CLA Formation By Lactobacillus Acidophilus Stains To Be Used In Dairy Industry

Conjugated linoleic acid (CLA) has attracted much attention as a novel type of biologically functional lipid in recent decades. The benefits that CLA produces cover the inhibition of carcinogenesis, improvement of atherosclerosis, and lowering of fat in body. CLA mainly synthesized throughout chemical isomerization of linoleic acid (LA) contains much unexpected isomers as by-products which are hardly employed in food and medicine sectors. Therefore, biology isomerization of LA into CLA with the use of food-grade microorganisms like lactic acid bacteria finds a new way for CLA-fortified foods. The objectives of this present study were: (1) to investigate the ability of Lactobacillus acidophilus strains usually used for the adjunct cultures as probiotics to metabolize LA to CLA;(2) to deal with the intrinsic and extrinsic factors affecting the production of CLA by this organism;and (3) to manufacture dairy products fermented with CLA-producing strains.In the present work, ultraviolet spectrophotometry and gas-liquid chromatography were adapted to analysis the level of CLA formed by L. acidophilus NCFM and L. acidophilus Lakcid used. It was seen that establishment of ultraviolet spectrophotometry was available for the assay of CLA concentration in laboratory case. A programmed-temperature gas chromatography (injector temperature at 200℃, maintenance of fire temperature at 60℃ for 5 min, and then elevation of temperature to 220℃ at a rate of 20℃/min within maintained time of 47 min;detector temperature at 250℃;injection volume with lμL;delay-time with 7 min;runtime with 60 min) was a good analytical method in determining the presence of CLA occurring in different media.It was proved that utilization of ultraviolet radiation and NTG to induce L. acidophilus NCFM and L.acidophilus Lakcid produced two mutants with higher ability to transform LA to CLA than those of original strains. The screened two mutants L. acidophilus NCFM-mt and L. acidophilus Lakcid-mt formed the maximum CLA with 28.67 μg/mL and 41.61 μg/mL respectively, whereas the original strains only produced CLA of 19.11 μg/mL for L. acidophilus NCFM or 31.4 μg/mL for L. acidophilus Lakcid.The optimal conditions for L. acidophilus NCFM-mt and L. acidophilus Lakcid-mt to transform LA into CLA were incubation temperature 37°C for 24h in a medium with pH 7.5 plus LA concentration of 0.2 g/L. A 5% (v/v) inoculation size was recommended in the maximum biosynthesis of CLA by the two mutants.L.acidophilus Lackid-mt preferred lactose as a carbon source to other sugars, and took peptone as the nitrogen source. The ration of lactose to peptone with 2:3 yielded the maximum CLA of 43.81 fig/mL by this mutant. L. acidophilus NCFM-mt liked saccharose as the best sugar and peptone as a nitrogen source. The ration of saccharose to peptone with 2:1 yielded the maximum CLA of 32.19 ug/mL by L. acidophilus NCFM-mt.BSA was proved to be the best agent in emulsifying LA than Tween 80 and monoglyceride. The two mutants grew good in MRS medium containing emulsified LA incubated at 37°C for 48h. pH values in medium cultured with L.acidophilus Lackid-mt and L.acidophilus NCFM-mt declined quickly from initial 6.5 to less than 3.7. Gas chromatography analysis indicated that the conversion rate of LA to CLA was 59% or 52% in the presence of L. acidophilus Lackid-mt or L. acidophilus NCFM-mt. The content of CLA isomers in total fatty acids was 36% in the presence of the mutant Lackid-mt or 32% in the presence of the mutant NCFM-mt.LA isomerase, as one of important enzyme able to convey LA to CLA, was extracted from the cells of L. acidophilus strains grown in MRS incubated at 37°C for 24h. Its purification and its properties were characterized in the following experiments. The molecular weight of LA isomerase from the two mutants L. acidophilus NCFM-mt and L. acidophilus Lakcid-mt was 60.5 KD. The LA isomerase showed the maximum activity in transformation of LA to CLA if the enzyme was incubated at 44°C for 10 h and maintained in a given pH of 6.0. It was proved that the enzyme was sensitive to heat relatively and never tolerated temperature higher than 60°C. Metal ions had clear effects on enzyme activity. Among metal ions used in the present study, Fe3+, Fe2+ and Na+ promoted positively the activity of LA isomerase compared to Mg2+ and K+. In fact, Mn2+, Zn and Cu + had a strong inhibition against LA isomerase. Gas chromatography analysis indicated that use the purified LA isomerase metabolized 67% LA to CLA.Skim-milk and whole milk cultured with CLA-producing L. acidophilus strains were sampled for the determination of CLA concentration after the termination of fermentation and subsequent 90-day refrigerated storage periods. Clearly, addition of LA to the cultured milks increased the production of CLA by the two mutants. An appropriateinclusion of carbon and nitrogen sources in cultured milks also improved CLA formation by L. acidophilus strains. It was noted that glucose was the best sugar in promoted the two mutants grown in milk to produce much more CLA, followed by lactose and saccharose. Compared to casein, urea and ammonium sulfate, inclusion of yeast extract in cultured milks was a good way to enhance L. acidophilus strains to form the most CLA. Addition of 0.2g/L LA to the cultured milks was appropriate for the biggest production of CLA by the two mutants. Utilization of L.acidophilus NCFM-mt and Lacidophilus Lackid-mt averagely produced as much 10 times CLA as those of plain yogurt products without inoculation of CLA-producing strains. In addition to the holding of higher CLA, the live cells reached 5.0x109 cfu/mL in the cultured milks. It was observed that CLA concentration in the cultured milks had little changed between the day 0 and day 16 of refrigerated storage, and then slowed down gently following the 16* days. CLA content in the cultured milks refrigerated declined by 21.5% from day 0 to day 30, and 44.31 % from day 30 to day 90.