and (Isl—casein concentration, hardness, elastic modulus, and viscosity decreased. The rheological parameters were correlated to both proteolysis indices, and to the concentration of (In-casein by linear regression equations. Hardness and viscosity had the highest correlation coefficients. Amantea et a1. (1986) studied rheological properties and a proteolysis parameter. They measured rheological properties from the force—compression curves, and the amount of amino groups in the TCA-soluble fraction (free amino groups) in Cheddar cheese, as a proteolysis parameter. As the cheese aged, free amino groups increased, and yield point of force- compression curves decreased. Hydrolysis of casein produces compounds that are largely soluble in water and that do not contribute to the protein network responsible for the cheese rigidity (Walstra and Vliet, 1982) . Lawrence (1987) explained that for this reason the cheese softened during maturation. As each peptide bond iscleaved, two new ionic groups are generated and each of them will compete for the available water in the system. The water previously available for solvation of the protein chains will become tied up with the new ionic groups. Relatively low moisture cheese, such as Cheddar, tends to become increasingly harder with age and more 10 resistant to slight deformation (Lawrence et a1. 1987) . Creamer and Olson (1982) reported that the amount of intact (Isl-casein in commercial Cheddar cheese was related directly to the yield force in a compression test. de Jong (1978) also found that the rheological properties of cheese were greatly dependent on the cleavage of (Isl-casein. High concentrations of intact oral-casein contribute to the rigidity of the protein matrix, and low concentrations of intact (151- casein contribute to the increased elasticity and softening of the cheese (Bertola et al., 1992). Ripening also affects the meltability of the cheeses. Oberg et al. (1991b) compared the melting characteristics of Mozzarella cheeses made with proteinase-deficient starters and with proteinase—positive starters. The cheeses made with paired proteinase-positive cultures melted better than cheese made with proteinase-deficient cultures throughout the storage period of 30 days. Oberg et a1. (1992) also studied the effects of milk—clotting enzymes on melting characteristics of Mozzarella cheese from one day to 28 days. The meltability of all cheeses increased over time; the sharpest increase was between days 7 and 14. The Mozzarella cheese made with calf chymosin had the largest increase in meltability, which was consistent with greater proteolysis of a-casein with calf chymosin than with the other milk-clotting enzymes. Cheese made with porcine pepsin had the least meltability over time. Porcine pepsin preferentially degrades B—casein over a-casein, 11 causing less weakening of the protein network. They concluded that, as the protein network of cheese was broken down by the enzyme, the cheese melted better. 2.3. Cheese Meltability Melting is defined as the change of a solid into a liquid when heat is applied (Gwinn, 1990). In a pure crystalline solid, this process occurs at a fixed temperature called the melting point. Amorphous (non crystalline) substances melt by gradually decreasing in viscosity as the temperature is raised, with no sharp transition from solid to liquid. Melting transition phenomena in polymers were described by Bever (1986) . The melting of polymers or flexible linear macromolecules differs from the melting of other materials only in aspects which involve the degree of connection of the flexible subunits of the molecule. Typical "polymer melting" is observed when there are no strong (covalent) bonds broken during melting. In many cheeses, especially Mozzarella, Cheddar, and process American, melting characteristics are prime factors in determining the quality for particular product applications (Park et a1. , 1984) . Meltability of cheese is a thermal phase change, characterized by the solid cheese and the flow characteristics of the melt (Park et al., 1984). Melted cheese is viscoelastic, which means it has both a property of flowing and a property of returning to its original shape 12 after deformation (Kindstedt et al., 1989). Characteristics of meltability are: (1) the cheese should melt in a certain temperature range, (2) it should remain homogenous both in appearance (no fat separation) and mouthfeel (no granular texture), and (3) the apparent viscosity should be low enough for the material to flow clearly and high enough to not flow too fast; hence there is an optimum viscosity (Rfiegg et al., 1991). There are many factors which affect the meltability of cheeses, such as: chemical composition (mineral, fat, and moisture), structure (fat dispersion and cheese structure), and other parameters (proteolysis and pH). Rfiegg and Moor (1988) showed the inverse proportional relationship between water content (on a fat-free basis) and the softening point temperature for various semi-hard type cheeses. Keller et a1. (1974) studied Mozzarella cheeses made by acidifying milk containing 2% fat. The meltability of cheeses was affected significantly by the type of acid and the pH at coagulation. In general, meltability increased. with a decrease in pH (within the range of 5.21 to 5.60) except for high meltability of all cheese made with citric acid, which caused low calcium/phosphate ratios of the cheeses. Meltability had a negative correlation with calcium in dty matter and viscosity of compression tests. Process cheese fat dispersion and meltability relationships were discussed by Savello et a1. (1989) and 13 Tatsumi et al. (1989). Savello at al. (1989) measured the meltability of model process cheese made with rennet and acid casein, and concluded that more complete emulsification resulted from decreased meltability in rennet casein cheese. The distribution of fat globule particles (by Scanning Electron Microscopy analysis) in acid casein process cheese was not related to cheese meltability. Tatsumi et al. (1989) tried to correlate meltability with fat globule size. They cooked Cheddar cheese in a twin screw extruder at 50 r.p.m. to 150 r.p.m.