HK1082901B - Method for altering fatty acid composition of milk - Google Patents

Description

Method for modifying fatty acid composition in milk

Technical Field

The present invention relates to a method for reducing the level of saturated fatty acids relative to the level of unsaturated fatty acids in milk. In particular, the invention relates to the genotyping and/or phenotyping of dairy cows based on the amino acid residue at position 67 of the beta-casein produced in their milk.

Background

Dietary saturated fatty acid intake is known to be a major risk factor for heart disease in humans, particularly in countries where the population is well-nourished. Animal products such as dairy products (especially milk) are a major source of dietary intake of saturated fatty acids in humans. The finding that the level of saturated fatty acids found in milk, particularly those with a chain length of less than 18 carbon atoms, is a risk factor for coronary heart disease, is widely accepted. In contrast, unsaturated fatty acids are considered beneficial. Therefore, it is preferred to consume oils of vegetable origin rather than animal products.

The medical community is also concerned with the consumption of fats found in milk, as it is rich in the saturated fatty acids C:14:0, which are believed to be responsible for atherogenesis. The dairy industry has also partially responded to this by producing "low fat" milk replacement products using chemical separation and extraction techniques.

The milk contains beta-casein A in addition to fat¹Specific protein components, including variants, are also health risk factors. There have been many reports of beta-casein A in humans¹Consumption is associated with certain diseases, in particular diabetes (Elliott et al, 1999, diabetes 42: 292-6; Wasmuth et al, 1999, diabetes 42 (suppl 1): A88 Proceedings of the Kongress derDiabetesgesellschaft 28 volume. -30.9 months 1999, brussel/belgium) and coronary heart disease (McLachlan, c.n.med. hypotheses 56 (2): 262-72, 2001) in a patient.

In addition to phenotyping dairy cows by identifying one or more variants of a particular beta-casein protein produced in the milk of a dairy cow, it is well known that a particular Single Nucleotide Polymorphism (SNP) genotyping may be performed on a dairy cow to determine which one or more beta-casein variants it will produce in milk. Selecting cows for production of beta-casein-free A based on the genotypic analysis method¹Variants, and preferably containing only beta-casein A²The method of producing cows of the variant is the subject of PCT/NZ96/00039 (published as WO 96/36239).

The applicant has now found that: there is a correlation between the ratio of saturated to unsaturated fatty acids in milk and the beta-casein variants in milk. Although there are known methods of altering the fatty acid composition in animal products, these methods generally include chemical extraction, special feeding and management systems, and quantitative genetic selection of specific fatty acid levels in milk. Each method is expensive and generally inefficient.

It is therefore an object of the present invention to provide milk or products derived therefrom having reduced levels of saturated fatty acids relative to unsaturated fatty acids, or to at least provide the public with a useful alternative.

Summary of the invention

In a first aspect of the invention, there is provided a method of reducing the level of saturated fatty acids relative to the level of unsaturated fatty acids in milk, the method comprising:

(a) determining which cows of the herd produce milk containing β -casein having a proline at position 67 by detecting the presence of DNA encoding β -casein having a proline residue at position 67 in the genetic material of each cow in the herd or by detecting the presence of β -casein having a proline at position 67 in the milk produced by each cow in the herd (or a product prepared from the milk), including cows producing milk containing β -casein having a proline at position 67 and cows producing milk containing β -casein having a histidine at position 67;

(b) selecting a cow having a DNA encoding a β -casein having a proline residue at position 67 or a cow producing milk containing a β -casein having a proline at position 67; and

(c) selected cows are milked to obtain milk having a reduced level of saturated fatty acids relative to the level of unsaturated fatty acids as compared to milk obtained from the herd.

It is preferred that the beta-casein having a proline at position 67 includes beta-casein A²、A³One or more of D, E and F. It is also preferred that the beta-casein having a histidine at position 67 comprises beta-casein A¹One or more of B and C.

In a preferred embodiment of the invention the beta-casein having a proline in position 67 is beta-casein A²Beta-casein with a histidine in position 67 is beta-casein A¹。

It is also preferred to reduce the level of short and medium chain saturated fatty acids containing 6 to 14 carbon atoms in each carbon chain (C6:0-C14:0) in addition to the level of saturated fatty acids relative to the level of unsaturated fatty acids in the milk produced by the herd of cows.

In another preferred embodiment of the invention, the determination of which cows of the herd produce milk containing β -casein having a proline at position 67 is made by testing the genetic material of cows for the presence of DNA encoding β -casein having a proline residue at position 67. In an alternative embodiment, the determination of which cows of the herd produce milk containing β -casein having a proline at position 67 is made by testing the milk produced by cows (or products made from the milk) for the presence of β -casein having a proline at position 67.

