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* Department of Food Science, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden
Department of Food Science, Danish Institute of Agricultural Sciences, Research Centre Foulum, DK-8830 Tjele, Denmark
Swedish Dairy Association, SE-223 63 Lund, Sweden
Department of Food Science, The Royal Veterinary and Agricultural University (KVL), DK-1958 Fredriksberg C, Denmark
1 Corresponding author: Anna.Wedholm{at}lmv.slu.se
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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S1-, ß-, and 
-casein (CN), -lactalbumin, and ß-lactoglobulin (LG) A and B were determined by reversed phase liquid chromatography. Cows of Swedish breeds were genotyped for genetic variants of ß- and -CN. Model cheeses were produced from individual skimmed milk samples and the milk clotting properties were evaluated. More than 30% of the samples were poorly coagulating or noncoagulating, resulting in weak or no coagulum, respectively. Poorly and noncoagulating samples were associated with a low concentration of -CN and a low proportion of -CN in relation to total CN analyzed. Furthermore, the -CN concentration was higher in milk from cows with the AB genotype than the AA genotype of -CN. The concentrations of S1-, ß-, and -CN and of ß-LG B were found to be significant for the cheese yield, expressed as grams of cheese per one hundred grams of milk. The ratio of CN to total protein analyzed and the ß-LG B concentration positively affected cheese yield, expressed as grams of dry cheese solids per one hundred grams of milk protein, whereas ß-LG A had a negative effect. Cheese-making properties could be improved by selecting milk with high concentrations of S1-, ß-, and -CN, with high -CN in relation to total CN and milk that contains ß-LG B.
Key Words: cheese yield • milk protein composition • milk clotting properties • poorly coagulating milk
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Genetic polymorphism of milk proteins has been associated with composition, production traits, and technological properties of milk. Concentration of ß-LG is higher in milk with the AA genotype than with AB or BB (McLean et al., 1984; Ng-Kwai-Hang et al., 1987; Graml et al., 1989), which results in a lower CN number in AA milk (Lunden et al., 1997; Schaar, 1984; van den Berg et al., 1992). Several workers have reported significantly higher concentrations of -CN in milk with the B allele (McLean et al., 1984; van den Berg et al., 1992; Bobe et al., 1999). The B allele of -CN has also been associated with improved milk clotting properties (Schaar, 1984) and a higher cheese yield (Schaar et al., 1985), whereas the E allele has been related to unfavorable milk clotting properties (Ikonen et al., 1999a; Caroli et al., 2000). Many studies have been carried out on the effects of genetic polymorphism on milk clotting properties and cheese yield (Ng-Kwai-Hang, 1998), whereas studies evaluating the actual milk protein composition in relation to cheese-making properties of individual milk samples are less frequent (Auldist et al., 2004). The CN to total protein ratio has decreased in Swedish bulk milk during the last decades, making it less suitable for cheese making (Lindmark-Månsson et al., 2003). Deteriorating trends like this may also occur in other countries, and therefore, it is important to identify suitable quality markers for cheese milk. In Sweden, milk is currently graded according to concentrations of total protein and milk fat. It could be an economical advantage to the dairy industry if a specific marker could be used to identify milk suitable for cheese making; that is, good milk clotting properties and high cheese yield. The object of this study was thus to evaluate the effect of protein composition on milk clotting properties and cheese yield from milk of individual cows.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Milk Composition
Samples of fresh milk were analyzed for concentration of milk fat, lactose, urea, and pH by a Milkoscan FT 6000 (A/S N., Foss Electric, Hillerød, Denmark) and for somatic cells using a Fossomatic 5000 (A/S N., Foss Electric) at Steins Laboratory (Holstebro, Denmark). Calcium content was determined using atom absorption spectrophotometry after drying to ash at 525°C for 6 h, followed by dissolving in acid and dilution in lanthanchloride solution. Concentrations of major milk proteins were determined using the reversed phase liquid chromatography (RP-HPLC) method modified by Wedholm et al. (2006) according to Bordin et al. (2001). No measurement was made for S2-CN. Concentration of total protein analyzed was defined as the sum of the concentrations of S1-CN, ß-CN, -CN, ß-LG A, ß-LG B, and -LA. Concentration of total CN analyzed comprised the sum of the concentrations of S1-CN, ß-CN, and -CN, and concentration of total whey protein analyzed comprised the sum of the concentrations of ß-LG A, ß-LG B, and -LA. Proteolysis in individual pasteurized skimmed milk samples were determined as the level of free amino terminals using the fluorescamine method modified for milk samples (Larsen et al., 2004). Level of free amino terminals was expressed as leucine equivalents (in mM) according to a standard curve for leucine.
