Cloning of a gene for D-sorbitol dehydrogenase from Gluconobacter oxydans G624 and expression of the gene in Pseudomonas putida IFO3738

We have cloned a novel gene for d-sorbitol dehydrogenase (SLDH), which efficiently converted D-sorbitol to L-sorbose, from Gluconobacter oxydans G624 (FERM BP-4415). A cosmid library of the genomic DNA was screened by assaying SLDH activity. The inserted DNA from a positive clone was downsized by subcloning into charomid and pUCP plasmid, successively. Sequencing analysis of the DNA responsible for SLDH activity revealed an open reading frame of 1455 bp coding for 485 amino acid residues with a calculated molecular mass of 53,642 Da. The amino acid sequence showed 42.2% identity with a NAD+-dependent mannitol dehydrogenase (MDH), which catalyzed conversion of d-sorbitol to d-fructose, from Pseudomonas fluorescens DSM50106. Since the intact SLDH was found to be very unstable during isolation and purification, this SLDH fused to 6 x His-tag was expressed in Pseudomonas putida IFO3738 and purified by immobilized metal affinity chromatography using cobalt-based resins. The 6 x His-tag SLDH catalyzed the oxidation of D-sorbitol to L-sorbose and exhibited 15 times higher activity in the presence of NADP+ than that of NAD+. These results indicate that the SLDH is a novel kind of dehydrogenase distinct from MDH previously reported.     

Purification and molecular characterization of a quinoprotein alcohol dehydrogenase from Pseudogluconobacter saccharoketogenes IFO 14464

We have cloned and verified a gene for a novel quinoprotein alcohol dehydrogenase (ADH) from Pseudogluconobacter saccharoketogenes IFO 14464 that has the ability to oxidize L-sorbose to 2-keto-L-gulonic acid (2-KLGA). The enzyme was purified from the soluble fraction of the bacterium and was estimated to be a monomeric protein with a molecular weight of 65 kDa from the analyses of SDS-PAGE and gel-filtration chromatography. An open reading frame of 1824 bp for 608 amino acid residues was estimated as the gene for ADH because of the consistency of the calculated molecular mass and the elucidated partial amino acid sequences of the native enzyme. Homology search revealed that the enzyme showed close similarity to quinoprotein alcohol dehydrogenases isolated from Methylobacterium extorquens and Acetobacter aceti, particularly in the tryptophan docking motifs in the alpha-subunits of those dehydrogenases. The ability to convert L-sorbose to 2-KLGA was found when the lysate of recombinant Escherichia coli DH10B transformed with the gene for ADH was mixed with CaCl2and pyrroloquinoline quinone (PQQ). These data indicate that the cloned DNA is the desired gene for the ADH in which CaCl2 and PQQ are essential for enzymatic activity.     

丁香酚对金黄色葡萄球菌抗菌作用的探究

为了寻找代替化学防腐剂的绿色安全天然添加剂,研究了丁香酚对金黄色葡萄球菌的抑菌作用。以丁香酚为原材料,采用96孔板微量稀释法和琼脂平板稀释法确定最低抑菌浓度（MIC）和最低杀菌浓度（MBC）,通过菌落数直观地确定丁香酚对金黄色葡萄球菌的MIC值和MBC值;并以分光光度计法探究不同质量浓度丁香酚对金黄色葡萄球菌中苹果酸脱氢酶（MDH）和琥珀酸脱氢酶（SDH）酶活性的影响。结果表明,丁香酚对金黄色葡萄球菌的最低抑制浓度（MIC）为600 μg/mL,最低杀菌浓度（MBC）为700 μg/mL,其可将金黄色葡萄球菌菌体蛋白质质量浓度、苹果酸脱氢酶（MDH）酶活、琥珀酸脱氢酶（SDH）酶活分别降至0.033 mg/mL、8.28 U/mg、2.40 U/mL,原因可能是抑制了菌体三羧酸循环和电子链传递中相关关键酶的活性。     