(screw rotation speed). The twin screw extruder modified fat globule size. There were few fat globules more than 5 microns in diameter in cheeses cooked at 130 r.p.m. or more. They observed a significant decrease of meltability from 100 r.p.m. to 130 r.p.m. They concluded that relatively large fat globules are necessary for meltability. Kalab et al. (1991) measured the meltability of a process cheese containing white cheese made by coagulating heated.milk with citric acid solution. They found that the white cheese did not melt when heated alone, but that the white cheese increased the meltability of the process cheese when it was used as an ingredient. On the other hand, an excessively heated process cheese, which does not melt alone, decreased the meltability of the process cheese (Kalab et al., 1987). Kalab et al. (1987) suggested that the structural difference between these cheeses, the excessively heated process cheese having a compact structure, and the white cheese having a 14 core-and-shell structure, caused.the different.me1tabilities. The core-and-shell structure characterizes milk products made by acid coagulation of hot milk. The effect of ripening on szzarella Cheese's melting properties was studied‘ by Kindstedt et a1.(l989) using helical viscometry. During one month of ripening, the melted cheese consistency changed from tough and fibrous to smooth and gelatinous, and the apparent viscosity of the melted cheese decreased. Arnott et al. (1957) investigated the meltability of commercial process cheeses and found that free tyrosine which showed the degree of proteolysis was related to meltability. As the free tyrosine increased, the meltability increased. Above certain tyrosine levels, which was approximately equal to the tyrosine level of 110 days for the average age of the Cheddar cheese, poor meltability was not observed. Researchers have been looking for objective methods to assess the melting patterns of cheeses (Park et al., 1984, Kindstedt et al., 1989) . The melting property cannot be described. by a single physical property. Instrumental measurement of melting characteristics is complicated by several factors, such as the heterogeneity of the cheese, "phase" separations (oiling off or wheying off), changes in specimen shape, and temperature gradients (Rfiegg et a1. , 1991). Conventional meltability tests are mainly based on 15 controlled heating of cylindrical samples and measuring time or changes in height or diameter (Park et al. ,1984) . The most traditionally reported methods are the Schreiber method (Kosikowski, 1982) and the Arnott method (1957) . Since neither procedure has been standardized, different researchers have used various specimen dimensions and heating conditions. Park et a1. (1984) compared the two meltability tests, the Schreiber method (232°C, 5 minutes) and the Arnott method (100°C, 15 minutes). They found a lack of correlation between the results of the two tests/and concluded that the two tests do not measure the same rheological attributes because of differences in temperature and exposure time. The reproducibility of the results of the conventional oven methods is not generally good. The relatively large experimental error may explain in part the absence of statistically significant correlations between meltability index and chemical composition in some studies (Riiegg et al. , 1991) . Olson and Price (1958) described the test designed for process cheese. In this test the cheese sample was heated in a sealed glass tube in an oven, and the distance flowed by the melted cheeses was measured as an index of meltability. A rotational viscosimeter has been used to evaluate meltability of cheeses. Lee et al. (1978) tried to determine meltability by recording the temperature at which flowability became measurable by a Brookfield viscometer. Olson and 16 Nelson (1980) measured Mozzarella cheese melting properties based on the Weissenberg effect, which is the tendency of a viscoelastic material to climb up a smooth rod that is rotated in the test material. An advantage of this test is that separate :measures of stretchabilityy elasticity, and meltability may be obtained, and a disadvantage is that the test requires considerable subjective assessment of Heated cheese texture and fracturing behavior by the analyst (Park.et al., 1984). Kindstedt and Kiely (1992) used apparent viscosity and free oil to evaluate the melting behavior of Mozzarella type cheeses. The apparent viscosity was measured by the helical viscometry test which included the addition of 25 mL of butter oil (60°C) to the sample surface. Adding exogenous butter oil standardized the surface conditions of samples that oiled off to varying degrees. The cheese sample was melted and tempered at 60°C for 60 minutes with the additional butter oil. A T- bar spindle was positioned near the bottom of a column of cheese. Resistance, exerted by the melted cheese on the rotating spindle as it was raised through the melted cheese column was recorded continuously. A profile of spindle resistance versus vertical distance was expressed as an " apparent viscosity“ profile, and the maximum peak of apparent viscosity was used as an index of melting properties. Park et al. (1984) used Differential Scanning Calorimetry (DSC) to differentiate thermal properties of American, 17 Mozzarella, and Cheddar cheeses in the range 10 to 70°C. The samples were heated at. 5°C per minute. Although slight differences were detected among the thermograms of the cheeses, they did indicate major differences in meltability patterns. Tunick et a1. (1990) measured the specific heat (at 60°C) of Cheddar and Cheshire cheese using DSC. The specific heat value of the Cheddar cheese was higher than that of the Cheshire cheese, which indicated a stronger cheese structure. 