Genotyping for - and ß-CN Genetic Variants
Blood samples were collected from SRB and SLB and the DNA was genotyped for genetic variants of ß- and -CN using pyrosequencing, as described by E. Hallén, T. Allmere, J. Näslund, A. Andrén, and A. Lundén, Departments of Food Science and Animal Breeding and Genetics, Swedish Univ. Agric. Sci., Uppsala, Sweden; personal communication).
Cheese Making
The model cheeses were produced from skimmed milk to reduce the number of variables influencing cheese yield. After 2 d of cold storage (4°C), individual milk samples were preheated to 40°C, defatted, and heated in a pilot plate heating apparatus (72°C for 15 s), as described by Allmere et al. (1998). Four liters of skimmed milk was inoculated with a commercial starter culture (0.1 g/L of Lactobacillus helveticus 174 and 0.1 g/L of Probat 404, Danisco, Sweden), and incubated at 30°C for 30 min. This was followed by addition of chymosin (1.25 mL/L of Chy-Max Plus, 190 International Milk Clotting Units/mL, Christian Hansen A/S, Denmark) and gentle stirring. After 30 min at 30°C, the gel formed was cut into 2-cm cubes. To allow syneresis, the curd was incubated at 50°C for another 30 min during gentle stirring. The whey was removed and the curd was pressed (0.04 kg/cm2) for 20 h at room temperature. After 2 wk of storage at 10°C, the cheeses were weighed to obtain the yield. To obtain dry weight from individual cheeses, 2 to 3 g was taken from the interior of the cheeses, grated, and mixed with a fixed amount of sand. Cheese samples were incubated at 105°C overnight and then placed in a desiccator for 1 h before weighing. The cheese yield was expressed in 3 different ways: in relation to amount of cheese milk used (as g of cheese/100 g of milk or as g of dry cheese solids/100 g of milk) or as a transition number (as g of dry cheese solids/100 g of milk protein), as recommended by Emmons (1993).
Rheological Measurements
Immediately after rennet addition, a 12-mL sample was transferred to the C25 measuring cup of a Bohlin VOR Rheometer (Malvern Instruments, Nordic AB, Uppsala, Sweden). An oscillating technique was used with a frequency of 1 Hz and the torsion bar 2 g·cm. Measurement temperature was 25°C. The elastic (storage) modulus, G', was determined at a constant strain of 0.0412 and plotted against time to obtain a gelation profile of the curd. Measurements were carried out for 15 min. The milk clotting time (MCT) was recorded as the time in minutes, from chymosin addition to the beginning of curd formation, to compare the clotting rate among the milk samples, and G' (Pa) at 15 min (G'15) was recorded to compare curd firmness.
Statistical Analysis
Multivariate Regression.
Partial least square regression analysis (PLS1) was carried out using the software Unscrambler (version 9.0, CAMO ASA, Oslo, Norway). The x-variables consisted of the descriptive variables of breed (SRB, SLB, and SDM), stage of lactation (early, mid, late, and very late), lactation number (1, 2, 3, and 4,) and sample occasion (1, 2, and 3), and the continuous regressors of milk fat, total CN analyzed, -CN, S1-CN, ß-CN, ß-LG A, ß-LG B, total ß-LG, LA, total whey protein analyzed, total protein analyzed (all g/100 g of milk), total CN per total protein analyzed, -CN per total CN analyzed, S1-CN per total CN analyzed, ß-CN per total CN analyzed, ß-LG A per total whey protein analyzed, ß-LG B per total whey protein analyzed, -LA per total whey protein analyzed, lactose (g/100 g), urea (mM), SCC (log/mL), free amino terminals (mM of leucine), and calcium concentration (g/100 g). The response variables (y-variables) analyzed were cheese yield expressed as grams of cheese per one hundred grams of milk, as grams of dry cheese solids per one hundred grams of milk, and as grams of dry cheese solids per one hundred grams of milk protein. Discriminant PLS1 was carried out to compare the milk composition of poorly coagulating and noncoagulating milk with normally coagulating milk (see definitions in first section of Results). Standardized (centered: µ = 0, and normalized: 1/SD) variables and full cross validation was used.