微液滴适应性进化强化大肠杆菌耐受高浓度L-山梨糖

前期研究构建了1株组成型表达山梨糖脱氢酶(sorbose dehydrogenase,SDH)的大肠杆菌工程菌,该菌株能利用L-山梨糖生产维生素C前体2-酮基-L-古龙酸(2-keto-L-gulonic acid,2-KLG),但对底物L-山梨糖的耐受性较差。为解决这一问题,对该菌株进行适应性进化,并强化了进化菌株发酵生产2-KLG的能力。首先,应用基于微流控技术的全自动高通量微生物液滴培养系统,将出发菌株在不同浓度梯度的L-山梨糖培养基中生长、传代,获得能够耐受高浓度L-山梨糖的进化菌株。在摇瓶上进一步验证,最终获得了1株能耐受高浓度L-山梨糖的进化菌株2-F6。然后在2-F6中共表达了能促进2-酮基-L-古龙酸积累的山梨酮脱氢酶(sorbosone dehydrogenase,SNDH),并在摇瓶水平上对接种量、发酵温度、SNDH诱导时间、IPTG诱导浓度以及L-山梨糖添加量进化了优化,在最优条件下,2-KLG的产量达6. 05 g/L。最终,将摇瓶发酵条件放大至5 L发酵罐后,2-KLG的产量为5. 70 g/L。研究结果为一菌一步发酵法生产维生素C前体2-KLG提供了参考。     

Purification and molecular characterization of a quinoprotein alcohol dehydrogenase from Pseudogluconobacter saccharoketogenes IFO 14464

We have cloned and verified a gene for a novel quinoprotein alcohol dehydrogenase (ADH) from Pseudogluconobacter saccharoketogenes IFO 14464 that has the ability to oxidize -sorbose to 2-keto- -gulonic acid (2-KLGA). The enzyme was purified from the soluble fraction of the bacterium and was estimated to be a monomeric protein with a molecular weight of 65 kDa from the analyses of SDS-PAGE and gel-filtration chromatography. An open reading frame of 1824 bp for 608 amino acid residues was estimated as the gene for ADH because of the consistency of the calculated molecular mass and the elucidated partial amino acid sequences of the native enzyme. Homology search revealed that the enzyme showed close similarity to quinoprotein alcohol dehydrogenases isolated from Methylobacterium extorquens and Acetobacter aceti, particularly in the tryptophan docking motifs in the α-subunits of those dehydrogenases. The ability to convert -sorbose to 2-KLGA was found when the lysate of recombinant Escherichia coli DH10B transformed with the gene for ADH was mixed with CaCl₂ and pyrroloquinoline quinone (PQQ). These data indicate that the cloned DNA is the desired gene for the ADH in which CaCl₂ and PQQ are essential for enzymatic activity.     

Construction of recombinants Pseudomonas putida BO14 and Escherichia coli QEFCA8 for ferulic acid biotransformation to vanillin

Recombinants Pseudomonas putida BO14 and Escherichia coli QEFCA8 capable of ferulic acid biotransformation to vanillin were constructed using homologous recombination and a PCR based cloning strategy, respectively. In the liquid culture of P. putida BO14, 26.81+/-2.30 microg vanillin ml(-1) of culture filtrate was detected. In the case of recombinant E. coli QEFCA8, 19.37+/-1.95 microg vanillin ml(-1) of culture filtrate was detected. Results indicate that the strains could be useful for the biotechnological production of vanillin, a very important flavoring substance.     

Heterologous expression of a gene encoding cholesterol oxidase in probiotic strains of Lactobacillus plantarum and Propionibacterium freudenreichii under the control of native promoters

To develop systems for the expression of heterologous genes in probiotic strains of Lactobacillus and Propionibacterium, we used Lactobacillus plantarum and Propionibacterium freudenreichii and a modified gene encoding cholesterol oxidase (choA) from Streptomyces sp. to generate working models. The acetyl coenzyme A carboxylase (acc) promoter derived from the acc operon of L. plantarum L137 and a previously constructed shuttle vector, pRN14, were used to construct vectors for the expression of heterologous genes in lactic acid bacteria. The concentration of cholesterol oxidase in recombinant L. plantarum carrying choA fused to the NH2-terminal region of the first open reading frame of the acc operon was 3.6 mU/mg of protein. Using the promoters from Propionibacterium, namely, P4, P8, and P138, which enabled high-level expression of choA in Escherichia coli, and a previously constructed shuttle vector pPK705, we constructed expression vectors for Propionibacterium. In recombinant P. freudenreichii subsp. shermanii IFO12426, the activities of cholesterol oxidase generated under the control of promoters P4, P8, and P138 were 1.6, 4.3, and 7.2 U/mg of protein, respectively. The expression of heterologous genes may facilitate the production of useful proteins in these economically important bacteria.     