2.4. .Applications of Dynamic Rheological Test Methods to Cheese Dynamic testing refers to the application of a continuously changing stress or strain. Normally, the form of the change is sinusoidal, and the response of the material to the varying stresses or strains is measured. Dynamic testing offers very rapid results with minimal chemical and physical changes (Shoemaker et al., 1992). In small—amplitude oscillatory shear experiments, the sample is contained in two parallel plates and undergoes oscillating deformation as the lower plate is rotated at a specified frequency and transient responses are recorded. Like most solid foods, cheese is viscoelastic in nature, meaning that it exhibits both solid (elastic) and fluid (viscous) behavior. In dynamic testing, the changes of cheese properties during heating can be continuously monitored using the same sample. The real and imaginary components of the 18 complex shear modulus (G*) are expressed as the storage modulus (G') and the loss modulus (G") which represent the elastic and viscous elements, respectively. The loss tangent (tan 8) is calculated as a measure of the energy lost compared with the energy recovered in cyclic deformation. (G*)2=(G')’+ (G")2 and tan 5 = G"/G’ The measurement of dynamic mechanical properties of food is a relatively new technique and has recently been applied to cheese. Nolan et al. (1989) studied dynamic rheological properties related to the meltabilities of low—moisture, part- skim—Mozzarella cheese, both natural and imitation. Imitation cheeses were made by adding calcium caseinate powder to fresh raw milk before pasteurization. Measurements of dynamic viscosities and shear viscoelastic moduli were determined at temperatures up to 70°C and frequencies from 0.10 to 100 rad/sec. in parallel plates. The experimental cheeses appeared to have a wide melting range. Initial softening was noticed at 35 to 40°C and continued up to 70°C. The addition of 1% calcium caseinate increased the elastic and viscous components of the shear modulus at 20°C over the corresponding values of natural Mozzarella cheese. At 2% calcium caseinate level, the elastic component of the modulus G' showed a decrease below the baseline values of natural cheese but the viscous component G" increased. Nolan et al. concluded that changes observed in cheese properties helped provide an 19 objective basis for distinguishing between imitation and natural low moisture, part skim Mozzarella cheeses. Taneya et al. (1979) measured the dynamic properties of Gouda, Cheddar, and processed cheese at temperatures between 5 and 90%:. The rigidity of the cheeses as expressed by the storage modulus (G’) declined as the temperatures increased. The decline accelerated at temperatures higher than approximately 30 to 40%:. However, plots of log(G’) versus temperature were smooth, and showed no sign of an abrupt decline that would indicate a phase change. Plots of the dynamic loss tangent (tan 5) versus temperature had features, that is, local maxima and minima, that could indicate structural transitions. Tunick et al. (1990) examined viscoelastic properties of Cheddar and Cheshire cheeses with a Rheometrics Dynamic Analyzer, and showed textural differences between these two cheeses. Proteolysis during ripening results in decreased viscosity and elasticity of cheese, and this effect was demonstrated in the downward shift of the G’ (the elastic modulus), G" (the viscous modulus), and. n* (the complex viscosity) curves at strain sweep tests. They estimated the values of G’, G", and n* at 0% strain from strain sweep tests curves, and found that these values of Cheddar were almost twice those of Cheshire. These finding are consistent with Cheshire being the crumblier of the two cheese types. Nolan et al. (1990) compared some properties of Cheddar 20 and.pasteurized process American cheese by dynamic rheological tests. The viscosity of both cheeses followed an Arrhenius- type relation, the natural logarithm of complex viscosity (1n n*) of the cheeses was indirectly proportional to the reciprocal of absolute temperature, at three different frequency values between 20 and 50°C. The results suggested that the particular sample of Cheddar cheese studied, about nine months old, was crumbly and melted with difficulty. The existence of inherent artifacts in the dynamic testing method are difficult to be ruled out, especially since fat separation takes place (Rfiegg et al., 1991). The fat separation from cheese causes slippage of the specimen in the apparatus. Nolan et al. (1989) observed inconsistencies in their measurements of cheese sample due to the slippage. They bonded the cheese specimen directly to the plates with cyanoacrylate ester adhesive, which effectively solved the problem of specimen slip. MATERIALS AND METHODS 3.1. Preparation of Cheese Milk Skim milk and cream were obtained from the Michigan State University Dairy Plant. The skim milk was pasteurized by a High Temperature Short Time pasteurizer (HTST) at 74°C for 18 sec., and cooled and stored below 4°C. The cream was batch pasteurized (60°C, 30 min.), and cooled and stored below 4°C until use. 3.2. Cheese Manufacture Cheddar cheeses with varying fat levels (34.3, 31.5, 26.8, 20.5, 12.6, and <0.l%) were manufactured from milk containing 4.0%, 3.2%, 2.4%, 1.6%, 0.8%, and approximately 0.03% milk fat. Milk for each treatment was standardized using the skim milk and cream to obtain the desired fat level. The standardized milk was warmed to 31°C, and inoculated with 0.02% of Direct to the vat (DVS) starter culture (DVS #980, CHR. Hansen’s Laboratory, Inc. , Milwaukee, WI). Milk was ripened for 1 h. Double strength chymosin (0.01% of the milk, Chymax-Double Strength, Pfizer, Milwaukee, WI) was diluted to 40 times with tap water and added to clot the milk in 30 min. Cheddar cheese was manufactured by the procedure outlined by Kosikowski (1982). Curd was salted (2.3% of the curd), 21 22 hooped, and pressed for 18 hours. Manufactured cheeses were vacuum packed the next day and ripened at 7°C up to 4 months. Samples were taken at 1 day, 2 weeks, 1,2,3, and 4 months of ripening for the appropriate analysis. 