Conjugated linoleic acid production by cells and enzyme extract of Lactobacillus delbrueckii ssp. bulgaricus with additions of different fatty acids

The objective of the study was to determine the effect of fatty acid additions to the cells and enzyme extract of Lactobacillus delbrueckii ssp. bulgaricus (CCRC14009) on CLA production. Washed cells of L. delbrueckii ssp. bulgaricus, obtained by cultivation in a MRS broth, were mixed with BSA and each of the three fatty acids: linoleic, oleic, and linolenic acids in sodium phosphate buffer at pH 6.5. After incubation at 37 °C for 108 h, CLA concentration was analyzed by HPLC. Enzyme extract from the culture was also reacted with each fatty acid at 50 °C for 10 min at pH 5 to test for CLA production. Results showed that linoleic acid addition to the culture improved CLA production, indicating the presence of linoleic acid isomerase activity in the culture. The crude enzyme extract from the culture was observed to be capable of oleic and linolenic acid conversions into CLA, demonstrating the possible presence of desaturase activity in the enzyme extract.     

Conjugated linoleic acid (CLA) and polyunsaturated fatty acids in muscle lipids of lambs from the Patagonian area of Argentina

The concentrations of fatty acids were measured in total lipids, triacyglycerol and phospholipid fractions of intramuscular fat (IMF) from the Longissimus dorsi (LD) muscle of 10 lambs reared to approximately 30kg live weight on natural pasture with their dams. Fatty acid composition was also measured in 25 (five of each) Semitendinosus (ST), Semimembranosus (SM), Rectus femoris (RF), Gluteus (GLU) and Tensor fascia latea (TFL) muscles. Intramuscular fat percentages were similar for all muscles. Aspects of the fatty-acid patterns of relevance to human nutrition tended to favor the leg muscles with lower saturated fatty acids (SFA %), n-6/n-3 fatty acid ratios (p<0.01) and higher concentrations of the conjugated linoleic acid (CLA) (p<0.05). The estimated fatty acid concentrations (mg/100g of meat) showed higher contribution of arachidonic (C20:4 n-6), eicosapentanoic (C20:5 n-3), docosapentanoic (C22:5 n-3) and docosahexanoic (C22:6 n-3) acids in leg compared to LD lipids.     

Growth and carcass fatty acid composition of beef steers fed soybean oil for increasing duration before slaughter

Duration of soybean oil (SBO) supplementation needed to enhance carcass conjugated linoleic acid (CLA) and trans-vaccenic (TVA) content was examined using 96 beef steers (293.6 ± 3.9 kg) fed a 78% corn-based diet supplemented with SBO for 0, 77, 137, or 189 days before slaughter. Duration of SBO supplementation had no effect (P ? 0.15) on animal performance or carcass traits, nor (P ? 0.15), total, total saturated, or total polyunsaturated fatty acids of Longissimus dorsi (LD). Concentrations of CLA in LD were not affected (P ? 0.18) by SBO supplementation. Concentrations of monounsaturated fatty acids (MUFA) decreased linearly (P = 0.03) in LD, whereas TVA increased (P = 0.04) in adipose tissue and tended (P = 0.07) to increase in LD with increasing duration of SBO supplementation. Supplementing SBO to a concentrate-based diet may enhance TVA without impacting CLA, while reducing the MUFA content of lean beef.     

Enzymatic synthesis of lysophosphatidylcholine containing CLA from sn-glycero-3-phosphatidylcholine (GPC) under vacuum

Lysophosphatidylcholine (LPC) was successfully synthesised by enzyme-catalysed esterification of sn-glycero-3-phosphatidylcholine (GPC) with the acid form of conjugated linoleic acid (CLA), using a vacuum system. GPC was prepared from phosphatidylcholine (PC) by alkaline deacylation using a methanolic tetra-butylammonium hydroxide solution. Five enzymes were tested as biocatalysts for direct esterification, and lipases were found to be more effective than phospholipases for production of LPC. Amongst the enzymes tested, Novozym 435, from Candida antarctica, gave the highest yield of LPC. The optimal temperature, molar ratio of substrate, and vacuum were 40 °C, 1:50 (GPC to CLA), and 1 mm Hg, respectively. The maximum yield (70 mol%) of LPC was obtained under optimal conditions after a 48 h reaction time.     

