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1 Minnesota-South Dakota Dairy Foods Research Center, Dairy Science Department, and
2 Department of Chemistry and Biochemistry, South Dakota State University, Brookings 57007
Corresponding author: Ashraf N. Hassan; e-mail: Ashraf.Hassan{at}sdstate.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Key Words: reduced-fat Cheddar cheese • proteolysis • exopolysaccharide-producing cultures
Abbreviation key: EPS = exopolysaccharide, FFC = full-fat control cheese, MNFS = moisture in the nonfat substance, RF-3534 = reduced-fat cheese made with the moderate ropy strain Streptococcus thermophilus CHCC 3534, RF-5842 = reduced-fat cheese made with the EPS-negative genetic variant Streptococcus thermophilus CHCC 5842, RFC = reduced-fat control cheese, RF-JFR1 = reduced-fat cheese made with the ropy culture Lactococcus lactis ssp. cremoris JFR1, RF-Slab = reduced-fat cheese made with the capsule-forming nonropy strain Streptococcus thermophilus Slab, WSN = water-soluble nitrogen
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Low- and reduced-fat cheeses are gaining popularity due to consumer awareness of the health benefits of low-fat diets. Low-fat cheeses typically have poor body, flavor, and functional properties (Mistry, 2001). The firm/rubbery texture of low-fat cheeses is due to the inadequate breakdown of casein (Mistry and Kasperson, 1998). To overcome rigidity in reduced-fat cheese, moisture level is increased by lowering cooking temperatures or draining whey at higher pH. However, such conditions result in a lower retention of chymosin and a negative influence on both flavor and texture of cheese (Mistry, 2001).
Exopolysaccharide (EPS)-producing cultures have been used in making fermented dairy products to improve their rheological properties, prevent syneresis, and replace stabilizers (Hassan et al., 1996; Adapa and Schmidt, 1998; Broadbent et al., 2003; Hassan et al., 2004). In addition, these cultures increase water retention and improve melting properties of low-fat Mozzarella cheese (Perry et al., 1998; Broadbent et al., 2003). In a current research project, several EPS-producing cultures have been used in making reduced-fat Cheddar cheese. These cultures produced reduced-fat cheese with physical characteristics similar to those of its full-fat counterpart (Awad et al., 2005). However, cheese made with some EPS-producing cultures developed bitterness after a few months of ripening. The objective of this work was to monitor proteolysis during ripening of reduced-fat Cheddar cheeses made with different EPS-producing and nonproducing cultures and to study relationships among moisture levels, chymosin activity, production of free amino acids, and accumulation of bitter peptides.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Cheese Making
Raw milk was obtained from the Dairy Research and Training Facility at South Dakota State University. Three replicates of Cheddar cheese were manufactured from standardized (reduced-fat, 2% or full-fat, 3.6%) pasteurized milk (heated to 63°C for 30 min and cooled to 31°C). Cheese milk (100 kg) was assigned to 2 double-O cheese vats (Kusel Equipment Co., Watertown, WI). The following 6 treatments of cheese were made: 1) FFC = full-fat cheese made using the commercial Cheddar starter culture (DVS 850; 0.015% wt/ wt); 2) RFC = reduced-fat cheese made using the commercial Cheddar starter culture (DVS 850; 0.015% wt/ wt); 3) RF-JFR1 = reduced-fat cheese made with the ropy strain Lactococcus lactis spp. cremoris JFR1 (2% vol/wt); 4) RF-Slab = reduced-fat cheese made with a capsule-forming nonropy Streptococcus thermophilus (0.4% vol/wt) plus the commercial culture (0.011% wt/ wt); 5) RF-3534 = reduced-fat cheese made with EPS-producing Streptococcus thermophilus CHCC 3534 (0.4% vol/wt) plus the commercial culture (0.011 % wt/ wt); and 6) RF-5842 = reduced-fat cheese made with the EPS-negative genetic variant of CHCC 3534 (Streptococcus thermophilus CHCC 5842; 0.4% vol/wt) plus the commercial culture (0.011% wt/wt). The inoculum size was selected, based on the preliminary experiment data, to give the same acidification rate and cheese making time (5 h) in all treatments.