3.3. Proximate Analysis Milk samples were taken after standardization. Milk was analyzed for fat, total nitrogen, and total solids. Cheese samples were taken from the center of the cheese block. Cheeses were analyzed for fat, total nitrogen, and moisture. Whey samples were taken at draining. Whey was analyzed for fat, total nitrogen, and pH. Milk samples were collected after standardization. Whey samples were collected at draining. Fat content was determined by the Babcock.procedure (Marshall, 1992). Total nitrogen.was determined.by the micro— Kjeldahl method (Marshall, 1992). Moisture content was determined by drying milk and whey at 100°C to 105°C for 3 h., and cheese for 24 h., cooling in a desiccator, and reweighing (Marshall, 1992). pH was determined by a Corning pH meter (Model 145, Halstead Essex, Medfield, MA). All analyses were done in duplicate. 3.4. Meltability Test (Arnott Method) Meltability of cheese was measured by the Arnott test (Arnott et al., 1957). Cheese samples were cut into cylinders (22mm in diameter and 17mm in height) with a corkborer and a knife. Each specimen was put in the center of a glass Petri dish. The Petri dish was heated in an oven at 100%: for 15 23 min. After cooling for 30 min. at approximately 25%:, the diameter of the cheese was measured. 3.5. Dynamic Rheological Test A.Rheometries Fluids Spectrometer (REFS-8400, Rheometrics Inc., Piscataway, NJ) was used to characterize the rheological properties of cheeses. To establish optimum instrumental parameters for dynamic testing, strain sweeps were performed at 25°C and 85%: using 1 day old cheese with 34.3% and less than 0.1% fat. At 25%: and 85%:, linear viscoelasticity was observed at less than 0.5% strain with both cheeses. At more than 0.5% strain, the machine stopped due to overload.when the cheese with less than 0.1% fat was tested. Hence, the tests were performed at a constant frequency of 1 rad/sec. and a constant strain of 0.1% in a parallel plate apparatus (2.5 cm radius). Cheese samples were cut to obtain disks approximately 6 cm in diameter and 1.0 mm to 2.0 mm in height using a meat slicer (Model 512, Hobart Co., Troy, OH) and a cookie cutter at 5°C. The samples were placed between the parallel plates. The temperatures of the samples were controlled by a temperature programmer (MTP-6 Micro processor, Neslab Instruments, Inc., Newington, NH) using silicon oil (poly dimethyl siloxane, Dow corning 200, Dow Corning Co., Midland, MI) as a medium. The temperature was raised from 25%: up to 85%: at a rate of 2°C per minute during the test. The data included the elastic (shear storage), G’ (Pa), and viscous 24 (shear loss), G" (Pa), components of the shear complex modules, G. (Pa), together with the complex viscosity, n. (Pa 5), and loss tangent, tan 5. These parameters are related as follows: ) 1 day after cheese manufacturing. Frequency, 1.0 rad/sec; strain, 0.1%. Fat level of cheeses; A) 34.3%, B) 31.5%; C) 26.8%; D) 20.5%; E) 12.6%; F). <0.1%. “Fwnumgt 10‘ 1 1 11,1 1 1 1 1 1 1 1 1 25 3O 35 40 45 50 55 60 65 7O 75 80 85 5 10 a. B On v H 4 E; 10 5M5; Q .6 e. ‘f‘ C) e. ‘nt 2 00. a .3 10’ ° t: fi‘. E3 ‘h 2% E A...“ ’ A z 102 ‘hw A E; 'Mms 10‘, 1 1 1 1 1 1 1 1 1 1 1 1 25 30 35 40 45 so 55 60 as 70 75 so 85 3 10 (3; A . k. 10‘ °55 w.~0~ K ‘\ I“. , wmfiA‘ 10 News.“ 1f 10‘, 1 1 1 1 1 1 1 1 1 1 1 1 25 3O 35 4O 45 50 55 60 65 70 75 80 85 39 10’ 1 1, 1 1 1 1 1 1 1 1 1 L1 25 30 35 40 45 50 55 60 65 7O 75 80 85 l l l l l l 1 25 30 35 40 45 50 55 60 65 7D 75 80 85 )0 F: ‘MJW‘ 10‘ Nun“ ~ lg”,,nd“’.dsfl~“ 10‘ 1 1 1 1, 1 1 1 1 1 r1, 1 1 25 30 35 40 45 50 55 60 65 70 75 80 85 TENHTHUNFURE(%D Figure 3. Effect of fat levels of cheeses on storage modulus, G'(A), and loss manufacturing. modulus, Frequency, Gt! (0) 1.0 rad/sec; after cheese strain, 0.1%. Fat 2 weeks level of cheeses; A) 34.3%, B) 31.5%; C) 26.8%; D) 20.5%; E) 12.6%; F) (0.1%. lllllJl ll 11 25 3O 35 40 45 50 55 50 55 70 75 80 85 10 B 2.“? 3 10‘ «t, Z) “N C) °T““v “u C) 2 ~. 35 2 10’ ~° “a. U '“s‘5 ‘ 0‘ E 3:“: 831° 3 g 10' .°5 A o. C) 10‘411111111111 25303540455055505570753035 10' c 10‘ “no.5 o °=‘:s . .. ’ 0...... M ' 00 .00 4“ IO oré’ efi'qg. ’ ”r ‘A “a: hang: 10' 10‘.1m111111111u 25 30 35 40 45 50 55 60 55 7O 75 BO 85 10 D ““866. 10‘ «9° 0......: .0. ¢“ s ‘ ‘ O o . We. “a b 103 . 6.“...3 102 10‘11'1111111111‘ 25 30 35 40 45 50 55 50 55 7O 75 30 85 1o” 1111111L1114 25303540455055506570753035 10’ F I A .4 10‘ “‘9'“, 4‘ ,0 A“ i. . ‘0” 0~.\‘.. 00 . 1‘...” 10‘741 1111111411 25303540455055506570753085 TEMPERATURE (°C) Figure 4. Effect of fat levels of cheeses on storage modulus, G'(A), and loss modulus, manufacturing; Frequency, G"(0) 1.0 rad/sec; 1 month after cheese strain, 0.1%. Fat level of cheeses; A) 34.3%, B) 31.5%; C) 26.8%; D) 20.5%; E) 12.6%; F) <0.1%. 412L 10 A 10 D . ‘4‘ . ‘A‘ 10‘ ‘lk‘fiu.‘- 10‘ °°°°°°‘5 “ m. 00. s g ‘5 ° ‘0’” ‘0 0 m 0‘ A“ a . ’ e 10 00 u 10 ‘3' . o A I! 3... ‘°‘ _ ”“9: 560%.. M J 102 ”“2““ ‘ 10a 000 A ‘ o O ” O 10‘711L111111111 10‘411111141111 25 30. 