嗜酸乳杆菌的分离、鉴定及其在发酵豆乳中生长特性的研究
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摘要
嗜酸乳杆菌是人体肠道内的重要微生物,与人体健康息息相关,当其达到一定数量时,对维护肠道健康和菌群平衡起着十分重要的作用,是目前乳酸菌家族中极为重视研究与开发的益生菌之一,被视为第三代酸乳发酵剂菌种。随着国内外众多学者对嗜酸乳杆菌的生物学特性、功能性及应用等方面研究的不断深入,人们越来越认识到嗜酸乳杆菌的益生特性,含有嗜酸乳杆菌的产品日益丰富,包括酸奶、胶囊、片剂、粉剂及口服液等,但用嗜酸乳杆菌发酵生产的功能性发酵豆乳制品尚未见商品化生产,关于嗜酸乳杆菌在豆乳中生长特性的研究报道甚为罕见。
在我国,豆浆既是一种传统食品,又是一种廉价易获取的资源,但由于受到传统习惯的影响,人们一直未能认识到豆浆的益处,加之豆奶(浆)较重的豆腥味,使得很多消费者,尤其是青少年、儿童对其的接受程度较低。因此,利用现代生物技术,通过乳酸菌的发酵处理,研制功能性的发酵豆乳饮料具有十分重要的意义,不仅可以解决豆奶的口感问题,而且可以为牛奶缺乏的地区提供优质价廉的功能性植物蛋白饮料。
本研究以健康婴儿的新鲜粪便为材料,经分离、纯化和分子生物学鉴定获得3株人源性嗜酸乳杆菌菌株,旨在为发酵豆乳提供生长能力旺盛、活菌数高、产酸能力强的发酵菌种,并探索供试菌株在发酵豆乳中的生长特性,为发酵豆乳的工业化生产提供一定的理论基础和科学依据。
通过比较3株嗜酸乳杆菌的生物学特性,包括生长曲线、产酸能力、耐胃酸能力、耐胆盐性、抗药性及抑菌能力,选择生物学性状较优的LA-5菌株作为供试菌株,其在MRS液体培养基中37℃培养14h,活菌数可达4.33×10~(12)cfu/mL;具有较强的耐酸和抗胆盐特性;对氯霉素高度敏感,对青霉素G、利福平和四环素中毒敏感,对卡那霉素不敏感;在体外对大肠杆菌、沙门氏菌、金黄色葡萄球菌和柠檬色葡萄球菌具有较强的拮抗作用。
为研制适合发酵豆乳的嗜酸乳杆菌发酵剂,本研究以豆乳为基础探索嗜酸乳杆菌LA-5的增菌培养基,得到最佳增菌培养基为:10%豆浆,1%葡萄糖,8%西红柿汁,5%玉米浸出汁,2%胡萝卜汁,2%平菇浸出汁。嗜酸乳杆菌LA-5在增菌培养基中培养时,增菌培养终止时间为10h,此时活菌数可达1.72×10~9cfu/mL。
嗜酸乳杆菌LA-5经增菌培养基活化后,按8%接种量接种于发酵豆乳基质中,37℃恒温、静置发酵20h后达发酵终点,此时发酵豆乳pH=3.57,滴定酸度为85.22°T,活菌数为3.62×10~8cfu/mL。将发酵豆乳产品转移至4℃条件下冷藏30天,pH降至3.45,滴定酸度升高到94.29°T,嗜酸乳杆菌活菌数减少至7.04×10~6cfu/mL。
Lactobacillus acidophilus is an important microorganism in human intestinal tract.When there exist certain number of Lactobacillus acidophilus in intestinal tract,they play an important role to maintain healthy intestinal flora balance.Lactobacillus acidophilus is one of probiotics which current scholar attaches great importance to research and development.It is considered as the third generation starter for fermented milk.With more and more in-depth studies on the biological characteristics,functionality and application of Lactobacillus acidophilus by many scholars at home and abroad, people have been growing awareness to this microorganism's prebiotic characteristics.There have been increasing products containing Lactobacillus acidophilus,such as yoghurt,capsule,troche, powder and oral liquid.However,functional fermented soymilk through the fermentation of Lactobacillus acidophilus haven't been commercial produced and there are few reports on the growth characteristics of Lactobacillus acidophilus in fermented soymilk.
