Production of cla-enriched dairy products by natural means

For use in product manufacture and spray drying, CLA-producing bifidobacteria should grow and produce CLA efficiently in milk based medium (RSM). CLA production was assessed in RSM, and found in general to be inefficient. The increased conversion of linoleic acid to CLA was assessed following addition of a selection of bifidogenic supplements to 20 % RSM. This was done by assessing the % conversion of linoleic acid to CLA in RSM containing a selection of supplements, including sugars, short chain fatty acids, and other bifidogenic agents. Of the supplements tested, sodium propionate and yeast extract were found to be the most effective, resulting in conversion rates from linoleic acid to c9, t11 CLA of >50% in RSM, while casein hydrolysate and sodium acetate also yielded satisfactory results, yielding conversion rates of 40-50%. A medium containing 200mM sodium acetate and 20mg mL-1 yeast extract was selected for further evaluation, and fermentation of B. breve NCIMB 702258 in this medium increased the percentage bioconversion of 0.35 mg ml-1 linoleic acid to the c9, t11 CLA isomer, from 15.5 +/- 1.1% (in 20% RSM) to 48.1 +/- 2.2 %. Inclusion of a further supplement into this medium, i.e. inulin (20mg mL-1), and optimization of inoculum and linoleic acid concentration resulted in further increases in the conversion rate up to 55.0 +/- 3.2%. This rate of conversion of linoleic acid to CLA was found to be comparable to that obtained in synthetic medium (cys-MRS), in which 58.8 +/- 2.7% of the linoleic acid was converted to c9, t11 CLA, under identical experimental conditions.

Certain bacterial species associated with food fermentations, including propionibacteria, lactobacilli, lactococci and streptococci have the ability to produce CLA from linoleic acid. Furthermore, bacterial inhabitants of the human intestine possess the ability to generate c9, t11 CLA from free linoleic acid (intestinal microflora of rats and bifidobacteria (Coakley et al.,2003; Rosberg-Cody et al.,2004). Bifidobacteria are common inhabitants of the human gastrointestinal tract (GIT) and are used for probiotics applications in Functional Foods. Such strains have the potential to enhance CLA levels in dairy products during fermentation, being used as either starters or adjunct cultures. In this study we assessed the ability of previously identified bacterial strains to generate CLA from linoleic acid. Of the strains tested, the propionibacteria and the bifidobacteria generated CLA from linoleic acid. A number of the bifidobacteria strains investigated produced mainly c9, t11 CLA isomer following incubation in 0.55 mg ml-1 linoleic acid. This screening programme indicated that there was considerable inter-species variation in the ability of bifidobacteria to produce CLA isomers. The t9, t11 CLA isomer was also generated, but at much lower levels. In an attempt to identify more robust and technologically viable strains of CLA-producing bifidobacteria, a study was conducted to isolate strains from the stools of (a) healthy human babies, (b) healthy adults and (c) elderly patients. The screening programme identified novel efficient CLA-producing Bifidobacterium strains, including B. breve, B. infantis, B. dentium, B. longum and B. catenulatum.