柠檬酸和氯化钠抑制地衣芽孢杆菌生物被膜的研究

地衣芽孢杆菌是食品中常见腐败菌。该研究发现地衣芽孢杆菌在常见培养基（如TSB、NB、LB、1/2LB）中均有较强的产生物被膜能力,地衣芽孢杆菌稀释100倍、在胰酪胨大豆肉汤（TSB）培养基中30 ℃静置培养更利于地衣芽胞杆菌在96孔板上产生物被膜。测得地衣芽孢杆菌对柠檬酸和氯化钠的最低抑菌质量浓度（MIC）分别为1.28 mg/mL和64 mg/mL。采用结晶紫染色法,研究不同质量浓度柠檬酸和氯化钠对地衣芽孢杆菌生物被膜的抑制效果。结果显示,1/2MIC的氯化钠即能显著抑制地衣芽孢杆菌生物被膜形成（P＜0.01）;而1/2MIC和1MIC的柠檬酸对地衣芽孢杆菌生物被膜形成无明显抑制效果（P＞0.05）。     

库巴德巴利酵母FBKL2.0130 D-阿拉伯糖醇脱氢酶基因的克隆表达及酶学性质研究

该研究从库巴德巴利酵母FBKL2.0130中克隆D-阿拉伯糖醇脱氢酶基因ARD,利用闪电克隆技术构建重组质粒Yep-PAK,并将重组质粒导入酿酒酵母（Saccharomyces cerevisiae）W303-1A中,得到重组菌株W303-ARD并成功表达。并对重组菌株和亲本菌株的生长性能进行测定,并对重组D-阿拉伯糖醇脱氢酶酶学性质进行研究。结果表明,与亲本菌株相比,重组菌株的D-阿拉伯糖醇脱氢酶的还原活力从（32.7±0.803） U/mL提高至（122.4±1.012） U/mL,氧化活力从（18.46±0.105） U/mL提高至（97.30±0.826）U/mL。重组D-阿拉伯糖醇脱氢酶的最适反应温度为30 ℃,在25～40 ℃范围内氧化、还原活力均保持稳定。还原反应的最适pH值为9.0,在pH 9.0～11.0范围内比较稳定;氧化反应的最适pH值为7.0,在pH 7.0～8.0的范围内能保持较高的活性。Cu2+、Ba2+、Zn2+均能抑制重组酶活力;Mg2+对重组酶活力无明显影响;Ca2+、Mn2+、Fe3+均能提高重组酶活力。     

Production of thermotolerant N-carbamyl-D-amino acid amidohydrolase by recombinant Escherichia coli

A plasmid, pNT4553, was constructed for high level production of N-carbamyl-d-amino acid amidohydrolase (DCase), the thermostability of which has been improved by amino acid substitution. The DCase activity and the stability of the plasmid in the host cells were dependent on the Escherichia coli strains used. E. coli HB101 was the most suitable host strain among the 13 types of E. coli tested. E. coli HB101 exhibited the highest activity, i.e. 6.36 units/ml of culture broth in 2YT medium (1.6% tryptone, 1.0% yeast extract, and 0.5% NaCl, pH 7.0), and the plasmid was stably maintained by cultivation in 5 types of E. coli including HB101. Casamino acids, NZ-amine, peptone, and protein extract (a mixture of hydrolyzates of corn gluten, wheat gluten and soybean), were found to be suitable as natural nitrogen sources for both enzyme activity and growth. When cultivation was carried out in the presence of high concentrations of glycerol (6.5%) as the carbon source, and protein extract (3.0%) as the nitrogen source, in a small volume of the medium (20 ml of medium in a 500-ml shaking flask), in which the aeration level was estimated to be high, growth and activity reached OD550=63.8 (17.1 mg of dry cell weight/ml of culture broth) and 22.9 units/ml of culture broth, respectively. The economical hyperproduction of DCase using only inexpensive constituents for the medium was achieved.     