ANOVA.
The GLM procedure from SAS (Version 8e, SAS Institute Inc., Cary, NC) was used to calculate least squares means and standard errors and for pairwise testing of significant differences in concentration of -CN among the genotype classes -CN-AA, AB, and AE. The model contained the fixed effects of - and ß-CN genetic variants, breed, sampling, stage of lactation and lactation number. Interactions between main effects were found to be insignificant and therefore were excluded from the statistical model. Concentration of total CN analyzed was included as covariate in the statistical model to adjust the main effects before ANOVA was accomplished. Significant differences between least squares means were evaluated based on F-values, using the option PDIFF. The GLM procedure was also used to test the overall differences in cheese yield between poorly coagulating and normally coagulating milk. The statistical model used included the fixed effect of coagulation class (0 or 1).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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). Milk samples that started coagulation within 15 min and had firm curd at cutting (30 min) were referred to as well-coagulating milk. Milk samples were assigned to the low clotting rate class if they did not start coagulation within 15 min, but formed a firm curd at cutting. Poorly coagulating milk had a pronounced weaker curd at cutting and a G'15 value of less than 15 Pa. Milk that did not start coagulation within 30 min was referred to as noncoagulating milk. Table 2 shows means and standard errors of cheese yield obtained from poorly coagulating and well-coagulating milk. Average cheese yields were higher for cheeses prepared from normally coagulating than from poorly coagulating milk, irrespective of the cheese yield formula used, but the difference between coagulation groups was not statistically significant (Table 2). Coagulation groups tended to differ significantly, however, when yield was expressed as grams of cheese per one hundred grams of milk (P = 0.055).
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and 4. Results from the PLS1 analyses are presented in Figures 1 and 2. Concentrations of lactose, urea, free amino terminals, pH, and SCC did not significantly affect milk clotting properties or cheese yield and did not improve the multivariate regression model when included. These variables were therefore excluded from the final model. The cheese yield, expressed as grams of cheese per one hundred grams of milk (Figure 1a), was improved at increasing concentrations of milk fat (P < 0.01), -CN (P < 0.001), S1-CN (P < 0.001), ß-CN (P < 0.001), total CN analyzed (P < 0.001), ß-LG B (P < 0.01), total ß-LG (P < 0.001), total protein analyzed (P < 0.001), and total whey protein analyzed (P < 0.001). The same list of variables was also important for the cheese yield when expressed as grams of dry cheese solids per one hundred grams of milk (results not shown). The significant effect of calcium concentration on cheese yield (Figure 1a,b) was very low. Cheese yield, in relation to exploitation of milk protein for cheese making (expressed as g of dry cheese solids/100 g of milk protein), was improved at increasing concentrations of ß-LG B (P < 0.001), total CN per total protein analyzed (P < 0.01), ß-LG B per total whey protein analyzed (P < 0.001), and -LA per total whey protein analyzed (P < 0.01). The ß-LG A concentration and the ß-LG A to total whey protein ratio analyzed were negatively associated with grams of dry cheese solids per one hundred grams of milk protein (P < 0.01 and P < 0.001, respectively). Cheese yield, expressed as grams of cheese per one hundred grams of milk (Figure 1a), was further positively associated with late lactation (P < 0.01) and lactation number 1 (P < 0.05), but negatively associated with early lactation (P < 0.01) and lactation number 4 (P < 0.01). Cheese yield was also positively associated with sampling occasion number 1 (in September; Figure 1a). No significant differences in cheese yield or milk clotting properties between the investigated breeds of SRB, SLB, and SDM were observed. Poorly coagulating and noncoagulating milk was associated with lower concentration of -CN (P < 0.001) and -CN per total CN analyzed (P < 0.001; Figure 2). Poorly coagulating and noncoagulating milk were most frequent within lactation number 1 (P < 0.05).
