微囊化清酒乳杆菌高密度培养及其活性保护技术研究
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摘要
我国发酵肉已有千年的历史,但直到本世纪才出现人工添加微生物发酵剂用于肉品加工。如今人们已经认识到了乳酸菌等微生物在发酵肉制品品质、风味及保存中的重要作用,因此在欧美等国,发酵剂在肉类工业中的应用越来越广泛。我国是一个肉类生产及消费大国,全国各地都有一些名优的发酵肉制品。随着人们生活水平的提高,对肉制品品种的多样性和肉制品质量的要求会更高,而传统的发酵肉制品工艺已不适应现代化的要求,直投式发酵剂加工发酵肉是当今发酵肉工业化生产的方向。因此,为了促进我国发酵肉制品产业的发展,有必要研制开发出安全、高效的肉品发酵剂。
本实验从制备高效浓缩型乳酸菌肉品发酵剂的关键技术入手:首先采用细胞固定化技术对从发酵肉中分离的清酒乳杆菌进行液芯微囊化处理;然后考察液芯微胶囊作为微反应器,通过单因素试验、响应曲面试验以及正交试验对微囊化清酒乳杆菌进行培养基和培养条件的优化,达到浓缩培养囊内细胞的目的;筛选出微囊化清酒乳杆菌深低温冷冻合适的保护剂来提高菌体的冷冻存活率,从而为研制新型的液芯微囊化乳酸菌肉品发酵剂奠定基础。主要研究结果如下:
1.微囊化清酒乳杆菌的制备
分别采用液芯包囊法和传统的海藻酸钙凝胶包埋法对从发酵肉中分离的清酒乳杆菌LS10进行微囊化处理,并对两种微胶囊囊内细胞密度进行比较,同样的增殖条件液芯微胶囊囊内细胞密度要明显高于凝胶珠囊内细胞密度,依据实验目的,确定在接下来的实验中采用液芯包囊法制备微胶囊。
2.微囊化清酒乳杆菌高密度培养的研究
将乳清粉作为基础培养基,补充适当碳、氮源,促生长因子,pH缓冲剂,通过单因素试验和响应曲面试验,确定了微囊化清酒乳杆菌的增殖培养基配方(L~(-1)):乳清粉60g,葡萄糖18.87g,蛋白胨21.98g,番茄汁98.62mL,CaCO_3 4.39g,MgSO_40.28g,MnSO_4 0.18g。通过正交试验得出微囊化清酒乳杆菌的最佳培养条件为:温度37℃、初始pH 7.0、培养时间20h、接种量1%。在优化条件下,其囊内细胞密度达到10.59 log(cfu/g)。
3.微囊化清酒乳杆菌深层冷冻发酵剂活性保持技术研究
对微囊化清酒乳杆菌在不同冷冻温度(-20℃和-70℃)下的存活率进行比较,确定深低温-70℃作为微囊化清酒乳杆菌的冷冻温度;对不同解冻温度(4℃、22℃、42℃)下菌体的存活率进行比较,选择42℃作为微囊化清酒乳杆菌深层冷冻发酵剂的解冻温度。
采用Plackett-Burman设计法,对蔗糖、乳糖、葡萄糖、果糖、麦芽糊精、甘油、甘露醇、山梨醇、明胶、脱脂奶粉、L-半胱氨酸盐酸盐、精氨酸、抗坏血酸以及谷氨酸钠14种材料对微囊化清酒乳杆菌深低温冷冻的保护效果进行评价。结果表明:在冷冻中有显著保护作用的是蔗糖、甘油、谷氨酸钠以及脱脂奶粉。进而用响应曲面法来确定清酒乳杆菌LS10的冷冻保护剂的浓度范围,得到其最佳保护剂组成为:蔗糖45.75g/L,甘油63.95mL/L,谷氨酸钠64.70g/L,脱脂奶粉92.93g/L,使清酒乳杆菌LS10的冷冻存活率达到92.54%。
在研制出高活性肉品发酵剂的基础上,研究了该发酵剂在不同条件(-20℃和-70℃)下的保存特性,结果表明在-70℃条件下保存菌数下降较慢,效果较好,6个月后菌体存活率为63.59%。
Fermented meat in our country has a long history encompassing more than thousand years,but the use of microbial starter in meat processing has started only recently.People have now comprehended the role of lactic acid bacteria and other microorganism in overall quality and preservation of fermented meat.In European and American countries,starter is applied more and more extensively in meat industry.Our country is the major producer and consumer of fermented meat and there are a range of different fermented meats available all over the country.The demand for supreme quality and diversified meat products is sharply increasing with the improvement in people's living standards,while traditional processing technology no longer meet the requirement of modernized fermented meat. Therefore,in order to promote the development of the fermented meat industry,it is highly imperative to develop safty and high-activity meat starter to give a boost to meat processing industry.