35 40 45 50 55 50 55 7O 75 80 85 25 30 35 40 45 50 55 50 55 7O 75 30 35 102' B 108 E ’3 e3 4. .— 10‘ A““ 10‘ K 5 “‘5 00 .o. I” Q .‘A Q i. O 0‘ A‘ .50” O .0 ..~...“: ’ :2 10' £3 ‘95, 10 ’ o A‘s 2 ° 0.3.0 ’4‘ A a 2 .5 .0 ‘445. ‘9‘ 0 fig: 10' 0 ° ‘° , 10' C) 10‘111111411111 10‘111111111111 25303540455055505570755035 25303540455055305570750035 10’ C 11)3 F ‘56 A“ 10° “‘3‘ 10° .“ 8‘ x N mg 3"...“ M ” a K A“ " '°.~...Wq. a u A \ 10' 10 . A‘ .‘ A‘s xaz.’.“~ A’O‘ad 10' e 10' (0311141111111110‘1111111L1111 25303540455055505570755055 25303540455055505570758035 o TENETRMHIHU3(C) Figure 5. Effect of fat levels of cheeses on storage modulus, G’ (A) , and loss modulus, G" (o) 2 months after cheese manufacturing. Frequency, 1.0 rad/sec; strain, 0.1%. Fat level of cheeses; A) 34.3%, B) 31.5%; C) 26.8%; D) 20.5%; E) 12.6%; F) <0.1%. 10 A'- is w‘ ‘ A A“‘ $0 .015 10' .0 ° 3 ”5‘!“ -‘:::‘ A 102 wkflfl‘“ 0 be...“ 10‘, 1 1 1 1 1 1 1 1, 1 1 1, 1 25 30 35 4O 45 50 55 50 55 7O 75 BO 85 105 B A pi.“ v . “‘Aa" t—t 10 ‘. _J 4 D “0..“ x‘~ C) s a, C) 10' ‘°°’~1 ‘° >2 ““3. o \. ’2‘ “awe“ .< ’T ‘ths EE ‘4 Q I 10 1 1 1 1 1 1, 1 1 1 1 1 1 25 30 35 4O 45 50 55 50 55 7O 75 BO 85 m' C: 10° °~H-~ihhhfik "‘~e5 5%“ 0.. 5A O Q... 10' “g “ W‘ “’06 \.~ if ‘ 10‘, 1 1 1, 1 ,1 1 1 1 1 1 1 1 25 3O 35 4O 45 50 55 50 55 7O 75 80 35 10 D 4 a to ‘44 N w. ”.00.. \ 00 0 e 0‘. . if a. A \o, if 10‘ 1 1, 1 1 1 1 ,1, 1 1 1 1 1 25 30 35 4o 45 50 55 so 55 7o 75 so 85 1111111 25303540455055505570758035 10 F 0‘ . {.4 1 . N‘ u 00* ‘°.i "o 0“... ”as “9‘“,009“ a la ‘ 10 “ 1f 10‘, 1, 1 1 1 1 1 1 1 1 1 1 1 2530354065055605570753085 TENETRMHIHUECC) Figure 6. Effect of fat levels of cheeses on storage modulus, G'(A), and loss modulus, manufacturingu Frequency, G“(0) 1.0 rad/sec; 3 months after cheese strain, 0.1%. Fat level of cheeses; A) 34.3%, B) 31.5%; C) 26.8%; D) 20.5%; E) 12.6%; F) <0.1%. AA 10‘ ‘N8 .é.° 9 ‘AA 5 1° $00.3. A ..’°o§2 AA ‘ A f“ 0 46A 1 ° '° 10' °° ° “.M‘ 6 19‘ 1 1 1 1 1 1 1 1 1,11 1 25 30 35 40 45 50 55 at) as 70 75 80 85 3 10 ’8 B e ““4 5° 10‘ *1 as. 5 9‘ ‘1 8 9 . o‘;§ 2 llila ‘8‘ 0 B a“. 2 A (c z °°§o ' g5 10 4&3. c:: Q 10‘ 111111111111 25 30 35 40 45 50 55 60 as 70 7s 80 as 5 )0 (2’ A 4 ~°“AAAMMI“ o o 9. 0 ””wa 10 “”96 “M A 5‘ 10, \“Mfltflm 0W“, 10’ 10‘ 1 1 1 1 1 1 1 1 1 1 11 25 30 35 4D 45 50 55 60 55 70 75 80 85 43 10 D AAA‘A A ‘0‘ M go NP 5°“ 06600.0 % “u 6.9 9 ‘ 0°11: ‘5‘ 103 °ee¢°¢°¢g 5. 8° '9 a axmn‘OgAA o 50.1.)». 2 0 10 10‘,11111111111u 25 30 35 4D 45 50 55 60 55 70 75 80 85 l l l l ,1, A, M I A 10 N‘s. “a“, u .4090 M ‘°.° .9 6. o “A 5° ' o\°°eO °° o. 10' ° N o‘ ‘ tA km 102 10‘ 1 1 1 1 1 1 1 1 Ll 1 25 30 35 40 45 50 55 60 65 70 75 80 55 TEMPERATURE (°C) Figure 7. Effect of fat levels of cheeses on storage modulus, G'(A), and loss manufacturing. modulus, Frequency, level of cheeses; A) 34.3%, B) 31.5%; C) 26.8%; D) 12.6%; F) <0.1%. G"(o) 4 months after cheese 1.0 rad/sec; strain, 0.1%. Fat 20.5%; E) 44 After two weeks, both the dynamic moduli of the high fat cheeses (34.3%, and 31.5%) decreased as the temperature increased (Figure 3). The storage modulus became lower than the loss modulus at approximately 60°C. The dynamic moduli of the low fat cheeses (12.6%, and <0.l%) did not decrease as much as that of the higher fat cheeses as the temperature increased. From 1 month to 4 months of ripening (Figures 4-7) , the dynamic moduli of the highest fat cheeses (34.3%) decreased as the temperature increased, but the storage moduli were higher than the loss moduli from 25 to 85°C. The storage modulus of the low fat cheese (12.6%, and <0.l%) decreased to around 66 to 70°C and then increased as the temperature was raised to 85°C. This phenomenon became more noticeable as the cheeses ripened. Figure 8 shows the mean loss tangent (tan 5) of four month old cheeses (n=4) . Loss tangent "(tan 8) represents the ratio of viscous properties to elastic properties of a viscoelastic material (Shoemaker et al., 1992) . At all fat levels the cheeses showed peaks at between 60 to 75°C. The lowest fat (<0.l%) cheese had a notable peak at 65°C. Hydrophobic interaction is one possible explanation for the hardening of the cheese at higher than 60°C. Below 60°C, as the temperature increased, fat melted and the hydrogen bonds decreased in the cheese, causing the cheese to become softer. Above 60°C, hydrophobic interactions are enhanced between protein molecules, and the protein matrix becomes more 03 02 OJ -0J -02 -03 ~04 -05 -06 -07 308 log (tan 6) 45 - A774 ,7?" -fi‘g’f ' ' «'47 l J l 1 J l L l L L L l L 25 30 35 40 45 50 55 60 65 70 75 80 85 TEMPERATURE (°C) Figure 8. Effect of temperature on loss tangent, tan 8 Cheddar cheeses 4 months after cheese manufacturing. Frequency, 1.0 rad/sec; strain, 0.1%. Fat level of cheeses: 34.3%, (I); 31.5%, (0); 26.8%, (A); 20.5%, (D); 12.6%, and <0.1%, (A). 46 rigid. If there is fat between the hydrophobic groups of the protein, the hydrophobic groups will interact with the fat, and interaction between protein molecules will be inhibited. As the cheese ripens, hydrophobic groups will be exposed due to protein degradation, which causes more hydrophobic interactions. Figure 9 shows the average of complex viscosity (n°) of four month old cheeses (n=4). The complex viscosity of the higher fat cheeses (34.3% to 20.5%) kept decreasing as the temperature increased, but the lower fat cheeses’ (12.6% and less than 0.01%) complex modulus decreased to around 65°C and then increased as the temperature went up. Nolan et al. (1990) showed that the viscosity of Cheddar followed an Arrhenius-type relationship between 20 to 50%:, and a plot of the natural log of complex viscosity (1n nl) versus the reciproca1.of absolute temperature became linear at three different frequency values 0m=1, 10, and 100 rad/s., strain=0.68%). Tunick et al. (1990) showed that plots of n. of Cheddar cheese and Cheshire cheese versus reciprocal of absolute temperature (1000/T) produced straight lines which followed the Arrhenius equation (temperature=20 to 40°C, (0:1 rad/8., strain=2.5%). Figure 10 shows the natural logarithm of complex viscosity (1n.n°) versus 1000 times the reciprocal of absolute temperature of four month old cheeses in this experiment. At a wider range of temperature (25 to 85°C, 1000/T=3.36 to 2.79) the plots of all fat level cheeses showed 47 5 4.5 - 4 .. 