The effect of pasteurisation temperature on the CLA content and fatty acid composition of white pickled cheese

In this study, the effect of pasteurisation temperature on fatty acid composition of cheese was investigated. The fatty acid composition of raw and different heat‐treated milk, salt and salt‐free cheese were determined using cheese made from raw milk at temperatures varying between 70 and 90°C for 5 min. Generally, C 16:0 palmitic acid was the major fatty acid present in all milk and cheese samples. C 18:1 t11 vaccenic acid was the major trans fatty acid (TFA) in all samples. C 18:2 cis‐9, trans‐11 (Rumenic acid) was the major CLA isomer in these samples. Pasteurisation temperatures had no effect on TFA, CLA and fatty acid composition of the milk and cheese samples.     

Augmentation of vaccenate production and suppression of vaccenate biohydrogenation in cultures of mixed ruminal microbes

To increase ruminal outflow of trans-vaccenic acid (t-VA), a new strain of Butyrivibrio fibrisolvens (MDT-10) was isolated that has a great ability to hydrogenate linoleic acid (LA) to t-VA. When strain MDT-10 was added to the batch cultures of mixed ruminal microbes (1% of the total number of viable ruminal bacteria), LA conversion to t-VA increased greatly; after 3 h, t-VA levels were > 4-fold higher than the control. By 10 h, all of the t-VA was hydrogenated to stearic acid. However, when a new strain of Bifidobacterium adolescentis (HF-11), which has a high capacity for incorporation of t-VA, was added in conjunction with MDT-10 (1% of the total number of ruminal bacteria), t-VA levels after 10 h were 6 times higher than with MDT-10 alone. These results suggest that t-VA produced by MDT-10 was incorporated into HF-11 cells, resulting in protection of t-VA from t-VA-hydrogenating microbes. Similar results were obtained in a continuous culture of mixed ruminal microbes in which addition of HF-11 simultaneously with MDT-10 increased the amount of t-VA in the effluent 2.5-fold. Both MDT-10 and HF-11 appeared to grow readily in the presence of mixed ruminal microbes. Sixty-two percent of t-VA incorporated by HF-11 was present in the free form, whereas 19, 15, and 3%, respectively, were incorporated into monoacylglycerol, glycerophospholipid, and diacylglycerol fractions. Because these lipids can be digested in the small intestine, it is likely that most t-VA in HF-11 cells is absorbed. Thus, introduction of MDT-10 and HF-11 simultaneously to the rumen might increase the amount of t-VA absorbed and might consequently increase the conversion of t-VA to conjugated linoleic acid in tissue.     

Chemical characteristics,fatty acid composition and conjugated linoleic acid (CLA) content of traditional Greek yogurts

Many studies with conjugated linoleic acid (CLA) indicate that it has a protective effect against mammary cancer. Because dairy products are the most important dietary sources of CLA, we have investigated the CLA concentrations and additionally the fatty acid profiles and chemical composition of several commercial, traditional, Greek yogurts from different geographical origin. The fat content of yogurts was in the order of goat < cow < sheep. Cow, sheep and goat milk yogurts contain respectively 0.128–1.501, 0.405–1.250 and 0.433–0.976 g CLA/100 g fat. Low-fat milk yogurts showed lower values of c-9, t-11 CLA content on lipid basis compared to full-fat yogurts. Samples from mountain areas showed average c-9, t-11 CLA content higher than those from prairie districts. The highest amounts of saturated fatty acids (SFA) were found in low-fat yogurts, of monounsaturated fatty acids (MUFA) in sheep milk yogurts and of polyunsaturated fatty acid (PUFA) in low-fat cow milk yogurts.     

