Free Access
Publication ahead of print
Journal
Dairy Sci. Technol.
DOI https://doi.org/10.1051/dst/2010033
Published online 02 August 2010

© INRA, EDP Sciences, 2010

1. INTRODUCTION

The polymorphism at the caprine αs1-casein locus has received considerable interest over the past years due to its impact on quality and important technological properties of the milk. The basis for these compositional and technological variations is a highly variable content of αs1-casein of the milk caused by considerable differences in casein synthesis [25].

To date, as many as 18 different variants of caprine αs1-casein have been identified, each associated with a specific protein concentration in the milk [6, 27]. Based on the concentration of αs1-casein present in the milk, the variants are commonly organized into four classes: (1) “strong” variants (A, B1, B2, B3, B4, Bk, C, H, L and M) synthesized at a high level (3.5 g·L−1 per allele), (2) “intermediate” variants (E and I) synthesized at a intermediate level (1.1 g·L−1 per allele), (3) “weak” variants (F and G) synthesized at a low level (0.45 g·L−1 per allele) and (4) “null” variants (01, 02 and N) not associated with protein synthesis (for review see [23, 25]).

In addition to a higher content of αs1-casein, milks containing these “strong” variants (“strong” milks) are characterized by higher content of total protein, milk fat, calcium, higher Ca-ion activity, lower pH and smaller casein micelles when compared to their “intermediate”, “weak” or “null” counterparts. Moreover, “strong” milks have more favourable cheese making properties (rennet coagulation properties, cheese yield and quality) [9, 10, 29] and are less prone to lipolysis and off-flavours [8] than milks of other αs1-casein classes.

In Europe caprine milk is mainly used for cheese manufacture, and in most economically significant breeds frequencies of “strong” and “intermediate” αs1-casein alleles dominate (for review see [25]). The occurrence of “weak” and “null” variants is reported to be very low in most European breeds, except from the Spanish Canaria and the Italian Frisa breeds in which the gene frequency of αs1-casein “null” is 20% and 10%, respectively [5, 22]. Moreover, a frequency of 23% of the “weak” F allele has recently been observed in the Italian Garganica breed [2]. The situation regarding frequencies of αs1-casein variants in Norwegian caprine milk is strikingly different. In a study by Hayes et al. [18] where 39 SNPs of the 4 casein genes were typed in 436 bucks, the “null” variant, unique to the Norwegian goat population, was found at a very high frequency (0.72). This particular mutation arising from a deletion in exon 12 of the αs1-casein gene (CSN1S1) and referred to as the D allele (referred to as allele 1 of exon 12 in Hayes et al. [18]) was found to decrease contents of protein and fat of the milk [1, 11].

The aim of this study was to provide more in-depth analysis on how this Norwegian “null” variant of αs1-casein affects other aspects of milk composition (contents of protein, casein, calcium and magnesium, pH, Ca-ion activity), mean size of casein micelles and rennet coagulation properties.

2. MATERIALS AND METHODS

2.1. Milk samples

Milk samples were obtained from 254 goats from nine farms in three different geographical regions of Norway. Herds 1 (69 animals), 2 (74 animals) and 3 (12 animals) were located in the North; Herds 4 (20 animals) and 5 (12 animals) in the South-West; Herds 6 (20 animals), 7 (20 animals), 8 (19 animals) and 9 (8 animals) in the South-East. The goats, aged 1–5 years, were of the Norwegian Dairy Goat breed, a landrace of ancient origin.

Milk samples from the animals belonging to the Herds 1, 2, 6 and 7 were collected once. To study changes in milk composition and renneting properties with time, milk samples from the animals in Herds 3, 4, 5, 8 and 9 were repeatedly collected three or four times in intervals of 2 or 3 weeks.

A sample of 40 mL was taken from each goat during the morning milking. The samples were preserved by addition of 0.02% (w/v) sodium azide and defatted by centrifugation at 3000× g as previously described [12]. Measurements of pH, Ca-ion activity, mean micellar size and renneting properties and preparation of the non-casein nitrogen fraction were performed on fresh milk. The milk samples were kept at −20 °C for further use.

2.2. Milk composition

The levels of total nitrogen (TN) and non-casein nitrogen (NCN) were determined using the Kjeldahl method (Kjeltec 1035 Analyser, Tecator, Höganäs, Sweden). Casein nitrogen was calculated as the difference between TN and NCN. The content of crude protein (CP) and casein (CN) was obtained using the conversion factor of 6.38, for N to protein [20].

