广谱乳酸菌素产生菌的筛选及其发酵研究
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摘要
随着经济与社会的不断发展,人们的生活水平在日益提高,从而对绿色食品、健康食品有了新的认识。现阶段绝大部分食品中添加的都是合成防腐剂,存在着安全隐患,然而乳酸菌素作为一种天然防腐剂正好迎合了人们的需要。乳酸菌素是一类由乳酸菌在代谢过程中产生的抑菌蛋白、多肽或前体多肽,主要用于抑制食品中腐败菌和致病菌的繁殖与生长。目前国内应用最广泛的Nisin只能抑制食品中革兰氏阳性细菌的生长,而不能抑制革兰氏阴性细菌,因此筛选能够抑制革兰氏阳性、阴性两类细菌的新菌株显得尤为重要。
本实验以安徽大学龙河校区农贸市场零售的发酵食品(豆角汁、黄瓜汁、醪糟汁、腐乳、豆瓣酱和泡菜汁)为原材料。首先通过脱脂乳培养基进行富集培养,然后用无菌水作梯度稀释,涂布,挑取单菌落,连续划线3次予以纯化并保存；对纯化菌株进行革兰氏染色、乳酸纸层析筛选出产乳酸的革兰氏阳性菌,同时进行试管斜面保存；最后以大肠杆菌(E.coli)、枯草芽孢杆菌(Bacillus subtills)、金黄色葡萄球菌(Stapylococcus aureus)、酿酒酵母(Saccharomyces cerevisiae)、青霉(Penicillium)和黑曲霉(Aspergillus niger)六种菌作为指示菌,采用牛津杯双层琼脂扩散法检测筛选菌株的抑菌谱大小,并经过有机酸排除、过氧化氢排除等试验,最终确定了来自黄瓜汁中的一株抑菌效果好、抑菌谱广的乳酸菌,本文命名为H1菌株。
实验中通过革兰氏染色、电镜拍照、传统的生理生化试验及16SrDNA分子水平的鉴定,确认所筛菌株H1为乳杆菌属(Lactobacillus)中的植物乳杆菌(Lactobacillus plantarum JDM1)。
随后实验以筛选并鉴定的H1菌株为出发菌株,进行500 mL三角瓶发酵研究,在此基础上,本实验以10 L小型机械搅拌式发酵罐为反应器,进行发酵动力学的研究。主要是测定菌体浓度(干重法)、葡萄糖含量(DNS法)和抑菌圈直径的大小(牛津杯双层琼脂扩散法)等随时间的变化。利用Luedeking-Piret方程和Logistic equation来描述各个指标的变化,并进行曲线拟合,最终成功构建了菌体生长、基质消耗和产物合成随时间变化的动力学数学模型。
本实验最后对海藻酸钠包埋植物乳杆菌的制备及其发酵进行了初步研究。优化得到固定化的最佳工艺条件为：海藻酸钠浓度为3.0%,CaCl2浓度为1.5%,离心后菌稀释比为1：5。外部因素对固定化细胞发酵的影响表明：发酵液最适pH为7.0,发酵培养最佳温度为36℃,培养基中添加0.1%的吐温80时,发酵液抑菌圈直径达最大,NaCl含量的多少对固定化细胞的发酵影响很大,应严格控制NaCl的浓度。固定化细胞的热稳定性研究结果显示,其热稳定性明显提高,连续发酵多批次效果良好。
With the development of economic and society, the level of people's life is gradually increasing, thus people put forward higher request about food. At present, more synthetic preservative were added in most of the food, so existing potential safety problems, however lactohancillin is a natural preservative, and it catering to the need of all people.Lactohancillin is a kind of protein、polypeptide and preproghrelin, they come from the course of lactobacillus metabolism, they mainly used for inhibit-ing corruption bacteria and pathogenic bacteria during their reproduction and growth. At present, Nisin is widely used at home,it not only inhibits Gram-positie bacteria in food, and also can inhibits Gram-negative bacteria.Therefore,screening a new strain that can inhibit Gram-positie bacteria and Gram-negative bacteria is especially impor-tant.