Cultures were added at 31°C to milk that was then ripened for 1 h. A 0.01% (vol/wt) chymosin (Chymax, Chr. Hansen) was added to clot milk in 30 min. The coagulum was cut and cooked to 39°C over 30 min and held at this temperature for 30 more minutes. After whey drainage, the curd was cheddared and then milled when the pH reached 5.4. Curd was salted at 1.7% in 3 equal applications over 15 min. The curd was hooped in rectangular blocks, pressed overnight at 2.5 kg/cm2, vacuum-packed, and ripened at 4°C for 6 mo.
Chemical Composition of Cheese
Cheese (1 d old) was analyzed for moisture by the oven method (method 926.08; AOAC, 2003), salt by chloride analyzer (model 926, Nelson Jameson Inc., Marshfield, WI), fat by Mojonnier (method 933.05; AOAC, 2003), and total protein by macro-Kjeldahl (method 920.123; AOAC, 2003). The pH was measured in a slurry prepared by macerating 20 g of grated cheese in 20 mL of deionized water.
Cheese yield was expressed as the ratio mass between the curd obtained after pressing stage and the weight of milk.
Microbiological Analyses
Cheese samples (10 g) were homogenized for 4 min with 90 mL of a sterile 2% sodium citrate solution in a laboratory blender 80 Stomacher (Seward 400, London, UK) and serially diluted using sterile 0.05% peptone. Appropriate dilutions were plated on lactose M17 agar. Duplicate dishes were incubated at 25 or 45°C for enumeration of lactococci and streptococci, respectively.
Determination of Residual Chymosin Activity
The residual coagulant activity was determined by the method described by Hurley et al. (1999) with some modifications. The HPLC analysis was conducted using a Varian Prostar composed of 2 Prostar pumps and photo diode array detector. The peptides resulting from hydrolysis of the substrate by chymosin were separated on an RP-C18-Lichrospher analytical column (250 x 4.6 mm, 5 µm; Perkin Elmer, Norwalk, CT) equilibrated with solvent A (0.1% trifluoroacetic acid in water) and solvent B (0.1% trifluoroacetic acid in acetonitrile). A gradient was generated by increasing the concentration of solvent B as follows: 15 to 50% solvent B over 20 min, 50% solvent B for 5 min, 50 to 95% solvent B over 3 min, maintaining at 95% solvent B for 2 min, and finally returning to equilibration conditions over 3 min. The flow rate was 1 mL/min. The photo diode array detector was used to monitor analyses at 300 nm. Integration of HPLC spectra was achieved using Star Software (Varian Chromatography Systems, Walnut Creek, CA). The concentration of residual coagulant activity was expressed as rennet activity units per kilogram of cheese using a standard curve of chymosin activity.
Proteolysis Assessments
Water-soluble nitrogen and free amino acids determination.
Water-soluble nitrogen (WSN) in the fat-free cheese homogenates was determined according to the method developed by Kuchroo and Fox (1982). Free amino groups were measured using the cadmium-ninhydrin method described by Folkertsma and Fox (1992).
Gel Electrophoresis Analysis
Urea-PAGE was conducted on whole cheese samples using a Protean II vertical slab gel unit (BioRad Laboratories, Hercules, CA) as described by Andrews (1983). Bands were scanned and quantified using a computerized densitometer and Image Quant 3.3 software (GE Healthcare, Sunnyvale, CA).