Earlier work in this project demonstrated unequivocally that cis-9, trans-11 conjugated linoleic acid (CLA) in milk is derived from cis-9, trans-11 CLA formed in the rumen and also in the mammary gland via the action of stearoyl-CoA desaturase on trans-11 C18:1. Research was further extended to evaluate the potential of specific lipids in the diet to enhance milk fat cis-9, trans-11 CLA content. Studies were conducted to examine the interaction between marine lipids in the diet that inhibit ruminal biohydrogenation and plant oils rich in C18:2 n-6 or C18:3 n-3 on the flow of fatty acids leaving the rumen and milk fatty acid composition. Fish oil (FO) was used as a source of long chain polyunsaturated fatty acids to inhibit ruminal biohydrogenation of trans-11 C18:1. Sunflower oil (SO) and linseed oil (LO) were chosen as sources of C18:2 n-6 and C18:3 n-3, respectively, which serve as substrates for trans-11 C18:1 synthesis in the rumen. Cows were fed grass silage based diets supplemented with no oil (control), 200 g FO/day, 200 g FO and 500 g SO/day or 200 g FO and 500 g LO/day. Addition of FO in the diet decreased intake and reduced milk fat output and milk fat content, but had no effect on milk yield or milk protein secretion. Feeding FO in combination with SO or LO further decreased intake and milk fat yield, and tended to reduce milk yield and milk protein content. Inclusion of oils in the diet induced significant changes in milk fatty acid composition, responses that were characterised as a reduction in the amount of fatty acids synthesised de novo (C6-C14), and an increase in the amount of trans C18:1, trans C18:2, total CLA, C20:5 n-3 and C22:6 n-3. Inclusion of FO in the diet increased total milk fat CLA content from 0.78 to 2.27 % fatty acids (FA). Feeding SO with FO did not increase milk fat CLA levels above those on the FO treatment (2.24 g/100 g FA), while FO and LO increased the levels of total CLA in milk yet further to 3.56 % FA. In all cases increases in milk fat CLA content to lipid supplements were associated with a decrease in milk C18:0 content and an increase in trans 18:1 concentrations. Increases in milk trans C18:1 to lipid supplements were isomer dependent, with levels of trans-11 C18:1 being comparable when FO and FO plus SO were fed, but significantly higher for diets supplemented with FO plus LO. For diets containing FO and SO, most of the increase in trans C18:1 was due to the elevated levels of the trans-10 isomer. The increase in milk fat cis-9, trans-11 CLA to FO was directly related to an increase in the supply of trans-11 C18:1 available for endogenous synthesis in the mammary gland and not due to an increase in preformed cis-9, trans-11 CLA. Feeding LO in combination with FO increased the flow of trans-11 C18:1 which was responsible for the further increase in milk fat cis-9, trans-11 CLA content on this dietary treatment. However, including SO to diets containing FO did not increase trans-11 C18:1 flow at the omasum above that with FO alone, but induced changes in ruminal metabolism causing trans-10 C18:1 to replace trans-11 C18:1 as the major biohydrogenation intermediate leaving the rumen. This is the first experiment to show the importance of the relative amounts and type of polyunsaturated FA when inhibitory marine lipids are included in the diet on ruminal lipid metabolism. Inclusion of FO in the diet is effective in enhancing milk fat CLA content. Increases in milk fat cis-9, trans-11 CLA when FO is fed on grass silage based diets can be further enhanced when LO is fed, but not when SO is used as oil supplement. This work showed that practical implementation of this nutritional approach to enhancing milk fat CLA content on-farm would need to be carefully managed to minimise the effects on intake, milk yield and milk fat content. Overall, this research has shown that when grass silage based diets contain marine lipids that inhibit the final reduction of trans C18:1 in the rumen, milk fat CLA concentrations can be further increased by linseed oil supplementation, but not when sunflower oil is fed. Use of a biohydrogention inhibitor and oils rich in C18:3 n-3 would also be expected to be a useful nutritional strategy to enhance milk fat CLA concentrations in grazing animals at pasture as well as cows fed dried grass, ensiled grass or legume forages. These findings are of importance in the formulation of dairy cow diets to enhance milk fat CLA content and indicate that a combination of lipids which inhibit the biohydrogenation of trans C18:1 in the rumen and are also a rich source of C18:3 n-3 is particularly effective. This research also serves to highlight the importance of ruminal lipid metabolism in the regulation of milk fat synthesis and milk fatty acid composition.