This paper take traditional fermented food from Anhui university farmers' markets as raw material, including acid fermented beans juice、cucumber juice、LaoZao juice、fermented bean curd、broad bean paste and pickles juice.First, enrich-ment training in skim milk medium, then I dilute enrichment broth by sterile water according to concentration gradient, coating,choosing the single colonies, scribing three times make it has been purified,and preservation.Purified strains were dyed by Gram stain, and lactic acid paper chromatography, screening Gram-positie strains, meanwhile preservation by cant. Finally,I take E.col、Bacillus subtilis、Staphylococcus aureus、Saccharomyces cerevisiae、Penicillium、Aspergillus niger for instructions bacteria.Using the oxford cup double agar diffusion method,detecting the diameter of bacteriostatic circle,after organic acids exclusion test and hydrogen peroxide alimina-tion test etc, I find a strain from cucumber juice, its antibacterial spectrum is wider, its bacteriostasis is also perfect, named it as H1 strain in this paper.
In this paper, gram's staining、Sem photos、traditional physiological and biochemical tests、16SrDNA molecular identify and so on,All these methods confirme-d the strain,the strain is lactobacillus genera and Plants Lactobacillus(JDM1).
Then I put H1 as original strains, researching fermentation in 500 mL triangle bottle, on this basis, based on 10 L mechanical agitation type fermentation tank, researching on fermentation dynamics.Mainly, measuring the bacteria content(dry weight method)、glucose content(DNS method)and the size of bacteriostasis circle(the oxford cup double agar diffusion method)whth the changing time.Adopting Luedeking-Piret equation and Logistic equation to describe each index changes, fitting curve,in the end, I successfully build the dynamic mathematical models of mycelium growth、substrates consumption and product generation.
Finally, I researched on the preparation of immobilized Lactobacillus plantaru-m by sodium alginate and fermentation. Results showed that the optimum technology conditions is 3.0% sodium alginate concentration,1.5% CaCl2 concentration and bacteria dilution ratio after centrifugation isl:5.Results of fermentation conditions of immobilized cell showed that the optimum pH of fermented filtrates is 7.0, the optimum temperature is 36℃, add 0.1% Tween80 to the medium, at this point, diameters of bacteriostatic circle is maximum, besides, NaCl concentration influenced the fermentation of immobilized cell greatly, So, NaCl concentration shuold be controlled strictly.Results of stability showed that the stability of immobilized cell have be improved greatly, the effect of continuous fermentation is good.
本实验以安徽大学龙河校区农贸市场零售的发酵食品(豆角汁、黄瓜汁、醪糟汁、腐乳、豆瓣酱和泡菜汁)为原材料。首先通过脱脂乳培养基进行富集培养,然后用无菌水作梯度稀释,涂布,挑取单菌落,连续划线3次予以纯化并保存；对纯化菌株进行革兰氏染色、乳酸纸层析筛选出产乳酸的革兰氏阳性菌,同时进行试管斜面保存；最后以大肠杆菌(E.coli)、枯草芽孢杆菌(Bacillus subtills)、金黄色葡萄球菌(Stapylococcus aureus)、酿酒酵母(Saccharomyces cerevisiae)、青霉(Penicillium)和黑曲霉(Aspergillus niger)六种菌作为指示菌,采用牛津杯双层琼脂扩散法检测筛选菌株的抑菌谱大小,并经过有机酸排除、过氧化氢排除等试验,最终确定了来自黄瓜汁中的一株抑菌效果好、抑菌谱广的乳酸菌,本文命名为H1菌株。
实验中通过革兰氏染色、电镜拍照、传统的生理生化试验及16SrDNA分子水平的鉴定,确认所筛菌株H1为乳杆菌属(Lactobacillus)中的植物乳杆菌(Lactobacillus plantarum JDM1)。
随后实验以筛选并鉴定的H1菌株为出发菌株,进行500 mL三角瓶发酵研究,在此基础上,本实验以10 L小型机械搅拌式发酵罐为反应器,进行发酵动力学的研究。主要是测定菌体浓度(干重法)、葡萄糖含量(DNS法)和抑菌圈直径的大小(牛津杯双层琼脂扩散法)等随时间的变化。利用Luedeking-Piret方程和Logistic equation来描述各个指标的变化,并进行曲线拟合,最终成功构建了菌体生长、基质消耗和产物合成随时间变化的动力学数学模型。
本实验最后对海藻酸钠包埋植物乳杆菌的制备及其发酵进行了初步研究。优化得到固定化的最佳工艺条件为：海藻酸钠浓度为3.0%,CaCl2浓度为1.5%,离心后菌稀释比为1：5。外部因素对固定化细胞发酵的影响表明：发酵液最适pH为7.0,发酵培养最佳温度为36℃,培养基中添加0.1%的吐温80时,发酵液抑菌圈直径达最大,NaCl含量的多少对固定化细胞的发酵影响很大,应严格控制NaCl的浓度。固定化细胞的热稳定性研究结果显示,其热稳定性明显提高,连续发酵多批次效果良好。