Reverse Phase-HPLC Analysis
The profile of peptides in water-soluble extract (Kuchroo and Fox, 1982) were separated using the C18-Lichrospher analytical column (250 x 4.6 mm, 5 µm; Perkin Elmer). Samples were eluted with a 4-step linear gradient over a period of 75 min according to Awad et al. (1999). Separation was conducted at 21°C and peptides were monitored at 214 and 280 nm. The areas of the chromatograms were divided into 2 regions according to the peptide elution time: hydrophilic peptides (retention time <35 min) and hydrophobic peptides (retention time >35 min). These peak areas of the different regions were statistically analyzed to evaluate the influence of the EPS-producing cultures on the relative amounts of peptides.
Statistical Analyses
Data reported are the average of 3 measurements per replicate. Cheeses were made 3 times. The SAS statistical analysis software package (SAS Institute, 1999) was used for ANOVA using the GLM procedure. Differences were considered significant at P < 0.05.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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. In agreement with previous findings (Fenelon and Guinee, 1999; Guinee et al., 2000), decreasing the fat content of cheese milk resulted in an increase in cheese moisture and protein and a decrease in cheese yield, level of fat in DM, moisture in the nonfat substance (MNFS), and salt-in-moisture. Cheese made with the EPS-producing culture JFR1 had higher (P < 0.05) moisture and yield than did all other reduced-fat cheeses. Cheese made with the capsule-forming nonropy culture (Slab) had higher (P < 0.05) moisture and yield than those made with the moderately ropy culture of S. thermophilus (RF-3534) and its EPS-negative variant (RF-5842). The yield of RF-JFR1 was 7.8% higher than that of cheeses made with the EPS-nonproducing culture (RF-5842). Fat in DM was similar (P > 0.05) among all reduced-fat cheeses. The MNFS was higher (P < 0.05) in RF-JFR1 than in all other reduced-fat cheeses. It is always the objective of the cheese manufacturer to produce reduced-fat cheese with similar MNFS to that of the full-fat counterpart. This requires modifications in cheese-making protocol that are always associated with textural and flavor defects. Interestingly, the use of JFR1 produced reduced-fat cheese with similar MNFS to that of the full-fat type without the need for modifying the cheese-making procedure used in making the full-fat type.
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). A drop in pH of all cheeses was observed during the first month of ripening. The relatively high MNFS in FFC and RF-JFR1 might have induced excessive growth of starter organisms, resulting in the most significant reduction in pH. Under our cheese-making conditions, viscosity of the whey was not affected by using the ropy culture JFR1. Preliminary experiments showed that ropiness produced by JFR1 increased as the growth temperature decreased. The cooking temperature used in this study (39°C) was much higher than the optimum temperature for production of ropiness (around 28°C). This might explain why the viscosity of the whey was slightly increased when Cheddar cheese curd was cooked at a relatively lower temperature (35°C; Dabour et al., 2005).
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. The activity was 18.4 rennet activity units/kg in full-fat Cheddar cheese and was in the range (11.1 to 20.1) found by Hurley et al. (1999) in commercial Cheddar cheese. The chymosin activity in reduced-fat Cheddar cheeses ranged from 20.2 to 25.5 rennet activity units/kg, which was significantly higher than that found in the full-fat type. The amount of coagulant retained in cheese depends on several factors, such as the pH of curd at cutting and whey drainage, cooking temperature, and final moisture content of cheese (Fox and McSweeney, 1996). Because the same procedure was used in making reduced- and full-fat cheeses and there were no significant differences in the amount of rennet used or the pH at d 1 of manufacture among all treatments, the differences in residual chymosin activity could be related only to differences in the moisture level.
Viability of Starter in Cheese During Ripening
The mean population of starter cultures is shown in Table 2. Numbers of mesophilic bacteria at d 1 of manufacture were similar (P > 0.05) in all cheeses. However, after 1 and 2 mo of ripening, counts of lactococci were higher in RF-JFR1 than in all other cheeses. The high moisture and low salt-in-moisture in RF-JFR1 might have increased survival of lactococci during the first 60 d of ripening. A gradual decline in numbers of lactococci was seen after 60 d of ripening in all cheeses, resulting in about a 2-log reduction after 6 mo. The results were in agreement with those reported by Sallami et al. (2004), who found a decline in the viable counts of lactococci from approximately 109 at d 1 to 107 cfu/g after 6 mo of Cheddar cheese ripening. Midje et al. (2000) also reported that populations of starter cultures reached a maximum during Cheddar cheese manufacture and decreased thereafter.