With the aim to develop CLA-rich dairy functional foods, several experiments were carried out using sheep and cow milk with different levels of CLA and using different production technologies. Experiment 1 - With the aim to optimize the production technology to obtain the best CLA recovery from sheep milk, the partitioning of CLA into different products and by-products during the manufacture of Pecorino Sardo and Ricotta cheeses has been assessed. The vaccenic and rumenic acid contents in milk, ripened cheeses and whey were similar. Experiment 2 - PDO Pecorino Sardo and Ricotta cheeses were manufactured from ovine milk having a low (18mg/g fat, low CLA milk) and a high (25mg/g of fat, high CLA milk) level of rumenic acid using two different rennet types: a liquid rennet which does not contain lipolytic enzymes and a rennet paste which on the contrary is characterised by an high level of the lipolytic enzymes. The effect of the rennet type on the vaccenic and rumenic acids partitioning into cheese at different ripening time (24-old cheese, 2M, 4M, 6M and 8M-old cheeses) and by-products such as ricotta cheese, whey and scotta whey was assessed. The rennet type did not affect the content of vaccenic and rumenic acids in ripened cheese from both high and low CLA-milk. Experiment 3 - PDO Pecorino Sardo and Ricotta cheeses were manufactured from milk without treatment (control milk) and from milk with homogenized cream. The cream homogeneisation brought some problems in the structure of the cheese. The particles of the curd had a poor cohesivness and in fact the paste of cheese tended crumbling. Gross composition was not affected by the treatment whereas a ripening effect was found for all determined parameters. FA composition also was not influenced by cream homogenisation, on the contrary several FA (like vaccenic acid and CLA) content changed significantly during ripening. The vaccenic and rumenic acids partitioning into different products (1 day and 30, 60, 90, 180 days old cheese) and by-products (ricotta cheese, whey and scotta whey) was not significant influenced by cream homogenisation. Experiment 4 - PDO Pecorino Sardo cheese were manufactured from bulk ovine milk using a liquid calf rennet. liquid calf rennet + esterase-lipase from Rhizomucor miehei and liquid calf rennet + esterase-lipase from Rhizopus oryzae. No significant effect was found in cheese gross composition related to the treatment and, as expected, a ripening effect was observed for these parameters. With reference to the FA profile, only butyric acid content in cheese was significantly influenced by the treatment. Ripening time significantly influenced several FA as oleic and linoleic acid. Experiment 5 - PDO Pecorino Sardo cheese were manufactured from bulk ovine milk using a natural whey culture and a freeze-dried culture (CO-02 Hansen). The results showed that gross cheese composition was not affected by treatment. On the contrary a ripening effect was observed for all parameters except for pH value. The use of different lactic cultures did not significantly influence the FA profile in cheese. As reported for the experiments above, a significant effect of ripening was found for several FA as oleic, linoleic and linolenic acid as far as CLA. CLA-enriched cheese has also been manufactured with CLA-enriched bovine milk from supplementation trials (1. sunflower oil anbd 2. sunflower and linseed oils). Product manufacture was not affected by the elevation of the CLA levels in the cheese milk and sensory or compositional properties of the product were not affected during ripening. Elevated CLA levels were maintained in the cheese during the ripening period. In conclusion the results demonstrate that the level of beneficial FAs in cheese depends form that of the milk. All studied technological parameters have not allowed enhancing the level beneficial FAs in the final products.

Nutritional quality is becoming a major issue in food choices because of growing consumer awareness of the link between diet and health. Numerous studies have established that cis-9, trans-11 conjugated linoleic acid (CLA) has a number of potential health benefits to man. Milk and dairy products are the main source of CLA in the human diet and therefore there is a need to develop effective strategies for enhancing milk fat CLA concentrations. Cis-9, trans-11 CLA is considered to be derived from ruminal biohydrogenation of C18:2 n-6 and also through endogenous conversion of trans-11 C18:1 in the mammary gland. Trans-11 C18:1 is produced as an intermediate of C18:2 n-6 and C18:3 n-3 metabolism in the rumen. Research in this project provided essential information on the relative importance of both ruminal and endogenous CLA synthesis in the dairy cow. Measurements of milk fatty acid composition in response to post-ruminal infusions of cis-9, trans-11 CLA and trans-11 C18:1 allowed the transfer of these fatty acids into milk and bioconversion of trans-11 C18:1 to cis-9, trans-11 CLA to be calculated. It was shown that 39.9 % of cis-9, trans-11 CLA at the small intestine was transferred into milk. Post-ruminal infusions of trans-11 C18:1 increased both the concentrations of trans-11 C18:1 and cis-9, trans-11 CLA confirming that endogenous synthesis is an important source of cis-9, trans-11 CLA in milk. Moreover, 8.1 and 21.4 % of the amount of trans-11 C18:1 infused was recovered in milk as cis-9, trans-11 CLA and trans-11 C18:1, respectively. Through examining the relationship between the output of cis-9, trans-11 CLA with the sum of trans-11 C18:1 and cis-9, trans-11 CLA in milk, it was possible to show that 27:8 % of trans-11 C18:1 available to the mammary gland was bio-converted to cis-9, trans-11 CLA. Overall the relative biological value of trans-11 C18:1 produced in the rumen was shown to be equivalent to 20 % of cis-9, trans-11 CLA synthesized in the rumen. This is the first study to show that endogenous synthesis of cis-9, trans-11 CLA in the mammary gland is not appreciably altered by relatively large changes in the availablity trans-11 C18:1 and directly compare ruminal versus endogenous synthesis of cis-9, trans-11 CLA in the dairy cow. These findings are of importance in the formulation of dairy cow diets to enhance milk fat CLA content and indicate that ingredients which alter ruminal lipid metabolism causing preformed cis-9, trans-11 CLA or trans-11 C18:1 to accumulate would be effective. More research is required to identify diets or feed ingredients that alter lipid metabolism in the rumen and enhance the amounts of trans-11 C18:1 and cis-9, trans-11 CLA available for absorption. Furthermore, these findings provide further evidence to suggest that trans-11 C18:1 in milk also serves as a substrate for endogenous cis-9, trans-11 CLA synthesis in humans.