With the development of economic and society, the level of people's life is gradually increasing, thus people put forward higher request about food. At present, more synthetic preservative were added in most of the food, so existing potential safety problems, however lactohancillin is a natural preservative, and it catering to the need of all people.Lactohancillin is a kind of protein、polypeptide and preproghrelin, they come from the course of lactobacillus metabolism, they mainly used for inhibit-ing corruption bacteria and pathogenic bacteria during their reproduction and growth. At present, Nisin is widely used at home,it not only inhibits Gram-positie bacteria in food, and also can inhibits Gram-negative bacteria.Therefore,screening a new strain that can inhibit Gram-positie bacteria and Gram-negative bacteria is especially impor-tant.
This paper take traditional fermented food from Anhui university farmers' markets as raw material, including acid fermented beans juice、cucumber juice、LaoZao juice、fermented bean curd、broad bean paste and pickles juice.First, enrich-ment training in skim milk medium, then I dilute enrichment broth by sterile water according to concentration gradient, coating,choosing the single colonies, scribing three times make it has been purified,and preservation.Purified strains were dyed by Gram stain, and lactic acid paper chromatography, screening Gram-positie strains, meanwhile preservation by cant. Finally,I take E.col、Bacillus subtilis、Staphylococcus aureus、Saccharomyces cerevisiae、Penicillium、Aspergillus niger for instructions bacteria.Using the oxford cup double agar diffusion method,detecting the diameter of bacteriostatic circle,after organic acids exclusion test and hydrogen peroxide alimina-tion test etc, I find a strain from cucumber juice, its antibacterial spectrum is wider, its bacteriostasis is also perfect, named it as H1 strain in this paper.
In this paper, gram's staining、Sem photos、traditional physiological and biochemical tests、16SrDNA molecular identify and so on,All these methods confirme-d the strain,the strain is lactobacillus genera and Plants Lactobacillus(JDM1).
Then I put H1 as original strains, researching fermentation in 500 mL triangle bottle, on this basis, based on 10 L mechanical agitation type fermentation tank, researching on fermentation dynamics.Mainly, measuring the bacteria content(dry weight method)、glucose content(DNS method)and the size of bacteriostasis circle(the oxford cup double agar diffusion method)whth the changing time.Adopting Luedeking-Piret equation and Logistic equation to describe each index changes, fitting curve,in the end, I successfully build the dynamic mathematical models of mycelium growth、substrates consumption and product generation.
Finally, I researched on the preparation of immobilized Lactobacillus plantaru-m by sodium alginate and fermentation. Results showed that the optimum technology conditions is 3.0% sodium alginate concentration,1.5% CaCl2 concentration and bacteria dilution ratio after centrifugation isl:5.Results of fermentation conditions of immobilized cell showed that the optimum pH of fermented filtrates is 7.0, the optimum temperature is 36℃, add 0.1% Tween80 to the medium, at this point, diameters of bacteriostatic circle is maximum, besides, NaCl concentration influenced the fermentation of immobilized cell greatly, So, NaCl concentration shuold be controlled strictly.Results of stability showed that the stability of immobilized cell have be improved greatly, the effect of continuous fermentation is good.
引文
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