Effect of a protein level in the diet on fatty acid profile in goat milk

The aim of the study was to evaluate the influence of feeding goats diets with different protein levels (11.4, 13.3 and 16.9%) and a small change of fatty acid contents on the concentrations of trans11C18:1 and conjugated linoleic acid (CLA) isomers in milk. The experimental design was 3 × 3 Latin Square. Each 22-day experimental period consisted of 14 days adaptation to the diets and 8 days for milk samples collection. Separation of methylated CLA isomers was achieved using GLC. Milk production was nearly the same when the lowand medium-protein diets were fed, while the high-protein diet resulted in a tendency to increase milk production. The concentrations of atherogenic (A-SFA), thrombogenic (T-SFA) and total saturated fatty acids (SFA) were similar in milk from goats fed the diets containing lowand medium-protein contents, whereas the diet containing the highest protein content resulted in an increase in the daily production of SFA and A-SFA in milk. The concentration and daily production of cis9trans11CLA, trans10cis12CLA, the sum of CLA isomers and usually trans11C18:1 and the sum of trans,transCLA isomers in milk increased as the dietary protein level increased. The higher dietary protein content resulted in the decrease in the concentration ratio of SFA and unsaturated fatty acids (UFA) (SFA/UFA) and tended to the increase in the capacity Δ9-desaturation.


INTRODUCTION
Milk and dairy products make a significant contribution to human nutrition, including numerous vitamins and minerals, conjugated linoleic acid (CLA) isomers and high-quality proteins.Although milk and dairy products provide essential nutrients, there is growing demand to increase the content of healthpromoting components in milk and dairy products (Andrade and Schmidely, 2006;Nudda et al., 2006;Czauderna et al., 2007b).Decreasing the milk concentration of atherogenic (A-SFA), thrombogenic (T-SFA) fatty acids through dietary manipulations has gained significant attention because of its implications for human health (Ulbricht and Southgate, 1991).Dietary manipulations should result in increasing the concentration of n-3 polyunsaturated fatty acids (n-3PUFA), cis11C18:1 (c11C18:1), trans11C18:1 (t11C18:1), CLA isomers and the value of the n-3PUFA/n-6PUFA ratio (Chilliard et al., 2003;Leiber et al., 2005;Rioux et al., 2005).Differences between goats and cows in milk fat content and fatty acid (FA) profile in response to dietary manipulations were recently reviewed (Chilliard and Ferlay, 2004;Chichlowski et al., 2005;Andrade and Schmidely, 2006).Indeed, goat milk is reported to contain more of the essential fatty acids, linoleic and arachnodonic acids, in addition to a higher proportion of short-chain and medium-chain fatty acids.Moreover, goat's milk may also have advantages when it comes to allergies.Goat's milk contains only trace amounts of an allergenic casein protein, alpha-S1, found in larger concentration in cow's milk.Goat's milk is also a very good source of calcium, magnesium, potassium, iron and tryptophan, as well as is also reach in phosphorus, riboflavin (vitamin B 2 ) and valuable proteins (e.g., the casein family of protein, the serum (whey) proteins).Milk proteins contain all 9 essential amino acids required by humans.
To our knowledge, there are few studies on the effect of dietary manipulation on the concentration of unsaturated fatty acids (UFA) and especially the CLA isomer profile in goats' milk (Czauderna et al., 2007b).Therefore, the objective of current investigation was to evaluate the effect of feeding different protein levels in the diet with minute changes of fatty acid contents on the concentrations of precursors of CLA isomers, CLA isomers, long-chain PUFA (LPUFA) and especially n-3LPUFA in milk of goats.

MATERIAL AND METHODS
The experiment was performed on 3 dairy primiparous Alpine goats (~12 months old) in similar lactation phases.The animals were housed and handled in accordance with protocols approved by the Local Animal Care and Use Committee (The Agricultural University of Warsaw, Poland).The experimental design was 3 × 3 Latin squares.Goats were housed in separate metabolic cages, fed every 6 h with three diets containing 11.4, 13.3 and 16.9% of crude protein in DM.Increasing the content of protein in the diets resulted also in slightly increasing the fatty acid content (Table 1).The 22-day period consisted of 14 days adaptation to the diets and 8 days for milk collection; water was freely available.Goats were milked twice a day at 06.00 and 18.00, milk was weighed and pooled for 24 h.Milk samples were stored at -20 o C until analysis.Thawed milk samples were warmed to 38 o C and sonicated for 1 min.Milk saponification, fatty acid (FA) extraction, FA methylation followed by separations of methylated FA using the GLC-FID method as previously described (Czauderna et al., 2007a).
The atherogenic SFA index (A index ) was calculated according to the equation (1) (Ulbricht and Southgate, 1991): The thrombogenic SFA index (T index ) was calculated according to the equation (2) (Ulbricht and Southgate, 1991): Statistical analyses of the effects of diets containing different protein levels were conducted using the non-parametric Mann-Whitney U test.The Statistica v. 6 package was applied (Statistica by StatSoft, 2002. Web:www.statsoft.pl).