The aims were, in dairy goats: i) to study feeding strategies to maximize milk CLA, ii) to evaluate the effect of feeding Vitamin E on milk fatty acid (FA) profile and sensory properties of CLA-rich cheeses (see ETIP 36839), and iii) to yield milk to make CLA-rich cheese for clinical evaluation in humans (see ETIPs 36846 and 36847). Three successive studies have been conducted to evaluate 16 different feeding strategies (in comparison with previous data obtained on 26 other diets, Int Dairy Fed, 2005, 0501/part 5: 297-304). The first feeding experiment was performed on 84 goats receiving one of 7 diets, to determine how forage/concentrate ratio and Vit E interact with linseed oil supplementation, and if extruded linseeds are efficient, compared to free oil, to increase milk CLA. The main results were: - Decreasing forage/concentrate ratio increased milk 10:0 to 14:0 and trans10-18:1 (% of total FA), did not change cis9,trans11-CLA (rumenic acid, RA), and decreased 18:0 and 18:3n-3 %; - Linseed oil supplementation increased milk fat yield and the % of 18:0, 18:3n-3 and several trans isomers of 18:1 and conjugated or non-conjugated 18:2, and decreased 10:0-16:0 and 18:2n-6 %. Increases of trans11-18:1 (vaccenic acid, VA) (from 0.9 to 7.5 %) and of RA (from 0.5 to 3.2 %) were large, as were decreases in atherogenicity index (3.9 to 1.8) and n-6/n-3 ratio (4.3 to 1.3); - Vit E addition increased milk fat yield, 18:0 %, and several trans isomers of conjugated or non-conjugated 18:2 and decreased 10:0-16:0 %. The increase of trans11-18:1 (from 7.6 to 8.8 %) was significant although RA was not changed (3.2 %); - Extruded linseeds, compared to free linseed oil, decreased 16:0 %, trans11-18:1 and RA (from 3.1 to 2.1 %) and increased milk protein content and 18:0, 18:2n-6, 18:3n-3 and cis9,trans13-18:2 %, and the n-3/n-6 ratio (from 0.9 to 1.3). The results after either 5 or 9 wk of dietary treatments were similar, showing that the responses were stable for at least 2 months. The plasma FA profile of the goats changed mostly in the same direction than observed for milk FA. The second feeding experiment was performed on 84 goats receiving one of 7 diets to determine: - How to maximize the milk CLA by combined effects of forage/concentrate ratio and high doses of either linseed oil (LO) or sunflower oil (SO)? - If the extrusion of a mixture of linseeds and sunflower seeds is efficient? - The kinetics of the response of CLA to dietary changes, compared to published data in cows in which CLA response is often unstable. Milk fat content was decreased by low (LF) vs. high forage (HF) diet, and strongly increased by lipid supplementation. Milk rumenic acid % rapidly increased to reach its maximum after 2 wk of lipid supplementation. The highest level (5%) was achieved with HF + SO, whereas other diets were around 3%. LO supplementation decreased 10:0 - 16:0 and increased 18:0, 18:3 n-3, and trans-18:1 and trans-18:2 isomers. SO supplementation to HF diet induced the greatest decrease in 10:0 - 14:0 and increased cis-9 18:1, trans10 + trans11-18:1, rumenic acid and 18:2 n-6. Extruded seeds maximized 18:0 and 18:3 n-3. The sum of non-trans11 18:1 and 18:2 was highest with LO, high with extruded seeds, medium with SO, and lowest with basal diets. The third feeding experiment was done to supply control and CLA-enriched milk for cheese making for studies in humans. Forty-eight goats received a diet based on alfalfa hay and supplemented, or not, with sunflower oil and Vit E. Milk CLA was increased from 0.3 to more than 4% of total FA (x13-fold) by lipid supplementation. A second objective of this trial was to compare milk FA composition, and responses to lipid supplementation, in goats differing by their genotype at the alpha-S1 casein locus (24 high- and 24 low-genotypes). In parallel, a retrospective analysis was done on milk FA composition from 33 Low- and 38 High-genotype goats. Low-genotype goats had a much lower (-6.8g/kg) milk fat content and much less medium-chain FAs, less stearic acid, more palmitic, oleic, linoleic and rumenic acid than High-ones. Furthermore, the delta-9 desaturation ratios of milk FAs were higher in Low-genotype goats for 10:0, 14:0, 17:0, 18:0, trans11-18:1 and trans13-18:1, strongly suggesting that delta-9 desaturase activity was increased, probably to insure the constancy of milk fat melting point despite the decrease in the synthesis of medium-chain FAs. In conclusion, the goat largely differs from the cow in its response to dietary factors for milk fat yield and FA profile. The goat is a very good, repeatable and sustainable responder for milk CLA. High forage diets supplemented with vegetable oil rich in polyunsaturated FA and Vit E, are suitable to yield high-CLA milk for cheesemaking.