In China,soymilk is a traditional food,but also a kind of cheap and easy accessible resource.But due to the impact of the traditional habits,people have been unable to recognize the benefits of soymilk. In addition,soymilk has heavier beany flavor,so that many consumers, especially teenagers and children have low level of acceptance. Therefore,the use of modern biological technique,it is of great significance to develop functional fermented soymilk beverages through the fermentation of lactic acid bacteria.These beverages can not only solve the problem of taste,but also provide high quality and inexpensive functional vegetable protein beverage for the regions which are short of milk.
In this study,Lactobacillus strains were isolated from fresh infant feces.There were three Lactobacillus acidophilus strains through molecularly biological identification.This study is aim to provide Lactobacillus acidophilus strains with strong growth capacity, high active number and strong acid-producing ability for fermented soymilk.Besides,we explored the growth characteristics of Lactobacillus acidophilus in fermented soymilk,providing a theoretical basis and a scientific basis for industrialized production of fermented soymilk.
By comparing the biological characteristics of the three strains, including the growth curve,acid-producing ability,gastric acid-resistant capacity,bile-resistant capacity,antibiotic resistance and antibacterial capacity,we choosed Lactobacillus acidophilus LA-5 as tested strain.The active number of Lactobacillus acidophilus LA-5 can reach to 4.33×10~(12) cfu/mL after cultivated in MRS liquid medium for 14 hours.Lactobacillus acidophilus LA-5 has strong acid-resistant and bile-resistant capacity.The strain is highly sensitive to chloramphenicol,penicillin G,rifampicin and acheomycin,but is't sensitive to kanamycin.In vitro,The metabolites of Lactobacillus acidophilus LA-5 are obvious antagonism on E.coli,Salmonella and Staphylococcus aureus and Staphylococcus citreus.
In order to develop a Lactobacillus acidophilus starter suitable for producing fermented soymilk,we studied on the growth promoting culture of Lactobacillus acidophilus LA-5 based on soymilk.The optimal growth promoting culture composition was as following:10% soymilk,1%glucose,8%tomoto juice,5%corn extract juice,2% carrot juice,2%juice of plerotus sapidus.According to the growth curve of Lactobacillus acidophilus LA-5 in the optimal growth promoting culture,the terminal time was at 10 hours.Lactobacillus acidophilus LA-5 got to 1.72×10~9 cfu/mL in the culture for 10 hours at 37℃.
After activated in the growth promoting culture for three times, Lactobacillus acidophilus LA-5 was inoculated in soymilk with 8% inoculum size.After fermented for 20 hours at 37℃,soymilk reached to the fermented end-point,and the parameters of fermented soymilk were as following:pH 3.57,titratable acidity 85.22°T,the cells reached to 3.62×10~8 cfu/mL.The finished product was stored for 30 days at 4℃,as a result,the parameters of fermented soymilk were as following:pH 3.45,titration acidity 94.29°T,the cells reached to 7.04×10~6 cfu/mL.
在我国,豆浆既是一种传统食品,又是一种廉价易获取的资源,但由于受到传统习惯的影响,人们一直未能认识到豆浆的益处,加之豆奶(浆)较重的豆腥味,使得很多消费者,尤其是青少年、儿童对其的接受程度较低。因此,利用现代生物技术,通过乳酸菌的发酵处理,研制功能性的发酵豆乳饮料具有十分重要的意义,不仅可以解决豆奶的口感问题,而且可以为牛奶缺乏的地区提供优质价廉的功能性植物蛋白饮料。
本研究以健康婴儿的新鲜粪便为材料,经分离、纯化和分子生物学鉴定获得3株人源性嗜酸乳杆菌菌株,旨在为发酵豆乳提供生长能力旺盛、活菌数高、产酸能力强的发酵菌种,并探索供试菌株在发酵豆乳中的生长特性,为发酵豆乳的工业化生产提供一定的理论基础和科学依据。
通过比较3株嗜酸乳杆菌的生物学特性,包括生长曲线、产酸能力、耐胃酸能力、耐胆盐性、抗药性及抑菌能力,选择生物学性状较优的LA-5菌株作为供试菌株,其在MRS液体培养基中37℃培养14h,活菌数可达4.33×10~(12)cfu/mL;具有较强的耐酸和抗胆盐特性;对氯霉素高度敏感,对青霉素G、利福平和四环素中毒敏感,对卡那霉素不敏感;在体外对大肠杆菌、沙门氏菌、金黄色葡萄球菌和柠檬色葡萄球菌具有较强的拮抗作用。
为研制适合发酵豆乳的嗜酸乳杆菌发酵剂,本研究以豆乳为基础探索嗜酸乳杆菌LA-5的增菌培养基,得到最佳增菌培养基为:10%豆浆,1%葡萄糖,8%西红柿汁,5%玉米浸出汁,2%胡萝卜汁,2%平菇浸出汁。嗜酸乳杆菌LA-5在增菌培养基中培养时,增菌培养终止时间为10h,此时活菌数可达1.72×10~9cfu/mL。
嗜酸乳杆菌LA-5经增菌培养基活化后,按8%接种量接种于发酵豆乳基质中,37℃恒温、静置发酵20h后达发酵终点,此时发酵豆乳pH=3.57,滴定酸度为85.22°T,活菌数为3.62×10~8cfu/mL。将发酵豆乳产品转移至4℃条件下冷藏30天,pH降至3.45,滴定酸度升高到94.29°T,嗜酸乳杆菌活菌数减少至7.04×10~6cfu/mL。