Although the genetic material of the cow may be any tissue containing nucleated cells, the genetic material is preferably obtained from blood, hair or milk.

In a second aspect of the invention, there is provided milk obtained by the method of the first aspect of the invention.

In a third aspect of the invention, there is provided a dairy product prepared from milk obtained by the method of the first aspect of the invention.

In a fourth aspect of the invention there is provided a method of altering the ratio of saturated and unsaturated fatty acids in a foodstuff by adding to the foodstuff an amount of beta-casein having a proline at position 67.

Preferably, the ratio of saturated fatty acids to unsaturated fatty acids is altered by reducing the level of saturated fatty acids in the food.

Preferably the food is milk or a dairy product prepared from milk. It is also preferred that the beta-casein having a proline in position 67 is added to the food by adding milk (or milk extract) obtained with the method of the first aspect of the invention.

Detailed Description

It is well known that animal genetic factors have a significant impact on yield levels and product quality, as well as health, environmental and animal welfare issues. Being able to determine the phenotype of animals by using genetic testing is a valuable tool that can be used to achieve rapid identification of animals and animal products with advantageous properties and to create groups of animals with improved yield and/or product quality. Animals may be grouped according to genetic differences related to animal or animal product characteristics that are related to economic interest.

In some cases, a gene (or variant of the gene) responsible for a particular physical characteristic of an animal can be identified by a Single Nucleotide Polymorphism (SNP). A SNP is a DNA sequence at a position in the genome of an animal that differs from the DNA sequence at the same position in the genome of another animal by only one nucleotide. Although the difference is so small, this may mean that the animal may exhibit specific physical characteristics not possessed by another animal.

Correlations between casein content and fat content of milk have been determined, but these correlations are variable in magnitude and direction of correlation. Therefore, the result is uncertain. Bovenhius and Weller (Genetics; 137 (10): 267-80, 1994) concluded that: if there is an association, it is due to linkage (in sire pedigree) or linkage disequilibrium (in the population) with the fat QTL on the same bovine chromosome (chromosome 6). The overall conclusion from the published data is: the total amount of fat in milk is independent of the genotype of the beta-casein, and one or more effects of the beta-casein on human health are independent of fat intake. However, the applicants have now determined that there is an unexpected relationship between the genotype of the beta-casein gene on bovine chromosome 6 and the fatty acid composition in milk.

The applicant has verified the previous finding that beta-casein A is contained¹The milk (A1 milk) is not containing beta-casein A¹Compared to milk (a2 milk) has a similar overall percentage of fat. Surprisingly and in contradiction to previous findings, applicants have found that milk a1 has a higher percentage of saturated fatty acids and a lower percentage of unsaturated fatty acids than milk a 2. Also surprising is the discovery that: in those of beta-casein A²The milk of homozygous cows has reduced levels of C6, C8, C10, C12 and C14 fatty acids. This important finding indicates that substantially beta-casein A is absent¹The milk of (a) may also produce milk fat containing lower levels of saturated and medium chain fatty acids (C6 to C14) and higher levels of unsaturated fatty acids. Thus, the milk has health benefits, namely a reduced risk of diseases associated with high intake of saturated fatty acids, such as atherosclerosis, obesity, coronary heart disease and diabetes.

Generally, cows produce beta-casein in their milk. However, different beta-casein variants may be present, including a¹、A²、A³B, C, D, E and F. The differences between these proteins are determined by sequence variations in the beta-casein gene. For example, one difference is: a. the²、A³The D, E and F variants have a proline residue at position 67, and A¹Variants B and C carry a histidine residue at position 67. This difference can be determined by substituting the nucleotide adenine with the nucleotide cytosine at position 200 of the gene coding region for beta-casein. The beta-casein variant phenotype of a cow can be indirectly determined by genotyping the SNPs responsible for distinguishing these variant types.

The applicant has found that: selection of animals based on the type of beta-casein variant or genetic variation of the beta-casein gene allows to determine groups of animals with significant differences in their milk fatty acid composition. For example, the milk of an animal homozygous for adenine nucleotide at position 200 of the β -casein gene-encoding region (a1) differs in fatty acid composition from the milk of an animal homozygous for adenine and cytosine nucleotides at that position (a1/a2), and from the milk of an animal homozygous for cytosine at that position (a 2).

More specifically, adenine at position 200 of the β -casein gene may increase the level of the saturated fatty acids C6:0, C8:0, C10:0, C12:0 and C14:0 and decrease the unsaturated fatty acid C18:1 in comparable amounts. The beta-casein genotype accounts for 15-20% of these specific fatty acid characteristic variations between animals, after removing the effects of herd, specific individuals in herd, breed, age 2-8+, days in milk, methylation group and sire.