The aims of this section of the BIOCLA project were i) to study the change in CLA content from goat milk to cheese according to different technologies, ii) to evaluate the sensory properties of CLA-rich cheeses, and iii) to manufacture goat cheeses, enriched in CLA, for clinical evaluation in human volunteers (see ETIP 36846 and 36847). Three successive studies have been conducted to choose an adequate combination of feeding strategies and processing methods for production of CLA-rich cheese with acceptable sensorial quality. During the first study, five diets have been used: three diets with high forage:concentrate ratio (HF), without or with linseed oil (LO, 4 % of diet dry matter, DM), this latter diet being supplemented or not with vitamin E; one diet with low forage:concentrate ratio (LF) supplemented with LO and vitamin E; one HF diet supplemented with extruded linseeds (ELS). Bulk milks obtained from these diets contained between 0.3 and 3.3% CLA in total fatty acids (FA) and have been pasteurized and transformed in 3 types of cheeses: lactic ripened cheese (St Maure type), lactic spread cheese, and soft cheese (camembert type). Cheese fat oxidation level was always weakest with the diets containing vitamin E, giving evidence of the antioxidant effect of vitamin E. The effects of cheese technologies were small, with increases in C10:0, C12:0 and C14:0 percentages and decrease in oleic acid, in cheese compared to processed milk. Very few changes were observed for polyunsaturated FA, including CLA. The sensory properties of cheeses were characterised at half shelf-life (30 days): - By a trained panel (8-10 experts) for the 15 cheeses coming from the five different diets (appreciation of the intensity of 22-26 descriptors of odour, flavour and texture on a linear continuous scale from 0 to 10). Low scores were observed for the goaty and the yeasty flavours with the spread technology. - By 60 French consumers for 12 of the 15 cheeses (except those from the LF+LO+vitamin E diet), with a global appreciation from very bad (note 1) to very good (note 7). The cheeses were considered by the consumers as medium or good enough, ranking in order spread < ripened lactic < soft cheese, without significant effect of dietary lipid supplementation. During the second study, CLA-rich milks were obtained from two HF diets with a high level (7 % of diet DM) of either LO or sunflower oil (SO). They were pasteurized and manufactured in 3 types of cheeses: two St. Maure types (one traditional, and one industrial ripened lactic cheese), and one high-fat spread cheese, made from either fresh or frozen curd. The high level of lipid supplementation resulted in very high CLA levels (up to 5.1% of total FA), but also in defects of flavour, especially with LO. This phenomenon could be amplified in high-fat spread cheese. Freezing had no effect on the FA profile (including CLA). During the third study, goats were fed a HF diet supplemented, or not, with SO and vitamin E, in order to maximize CLA in the milk, and simultaneously to minimize the oxidation of cheese fat and the risk of flavour defects. The cheeses were manufactured from 1 to 8-month stored frozen curd, into high-fat spread cheese. This allowed to yield goat cheeses for the clinical trial in Italy (see ETIP 36846 and 36847), with CLA content of 0.3% or 3.9% of total FA for control or CLA-rich goat cheese, respectively, and with acceptable flavour (as evaluated by two different panels in France) despite a decrease in sensorial quality with curd ageing, particularly for CLA-rich cheese. In conclusion, these results provide novel information on strategies to produce CLA-enriched goat dairy products that could be marketed as a functional food per se. Furthermore, CLA enriched-milk could be a valuable ingredient for the manufacture of new healthy goat products.