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) and in cheese yield expressed as grams of dry cheese solids per one hundred grams of milk protein (Figure 1b). This was indicated by the correlation coefficient (R2) between measured and predicted y-variables, which were less than 0.50 (Figure 1b and 2). A larger amount of the total variation could be explained by the model when describing variations in cheese yield expressed as grams of cheese per one hundred grams of milk with R2 = 0.65 (Figure 1a).
Influence of -CN Genotype on -CN Concentration and Milk Clotting Properties
Table 5 shows least squares means and standard errors of concentration of -CN in milk from SRB and SLB cows with the -CN AA, AB, and AE genotypes. Concentration of -CN was significantly higher in milk with the AB genotype compared with milk with the AA genotype (P < 0.05). No difference in -CN concentration was found between the AB and AE genotypes or between the AA and AE genotypes. Furthermore, no effect of ß-CN genotype on -CN concentration was found. Comparing poorly and noncoagulating milk with well-coagulating milk, the AE genotype was more frequent within the poorly and noncoagulating group (Table 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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It has been shown that milk with impaired clotting properties was not improved by mixing it with an equal amount of well-coagulating milk (Okigbo et al., 1985b). The same authors showed that the milk clotting properties of poorly coagulating milk were improved by addition of calcium chloride and by reduction of milk pH. However, the clotting properties were not improved to the same level as in well-coagulating milk. The association we found between poorly coagulating milk and lactation number 1 (Figure 2) is in line with the results of Schaar (1984). He observed that milk samples from cows within lactation number 1 and 2 formed weaker curds than milk from cows in later lactations and suggested that this effect could be due to differences in composition and distribution of milk salts.
We found that the -CN concentration and its proportion in relation to S1-CN and ß-CN were significantly lower in poorly coagulating and noncoagulating milk. This finding is in line with the results of St-Gelais and Haché (2005), who showed poorer milk clotting properties of milk enriched with ß-CN powder. They also found that cheeses produced from ß-CN enriched milk were harder. The higher -CN concentration found in milk from cows with the -CN AB genotype compared with the AA genotype (McLean et al., 1984; van den Berg et al., 1992; Bobe et al., 1999; Table 5) and the positive association between -CN concentration and well-coagulating milk indicate that the AB genotype can influence milk clotting properties, indirectly, via the -CN concentration. However, the higher frequency of -CN AE in poorly and noncoagulating milk (Table 5) suggests that functionalities of -CN, other than solely the concentration, can contribute to the milk clotting properties. The high frequency of -CN AE observed in poorly coagulating milk (Table 5) also supports the significant correlation between the -CN E allele and poor milk clotting ability reported by Ikonen et al. (1999a).
Noncoagulating milk has been observed in several milk clotting studies (Okigbo et al., 1985a; Ikonen et al., 1999a; Nsofor, 2000). It has been suggested that noncoagulating milk could be due to genetic parameters because high heritability estimates for milk clotting properties have been observed (Ikonen et al., 1999a). We found that the average calcium concentration among noncoagulating milk samples was 0.10 g/100 g of milk (results not shown); that is, below the average of all analyzed milk samples (0.12 g/100 g; Table 3). It is thus possible that the lower calcium concentration in combination with a low concentration of -CN (Figure 2) was responsible for the noncoagulating milk samples in this study. However, only 4 noncoagulating milk samples were observed (Table 1), which is not enough observations for a statistical evaluation. Hence, it would be interesting to evaluate milk composition in a substantial number of noncoagulating milk samples.