Factors influencing proportion and composition of CLA in beef

Bovine meat is criticised for the bad nutritional image of its lipids and fatty acids. However, with dairy products, beef is the major source of conjugated linoleic acid (CLA) which could have several human health benefits. The present study compared, from data of five nutritional experiments on bovine animals performed by the laboratory, the impact of factors linked to the animals (breed, age, sex, type of muscle) and to feeding conditions (basal diet, lipid supplements) on the CLA proportion and composition in muscles. Among these factors, linseed supplementation was an efficient way to increase CLA proportion in beef (+22% to +36%) but was highly modulated by the nature of the basal diet, and by intrinsic factors (breed, age/sex, type of muscle) since these ones could modulate CLA proportion in beef from 24% to 47%. Moreover, these factors modified also the proportion of cis,trans-CLA, related to cis,cis- and trans,trans-isomers. Specific biological properties of these latter isomers should be determine to understand the consequences of intramuscular CLA isomer variations for the health of consumers.     

Isoamylacetate production by entrapped and covalently bound Candida rugosa and porcine pancreatic lipases

Candida rugosa lipase (CRL) and porcine pancreatic lipase (PPL) were immobilised by entrapping and also by covalent binding for use in synthesis of isoamyl acetate (IAAc), which has a typical banana flavour. Lipase entrapment was carried out by dripping sodium alginate (Na-Alg)-chitosan (Chi)-lipase mixture into CaCl₂-glutaraldehyde (GAL) solution to obtain Ca-Alg/Chi_CRL/PPL. Immobilisation conditions were optimised as 1.5% Na-Alg, 1.5% chitosan and 0.15% GAL. Ca-Alg/Chi_CRL/PPL samples showed the highest activity when they were dried upon reaching 27% of their initial weights. Covalent binding was achived with Chi modified with spacerarm via glutaraldehyde to get Chi_CRL/PPL. The highest IAAc production was observed when 1,3-diaminopropane was used as a spacer arm. The best ester yield was achieved in heptane, at 40 and 45 °C reaction temperatures, 50 mM IAA and 50 or 75 mM AA concentrations. The amount of IAAc was nearly 10 times higher for the batch type than for the continuous packed bed column reactor.     

Production of conjugated linoleic acid by Propionibacterium freudenreichii

Two strains each of Propionibacterium freudenreichii ssp. shermanii and P. freudenreichii ssp. freudenreichii were tested for their ability to produce conjugated linoleic acid (CLA) in sodium lactate medium (SLM), De Man-Rogosa-Sharpe (MRS) medium and skim-milk. Data showed that both strains were able to produce CLA in three media supplemented with different concentrations of sunflower oil. Maximum production of CLA (78.8 μg/ml) was observed after 36 h of incubation in MRS containing 12 mg/ml of sunflower oil by P. freudenreichii ssp. shermanii. Moreover, the growths of both strains were inhibited by sunflower oil and a positive relationship between CLA production and ability to tolerate sunflower oil was observed. At the same time, it was also observed that the inhibitory effects on P. freudenreichii ssp. shermanii and P. freudenreichii ssp. freudenreichii in three media follow the order SLM > skim-milk > MRS and SLM > MRS > skim-milk, respectively. Micro aerobic conditions were in favour of increasing the amounts of CLA. The amounts of CLA increased from 0 to 36 h under micro aerobic conditions and no significant (p > 0.05) increases in total CLA levels were observed after 80 h of incubation. Results showed that P. freudenreichii may have potential for producing CLA.     

Direct quantification of fatty acids in dairy powders with special emphasis on trans fatty acid content

In this study a validated procedure for accurate determination of fatty acids in dairy products, with special emphasis on total trans fatty acids (TFA) content is presented. Dairy fat naturally contains 4–6% of trans fatty acids, mainly trans-octadecenoic acids (i.e. vaccenic acid), and 0.3–1.5% of conjugated linoleic acids (CLA). The proposed procedure does not require lipid extraction, and transesterification of lipids could be carried out directly on dairy products. Optimal analytical conditions have been developed to allow accurate determination of TFA content without prior fractionation of cis/trans FAME isomers by thin-layer chromatography. The methodology requires the use of a highly polar open tubular capillary column having at least 100 m length. CLA and other fatty acids from C4:0 (butyric) acid to long-chain polyunsaturated fatty acids (LC-PUFAs) could also be analyzed. Therefore, the methodology presented is versatile and could be used for both targeted analysis (e.g. determination of TFA in dairy products) and determination of the broad fatty acid profile in dairy products.     