RESULTS AND DISCUSSION
The data summarized in Table 1 showed that there were substantial differences between groups in crude protein intake.Milk production was practically the same when the low-and medium-protein diets were fed, whereas the high protein diet resulted in a tendency (P<0.083) to increase milk production in comparison with animals fed the low-protein diet.
The obtained results (Table 2) documented that various protein levels in the diet resulted in inconsistently changes in daily production of C16:0 in milk, whereas the increase of the concentration of protein in the diet reduced the concentration of C16:0 in milk.Indeed, feeding the diet containing the lowest content of protein resulted in the increase in the concentration of C16:0 in milk, while the highest content of protein in the diet most efficiently decreased the concentration of C16:0 in milk (P<0.05).As C16:0 to be particularly linked with the incidence of coronary heart disease (CHD), these results constitute valuable information for nutritionists in the context of carrying out research to improve the nutritional quality of food for humans and domestic animals.
We decided to analyse whether the protein level in the diet may affect the milk atherogenic SFA concentration and interaction of some fatty acids that may have atherogenic and thrombogenic properties.The results of our studies confirmed that the concentration of protein in the goat diet negatively correlated with the values of the atherogenic and thrombogenic indexes (i.e.A index and T index ) in milk.Therefore, we argue that increasing the protein concentration in the diet resulted in improving the nutritional quality of milk due to decrease in the values of A index and T index A i, particularly A index .
The increase of the content of dietary protein (from 11.4 to 16.9%) caused an increase in the concentration and daily production of t10c12CLA and ttCLA (P<0.05).Therefore, we stated that increasing the protein concentration in the diet resulted in improving the nutritional quality of milk due to increases in the concentration of CLA isomers, particularly c9t11CLA.The highest protein level (16.9%) in the diet caused highest increase in the concentration ratio of c9t11CLA to t10c12CLA.Observed increased the concentration ratio of c9t11CLA to t10c12CLA was probably accounted for by a lowest yield of linoleic acid isomerization to t10c12CLA in a rumen of goats fed the diet containing the highest concentration of protein.
The results of the present work confirmed that the protein level in the diet has a minor influence on the concentration and daily production of LPUFA as well as n-3LPUFA in milk.Observed small decrease in the ratios of n-3LPUFA/ΣFA and LPUFA/ΣFA in milk, especially when 16.9% protein was supplemented, might be an effect of ΣFA elevation in the milk and a minor reduction of a capacity of ∆6-desaturase and elongase (Table 2).Therefore, the concentration of LPUFA (the products of Δ4-, Δ6-desaturates and elongase) in milk decreased when the diet with the highest protein level (16.9%) was fed to goats.The increase in the protein level in the diet caused increase (P<0.05) in the concentration and daily production of unsaturated fatty acids (UFA) in milk.Moreover, in our study significant decreased (P<0.05) the SFA/UFA ratio in milk was stated when the diet was supplemented with 13.3 or 16.9% protein in comparison with the lowest protein treatment.Considering above observations, we suggest that the increase of the amount of protein in the diet was reflected in the decreasing the yield of the biohydrogenation of unsaturated fatty acids in goat rumen.

CONCLUSIONS
Increasing the protein content in the diet improves the health promoting properties of milk, because milk fat contains higher concentrations of unsaturated fatty acids (UFA), especially c9t11 and cc isomers of conjugated linoleic acid.We hypothesized that decreasing the yield of biohydrogenation in a rumen of goats fed diets containing higher content of protein is mainly responsible for an increase of the concentrations of these fatty acids.In consequence, the concentration of c11C18:1 and t11C18:1 (incomplete biohydrogenation products of UFA) in milk is positively correlated with the content of protein in the goat diet.We suggest that increasing the dietary protein decreased the capacity of desaturations; this effect resulted in lower concentrations of pro-healthy LPUFA in milk.The protein level in the diet has a negligible influence on the concentration pro-healthy n-3LPUFA in milk.

Table 1 .
Ingredient and chemical composition of diets, diet intake and milk production calculated according to Energy Allowances and Feeding System for Ruminants, MAFF, London Tech.Bull., p. 33; 2 a,b -values sharing different letters differed at P<0.05

Table 2 .
Fatty acid profile in milk of goats fed diets containing various protein concentrations 1