Milk, together with ruminant meat, represent the major dietary source of CLA. Milk CLA has two origins, - Ruminal biohydrogenation of linoleic acid into CLA followed by its uptake and secretion by the mammary gland, - Mammary synthesis by action of the enzyme stearoyl-CoA desaturase (SCD) on circulating vaccenic acid (trans-11-18:1) coming from ruminal biohydrogenation of C18 unsaturated FA. The latter pathway contributes to more than 90% of milk CLA. Then, it is of major importance to study the regulation of genes involved in lipogenesis and CLA synthesis (particularly SCD gene expression and activity) in ruminant mammary gland, both in vitro and in vivo, in response to nutritional factors. In this context, the effect of diets supplemented with lipids rich in C18:2 (n-6) and differing by the forage:concentrate ratio and/or degradable starch content in concentrate, on milk fatty acid composition and in vivo mammary metabolism, was examined in dairy goats. Regarding milk composition, the high starchy concentrate diets led to - An increase in the trans-10 isomers at the expense of the trans-11, - A decrease in milk cis-9, trans-11 CLA and - An increase in the level of medium-chain fatty acids at the expense of the long-chain fatty acids. These effects were more pronounced with the concentrate rich in rapidly degradable starch. Regarding mammary metabolism, the responses of the four studied lipogenic genes (LPL, ACC, FAS, SCD) were not always related to the correspondent milk fatty acid secretion. ACC activity was not significantly different between the dietary treatments, due to the individual variability, but was related to the variation of secretion of medium-chain fatty acids. SCD mRNA and activity did not vary under these dietary conditions, which is in accordance with milk delta-9 desaturation ratios that represent a proxy for SCD activity. This suggests that the amount of lipogenic enzymes or mRNA is only one regulator of lipogenesis together with the availability of substrates and cofactors. Furthermore, the trans-10 C18:1 secretion was not related in this goat experiment to lipogenic enzymes and fat yield responses, conversely to what was observed in cows. In vitro trial was performed in parallel by the development and characterization of a mammary explant culture technique on tissue of lactating goats. Mammary explants were incubated for 20 hours in 9 media supplemented with three different C18 FAs (18:1 n-9, 18:2 n-6 and 18:3 n-3). These conditions led to convergent data using different approaches (14C-acetate incorporation representing total lipogenic activity, FAS and G6PDH activities, ACC, FAS and SCD mRNA levels) to estimate mammary lipid synthesis. Then, was investigated on 5 lactating goats, the effect of 2 CLA isomers (cis9, trans11-18:2, trans10, cis12-18:2), 2 major trans-18:1 (trans11-18:1, trans10-18:1) and of unsaturated C18 FAs (18:1 n-9, 18:2 n-6 and 18:3 n-3), on mammary lipid metabolism. The 7 FAs had less effect on goat mammary FAS and G6PDH activities and on ACC, FAS and SCD mRNA levels than previously published in cows. These results suggest that in goats the nature of FA brought to mammary tissue is not as important as in cow to regulate mammary lipogenesis and CLA synthesis, in accordance with the determinant role of the availability of the substrates suggested by the in vivo experiment. Whole results from this study are still under analysis.

Cheddar cheese was manufactured in duplicate trials at pilot scale with adjunct culture B. breve NCIMB 702258 (an efficient CLA-producing bifidobacteria). This adjunct culture B. breve NCIMB 702258 survived cheese-making following inoculation into the cheese milk at 10e7 cfu/g. Some of these bifidobacteria were lost in the whey at draining, but upon concentration of the curd at draining, bifidobacteria numbers in the curd increased to 10e8 cfu/g and survived at this level during milling. All cheeses were then ripened for 6 months at 8oC, and microbiological analyses conducted throughout. Bifidobacteria numbers were recorded up to 10e8 cfu/g at Day 1, and these decreased to 10e7 cfu/g at Week 1 and further decreased to 10e4-10e6 cfu/g bifidobacteria by Month 1. From Month 1 to month 3, bifidobacteria numbers remained constant at 10e4 cfu/g, but by Month 4 these had decreased to 10e2 cfu/g with further viability losses observed up until Month 6. Such low numbers of bifidobacteria at the end of ripening are unsatisfactory for probiotic product development, where ideally bifidobacteria numbers should be at 10e7 - 10e8 cfu/g cheese, for successful development of a probiotic cheese. Because of the poor survival of the bifidobacteria in cheese during ripening, the ability of CLA-producing propionibacteria to survive cheese manufacture and ripening was evaluated. A Swiss-type cheese was manufactured at laboratory scale with added Propionibacterium. Microbiological analysis was performed up to 16 weeks of ripening, and it was found that the Propionibacterium strain survived very well in the cheese during ripening, reaching >10e7 cfu/g cheese after 16 weeks, which meets recommended bacterial numbers for a probiotic product. This demonstrates that cheese is a suitable medium for the delivery of CLA-producing propionibacteria.