Lactobacillus acidophilus is an important microorganism in human intestinal tract.When there exist certain number of Lactobacillus acidophilus in intestinal tract,they play an important role to maintain healthy intestinal flora balance.Lactobacillus acidophilus is one of probiotics which current scholar attaches great importance to research and development.It is considered as the third generation starter for fermented milk.With more and more in-depth studies on the biological characteristics,functionality and application of Lactobacillus acidophilus by many scholars at home and abroad, people have been growing awareness to this microorganism's prebiotic characteristics.There have been increasing products containing Lactobacillus acidophilus,such as yoghurt,capsule,troche, powder and oral liquid.However,functional fermented soymilk through the fermentation of Lactobacillus acidophilus haven't been commercial produced and there are few reports on the growth characteristics of Lactobacillus acidophilus in fermented soymilk.
In China,soymilk is a traditional food,but also a kind of cheap and easy accessible resource.But due to the impact of the traditional habits,people have been unable to recognize the benefits of soymilk. In addition,soymilk has heavier beany flavor,so that many consumers, especially teenagers and children have low level of acceptance. Therefore,the use of modern biological technique,it is of great significance to develop functional fermented soymilk beverages through the fermentation of lactic acid bacteria.These beverages can not only solve the problem of taste,but also provide high quality and inexpensive functional vegetable protein beverage for the regions which are short of milk.
In this study,Lactobacillus strains were isolated from fresh infant feces.There were three Lactobacillus acidophilus strains through molecularly biological identification.This study is aim to provide Lactobacillus acidophilus strains with strong growth capacity, high active number and strong acid-producing ability for fermented soymilk.Besides,we explored the growth characteristics of Lactobacillus acidophilus in fermented soymilk,providing a theoretical basis and a scientific basis for industrialized production of fermented soymilk.
By comparing the biological characteristics of the three strains, including the growth curve,acid-producing ability,gastric acid-resistant capacity,bile-resistant capacity,antibiotic resistance and antibacterial capacity,we choosed Lactobacillus acidophilus LA-5 as tested strain.The active number of Lactobacillus acidophilus LA-5 can reach to 4.33×10~(12) cfu/mL after cultivated in MRS liquid medium for 14 hours.Lactobacillus acidophilus LA-5 has strong acid-resistant and bile-resistant capacity.The strain is highly sensitive to chloramphenicol,penicillin G,rifampicin and acheomycin,but is't sensitive to kanamycin.In vitro,The metabolites of Lactobacillus acidophilus LA-5 are obvious antagonism on E.coli,Salmonella and Staphylococcus aureus and Staphylococcus citreus.
In order to develop a Lactobacillus acidophilus starter suitable for producing fermented soymilk,we studied on the growth promoting culture of Lactobacillus acidophilus LA-5 based on soymilk.The optimal growth promoting culture composition was as following:10% soymilk,1%glucose,8%tomoto juice,5%corn extract juice,2% carrot juice,2%juice of plerotus sapidus.According to the growth curve of Lactobacillus acidophilus LA-5 in the optimal growth promoting culture,the terminal time was at 10 hours.Lactobacillus acidophilus LA-5 got to 1.72×10~9 cfu/mL in the culture for 10 hours at 37℃.
After activated in the growth promoting culture for three times, Lactobacillus acidophilus LA-5 was inoculated in soymilk with 8% inoculum size.After fermented for 20 hours at 37℃,soymilk reached to the fermented end-point,and the parameters of fermented soymilk were as following:pH 3.57,titratable acidity 85.22°T,the cells reached to 3.62×10~8 cfu/mL.The finished product was stored for 30 days at 4℃,as a result,the parameters of fermented soymilk were as following:pH 3.45,titration acidity 94.29°T,the cells reached to 7.04×10~6 cfu/mL.
引文
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