6? {U as V 3.5 - .A 5 E? 3 — i = = . 2.5 — 2 L l J 41 l l l l_ I L l l l 25 30 35 4o 45 so 55 60 65 7o 75 80 85 TEMPERATURE (°C) Figure 9. Effect of temperature on complex viscosity, 1]., of Cheddar cheeses 4 months after cheese manufacturing. Frequency, 1.0 rad/sec; strain, 0.1%. Fat level of cheeses: 34.3%, (I); 31.5%, (4); 26.8%,. (A); 20.5%, (D); 12.6%, (o); and <0.1%, (A) . 48 11 In (11') (Pa 8) s l 4 l l l l J l 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 1,000/T (°Kelvin)’1 Figure 10. Natural logarithm of complex viscosity, 'n', of Cheddar cheeses with l/T (° Kelvin)‘1 4 months after cheese manufacturing. Frequency, 1.0 rad/sec; strain, 0.1%. Fat level of cheeses: 34.3%, (I); 31.5%, (0); 26.8%, (A); 20.5%, (D); 12.6%, (0); and ~ 5" S m 4— <1: E-4 .4 § 3‘ 2.— 1 l I l I l I l 15 2 2.5 3 3.5 4 45 log (G*) (Pa) Figure 11. Relationship between the minimum complex modulus, G*, and the meltability of Cheddar cheeses during ripening from 1 day to 4 months. Regression equation: (Meltability):— O.52(log G*)+4.45. Frequency, 1.0 rad/sec; strain, 0.1%. 52 electrophoretogram of cheeses during ripening from 1 day to 4 months and.molecu1ar weight standards are shown in Figures 12 to 17. Lanes 1 to 6 represent cheeses with 34.3%, 31.5%, 26.8%, 20.5%, 12.6%, and <0.1% fat, and lane 7 represent the molecular weight standards. In general, the levels of all of the caseins appeared to decrease with time. In agreement with previous reports (Grappin et al.,1985), a,-casein was more extensively degraded compared to B-casein. B-Casein underwent slight proteolysis over the ripening period of 4 months. In 1 day old cheese (Figure 12), thick bands of’og-casein and B— casein were observed. After 2 weeks (Figure 13), the amount of og-casein and B-casein started to decrease. From 1 month to 4 months (Figures 14, 15, 16, and 17), a significant level of a.-casein decreased, B-casein also decreased, but not as much as that observed for a.-casein, and low molecular fraction bands (<24kDa) became stronger. The same amount of protein (65ug) was applied in every lane to compare the effect of fat levels (data not shown). One day after cheese production, there was no difference among cheeses with six different fat levels. In 4 month old cheeses, the fat level affected the breakdown of proteins. The degradation of a.-casein was slower in the low fat cheeses (12.6% and <0.l%), than in the higher fat cheeses. The slow proteolysis could be explained by the low moisture in.non-fat solids of these cheeses (Table 3). The lower moisture in non-fat solids lead to a decrease Molecular Weight — —66 -45 —34.7 (g— . B- - ~-24 -18.4 .5 -14.3 (kDa) Figure 12. The SDS~PAGE of the Cheddar cheeses with various fat levels 1 day after cheese manufacturing. Lanes: 1) 34.3% fat cheese; 2) 31.5%; 3) 26.8%; 4) 20.5%; 5) 12.6%; 6) <0.l%; 7) molecular weight standards. 54 s- caseins _. g- casein— Figure 13. The SDS—PAGE of the Cheddar cheeses 2 weeks after Cheese manufacturing. Lanes: 1) 34.3% fat cheese; 2) 31.5%; 3) 26.8%; 4) 20.5%; 5) 12.6%; 6) <0.l%; 7) molecular weight standards. 55 (Is- caseins _ Figure 14. The SDS—PAGE of the Cheddar cheeses 1 month after cheese manufacturing. Lanes: 1) 34.3% fat cheese; 2) 31.5%; 3) 26.8%; 4) 20.5%; 5) 12.6%; 6) 0.01 1 day 5.51"“1 5.51M 5.1“ 4.4“0 4.1““ 3.3° (1.13) (0.93) (1.00) (0.87) (0.87) (1.13) 2 weeks 9.3““ 8.9“ 8.5J 6.8’“1 6.31'“ 5.61”“ (0.27) (0.53) (0.85) (0.59) (0.27) (0.94) 1 month 13.1"“h 13.2“”h 11.9h 10.01 9.7L“ 8 3"1‘ (0.89) (2.20) (1.83) (1.45) (0.99) (0.80) 2 months 6.9“ 16.6“ 14.7“ 13.9““‘ 13.0“"h 9.7‘“1 (0.93) (1.81) (1.40) (1.70) (1.05) (0.52) 3 months 19.0""c 18.6‘”c 17.6““ 16.6“ 15.2“ 12.3h (1.19) (2.11) (0.83) (0.86) (0.91) (0.64) 4 months 19.5b 21.3“ 19.4b 17.8““ 16.4“ 14.6“” (1.60) (2.70) (2.23) (1 22) (1.24) (1.37) 1Means with standard deviations in brackets. n=4. Nitrogen % of total cheese nitrogen. Means in the same column as well as the same raw with different superscripts are different (P<0.05). 62 25.0 r T ' I T I ' l ' l f T ' I N .O O I 1 15.0 10.0 WATER SOLUBLUE NITROGEN (%) 5.0 0'00 4 L L 8 L 12 16 WEEKS \ Figure 18. Water soluble nitrogen of Cheddar cheeses as influenced by ripening time and fat level. Fat level of cheeses: 34.3% (0), 31.5% (D), 26.8% (A), 20.5% (o), 12.6% (<), and <0.1% (v). 63 ripening) . Farkye and Fox (1991) showed that the WSN of full— fat stirred-curd Cheddar increased from 6% at one day to 20.61% after three months of ripening at 10°C. CONC LUS ION Both the fat levels of cheese and the ripening periods significantly affected the melting properties of cheeses manufactured. The higher fat cheeses (from 20.5% to 34.3% fat) had higher meltability' than the lower fat cheeses. Cheeses with higher fat‘ content also improved their meltability during ripening, particularly during the first two weeks of ripening; however the cheeses with lower fat content (12.6%, and <0.1% fat) did not improve their meltability through the ripening period as the higher fat cheeses did. SDS—PAGE results showed that degradation of caseins, particularly the (g~caseins, was slower in the lower fat cheeses (12.6%, and <0.l% fat) compare to the higher fat cheeses (from 20.5% to 34.3% fat). The results of WSN also showed slower protein degradation in the lower fat cheeses. The non—destructive dynamic testing was sensitive for detecting