Opportunities to improve the lipid composition of ruminant products, whether reducing saturated fatty acids or increasing beneficial PUFA, are governed by microbial biohydrogenation of unsaturated fatty acids. Altering microbial lipid metabolism in the rumen for the production of higher levels of PUFA and other interesting fatty acids such as conjugated linoleic acid (CLA) has been a major goal for animal scientists for over thirty years. Numerous studies have shown that the milk from cows grazing fresh pasture had significantly higher levels of the beneficial PUFA and CLA than cows fed on conserved forage. Previous studies have reported the nature of the volatile components released during the cutting of grass. This green odour consists of the products of fatty acid oxidation carried out by plant lipoxygenases. These enzymes catalyse the oxygenation of PUFA forming a mixture of volatile hydroperoxides, alcohols, aldehydes and ketones. These compounds have been shown to have antimicrobial activities and recently have been shown to have an effect on ruminal lipid metabolism. We have shown the effect of two typical fatty acid oxidation products (hydroperoxides and long chain aldehydes) on the biohydrogenation of grass silage C18:2 (n-6) and C18:3 (n-3) when incubated in vitro with strained rumen fluid. The increase in the biohydrogenation of C18:2 (n-6) and C18:3 (n-3) and the associated increase in trans-vaccenic acid (TVA, a pre-requisite for the formation of CLA in animal tissues such as the mammary gland) may be attributable to the proliferation of biohydrogenating micro-organisms as a consequence of the toxic nature of the green odour compounds to competing micro-organisms reducing inter-specific competition. This green odour effect may offer a partial explanation for the changes in milk fat profiles when animals graze fresh pasture as opposed to conserved forage.

Dairy products have had a bad reputation from a nutritional point of you because of the presence of TFA. In the last 20 years several evidences have been published that TFAs possess detrimental effects, even more than SFA, because they not only increase LDL cholesterol but also decrease HDL cholesterol when the daily intake is higher than 2 % of en. Several studies have even elucidated a possible mechanism by which saturated and tFAs may influence cholesterolemia. The detrimental effects of tFAs, however, have also been extended to those present in animal fat. In fact, tFAs are formed not only during industrial partial hydrogenation but also by a biohydrogenation performed by anaerobic bacteria present in the rumen through a sequence of enzymatic reactions. However, there are substantial differences between tFAs from these two types of fat. The formation by enzymatic reactions leads only to a few isomers of tFAs from animal origin. During industrial hydrogenation randomly distributed isomers are produced. Furthermore, the major product of biohydrogenation, vaccenic acid (t11-18:1), has been shown to be an efficient precursor of rumenic acid (c9,t11 CLA) both in animals and humans,. and therefore can be regarded as an intermediate product. One of the aim of our study was to evaluate the contribution of vaccenic acid present in the CLA-enriched cheese to CLA plasma levels in humans. At this scope we first evaluate the bioavailability of c9,t11 CLA with the administration to human volunteers increasing concentration of synthetic pure c9,t11 CLA in triglyceride form. The equation of the curve built with the value of synthetic c9,t11CLA daily intake and its corresponding plasma levels, allowed us to determine VA contribution to c9,t11 CLA plasma levels in the same volunteers with a known amount of CLA-enriched cheese. Our data show that VA significantly contributed to c9,t11 CLA plasma levels within a range of conversion between 15 to 18%.