Effects of dietary replacement of fish oil by conjugated linoleic acid on some meat quality traits of Pacific white shrimp Litopenaeus vannamei

This experiment was conducted to evaluate the effects of fish oil replacement by conjugated linoleic acid (CLA) on body proximate analysis and thiobarbituric acid-reactive substances, fat content, shear force and fatty acid composition in musculature of Pacific white shrimp (Litopenaeus vannamei). Graded levels of CLA (0%, 0.5%, 1% and 2%) were added to the basic diet of shrimp at the expense of fish oil. Results showed that fat content (p = 0.036) and shear force (p = 0.001) in shrimp musculature were enhanced with increasing dietary CLA inclusion. Fish oil replacement by CLA significantly promoted the incorporation of cis-9, trans-11 CLA (p = 0.0001) and trans-10, cis-12 CLA (p < 0.0001) into shrimp musculature; moreover, the polyunsaturated fatty acid was elevated (p = 0.020) and monounsaturated fatty acid was reduced by CLA inclusion (p = 0.024). It was concluded that replacement of fish oil by CLA could improve some meat quality traits of shrimp and 1% CLA was an appropriate amount.     

Biocatalytic production of lactose ester catalysed by mica-based immobilised lipase

Enzyme immobilisation technology is an effective means to improve sugar ester production through the employment of biocatalysts. In the present study, immobilisation of Candida rugosa (CRL) lipase onto amino-activated mica is performed via covalent bonding (namely Amino-CRL) and the cross-linking of lipases into nano-reactors through physical adsorption (namely NER-CRL). Free and immobilised lipases were tested for their esterification activities. Specific activities for Amino-CRL and NER-CRL increased by 2.4 and 2.6-fold, respectively, upon immobilisation. Extending this work, immobilised lipases have novel capabilities in the synthesis of sugar esters. The optimised conditions for sugar fatty acid ester syntheses are 48 h at 2:1 of molar ratio of lactose sugar to capric acid at 55 °C. Furthermore, a high operational stability with half-lives of over 13 and 10 runs was achieved for NER-CRL and Amino-CRL, respectively, indicating the efficiency of the immobilisation process.     

Fatty acid profile, trans-octadecenoic, α-linolenic and conjugated linoleic acid contents differing in certified organic and conventional probiotic fermented milks

Development of dairy organic probiotic fermented products is of great interest as they associate ecological practices and benefits of probiotic bacteria. As organic management practices of cow milk production allow modification of the fatty acid composition of milk (as compared to conventional milk), we studied the influence of the type of milk on some characteristics of fermented milks, such as acidification kinetics, bacterial counts and fatty acid content. Conventional and organic probiotic fermented milks were produced using Bifidobacterium animalis subsp. lactis HN019 in co-culture with Streptococcus thermophilus TA040 and Lactobacillus delbrueckii subsp. bulgaricus LB340. The use of organic milk led to a higher acidification rate and cultivability of Lactobacillus bulgaricus. Fatty acids profile of organic fermented milks showed higher amounts of trans-octadecenoic acid (C18:1, 1.6 times) and polyunsaturated fatty acids, including cis-9 trans-11, C18:2 conjugated linoleic (CLA-1.4 times), and α-linolenic acids (ALA-1.6 times), as compared to conventional fermented milks. These higher levels were the result of both initial percentage in the milk and increase during acidification, with no further modification during storage. Finally, use of bifidobacteria slightly increased CLA relative content in the conventional fermented milks, after 7 days of storage at 4 °C, whereas no difference was seen in organic fermented milks.     