the thermal change of Cheese’s rheological properties. All cheeses became softer as the temperature increased, but at approximately 60 to 70°C they became harder as the temperature increased to 85%L This thermal transition was more significant as the fat content of the cheeses decreased. The minimum value of complex modulus (G') of the 64 65 cheese correlated with the meltability obtained by the Arnott test, a traditional oven heating method. From this research it can be summarized that the reasons why reduced-fat Cheddar cheese does not melt well are the following: 1. slow degradation of og-caseins, 2. hydrophobic interaction between hydrolyzed. protein, and 3. chemical composition (high. protein, 10W' moisture in the non-fat substance) . To control meltability of Cheddar cheese, the fat level of the cheese, ripening period, (It-caseins degradation, and moisture are important. FUTURE RESEARCH (1)To measure quantitatively the change of the a.-casiens during ripening, which will make clear the relationship between the (Is-casein degradation and meltability of Cheddar cheese. (2)To clarify the selection of milk-clotting enzymes or starter culture which have strong (lg—casein proteolysis activity, and to apply the enzymes or starter to reduced-fat cheese production. (3)To select cheese manufacturing conditions which will produce high-moisture reduced-fat cheese in order to improve meltability and to stimulateeog-casein degradation. (4)To analyze the relationship between hydrophobic interaction and cheese meltability; To study the utilization of some substance such as emulsifier which binds to hydrophobic groups of caseins which inhibit hydrophobic interaction between protein molecules. 66 LIST 0? REFERENCES Amantea, G. F., and B. J. Skura, and S. Nakai. 1986. Culture effect on ripening characteristics and rheological behavior of Cheddar cheese. J. Food Sci. 51:912. AHA. 1986. Dietary guidelines for healthy adult Americans. Am. Heart Assn. Circulation. 74:1465A. Arnott, D. R., H. A. Morris, and W.B. Combs. 1957. Effect of certain chemical factors on the melting quality of process cheese. J. Dairy Sci. 40:957. Banks, J. M., E. Y. Brechany and W. W. Christie. 1989. The production of low fat Cheddar-type cheese. J. Soc. Dairy Tech. 42:6. Bertola, N. C., A. E. Bevilacqua, and N. E. Zaritzky. 1992. Proteolytic and rheological evaluation of maturation of Tybo Argentiono cheese. J. Dairy Sci. 75:3273. Bassette, R. and J. S. Acosta. 1988. Composition of milk products. Page 39 in FUndamentals of Dairy Chemistry. N. P. Wong, ed. van Nostrand Reinhold, New York. NY. Best, D. 1991. The challenges of fat substitution. Prepared Foods, May:72. Bever, M. B. 1986. Encyclopedia of Materials Science and Engineering. 4:2939. The MIT press, Cambridge, MA. Bringe, N. A., and J. E. Kinsella. 1985. Forces involved in the enzymatic and acidic coagulation of casein.micelles. Page 159 in Developments in Food Proteins-5. B. J. Hudson, ed. Elsevier.Applied Science, Barking, England. Campbell, J. R., and R. T. Marshall. 1975. The Science of Providing Milk for Man. McGraw Hill, New York, NY. Chen, C. M., M. E. Johnson, and N. F. Olson. 1992a. Optimizing manufacturing parameters in 33% reduced«fat Cheddar cheese. J. Dairy Sci. 75 (Suppl. 1): 104. Chen, C. M., A. C. Macedo, M. E. Johnson, and N. F. Olson. 1992b. Analysis of four starter cultures in the ripening 67 68 of reduced-fat Cheddar cheese. J. Dairy Sci. 75 (Suppl. 1): 103. Creamer, L. K., and.N. F. Olson. 1982. Rheological evaluation of maturing Cheddar cheese. J. Food Sci. 47:631. de Jong, L. 1978. The influence of the moisture content on the consistency and protein breakdown of cheese. Neth. Milk Dairy J. 32:1. DiMatteo, M., G. Chiovitti, and F. Addeo. 1982. variation in the composition of Mozzarella cheese during storage. Sci. Tec. Latt. Cas. 33:197. El—Neshawy, A. A., A. A. Abdel Baky, A. M. Rabio and M. M. Ashour. 1986. An attempt to produce low fat Cephalotyre (Ras) cheese of acceptable quality. Food Chem. 22:123. Emmons, D. B., M. Kalab, and E. Larmond. 1980. Milk gel structure. x. Texture and microstructure in Cheddar cheese made from whole milk and from homogenized low-fat milk. J. Texture Stud. 11:15. Farkye, N. Y., and P. F. Fox. 1991. Preliminary study on the contribution. of plasmin. to proteiolysis in_ Cheddar cheese: cheese containing plasmin inhibitor, 6- aminohexanoic acid. J. Agric. Food Chem. 39:786. Fox, P. F. 1989. Proteolysis during cheese manufacture and ripening. J. Dairy Sci. 72:1379. Grappin, R., T. C. Rank, and N. F. Olson. 1985. Primary proteolysis of cheese proteins during ripening. A review. J. Dairy Sci. 68:531. Green, M. L., A. Turvey, and D. G. Hobbs. 1981. Development of structure and texture in Cheddar cheese. J. Dairy Res . 48:343. Grundy, S. M. 1991. Recent nutrition research: Implications for foods of the future. Annals Med. 23:187. Gwinn, R. P. 1990. The New Emcyclopedia Britannica. 8:700. Encyclopedia Britannica, Inc., Chicago,IL. Jameson, G. W. 1990. Cheese with less fat. Aust. J. Dairy Tech. 45:93. Johnson, M. E., and C. Chen. 1991. Making quality reduced-fat cheese. CDR Cheese Research and Technology. Conference Proceedings March 6-7, 1991. p35. 69 Jones, L. 1992. Low-fat cheese. JNutrition. Action. Health Letter. April:10. Kalab, M., H. W. Modler, M. Caric, and S. Milanovic. 1991. Structure, meltability, and firmness of process cheese containing white cheese. Food Structure. 10:193. Kalab, M., J, Yun, and S. H. Yiu. 1987. Textural properties and microstructure of process cheese food rework. Food Microstruc. 6:181. Keller, B., N. F. Olson, and T. Richardson. 1974. Mineral retention and rheological properties of Mozzarella cheese made by direct acidification. J. Dairy Sci. 57:174. Kindstedt, P. S., and.L. J. Kiely. 