The presence of histidine in beta-casein at position 67 allows the formation of beta-casomorphin-7 by the action of the enzyme. Beta-casomorphin-7 is a heptaamino acid peptide consisting of beta-casein A only¹B and C. Casomorphins are known to act as opioids. Data from Lin et al (1998, Peptides 19 (2): 325-31) show that beta-casomorphin-7 regulates dietary fat intake. Beta-casomorphin stimulates the intake of dietary fat in rats, while enterostatin (enterostatin) inhibits its intake. In addition, peptides with tyrosyl end groups from casein hydrolysates, such as beta-casomorphin-7, have also been found to promote peroxidase-dependent oxidation of human LDL (low density lipoprotein). Thus, in its relationship to factors that are dangerous to human health, beta-casein A is currently being treated¹Is related to the effect of casein and peptides derived therefrom on the fat metabolism of the consumer and is not related to differences in the fat composition of milk from different beta-casein genotype animals.

The mechanism by which beta-casein affects milk fatty acid composition is not necessarily due to the linkage of genes. This is due to the magnitude and consistency of the effects observed by sires. It is speculated that this finding is related to the direct influence of β -casein on lipid biosynthesis in mammary tissue, but this does not constitute any limitation of the present invention. Alternatively, the findings may be the direct result of the interaction of casein with lipids in milk. If the latter is correct, the product is changedThe fatty acid character of the product is possible. Thus, a variant form of casein derived from the selection (e.g.without. beta. -casein A) will be obtained under the process conditions described¹) The addition of animal beta-casein to the product may beneficially alter the fatty acid profile of the product.

The beta-casein assay may be used to select animals for inclusion in a dairy herd or may be used to select animals for inclusion in a dairy herd for use as sires, dams, or tissue donors for artificial propagation or cloning to propagate the offspring of the animals. In this way, it can be formed that the produced milk does not have beta-casein A in the protein fraction of the milk¹The protein (or the only beta-casein contained is beta-casein A)²) And a reduced level of specific saturated fatty acids and an increased level of specific unsaturated fatty acids in the fat portion of the milk. Selecting cows to produce a dairy product free of beta-casein A based on said genotypic analysis¹Variants and preferably containing only beta-casein A²The method of dairy herd of variant cow's milk is the subject of PCT/NZ96/00039 (published as WO 96/36239).

Another feature of the invention is: once an animal with a particular genotype has been selected and milk produced therefrom, the source of the milk or other products, such as milk powder and refined milk products, can be demonstrated to be produced by the selected animal. This can be achieved by determining the fatty acid composition of the milk product. Thus, the consumer can be confident that the milk is indeed from an animal of the desired genotype.

The benefits of the milk of the invention are considerable:

(1) absence of beta-casein A¹Protein and the presence of beta-casein A only²The risk of coronary heart disease and type 1 diabetes is lower;

(2) the substitution of unsaturated fats for saturated fats may result in lower risk of coronary heart disease, obesity and other diseases;

(3) reducing the consumption of C14:0, which is thought to be atherogenic.

Casein affecting milkThe mechanism of fatty acid composition is not clear, but it may be mediated by the formation of casomorphin from casein. At this point and in humans consuming beta-casein A¹There may be a mechanistic relationship between the effects of (a). However, the direct effect of casein genotype on milk fatty acid profile has a distinct utility from the direct effect of casein and casein metabolites on consumer metabolism. There may also be a direct effect of beta-casein (or a specific variant thereof) directly altering the fatty acid composition of milk.

Examples

DNA was extracted and fatty acid composition of milk from 1114 offspring of six breeders of the 200-nucleotide hybrid A/C of the beta-casein gene was determined.

DNA was extracted from milk by the following method. The milk was mixed well by inversion and 1.0ml was pipetted into a 1.5ml microcentrifuge tube. The centrifuge tube was centrifuged at 8,000rpm for 10 minutes and a 100. mu.l aliquot of the supernatant (containing crude DNA) was pipetted from each sample into a new 1.5ml tube. The crude DNA extract was stored frozen at-20 ℃ and 1-5. mu.l was used for genotyping without further purification.

The genotyping method used was previously described in detail in PCT/NZ96/00039 (published as WO 96/36239).

The samples for fatty acid analysis were centrifuged at 15,000rpm for 15 minutes. An aliquot of the upper lipid was removed from each sample. The lipid sample was heated to 60.0 ℃, the thawed lipid was removed and stored frozen. The sample was subsequently methylated and analyzed by gas chromatography. The peak areas on the chromatogram were integrated to quantify the level of each fatty acid. The identity of each fatty acid was determined by comparing the retention time of each peak to known standards.