According to the literature, associations between milk clotting properties and cheese yield vary. Aleandri et al. (1989) and Martin et al. (1997) suggested that a firm curd at cutting was associated with high cheese yield, whereas Ikonen et al. (1999b) did not find a significant difference in yield between cheeses made from poorly coagulating and well-coagulating milk. Riddell-Lawrence and Hicks (1989) reported that curd-healing time affected cheese yield more than coagulum strength at cutting. However, it has also been suggested that curd firmness at cutting is important for the sensory attributes of cheeses. Johnson et al. (2001) reported that an increase in cheese moisture content, due to firmer curd at cutting, resulted in softer and smoother texture of reduced-fat Cheddar cheeses. In the present study, the difference (although not significant) in cheese yield between the poorly coagulating and the well-coagulating milk was more pronounced when expressing yield as grams of cheese per one hundred grams of milk than as grams of dry cheese solids per one hundred grams of milk (Table 2). This suggests that an increase in cheese yield due to a firmer curd at cutting could be associated with an increase in the water-holding capacity of cheeses prepared from well-coagulating milk, as reported by Johnson et al. (2001).
The significantly positive correlation between ß-LG B and cheese yield (Figure 1) was expected because it has been suggested that the B allele correlates with higher CN number (Schaar, 1984; van den Berg et al., 1992; Lunden et al., 1997) because of the lower concentration of ß-LG in this milk (McLean et al., 1984; Ng-Kwai-Hang et al., 1987; Graml et al., 1989). In our study, the CN to total protein ratio analyzed did not affect the cheese yield expressed as grams of cheese per one hundred grams of milk (Figure 1a), whereas the yield expressed as grams of dry cheese solids per one hundred grams of milk protein was affected (Figure 1b). The latter expression would thus be the best to explain the effect of CN to total protein ratio analyzed on cheese making properties of milk. The milk fat was important for the cheese yield even though the milk was defatted before cheese making. This was probably indirectly due to the strong significant correlation (P < 0.001; results not shown) between concentration of total protein and milk fat.
The small amount (less than 50%) of the total variation in milk clotting properties (Figure 2) and grams of dry cheese solids per one hundred grams of milk protein (Figure 1b) explained by milk protein composition implies that other variables not measured in our study contributed significantly to these traits. Auldist et al. (2004) evaluated the effect of milk composition on milk clotting properties from Friesian and Jersey dairy cows. They also found that milk protein composition only partly explained the total variation in milk clotting properties. However, the process from milk to cheese is very complex with several significant variables, which makes it difficult to find only one or a few markers for the cheese-making properties.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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-CN and a low proportion of -CN in relation to total CN analyzed were associated with poorly coagulating and noncoagulating milk. However, the analyzed milk protein composition only partly explained the total variation in milk clotting properties. Nevertheless, our results suggest that milk superior for cheese making is high in concentrations of S1-, ß-, and -CN, has high -CN in relation to total CN, and contains ß-LG B. Furthermore, a high concentration of CN to total protein analyzed was significant for the transfer of proteins from milk to cheese.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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-CN. At the same university, Toomas Allmere, Department of Food Sciences, is acknowledged for planning and supervision of this project, and Roger Andersson, Department of Food Sciences, for good advice regarding the statistical analyses. Gudrun Franzén and Lena Hagenvall are acknowledged for the milk sample collection. At the Danish Institute of Agricultural Sciences, Hanne Damgaard Poulsen, Department of Animal Health, Welfare and Nutrition is acknowledged for performing the calcium analyses and Stina Greis Handberg and Helle Louise Christensen, Department of Food Quality, for excellent technical assistance. The Chy-Max Plus was a kind gift from Marianne Harboe, Christian Hansen A/S, Denmark. The financial support from the Swedish Farmers’ Foundation for Agricultural Research, Danish Dairy Research Foundation, and the Innovation Law is gratefully acknowledged. Received for publication January 13, 2006. Accepted for publication March 12, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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-casein. J. Agric. Food Chem. 46:3004–3008.-casein E allele on clotting aptitude of Italian Friesian milk. Zootec. Nutr. Anim. 26:127–130.S1-casein locus in cattle. Genet. Sel. Evol. 21:547–554.-casein genetic variants and lactation number on the renneting properties of individual milks. J. Dairy Res. 51:397–406.-casein and ß-lactoglobulin on cheesemaking. J. Dairy Res. 52:429–437.-casein and ß-lactoglobulin in relation to milk composition and processing properties. Neth. Milk Dairy J. 46:145–168.This article has been cited by other articles:
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