The NCN fraction was obtained from the skimmed milk by isoelectric precipitation of the caseins, essentially as described by Aschaffenburg and Drewry [4] using smaller volumes of milk and reagents and a premixed solution of acetic acid, sodium acetate and water (1:1:4 v/v). In brief, 4 g of milk were mixed with 6 g of the acetic acid-sodium acetate-water solution to obtain a pH of 4.6. The NCN fraction was separated from the precipitated caseins by centrifugation at 3000× g for 20 min at room temperature.

Milk pH and Ca-ion activity were measured using a pH and ion meter (Radiometer, Copenhagen, DK) equipped with a combined pH electrode or an ion selective electrode and a calomel reference electrode, respectively. The apparatus was calibrated using calcium-containing standard solutions [19]. The contents of total calcium and magnesium were measured by atomic absorption [12].

2.2.1. Identification of αs1-casein variants by isoelectric focusing

The genetic variants of αs1-casein in milk from individual animals were identified by isoelectric focusing (IEF) using ultrathin (0.3 mm) urea polyacrylamide gels according to a modified method of Erhardt [14]. A mixture of carrier ampholytes was chosen to give maximum resolution of the caprine αs1-casein complex; Ampholine, narrow range pH 3.5–5.0, Pharmalyte, narrow range pH 4.2–4.9 and Pharmalyte narrow range pH 5.0–6.0 (Amersham Biosciences, Uppsala, Sweden) were used at a volume ratio of 3:4:1. The protein load was increased three-fold from that of the original protocol to obtain a more reliable identification of “weak” and “null” variants of αs1-casein. The gels were stained with Coomassie Brilliant Blue R-250.

The αs1-casein variants were identified by the use of reference samples of known identity; AA, BF, CC, EE or 00 (kindly provided by Prof. F. Grosclaude, INRA, France) and Norwegian “null” or F-like (selected samples from Herds 1 and 2) [1].

2.2.2. Identification of caseins by MALDI peptide mass fingerprinting

Proteins were separated by IEF and visualized by staining with Coomassie Brilliant Blue. Identification of dissected protein bands by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was carried out essentially as described in Devold et al. [13]. Resulting tryptic peptides were desalted and concentrated on a Zip-tip column (Millipore, MA, USA) and successively dried on the MS target in presence of acidified α-cyano-4-hydroxycinnamic acid and trifluoroacetic acid. MALDI MS spectra of the peptide mixtures were obtained in positive reflector-ion mode on a Voyager DE PRO mass spectrometer (Applied Biosystems, MA, USA). Monoisotopic peptide masses were assigned and used in database searches against the NCBI and Swissprot databases.

2.2.3. Genotyping of αs1-casein variants by PCR

The goats from Herds 1 and 2 were genotyped using a PCR method that allowed identification of three alleles in exon 12 of CSN1S1: here termed D, G and A. This method allowed identification of Norwegian “null” and F-like αs1-casein variants (encoded by the D and G alleles, respectively) [1]. The exon 12 A allele is found in all αs1-casein variants other than “null” and F-like. DNA for genomic analysis was isolated from blood samples according to Rogne et al. [36]. The PCR analysis was peformed as previously described by Ådnøy et al. [1]. In brief, a typical 40 PCR cycles was performed in a PCR mixture containing: 10 μL 10 X PCR buffer (500 mmol·L−1 KCl, 200 mmol·L−1 Tris-HCl, 1.5 mmol·L−1 MgCl2, pH 8.4 and 0.01% gelatin), 0.2 mmol·L−1 of each dNTP, 2.5 U AmpliTaq DNA polymerase, 40 pmol of each primer, 50 ng DNA and H2O to a final volume of 100 μL. Denaturation; 1 min at 95 °C, annealing; 45 s at 58 °C, extension; 2 min at 73 °C. Sequencing was carried out with ABI PRISM®BigDye Terminator Cycle Sequencing Ready Reaction Kit and the automatic DNA-sequencer Model 377 (PE Applied Biosystems, Wellesley, MA, USA).

2.3. Mean size of native and heated casein micelles

The mean size of native and casein micelles in unheated (raw) and heated milks was determined by photon correlation spectroscopy on skimmed milks as previously described by Devold et al. [12]. The effect of heat treatment on mean micelle size was measured after heating the skim milk samples at 70, 80 and 90 °C for 30 min. Prior to particle size analysis, samples were diluted in simulated milk ultrafiltrate [21] which had been filtered through a 0.2 μm filter (Millipore, Bedford, MA, USA). Size measurements were performed using either the Coulter Model N4MD apparatus (Coultronics, Hialeah, FL, USA) or the ZetaSizer 3000 HS (Malvern Instruments, Malvern, UK).