Feeding dairy sheep relies substantially on grazing. Native and cultivated grass and legume based pastures are the main feed sources being either grown as monocultures or mixtures. With reference to this result, the BIOCLA consortium has recently worked to: - Identify the FA composition of some relevant Mediterranean forage species during vegetative and reproductive phases with particular reference to CLA precursors; - Identify the milk CLA profile of sheep fed these forages either as monocultures or mixtures; - Find out periods in which CLA concentration in milk falls and the factors that are underlying these drops; - Envisaging and testing feeding techniques such as supplementation with fat-enriched concentrates or complementary grazing of CLA precursor-rich forages able to fill the above gaps. The body of knowledge accumulated so far allows us to point out that the concentration of milk CLA of pasture-fed sheep tends to increase along with the proportion of green herbage in the diet. In sheep fed fresh forages only, milk CLA content is related to two main factors: the forage genotype (species and cultivar) and the phenological phase of forages which in turn affects the dietary proportion of leaves. Both these factors are under the control of grazing management. Four experiments and a survey have been undertaken within this project to investigate this. On the basis of the results, a feeding and management strategy aimed at maximizing CLA concentration in ovine milk is put forward. It lays on the following guidelines: - Choose preferably mixtures of grass and legumes or sequential grazing of legume and grass pastures. Legumes such as burr medic and subclover can be preferable to sulla. Daisy forbs rich in linoleic acid such as safflower or Chrysanthemum coronarium can be conveniently included in lactating sheep diets. - Graze at moderate to high intensity, particularly during the fast growth spring period in order to keep pasture leafy as long as possible, hence maintaining at good level the CLA precursors concentration in the grazed herbage. - Minimize the use of use of non-fat enriched supplements unless the quality of the pasture forage is poor. They usually have substitution effects and may also exert a dilution effect on most beneficial PUFA. - Consider offering fat-enriched supplements, particularly when the intake of green forage is expected to be low. This is an area, which deserves further multi-disciplinary studies involving the evaluation of technological and sensory properties of ovine dairy produce. All this information is currently being submitted for publication in peer-refereed journals and will be disseminated at farm scale via technical papers and ad hoc meetings with farmers, advisers and cheese manufacturers. Technological implementation could include compound feed manufacture companies being interested in the development of novel concentrate production lines based on the above outcome.

The result consists of a set of blueprints to allow dairy farmers to produce milk with an improved fatty acid composition. The improved milk fat is characterised by having a higher content of omega-3 fatty acids and cis-9, trans-11 conjugated linoleic acid and a lower content of saturated fatty acids. This pattern of change, on the basis of existing literature, would be expected to improve the health properties of milk (and dairy foods made from it) in terms of reduced rates of cardiovascular disease and potentially cancer. Human nutrition studies (see Result 36846 in this project) show that the changes in milk fatty acids achieved are sufficient to significantly alter human blood fatty acid levels in volunteers consuming 50 g/d of "healthy" compared to 50 g/d of standard cheese. The result would allow extention services in the different dairying areas of the EU to advise farmers as to how they can produce "healthy" milk. This may involve modification of the pasture management and forage conservation systems and the use of particular feed supplements. The precise advice depends on the local dairy system. Each system has been extensively tested in one case for a continuous period of two years.

Milk of improved fatty acid composition was used to produce semi-hard cheese on a pilot scale. Full chemical and sensory evaluation of the cheese over its intended shelf life showed that there were no adverse effects due to the use of milk of altered fatty acid composition. The resulting cheeses had levels of CLA and omega-3 polyunsaturated fatty acids that were approximately three times the levels in cheese made from the standard (control) milk. The particular cheeses were evaluated in a human nutrition study, which showed that consumption of 50g/d of the cheese significantly raised levels of CLA and omega-3 polyunsaturated fatty acids compared to values for people consuming the same quantity of control cheese. The result provided the basis for marketing semi-hard cheese with an improved, "healthy" fatty acid composition. It defines the types of cheese that can be made and the claims that might be used in its marketing.

Several lines of evidences have shown that c9,t11 CLA either in natural or synthetic forms possesses strong anticarcinogenic and antiatherogenic activities in animal models. Unfortunately, these properties are very difficult to demonstrate in humans because they could be evaluated only with a long period of supplementation and in a large population. However, some observational studies have shown that an increased intake of dairy products, with a supposed increase of c9,t11 CLA, may lower the incidence of mammary cancer and cardiovascular events. One of the mechanisms by which c9,t11 CLA may exert its effects is by its metabolism and consequent deposition of its metabolites in tissues which may influence eicosanoid production, deeply involved in cell proliferation and inflammation, two major events in carcinogenesis and atherosclerosis. In this project we therefore aimed to verify whether a relative low intake of CLA-enriched cheese which corresponded to the daily average intake of the Italian population, was sufficient to significantly increase plasma levels of c9,t11 CLA and its metabolites. Our data showed that an increased of cheese c9,t11 CLA content from about 0.5-1%, which is the average concentration of c9,t11 CLA in cows cheese in the market, to 2-4 % of the CLA enriched cheese was sufficient to increase significantly the plasma levels of CLA and its metabolites in subjects who had a daily intake of 50 g of CLA enriched cheese. We therefore demonstrate that it would be envisaged to increase c9,t11 CLA at least up to 2 % in dairy products in order to have a significantly change in plasma fatty acid profile.

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