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Proteolysis During Ripening
WSN.
The concentration of nitrogen soluble in water expressed as either a percentage of total nitrogen or grams of N/100 grams of cheese increased in all cheeses during ripening (Table 3). Among 6-mo-old cheeses, the WSN as a percentage of total N was highest in RF-JFR1 followed by FFC and RF-Slab, whereas the lowest levels were found in reduced-fat cheeses made with no EPS. The level of WSN as a percentage of total N increased with increasing MNFS and the ratio of the residual chymosin activity to cheese proteins (Table 1). Such factors were reported by other researchers to affect WSN as a percentage of total N (Fenelon et al., 2000). The soluble nitrogen expressed as grams of N/100 grams of cheese was higher in RF-JFR1 than in all other cheeses, which were not different.
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. The level of free amino groups significantly increased as ripening progressed. However, their release occurred at a much slower rate in RF-JFR1 than in all other cheeses. The Slab culture produced higher levels (P < 0.05) of free amino groups than all other cultures used in this study. Starter peptidases are mainly responsible for the production of free amino acids in cheese (Visser, 1977), and rennet and plasmin are not able to contribute to the further degradation of the larger peptides in the soluble nitrogen fraction (Berg and Exterkate, 1993). The high level of free amino acids produced by Slab along with the decline in their numbers during the first few weeks and the relatively sharp increase in pH of cheese made with this culture after 2 mo of ripening might indicate extensive autolysis.
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S1- and ß-caseins. The values of individual casein fractions were expressed as a percentage of the values of the corresponding casein in the same cheese at d 1. The relative proportions of the residual ß- and S1-caseins and the level of S1-1-casein as a percentage of the corresponding S1-caseins at d 1 are shown in Table 5. Generally, the trend observed with urea-PAGE was consistent with that for WSN data, with the overall level of proteolysis in RF-JFR1 being higher than in all other cheeses. The S1-casein was hydrolyzed more extensively than ß-casein during ripening. After 6 mo of ripening, the S1-casein was almost completely degraded in all cheeses. The intensity of the band corresponding to ß-casein decreased slightly throughout ripening with a concomitant increase in the bands corresponding to the 
-caseins (data not shown). Cheese RF-JRF1 showed the most significant reduction in the level of ß-casein (Table 5). Peptides produced by chymosin action on ß-casein have been widely reported as the major cause of bitterness in cheeses (Lemieux and Simard, 1992; Broadbent et al., 1998). No differences in the levels of -caseins were found among all cheeses (data not shown).
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S1-1 casein (f24-199) during the 2 mo of ripening was higher in RF-JFR1 than in all other cheeses, which could be due to its higher residual chymosin activity and moisture content. The concentration of S1-1 casein (f24-199) decreased after 60 d of ripening in all cheeses. It was reported earlier that the relationship between the amounts of S1-casein and S1-1 peptides in cheese is very weak, probably because S1-casein can be hydrolyzed to other products of low molecular weight (less than 11 kD; Marcos et al., 1979). In addition, S1-1 casein undergoes further degradation by rennet or other proteases (Grappin et al., 1985).