2.4. Rennet coagulation properties

Rennet coagulation properties were measured using the Formagraph method (Foss Electric A/S, Hillerød, DK). Chymosin (Naturen Standard 190, Chr. Hansen Laboratorium, Hørsholm, DK) was diluted in acetate buffer (1:50) and added to milk at a level of 20 μL·mL−1. Four parameters were derived from the resultant traces: rennet clotting time (RCT; time from addition of rennet to starting point of curd formation), curd firming times (K12, K20; time from starting point of curd formation until curve widths of 12 or 20 mm width, respectively) and gel strength (A30; width of curve 30 min after rennet addition).

2.5. Statistical analyses

For statistical evaluation, the milk samples were divided into four classes: “strong”, “intermediate”, “weak” and “null” based on their IEF “αs1-casein profile”; e.g. all homozygous “strong” samples and heterozygous samples containing one “strong” genetic variant were merged into the “strong” class.

Some samples exhibited poor renneting properties and did not coagulate. Other samples coagulated but did not reach the gel strengths of 12 and/or 20 mm. In cases where no coagulation was observed after the 30 min experimental period a RCT of 50 min was used for further evaluation of the results. Samples that did not reach K12 and K20 were given fixed values of 20 and 40 min, respectively.

The response variables were analysed by using the mixed model procedure (proc mixed) of SAS (SAS release 8.3, SAS Institute, Cary, NC, USA), based on the following model:

The explanatory fixed effects included were: group of αs1-casein: “strong”, “intermediate”, “weak” and “null”; age of goat, either 1, 2, 3 or 4 or > 4 years; day and herd: test day in herd (4 different test days, 9 herds). The random effects were goat + residual.

Differences between treatment means were considered significant when resultant P values were > 0.05.

Proportions of samples in each αs1-casein class without proper coagulating properties (i.e. samples in which RCT, K12 and K20 were not achieved during the measurement period as described above) were compared using Fisher’s Exact test SAS (SAS release 8.3, SAS Institute, Cary, NC, USA). Significance was set at the 5% level (two-tailed P). Regarding the animals in herds 3, 4, 5, 8 and 9 from which samples were repeatedly collected, milks from the first sampling were included in this dataset.

3. RESULTS AND DISCUSSION

3.1. αs1-casein polymorphism and milk composition

Our results showed a remarkable high frequency (70%) of homozygous “null” goats. These results are in agreement with the previously published results of Hayes et al. [18]. Frequencies “strong”, “intermediate” and “weak” goats in our study were 17%, 2% and 11%, respectively. αs1-casein variants in milk samples from Herds 1 and 2 were determined by both IEF and PCR methods. The results based on the latter method were particularly noteworthy; animals devoid of the exon 12 D allele (“null” allele) were found at very low frequencies (2%). Frequencies of individuals homozygous and heterozygous for this allele were 73% and 25%, respectively [1].

Isoelectric focusing protein profiles of a selection of caprine milks with different genetic variants of αs1-casein are presented in Figure 1. From this figure it is evident that some milk samples exhibited an atypical band pattern; in the “null” milks (genotype 00) seen in Lanes 1 and 3 high concentration of proteins appeared in the αs1-casein area. The samples shown in Lanes 2, 4 and 5 represent F-like, “null” and A-like variants (genotype F0, 00 and X0), respectively. MALDI peptide mass fingerprinting was used to identify two protein bands taken from one of the atypical “null” milks (Lane 1). No traces of αs1-casein were detected in these bands. Analysis of the band co-migrating with the upper band of αs1-casein A (highest pI) revealed the presence of κ-casein. The identity of a second protein band corresponding to the αs1-casein A middle band could not be established. The intensities of these non-αs1-casein protein bands were highly variable between samples (data not shown). IEF profiles of most “strong” milks in this study resembled the A variant of αs1-casein standard. Such “A-like” variants (Lane 5 in Fig. 1) may correspond to “new” protein variants (encoded by αs1-casein haplotype 4) recently identified in the Norwegian goat population [18, 28]. This particular haplotype found at a frequency of 6% and with a positive impact on protein content in the milk was suggested as the “link” between the A variant and the Norwegian “null” variant (referred to as haplotype 1).

thumbnail Figure 1.

IEF protein profiles of caprine milks with different αs1-casein variants (genotypes are given in parentheses). Lanes 1 and 3: Atypical αs1-casein “null” milks (00), Lane 2: αs1-casein F-like milk (F0), Lane 4: Typical αs1-casein “null” milk (00) and Lane 5: αs1-casein A-like milk (X0).