Invertase production by Saccharomyces cerevisiae protoplasts immobilized in strontium alginate gel beads

The invertase productivity of Saccharomyces cerevisiae IFO 0309 protoplasts in a static culture was 35 times (extracellular) and 9 times (extracellular + intracellular) higher than those of cells. When S. cerevisiae protoplasts were immobilized in 1.0% strontium alginate gel as an artificial cell wall, the protoplasts could be cultivated in a shake flask without disruption, and invertase was secreted into the broth. However, cell wall regeneration in the immobilized protoplasts was detected at about 24 h of cultivation. This implies that prevention of cell wall regeneration is a prerequisite for long term process with protoplasts. When 0.5 microg/ml aculeacin A (inhibitor of beta-1,3 glucan synthesis) was added to the broth, active protoplasts were maintained without cell wall regeneration for more than 24 h and invertase was produced extracellularly. Immobilized S. cerevisiae T7 protoplasts (this strain produces twice as much invertase as S. cerevisiae IFO 0309) were used for invertase production in a bubble column reactor, and a high and stable level of invertase (45 U/ml) was maintained for 72 h.     

Expression and purification of goat lactoferrin from Pichia pastoris expression system

ABSTRACT:  The recombinant goat lactoferrin (rGLF) was expressed in the methylotropic yeast Pichia pastoris using pGAPZαC vector, GAP as promoter, and Zeocin as the selective marker. After transformation of the GLF-pGAPZαC into Pichia pastoris X-33 expression host, the GLF-pGAPZαC vector was integrated into the GAP promoter locus of Pichia pastoris X-33 chromosome. The rGLF was expressed and secreted into the broth using α-factor preprosequence. SDS-PAGE and PAS staining analysis indicated that the rGLF could be purified to electrophoretic homogeneity by heparin-Sepharose 6 Fast Flow affinity chromatography and glycosylated by the expression host. The yield of purified rGLF was approximately 2.0 mg/L of culture broth. The N-terminal sequence was identical to the native goat lactoferrin (nGLF). The iron-binding behavior, papain-inhibiting property, and thermal stability of the purified rGLF were comparable to nGLF. This is the 1st report of intact goat lactoferrin expression using the P. pastoris system.     

Cloning and sequencing of a gene encoding of aldehyde oxidase in Pseudomonas sp. AIU 362

We have cloned a gene encoding an aldehyde oxidase (ALOD) oxidized glyoxal but not glyoxylic acid from Pseudomonas sp. AIU 362. The ALOD gene contained an open reading frame consisting of 888 nucleotides corresponding to 295 amino acid residues. The deduced amino acid sequence exhibited a high similarity to those of 3-hydroxyisobutyrate dehydrogenases (3-HIBDHs). We expressed the cloned gene as an active product in Escherichia coli BL21 cells. The productivity (total units per culture broth volume) of the recombinant ALOD expressed in E.?coli BL21 was 20,000-fold higher than that of ALOD in Pseudomonas sp. AIU 362. The recombinant ALOD exhibited ALOD activity and 3-HIBDH activity. The 3-HIBDH from Pseudomonas putida KT2440 also exhibited ALOD activity. Thus, the ALOD from Pseudomonas sp. AIU 362 and 3-HIBDH from P.?putida KT2440 were classified into the same enzyme group.     

Utilization of fluorogenic assay for rapid detection of Escherichia coli in acidic fruit juice

This study was undertaken to investigate interference by acids commonly found in fruit juice in Escherichia coli assays involving the use of 4-methylumbelliferyl-beta-D-glucuronide (MUG) as a fluorogenic substrate for enzyme reaction. Fluorescence intensity was negatively correlated (P < 0.001) with the volume of fresh citrus juice tested by the lauryl tryptose broth (LST)-MUG assay, and the permissible sample sizes were limited to 0.3 and 0.5 ml for fresh citrus juices with pHs of 3.3 and 3.9, respectively. In addition, false-negative results were visually observed under UV light when the E*Colite assay was used to test large volumes (5 to 10 ml per test) of fresh citrus juice or when the test broth used for the LST-MUG assay was supplemented with citric, malic, or tartaric acid at 2 to 4 g/liter. These results suggest that the size and pH of acidic samples should be controlled in MUG-based fluorogenic assays. The inhibitory effect on fluorescence was due to high acidity, which reduces fluorescence from 4-methylumbelliferone. Buffering improved the assays. When sodium bicarbonate was incorporated in the enrichment broth at 10 g/liter, the permissible sample sizes for fresh grapefruit juice (pH 3.1) increased from 0.3 to 1 ml for the LST-MUG (with 9.9 ml of broth) assay and from 3 to 10 ml for the E*Colite (with 99 ml of broth) assay.     

