【作者】 李兴峰；

【导师】 潘蓓蕾； 江波；

【作者基本信息】 江南大学 ， 食品科学， 2008， 博士

【摘要】 苯乳酸（phenyllactic acid,PLA）是乳酸菌产生的一种新型抑菌物质,它不仅能够抑制多种食源性致病菌,而且对引起食品腐败的真菌具有广泛的作用。此外,苯乳酸稳定性高,安全无毒,有望开发成一种新型生物防腐剂应用于食品工业。国外对苯乳酸的研究尚处于初始阶段（1998-2008）,存在诸如乳酸菌种产量低、底物成本高和发酵时间长等问题。本文在国内率先对乳酸菌合成苯乳酸进行了系统的研究,主要内容包括:筛选高产苯乳酸的乳酸菌;研究其产生苯乳酸的途径和机理;优化乳酸菌生长细胞产生苯乳酸的发酵条件;研究影响乳酸菌静息细胞合成苯乳酸的因素;乳酸菌合成苯乳酸的酶进行分离、纯化并研究其酶学性质。利用MRS培养基发酵和HPLC检测,从中国传统发酵食品——泡菜中分离出一株高产苯乳酸的乳酸菌菌株,30℃培养72 h最高可产生91.3 mg/L的苯乳酸。通过形态学、生理生化及16S rDNA,初步鉴定为乳杆菌,命名为Lactobacillus sp.SK007,GenBankaccession number:DQ534529。系统发育分析表明,和植物乳杆菌亲缘关系最近。研究利用苯丙氨酸途径合成苯乳酸,提高苯丙氨酸的浓度可以增加苯乳酸的产量。以1.5g/L苯丙氨酸为底物的转化过程中发现:发酵72 h苯乳酸达到最高,而此时苯丙氨酸剩余94%、检测不到中间产物苯丙酮酸,这表明转氨反应是苯乳酸合成的限速步骤。进一步对影响转氨反应的因素进行研究,确定氨基受体α-酮戊二酸的缺乏是主要因素。研究了通过促进转氨反应提高苯乳酸的途径,分别采用添加α-酮戊二酸、谷氨酸以及柠檬酸和谷氨酸,苯乳酸产量有一定提高。苯丙氨酸的转氨反应已成为苯乳酸合成的瓶颈。在此基础上,使用苯丙酮酸替代同浓度的苯丙氨酸作为底物合成苯乳酸,结果苯乳酸产量提高了11倍、发酵时间缩短了48 h、原料成本降低了2/3。这表明,以苯丙酮酸为底物是苯乳酸合成的有利途径,为规模化生产提供了可能性。对乳杆菌的发酵条件进行了优化,用廉价的玉米浆代替昂贵的蛋白胨作为氮源。利用响应面法对培养基成分进行了优化,得到的培养基成分（g/L）:为葡萄糖30,玉米浆47,酵母粉5,磷酸氢二钟3,醋酸钠5,柠檬酸三铵2,硫酸镁0.58,硫酸锰0.25,吐温-80 3 mL,苯丙酮酸5。最适的培养温度30℃、接种量3%、静置培养。发酵过程中pH变化对苯乳酸影响很大,可采用添加0.5%的缓冲盐及中和剂进行内源调节。在发酵罐中,采用后期流加碱液控制pH的方法有利于苯乳酸合成。在此基础上进行了300 L发酵罐中试,苯乳酸产量达到2.81 g/L。利用乳酸菌静息细胞也可以合成苯乳酸,HPLC分析了静息细胞内、外的苯乳酸含量,结果表明绝大部分苯乳酸释放到转化液中。研究了细胞浓度、底物浓度、表面活性剂以及环境因素对苯乳酸产生的影响,得到的转化条件为:菌体静置培养24 h、用pH6.0的缓冲液收获细胞调节浓度为20g/L,加入2g/L苯丙酮酸,35℃、120 rpm反应2 h,苯乳酸产量可达0.98 g/L。静息细胞重复利用实验表明:第二次使用时,细胞活力仅为首次使用的20%,第三次已没有苯乳酸产生。进一步研究表明还原型辅酶NADH的缺乏是主要影响因素。针对反应过程中辅酶缺乏这一问题,构建了基于辅助底物的酶法再生体系。研究了葡萄糖、乙醇等辅助底物对苯乳酸合成的影响,确定葡萄糖是最适的辅助底物。在添加1%的葡萄糖,细胞重复利用5次活力仍能保持80%,细胞冷藏10天仍有60%的活力。经过超声波破碎、硫酸铵沉淀、DEAE-Sepharose Fast Flow离子交换色谱和AKTAExplore Sephacryl S-200凝胶过滤等分离步骤,纯化得到乳酸脱氢酶（LDH）,SDS-PAGE呈现单一条带,HPLC检测纯度达98%。比酶活提高34.29倍、达到203.30 U/mg,回收率19.27%。在各纯化步骤中,该酶作用丙酮酸和苯丙酮酸的比值恒定,说明此酶是将苯丙酮酸还原成苯乳酸的主要酶。由SDS-PAGE得到LDH相对分子质量为39 kDa,而凝胶过滤色谱求得LDH的相对分子量约为80 kDa,因此,推断乳杆菌LDH分子内有2个亚基,是一种二聚体酶。圆二色性分析表明,LDH二级结构中α-旋占14.9、β-折叠35.7%、回转结构由为28.6%、无规卷曲为20.8。纯化的LDH作用苯丙酮酸的最适作用温度是40℃,超过60℃迅速下降;最适pH值为6.0、在4-8之间保持稳定;Mg²⁺、Mn²⁺对酶活力有促进作用,Cu²⁺、Hg²⁺对酶活力有不同程度的抑制作用。LDH对丙酮酸和苯丙酮酸的亲和力差别很大。采用Lineweaver-Burk法作图法,求得LDH作用丙酮酸和苯丙酮酸的米氏常数K_m分别为0.31、1.69 mM。对10株乳杆菌的乳酸脱氢酶活力与其合成苯乳酸的能力进行了初步探讨,结果表明乳杆菌之间LDH对丙酮酸和苯丙酮酸的差别很大,对苯丙酮酸高的LDH乳杆菌产生了高的苯乳酸含量。因此,乳酸脱氢酶的活力可以作为筛选乳酸菌产生苯乳酸的标准之更多还原

【Abstract】 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.更多还原