Mean values for compositional factors, casein micelle size and rennet coagulation characteristics of the different αs1-casein classes and statistical analysis of these data are given in Table I. These results showed that class of αs1-casein (“null”, “weak”, “intermediate” and “strong”) had a statistically significant effect on contents of CP and casein, pH, Ca-ion activity (P = 0.06), content of magnesium, mean size of native micelles and gel strength (A30).

Table I.

Effect of class of αs1-casein on milk composition, mean micellar size and rennet coagulation properties. Significant effects of casein class are indicated by P < 0.05 (bold letters). Results are given as mean values (ls means).

In the present study the contents of CP and casein were significantly lower in “null” milks than in milks of the “strong” group. In a recent study the exon 12 D allele of αs1-casein was found to reduce the content of protein in Norwegian caprine milk [11]. These observations are in good agreement with previous results [9, 16, 30, 34, 37]. In these aforementioned studies the difference in casein content between “weak” and “strong” milks was smaller than the actual difference in their αs1-casein content (i.e. ~3 g per allele L−1 between homozygous A and F milks). Grosclaude and Martin [17] pointed out that this may be due to an increased production of other caseins, such as β-casein in low αs1-casein milks. These observations conflict with the work of Chanat et al. [7], who reported that intracellular transport of casein in secretory cells was impaired in milks devoid of αs1-casein.

The difference in casein content between “strong” and “null” milks in our study (1.2 g·L−1, respectively) was less pronounced than that reported in the aforementioned studies. The high frequency of heterozygous “null-strong” αs1-casein goats in the “strong” group as confirmed by the PCR analysis of the animals in Herds 1 and 2 may explain this trend. The contents of CP for the “strong” and “null” classes were 2.98% and 2.87%, respectively. No effect of class of αs1-casein was seen on the content of NCN (i.e. difference between CP and casein content). These results may indicate that the reduced rate of αs1-casein synthesis in “null” goats is compensated for by synthesis of other caseins and not by other nitrogen-containing compounds such as whey proteins.

Our results showed that the pH in milk of the αs1-casein “strong” group was significantly lower than that of “null” milks. Hence, our results are in keeping with previous findings in that αs1-casein polymorphism was correlated with milk pH [3, 30, 34]. However, the link between αs1-casein polymorphism and calcium content distribution in the milk was less clear; both the fraction of Ca (i.e. total amount, soluble content and/or ion activity) seemed to be affected, as well as the magnitude of these variations between “strong” and “weak” milks among the reports [3, 30, 34, 37]. No difference was found in total calcium content between “strong” and “null” milks in our study. Thus, the higher Ca-ion activity observed in “strong” milks is explained by their lower pH compared to their “null” counterparts. When pH is reduced, the equilibrium between insoluble colloidal calcium phosphate associated with casein micelles and soluble complexes is shifted towards the latter [15]. A pH shift in bovine milk similar to the difference between “null” and “strong” milks in our study (6.73 vs. 6.66) gave an increase in Ca-ion activity corresponding to the differences observed between these milks (2.37 vs. 2.78 mmol·L−1) [24].

3.2. αs1-casein polymorphism and mean size of casein micelles in unheated and heated milks

Our results showed that individual milk samples devoid of αs1-casein (“null” class) were characterized by larger micelles than αs1-casein “strong” milk samples. These differences were statistically significant and in good agreement with previous studies [29, 31, 33, 38], all of which concluded that the mean size of casein micelles increased as the level of αs1-casein decreased. Casein micelles in caprine milks with a low content of αs1-casein have been described as more highly hydrated than those from milks having a high content of this protein [26]. This may in part explain some of the differences in mean size between casein micelles from milks with “strong” and “weak” variants of αs1-casein.

Previously reported differences in mean size between αs1-casein “null” and “strong” micelles were much more pronounced than those reported in the current study (216 ± 26 vs. 207 ± 35 nm); 280 vs. 200 nm [31] and 260 vs. 220 nm [32]. These previous studies included only three αs1-casein “null” goats, compared to the present study including 150 homozygous αs1-casein “null” goats. The smaller differences in mean size observed in our study are probably related to the smaller differences in casein content between “strong” and “null” milks compared to the studies mentioned above in which the variation in casein content between “strong” and “null” milks was much more pronounced.

The studies by Pierre et al. [31, 32] concluded that the content of αs1-casein was negatively correlated to the mean size of casein micelles in caprine milks.