Changes in Peptide Profile During Cheese Ripening
The reverse phase-HPLC chromatograms of water-soluble peptides from cheeses during ripening are shown in Figure 2, panels A to F. The chromatograms were divided into 6 zones (I, II, III, IV, V, and VI), each of which contained one peak or more. Similar to previous findings for Cheddar cheese (Fenelon et al., 2000), the chromatograms for all cheeses showed age-related changes in the area and distribution of different peaks. This trend is consistent with the increase in WSN in all cheeses and suggests the progressive breakdown of casein by residual coagulant, plasmin, and microbial proteinases, resulting in the formation of peptides of different molecular masses and free amino acids. The total peaks area, and in particular, that of the early eluting peaks increased during storage. The late eluting peaks increased during the first 4 mo of ripening and decreased thereafter. The results indicated the accumulation of new proteolytic products and increasing contribution of bacterial enzymes with ripening time.
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), suggesting that peptides corresponding to these peaks were produced by proteinases from commercial culture used in all cheeses but RF-JFR1. The retention times of peaks I and II were very similar to those of aS1-casein (f 1-9) and aS1-casein (f 1-13) produced in Cheddar cheese by proteinases from Lactococcus (Singh et al., 1994).
At all stages of storage, the reverse phase-HPLC profile for the WSN of RF-JFR1 differed markedly from those of all other cheeses. The WSN of RF-JFR1 showed the greatest number and widest distribution of late-eluting peaks (retention time >45 min), denoted collectively as zones IV, V, and VI. Such peaks correspond to high molecular weight and hydrophobic peptides (Lemieux and Simard, 1992). The larger area of late-eluting peaks in RF-JFR1 (Figure 2C) might indicate accumulation of peptides produced by rennet (as indicated by the high level of WSN found in this cheese), which were not degraded further to peptides of lower hydrophobicity and molecular mass, due to the absence or weak starter culture peptidases (Lemieux and Simard, 1992; Awad et al., 2000). At the same time, the WSN of RF-JFR1 showed a lower concentration of early-eluting peaks, especially the peak with retention time of 18 min, denoted collectively as zone I. The total peak area in zones I and II increased during storage in all cheeses except RF-JFR1, which contained the lowest ratio of hydrophilic to hydrophobic peptides (Table 6). The WSN of RF-Slab (Figure 2D) showed a high number and wide distribution of the early and middle-eluting peaks (retention time < 35 min), which probably correspond to low molecular weight and hydrophilic peptides (Lemieux and Simard, 1992). The total area of late-eluting peaks (zones V and VI) and the ratio of hydrophobic to hydrophilic peptides were generally smaller in RF-Slab than in all other reduced-fat cheeses made with EPS-producing cultures after 60 d of ripening (Table 6). Cheese RF-JFR1 contained the highest levels of the late-eluting peptides and lowest ratio of hydrophilic to hydrophobic peptides among all cheeses. Bitter peptides are rich in hydrophobic amino acids residues (Lemieux and Simard, 1992) and exhibit higher retention times on reverse phase-HPLC columns (Cliffe and Law, 1990; Awad et al., 1999). In contrast, Lee and Warthesen (1996) found little correlation between retention time on the reverse phase-HPLC column and peptide hydrophobicity or suspected bitterness. The high residual chymosin activity and moisture content and low salt-in-moisture in RF-JFR1 might have increased the level of the hydrophobic peptides. Cell envelope proteinases from JFR1 could have accumulated bitter peptides (Broadbent et al., 2002).
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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FOOTNOTES |
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Current address: Department of Dairy Science and Technology, Faculty of Agriculture, Alexandria University, Egypt.
Received for publication May 1, 2005. Accepted for publication July 18, 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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= FFC (Full-fat control); 
= RFC (reduced-fat control); 
= RF-JFR1 (reduced-fat cheese made with the ropy culture Lactococcus lactis ssp. cremoris JFR1); 
= RF-Slab (reduced-fat cheese made with the capsule-forming nonropy strain Streptococcus thermophilus Slab); •= RF-3534 (reduced-fat cheese made with the moderate ropy strain Streptococcus thermophilus CHCC 3534); and 
= RF-5842 (reduced-fat cheese made with the exopolysaccharide-negative genetic variant Streptococcus thermophilus CHCC 5842).