1992. Revised protocol for the analysis of melting properties of Mozzarella cheese by helical viscometry. J. Dairy Sci. 75:676. Kindstedt, P. S., J. K. Pippe, and C. M. Duthie. 1989. Measurement of Mozzarella cheese melting properties by helical viscometry. J. Dairy Sci. 72:3117. Konstance, R. P., and V; H. Holsinger. 1992. Development of rheological test.methods for cheese. Food.Tech. January: 105. Kosikowski, F. V; 1982. Cheese and fermented.milk foods. 2nd ed. Edward Bros., Inc., Ann Arbor, MI. Kuchroo, C. N., and P. F. Fox. 1982. Soluble nitrogen in Cheddar cheese: comparison of extraction procedures. Milchwissenschaft 37:331. Laemmli, U. K. 1970. Cleavage of structural proteins during assembly of head of bacteriophage T4. Nature 227:680. Law, J., G. F. Fitzgerald, C. Daly, P. F. Fox, and N. Y. Farkye. 1992. Proteolysis and flavor development in Cheddar cheese made with the single starter strains Lactococcus lactis ssp. lactis ‘UC317 or' Lactococcus lactis ssp. cremoris HP. J. Dairy Sci. 75:1173. Lawrence, R. C., L. K. Creamer, and J. Gilles. 1987. Texture development during cheese ripening. J. Dairy Sci. 70:1748. Lawrence, R. C., J. Gilles and L. K. Creamer. 1983. The relationship between cheese texture and flavor. N.Z. J. Dairy Sci. Tech. 18:175. 70 Lee, C., E. M. Imoto, and C. Yun. 1978. Evaluation of cheese texture. J. Food Sci. 43:1600. Marshall, R. T., ed. 1992. Standard Methods for the Examination of Dairy Products. 16th ed..Amm Publ. Health Assoc., Inc., Washington, DC. Masi, P. and F. Addeo. 1986. An examination of some mechanical properties of a group of Italian cheese and their relation to structure and conditions of manufacture. J. Food Eng. 5:217. Lau, K. Y., D. M. Barbano, and R. R. Rasmussen. 1991. Influence of pasteurization of milk on protein breakdown in Cheddar cheese during aging. J. Dairy Sci. 74:727. Nolan, E. J., V. H. Holsinger, and J.J. Shieh. 1989. Dynamic rheological properties of natural and imitation Mozzarella cheese. J. Texture Stud. 20:179. Nolan, E. J., J. J. Shieh, and V. H. Holsinger. 1990. A comparison.of some rheological properties of Cheddar and pasteurized process American cheese. Page 370 in Engineering and Food: Physical Properies and Process Control Vol.1. W. E. L. Spiess, and H. Schubert, ed. Elsevier Applied Science Publishers, London. Oberg, C. J., R. K. Merrill, R. J. Brown, and G. H. Richardson. 1992. Effects of ndlkrclotting enzymes on physical properties of Mozzarella cheese. J. Dairy Sci. 75:669. Oberg, C. J., R. K. Merrill, L. V; Moyes, R. J. Brown, and G. H. Richardson. 1991a. Effects of Lactobacillus helveticus culture on physical properties. J. Dairy Sci. 74:4101. Oberg, C. J., A. Wang, L. V. Moyes, R. J. Brown, and G. H. Richardson. 1991b. Effects of proteolytic activity of thermolactic cultures on physical properties of Mozzarella cheese. J. Dairy Sci. 74:389. Olson, N. F. 1991. A report from the conference on fat and cholesterol reduced foods. Dairy Field. Junez34. Olson, N; F., and.M. E. Johnson. 1990. Light cheese products: Characteristics and economics. Food Tech. October:93. Olson, N. F., and D. L. Nelson. 1980. A new method to test the stretchability of Mozzarella cheese on pizza. Proc. 17th Ann. Marschall Invit. Ital. Cheese Sem. Madison, WI. 71 Olson, N. F., and.W. V; Price. 1958. A melting test for pasteurized process cheese spreads. J. Dairy Sci. 41:999. Park, J., J. R. Rosenau, and M. Peleg. 1984. Comparison of four procedures of cheese meltability evaluation. J. Food Sci. 49:1158. Rank, T. C., and R. Grappin, and N. F. Olson. 1985. Secondary proteolysis of cheese during ripening: A review. J. Dairy Sci. 68:801. Rfiegg, M., P. Eberhard, L. M. Popplewell, and M. Peleg. , 1991. Melting properties of cheese. In "Bulletin of the International Dairy Federation No.268,1991.' Chapter 6 p.36. Rfiegg, M. and U. Moor. 1988. Softening and dropping point temperatures of semi-hard and hard cheese varieties. Milchw. Forsch. 17: 69. Savello, P. A., C. A. Ernstrom, and M. Kalab. 1989. Microstructure and meltability of model process cheese made with rennet and acid casein. J. Dairy Sci. 72:1. Simard, R. E. 1991. Evaluation of lowfat cheese problems. Cheese Research and Technology. Conference Proceedings March 6-7, 1991. p37. Shoemaker, C. F., J} Nantz, S. Bonnans, and A, C. NOble. 1992. Rheological characterization of dairy products. Food Tech. January:98. Taneya, S., T. 'Izutsu, and T. Sone. 1979. Dynamic viscoelasticity of natural cheese and processed cheese. In “Food Texture and Rheology" ed. Sherman, P. p.39. Academic Press, London. Tatsumi, K, T. Nishiya, H. Yamamoto, K. Ido, N. Hanawa, K. Itoh, and K. Tamaki. 1989. Functional properties of cheese cooked without emulsifying salts in a twin screw extruder. Snow Brand Milk Products Annual Reports. 88:73 . Tieleman, A. E. and J. J. Warthesen. 1991. Comparison of three extraction procedures to characterize Cheddar cheese proteolysis. J. Dairy Sci. 74:3686. Tunick, M. H., J. J. Basch, B. E. Maleeff, J. F. Flanagan, and V. H. Holsinger. 1989. Characterization of natural and imitation Mozzarella cheeses by differential scanning calorimetry. J. Dairy Sci. 72:1976. 72 Tunick, M. H., K. L. Mackey, P. W. Smith and V. H. Holsinger. 1991. Effects of composition and storage on the texture of Mozzarella cheese. Neth. Milk Dairy J. 45:117. Tunick, M. H., E. L. Malin, J. J. Shieh, P. W. Smith, K.L. Mackey, and V. H. Holsinger. 1992. Comparison of low-fat and full-fat Mozzarella cheeses prepared from homogenized milk. J. Dairy Sci. 75(Suppl. 1): 130. Tunick, M. H., E. J. Nolan, J. J. Shieh, J. J. Basch, M. P. Thompson, E. E. Maleeff, and V. H. Holsinger. 1990. Cheddar and Cheshire cheese rheology. J. Dairy Sci. 73:1671. Walstra, P., and T. van Vliet. 1982. Rheology of cheese. Int. Dairy Fed. Bull. Doc. 153. MICHIGQN STQTE UNI (I) 31i'fla30m1' 051 will)? *