食品中柠檬酸快速检测试剂盒的研制

建立了一种酶法检测食品中柠檬酸含量的快速检测方法。将6 U L-苹果酸脱氢酶、9.13 U L-乳酸脱氢酶、0.5 mg烟酰胺腺嘌呤二核苷酸、2 mg硫酸镁、3.783 mg海藻糖、5 mg聚乙二醇6000在0.2 mL 0.082 g/mL双甘肽质量浓度下-60 ℃冷冻5 h,室温条件下抽真空18 h得到试剂Ⅰ,0.468 U柠檬酸裂解酶在0.2 mL水体系冷冻干燥得到试剂Ⅱ。结果表明,该方法标准曲线线性关系良好,相关系数R2=0.995,线性范围1～80 μg/L;灵敏度0.25 mg/L;检测限0.5 mg/L;各样品回收率均在90%～110%。该试剂盒方法可以在20 min实现样品中柠檬酸的快速检测,方法灵敏度高、特异性高、快速简便,适合于食品生产企业对食品中的柠檬酸进行快速检测及对食品的质量和品质进行评价。     

Production of recombinant human antithrombin by Pichia pastoris

This paper deals with the production of recombinant human antithrombin (rAT) by the methylotrophic yeast Pichia pastoris. In preliminary methanol-limited fed-batch fermentation, the rAT concentration reached 324 mg/l at 192 h of cultivation, but the specific heparin cofactor (HC) activity of rAT in the culture supernatant was 10% of that of plasma-derived antithrombin (pAT). To improve the specific HC activity of rAT, effort was first focused on the optimization of culture pH and media composition, resulting in protection of rAT against pH-dependent instability and proteolytic degradation. However, even in the optimized methanol-limited fed-batch fermentation, the specific HC activity of rAT in the culture supernatant was still 20% that of pAT. To investigate the unknown mechanisms involved in the decreased specific HC activity of rAT, the culture supernatant of mock-transfected cells was prepared by methanol-limited fed-batch fermentation. When pAT was added to this supernatant, a rapid decrease in HC activity was observed; the residual HC activity was 26% after 24 h of incubation at 25 degrees C. The loss of pAT activity was prevented by addition of a formaldehyde scavenger, amino urea, to the supernatant. In addition, alcohol oxidase activity was observed in the supernatant, resulting in the accumulation of formaldehyde in the culture broth. These results suggest that the formaldehyde produced by methanol oxidation in the culture broth of P. pastoris might decrease the HC activity of rAT during fermentation. Replacing the methanol with glycerol as the carbon source improved the specific HC activity of rAT from 20% to above 40% of that of pAT. In the glycerol-limited fed-batch fermentation, rAT is expressed at 100 mg/l under the control of the truncated mutated AOX2 promoter.     

丁香酚对水产品中普罗威登斯菌的抑制作用

以从自然腐败变质的香辣蟹中分离鉴定的一株普罗威登斯菌(Providencia vermicola)为对象,研究丁香酚对其抑菌作用及其机理。结果表明,丁香酚对普罗威登斯菌的最小抑菌浓度为0.25 mg/mL,与对照组相比,最小抑菌浓度剂量的丁香酚处理条件下,普罗威登斯菌菌体严重变形,出现大量的褶皱,胞内物质流出,胞外可溶性蛋白含量提高,苹果酸脱氢酶(MDH)和琥珀酸脱氢酶(SDH)酶活分别降低了33.01%,19.81%。表明丁香酚可以通过破坏普罗威登斯菌细胞壁,改变细胞膜通透性,引起细菌胞内物质泄露,以及细菌正常新陈代谢失调等作用,从而抑制普罗威登斯菌正常生长繁殖,发挥抑菌活性。     

灵芝菌液体发酵玉竹产水溶性多糖的工艺优化和抗氧化性研究

为探究灵芝菌发酵玉竹产水溶性多糖工艺及多糖的抗氧化能力,该研究利用灵芝菌液体发酵玉竹,以多糖提高率为评价指标,通过单因素试验及响应面试验对其发酵工艺进行优化,并测定发酵前后多糖的抗氧化能力。结果表明,最佳发酵条件为玉竹添加量6%、豆粕添加量为2%、KH₂PO₄添加量0.15%、Mg SO₄·7H₂O添加量0.15%、接种量8.7%、发酵时间4 d及发酵温度30℃。在此优化条件下,发酵液多糖含量达到5.91 mg/m L,较未发酵的培养基(3.41 mg/m L)提高了73.48%。抗氧化试验表明,在相同浓度下,发酵后多糖(PAF)对比发酵前多糖(PBF)的ABTS+·、DPPH·、OH·清除率均有所提高。其中,PAF对DPPH和OH·的半抑制浓度(IC50)值分别为2.77 mg/m L、0.54 mg/m L,PBF对DPPH·和OH·的IC₅₀值分别为7.94 mg/m L、1.57 mg/m L。结果表明,PAF具有更强的体外抗氧化能力。     