The effect of solids dilution rate and oil source on trans C18:1 and conjugated linoleic acid production by ruminal microbes in continuous culture

The objective of this study was to evaluate the effect of solids dilution rate (SDR) and oil source [soybean oil (SBO) or linseed oil (LSO)] on the ruminal production of trans C18:1 and conjugated linoleic acid (CLA). A dual-flow continuous culture system consisting of 4 fermenters was used in a 4 × 4 Latin square design with a factorial arrangement of treatments over 4 consecutive periods of 10 d each. Treatment diets (50:50 forage to concentrate) were fed at 120 g/d of dry matter (DM) in 3 equal portions. The concentrate mix contained 1% fish oil and either 2% SBO or 2% LSO on a DM basis. Treatments were as follows: 1) SBO at 6%/h SDR, 2) SBO at 3%/h SDR, 3) LSO at 6%/h SDR, and 4) LSO at 3%/h SDR. The oil source by SDR interaction was not significant for trans C18:1 and CLA fatty acids. The concentrations of trans C18:1 and vaccenic acid were greater in effluents when diets were supplemented with SBO vs. LSO (37.11 vs. 34.09 and 32.71 vs. 29.70 mg/g of DM, respectively) and at high SDR than low SDR (37.60 vs. 33.61 and 32.72 vs. 29.61 mg/g of DM, respectively). The concentration of cis-9, trans-11 CLA in effluents was also greater with SBO than LSO (0.81 vs. 0.40 mg/g of DM) supplementation and at high SDR than low SDR (0.68 vs. 0.54 mg/g of DM). Biohydrogenation of linoleic acid and linolenic acid increased at higher SDR within each oil treatment. Based on these results, SBO supplementation at high SDR enhances ruminal production of vaccenic acid, and therefore could potentially enhance cis-9, trans-11 CLA in milk fat through synthesis by Δ⁹-desaturase.     

Effect of high or low protein ration combined or not with rumen protected conjugated linoleic acid (CLA) on meat CLA content and quality traits of double-muscled Piemontese bulls

A trial was carried out on double-muscled Piemontese bulls to evaluate the effects of two rations differing in crude protein density (HP=14.5% DM and LP=10.8% DM) and top dressed or not with 80 g/d of rumen protected CLA (rpCLA) for a long period (336 d) on meat quality traits and CLA content. Forty-eight bulls were fed one of the four experimental diets based on corn silage and cereals and were slaughtered at an average age and body weight (BW) of 562 ± 18 d and 668 ± 56 kg, respectively. After slaughter the 5th rib cut was dissected into Longissimus thoracis (LT), other muscles (OM), inter-muscular fat (IF), cover fat (CF), and bones. Muscles and fatty tissues were analyzed for proximate composition and fatty acid (FA) profiles. Rib was composed by 81.1, 3.7, 1.6 and 13.6% of muscles, IF, FC and bone, respectively; LT and OM contained only 0.8 and 1.4% of lipid, respectively. The treatments did not influence these values, but rpCLA increased, compared to control, both c9,t11-CLA and t10,c12-CLA concentrations in all the tissues (P<0.01); t10,c12-CLA concentration was increased much more in muscles (+20 times) than in fatty tissues (from +0.2 to +0.9 times). This suggests that in the muscle this isomer is preferentially stored and/or less combusted with respect to other fatty acids. Low protein rations did not exert any influence on carcass and meat quality, as on growth performance, but reduced nitrogen excretion, their use for improving the environmental impact (process quality) of this meat production system is recommended.     

Strategy for simultaneous saccharification and fermentation using a respiratory-deficient mutant of Candida glabrata for bioethanol production

High temperature and agitation are advantageous for ethanol production from insoluble feedstock when using the simultaneous saccharification and fermentation (SSF) technique. To construct an effective SSF system, a respiratory-deficient mutant was isolated from the thermotolerant yeast Candida glabrata. Our results suggest that this respiratory-deficient mutant has higher ethanol production abilities in SSF.