To date, no information on the effect of heat treatment on the mean size of caprine casein micelles has been reported. In this study, the milk samples were heated at 90 °C for 30 min. No changes in micellar size were observed when milk samples were heated at lower temperatures, such as 70 and 80 °C (data not shown). There was no significant relationship between class of αs1-casein and the mean size of heated casein micelles (P = 0.47). However, the heat-induced changes were smaller in “null” than in “strong” micelles (10 vs. 17 nm).

3.3. αs1-casein polymorphism and rennet coagulation properties

The results in Table I showed that class of αs1-casein significantly affected the gel strength (A30) with the “strong” milks giving firmer gels than the “null” milks.

It is generally accepted that αs1-casein polymorphism has a strong impact on renneting properties of the milk with “strong” milks giving firmer gels and higher cheese yield than milks with “weak” or “null” αs1-casein variants. However, much remains uncertain regarding the nature and magnitude of this relationship (the type of renneting property to be affected, as well as the magnitude of variations). In a recent study by Albenzio et al. [2] no significant differences in coagulation properties were observed between two groups of goats characterized by the highest frequency of strong or weak alleles at CSN1S1 locus. In a study of Ambrosoli et al. [3] RCT was significantly affected by αs1-casein variant (“high type” vs. “low type”). Pirisi et al. [34] found only minor differences in the RCT between the milks of two groups of goats classified “high” and “zero” using a turbidimetric method.

In the studies by Clark and Sherbon [9, 10] the presence of “strong” αs1-casein variants positively influenced K20, however, the authors concluded that rennet coagulation properties were better correlated to contents of casein and αs1-casein than to the actual variant of αs1-casein. In the studies described above, longer RCTs were observed with “strong” milks, thus they conflict with our results which showed the opposite trend.

The composition of “strong” milks (lower pH, higher content of casein and calcium and higher Ca-ion activity) in our study was more favourable regarding renneting properties compared to “null” milk samples. These findings correspond with previously published results [3, 9, 29, 34] and may in part be responsible for the better rennet coagulation properties of milks of the “strong” αs1-casein group. By manipulating these compositional factors Remeuf et al. [35] considerably improved the rennet coagulation properties of caprine milk containing “weak” αs1-casein variants. Adjustments of pH and protein content proved to be the most successful approach.

Some of the milk samples in the current study had poor coagulation properties and did not give recordable Formagraph values of K12 and K20. The proportions of such samples in each class of αs1-casein are shown in Table II. A considerable fraction of samples regardless of group failed to form strong gels (K20); these samples accounted for 11.4% and 5.6% in the “null” and “strong” groups, respectively. However, the proportions of poor renneting samples were not significantly different between αs1-casein classes. It should be noted that this analysis was done on a dataset including milks from the first sampling in cases where samples were repeatedly collected. It should be noted that in these cases of repeated sampling, the proportions of poor renneting samples remained constant as stage of lactation increased.

Table II.

Proportion of milk samples (given in %) in each αs1-casein class in which gel strengths of K12 and K20 were not achieved during the Formagraph measurement.

4. CONCLUSION

Norwegian Dairy Goats are exceptional in having a remarkable high frequency of an αs1-casein “null” allele. This allele is found at high frequencies in homozygous (70%) and heterozygous forms, and goats carrying only “non-null” αs1-casein alleles are found at very low frequencies. The low frequencies of the latter in the “strong” αs1-casein class in this study may explain the smaller than expected differences in milk composition and renneting properties between our “strong” and “null” milks. The potential for improving milk composition and thereby its coagulation properties by selecting for “strong” αs1-casein variants may be significant.

Further research on Norwegian goats’ milk should focus on the phenotypic differences between the recently identified casein haplotypes many of which are unique to this national goat population. Emphasis should be on compositional factors which are likely to influence quality of the milk and products made therefrom, such as the contents of total casein and individual caseins, rate lipolysis and content of free fatty acids.

Acknowledgments

We are grateful to T. Ulleberg for his help and advice in the statistical analysis of the data (IF Forsikring, Norway).

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All Tables

Table I.

Effect of class of αs1-casein on milk composition, mean micellar size and rennet coagulation properties. Significant effects of casein class are indicated by P < 0.05 (bold letters). Results are given as mean values (ls means).

Table II.

Proportion of milk samples (given in %) in each αs1-casein class in which gel strengths of K12 and K20 were not achieved during the Formagraph measurement.

All Figures

thumbnail Figure 1.

IEF protein profiles of caprine milks with different αs1-casein variants (genotypes are given in parentheses). Lanes 1 and 3: Atypical αs1-casein “null” milks (00), Lane 2: αs1-casein F-like milk (F0), Lane 4: Typical αs1-casein “null” milk (00) and Lane 5: αs1-casein A-like milk (X0).

In the text