In the samples analyzed, animals were tested for CC (A2), AC (A1/A2) or AA (A1) at position 200. The differences between genotypes were compared using a generalized linear model, in which the raw data were adjusted for other factors that may affect fatty acid composition. The pre-adjustment is made for the following factors: herd, specific individual in herd, breed, age 2-8+, days of milk production, and methylation group in herd. Finally, sire, genotype, and sire-genotype interactions were fitted.

The results of this study are given in table 1 and show that the a2 genotype has a significant effect on fatty acid composition. The level of statistical significance varied from one fatty acid to another: (^＊＝p＜0.05，^＊＊＝p＜0.1，^＊＊＊P < 0.001). Milk of animals with the a2 genotype had a significantly higher percentage of long chain unsaturated fatty acids (C18:1) and a lower percentage of saturated and medium chain fatty acids (C6:0-C14:0) than a 1; while those for the A1/A2 individual were centered.

The milk from a2 contained about 3% more C18:1 than the milk from a1 for a total C18:1 percent. C18:1 accounts for about 15% in milk fat, so the overall effect is about 0.5% more for C18:1 as a proportion of total milk fat. The reduction in the percentage of saturated fatty acids is similar to the increase in unsaturated fatty acids. The beta-casein genotype constitutes 15-20% of the variation in these specific fatty acid compositions between animals after removal of the effects of herd, specific individuals in herd, breed, age 2-8+, days of milk production, methylation group and sire by the model.

Table 1: summary of beta-casein genotype analysis of fatty acid percentage in milk fat

TABLE 1 (continuation)

Although the invention is illustrated by way of example, it should be understood that: variations and modifications may be made without departing from the scope of the invention. Furthermore, if there are known equivalents to a particular feature, then such equivalents are incorporated as if specifically referred to in this specification.

INDUSTRIAL APPLICABILITY

Milk containing low levels of saturated fatty acids compared to unsaturated fatty acids can be used to avoid certain diseases and conditions. Dietary fatty acid intake is a major risk factor for heart disease, and many of this dietary fatty acid intake comes from milk and dairy consumption. Milking only those cows that have been genotyped or phenotyped based on their ability to produce a beta-casein variant with a proline, but not a histidine, at position 67 to obtain milk having a low level of saturated fatty acids relative to unsaturated fatty acids is a useful method for producing milk that is beneficial to human health.

Claims (10)

1. A method of reducing the level of saturated fatty acids relative to the level of unsaturated fatty acids in milk, the method comprising:

(a) determining which cows of the herd produce milk containing β -casein having a proline at position 67 by detecting the presence of DNA encoding β -casein having a proline residue at position 67 in the genetic material of each cow in the herd or by detecting the presence of β -casein having a proline at position 67 in the milk produced by each cow in the herd or in a product prepared from the milk, the herd including cows producing milk containing β -casein having a proline at position 67 and cows producing milk containing β -casein having a histidine at position 67;

(b) selecting a cow having a DNA encoding a β -casein having a proline residue at position 67 or a cow producing milk containing a β -casein having a proline at position 67; and

(c) selected cows are milked to obtain milk having a reduced level of saturated fatty acids relative to the level of unsaturated fatty acids as compared to milk obtained from the herd.

2. The method of claim 1, wherein the beta-casein having a proline at position 67 comprises beta-casein a²、A³One or more of D, E and F.

3. The method of claim 2, wherein the beta-casein having a proline at position 67 is beta-casein a²。

4. The method of claim 1, wherein the beta-casein having a histidine at position 67 comprises beta-casein A¹One or more of B and C.

5. The method of claim 4, wherein the beta-casein having a histidine at position 67 is beta-casein A¹。

6. A method as claimed in any one of claims 1 to 5 wherein the level of short and medium chain saturated fatty acids C6:0-C14:0 containing 6 to 14 carbon atoms in each chain is reduced compared to milk obtained from the herd.

7. The method of any one of claims 1 to 5 wherein the determination of which cows in the herd produce milk containing β -casein having a proline at position 67 is made by testing the genetic material of cows for the presence of DNA encoding β -casein having a proline at position 67.

8. The method of any one of claims 1 to 5 wherein the determination of which cows of the herd produce milk containing β -casein having a proline at position 67 is made by testing the milk produced by cows or products prepared from the milk for the presence of β -casein having a proline at position 67.

9. The method of any one of claims 1 to 5, wherein the genetic material of the cow is any tissue containing nucleated cells.

10. The method of claim 9, wherein the genetic material is obtained from blood, hair or milk.