Up-cycling of PET (polyethylene terephthalate) to the biodegradable plastic PHA (polyhydroxyalkanoate)

The conversion of the petrochemical polymer polyethylene terephthalate (PET) to a biodegradable plastic polyhydroxyal-kanoate (PHA) is described here. PET was pyrolised at 450 degrees C resulting in the production of a solid, liquid, and gaseous fraction. The liquid and gaseous fractions were burnt for energy recovery, whereas the solid fraction terephthalic acid (TA) was used as the feedstock for bacterial production of PHA. Strains previously reported to grow on TA were unable to accumulate PHA. We therefore isolated bacteria from soil exposed to PET granules at a PET bottle processing plant From the 32 strains isolated, three strains capable of accumulation of medium chain length PHA (mclPHA) from TA as a sole source of carbon and energy were selected for further study. These isolates were identified using 16S rDNA techniques as P. putida (GO16), P. putida (GO19), and P. frederiksbergensis (GO23). P. putida GO16 and GO19 accumulate PHA composed predominantly of a 3-hydroxydecanoic acid monomer while P. frederiksbergensis GO23 accumulates 3-hydroxydecanoic acid as the predominant monomer with increased amounts of 3-hydroxydodecanoic acid and 3-hydroxydodecenoic acid compared to the other two strains. PHA was detected in all three strains when nitrogen depleted below detectable levels in the growth medium. Strains GO16 and GO19 accumulate PHA at a maximal rate of approximately 8.4 mg PHA/l/h for 12 h before the rate of PHA accumulation decreased dramatically. Strain GO23 accumulates PHA at a lower maximal rate of 4.4 mg PHA/l/h but there was no slow down in the rate of PHA accumulation over time. Each of the PHA polymers is a thermoplastic with the onset of thermal degradation occurring around 308 degrees C with the complete degradation occurring by 370 degrees C. The molecular weight ranged from 74 to 123 kDa. X-ray diffraction indicated crystallinity of the order of 18-31%. Thermal analysis shows a low glass transition (-53 degrees C) with a broad melting endotherm between 0 and 45 degrees C.     

Production of free conjugated linoleic acid by Lactobacillus acidophilus and Lactobacillus casei of human intestinal origin

A gas chromatographic procedure was used for analysis of conjugated linoleic acid (CLA) isomers cis-9, trans-11-octadecadienoic; trans-10, cis-12 octadecadienoic; and trans-9, trans-11-octadecadienoic (c9t11, t10c12, t9t11) produced by lactobacilli. Four different cultures, two strains each of Lactobacillus acidophilus and Lactobacillus casei were tested for their ability to produce CLA from free linoleic acid in MRS broth supplemented with linoleic acid. Different concentrations of linoleic acid (0, 0.05, 0.1, 0.2 and 0.5 mg/ml) were added to MRS broth, inoculated with the lactobacilli, and incubated at 37 degrees C. Viable counts and amounts of individual isomers of CLA (c9t11, t10c12, t9t11) were measured at 0, 24, 48, and 72 h. All the cultures were able to produce free CLA in media supplemented with linoleic acid. Maximum production of CLA (80.14 to 131.63 microg/ml) was observed at 24 h of incubation in broth containing 0.02% of free linoleic acid. No significant (P > 0.05) increases in total CLA levels were observed after 24 h of incubation. The ability of the cultures to produce CLA in skim milk supplemented with 0.02% free linoleic acid also was studied. In this medium, the total amounts of free CLA after 24 h of incubation ranged from 54.31 to 116.53 microg/ml. The use of lactic acid bacteria able to form free CLA in cultured dairy products may have potential health or nutritional benefits. Free CLA in the products likely would be more readily available for absorption from the digestive tract than if it were incorporated into the cells of the starter culture.