Production Of Phenyllactic Acid (PLA) By Lactobacilli

Phenyllactic acid(PLA) is a novel antimicrobial compound,which has been recently found in culture of lactic acid bacteria(LAB).The inhibitory properties of PLA have been demonstrated against both gram-positive and gram-negative bacteria.Moreover,PLA has an inhibitory activity against a wide range of fungi including some mycotoxigenic species.Due to its broad inhibitory activity,higher stability and safety, PLA has interesting potential for practical application as a novel bio-preservative in the food industry. International research on PLA is quite new(1998-2008),and sevaral problems are being faced in PLA production,such as low yield,expensive substrate and long fermentation time.In this study, PLA-producing LAB strains were isolated and screened.PLA production from Phe(phenylalanine) and PPA(phenylpyruvic acid) by LAB was investigated.Fermentation conditions were optimized for PLA production by growing cells.The bioconversion of PLA from PPA by resting cells was studied.LDH from LAB was purified and characterized.The strain B7,which was isolated from Chinese traditional pickles using MRS medium,exhibited the highest PLA yield(91.3 mg/L) after growing in MRS broth at 30℃for 72h.The strain was identified and named as Lactobacillus sp.SK007,according to its cell morphology,metabolism characters and 16S rDNA sequence.The sequence has been deposited in GenBank under the accession number DQ534529.PLA production from Phe by Lactobacillus sp.SK007 was investigated.PLA production was improved in MRS broth using increased concentrations of Phe,but the conversion inclined drastically.It was found that 94%of Phe remained after fermentation for 72h,but the intermediate metabolite PPA stayed below the detection level.These results indicated that the transamination of Phe to PPA is a rate-limiting step.A further study showed that the deficiency in amino acceptorα-ketoglutarate(α-KG) is the main factor during transamination.PLA yield was increased by improving Phe transamination,including the addition ofα-KG,glutamine,and the combination of both citric acid and glutamine.Phe transamination is the bottleneck in PLA production by Lactobacillus sp.SK007.Therefore,PLA production from PPA was investigated.When 1.5 g/L PPA was used to replace Phe as substrate at the same concentration,PLA yield increased 11-fold,the fermentation time decreased from 72 h to 24 h and the cost of substrate decreased to 2/3.The limiting factor was overcome using PPA to replace Phe as substrate and this finding provided the possibility of utilizing PPA as substrate in PLA production using growing cells of Lactobacillus sp.SK007.The fermentation process for PLA production by LAB was carried out by replacing the nitrogen peptone with cheaper corn steep liquor.The obtained optimal medium consisted of 3%glucose,0.5%yeast extract,4.7%corn steep corn,0.3%Tween-80,0.5%PPA and some salt(0.5%acetate,0.3%K₂HPO₄,0.2% citric acid ammonium,0.058%MgSO₄ and 0.025%MnSO₄) using response surface method.PLA production was effective when Lactobacillus sp.SK007 was cultured at 30℃for 24 h without shaking.The initial pH is one of important factors for PLA production and pH could be controlled by two methods: addition of buffer salts or neutralizer,and feeding NaOH in bioreactor.Based on previous results,the fermentation process was enlarged and PLA yield achieved 2.81 g/L in 300 L bioreactor.PLA production by resting cells of Lactobacillus sp.SK007 was also investigated.HPLC analysis showed that almost all PLA is extracellular after biotransformation.The effects of bioconversion factors on PLA production were also studied and the obtained conditions were 20 g/L for initial cell concentration,2 g/LPPA,pH 6.0,35℃and 120 rpm of shaking spped.It was found that PLA content in the second batch is only 20%of that in the first batch,and no PLA is in the third batch for repetitive conversions by resting cells.Further tests indicated that the reduced co-enzyme NADH is not sufficient for repetitive batch.In order to achieve the coenzyme regeneration,the effects of various cosubstrates on the efficiency in repetitive batch were investigated.Results indicated that glucose was the best cosubstrate and the optimal concentration was 10 g/L.In the presence of glucose,the whole cells could be reused for 5 times and PLA yield reached 80%,and resting cells could maintain 60%of their activity after storage at 4℃for 10 days.A lactate dehydrogenase(LDH),which catalyzes the reduction of PPA to PLA,has been purified to homogeneity from a cell-free extract of Lactobacillus sp.SK007 by precipitation with ammonium sulfate, ion exchange and gel filtration chromatography,respectively.Purified LDH averaged 19.27%of yield with a 34-fold purification level.The specific activities of purified LDH with pyruvate and PPA were 203.3 and 22.48 U/mg,respectively.However,the ratio of enzyme activity with PPA to that with pyruvate was almost invariable at every purification step.These results indicated that,in Lactobacillus sp.SK007,LDH is responsible for the conversion of PPA into PLA.HPLC profiles of PPA transformation into PLA by growing cells,cell-free extract,and purified LDH of Lactobacillus sp.SK007 were also investigated.The molecular mass of purified LDH was estimated at 78 kDa by size exclusion chromatography and 39 kDa by SDS-PAGE.Results from Circular Dichroism showed that the enzyme has a-helix,35.7%β-sheet,28.6% turn structure and 20.8%randon coil.Purified LDH displayed optimal activity for PPA at 40℃and could retain 50%of its relative activity at 60℃.The optimal pH was 6.0 and the stability was observed from pH 4.0 to 8.0.LDH activity was stimulated by Mg²⁺,Mn²⁺ and inhibited by Cu²⁺,Hg²⁺.The apparent Michaelis-Menten constant(K_m) of the enzyme for PPA and pyruvate were 1.69 and 0.32 mM,respectively.The activities of LDH from 10 isolated LAB strains and their abilities to produce PLA in MRS broth were investigated.Results showed that the relative activity(the percentage of enzyme activity with PPA to that with pyruvate) varies from 1.08 to 12.51%within strains and high LDH activity towards PPA correlates with high PLA production.Therefore,LDH activity for PPA could be used as a screening marker for PLA-producing LAB.