Starting with the crux technology of preparing high-activity concentrated lactic acid bacteria as meat starter.The present research used liquid-core capsule immobilized technology on Lactobacillus sake LS10 which was isolated from fermented meat.The concentration of L.sake LS10 was studied using the single factor experiments,response surface methodology and orthogonal designs to optimize the enrichment media and culture conditions.Screening the optimal deep-frozen protective for encapsulated L.sake LS10 was done to improve the cell viability.The study provided a theoretical basis for preparing new immobilized lactic acid bacteria as meat starter.The main results were as follows:
1.Preparation of microencapsulated L.sake LS 10
Lactobacillus sake LS10 was immobilized in calcium alginate capsules by using liquid-core method and conventional embedding method,respectively.The capsulated cell density of liquid-core microcapsule was obviously higher than that of gel bead under the same culture condition.According to the test purpose,the liquid-core method was chosen.
2.Study on high-density cultivation of microencapsulated L.sake LS10
Based on whey culture medium,and supplemented with carbon source,nitrogen source,growth factors,pH-buffer,the optimum culture medium was determined by single factor experiments and response surface methodology.It was found that the strain LS10 required the following ingredients for the optimal growth(L~(-1)):whey powder 60g,glucose 18.87g,peptone 21.98g,tomato juice 98.62mL,CaCO_3 4.39g,MgSO_4 0.28g and MnSO_4 0.18g.The results of orthogonal designs showed that the optimal culturing conditions were temperature 37℃,pH 7.0,culture time 20h,inoculum 1%.At the optimized condition,the cell concentration in the liquid-core capsules was increased to 10.59 log(cfu/g).
3.Study on activity protection of microencapsulated deep-freezing starter
Cell viability of Lactobacillus sake LS10 was compared under different freezing temperature(-20℃and -70℃) and different thawing temperature(4℃,22℃and 42℃). The optimum freezing temperature was found to be -70℃while the optimum thawing temperature 42℃.
Plackett-Burman design was used to evaluate the deep-frozen protective effect of sucrose,lactose,glucose,fructose,maltodextrin,glycerol,mannitol,sorbitol,gelatin,skim milk,L-cysteine hydrochloride,arginine,ascorbic acid and sodium glutamate on encapsulated L.sake LS10.Results showed that sucrose,glycerol,sodium glutamate and skim milk were the effective deep-frozen protectants for L.sake LS10.Then the central composite design and response surface analysis were used to determine the optimal levels of these media.The results showed that the optimal protective medium for enhancing the viability of deep-frozen L.sake LS10 was sucrose 45.75g/L,glycerol 63.95 mL/L,sodium glutamate 64.70g/L,and skim milk 92.93 g/L,which allowed the cell viability of L.sake LS10 up to 92.54%after deep-freezing.
On the basis of preparation of deep-frozen concentrated meat starter,the change of bacterium count under different storage conditions(-20℃and -70℃) was studied.The results showed that frozen storage at -70℃contributed more to L.sake LS10 protection than that at -20℃,the cell viability was 63.59%six months later.
本实验从制备高效浓缩型乳酸菌肉品发酵剂的关键技术入手:首先采用细胞固定化技术对从发酵肉中分离的清酒乳杆菌进行液芯微囊化处理;然后考察液芯微胶囊作为微反应器,通过单因素试验、响应曲面试验以及正交试验对微囊化清酒乳杆菌进行培养基和培养条件的优化,达到浓缩培养囊内细胞的目的;筛选出微囊化清酒乳杆菌深低温冷冻合适的保护剂来提高菌体的冷冻存活率,从而为研制新型的液芯微囊化乳酸菌肉品发酵剂奠定基础。主要研究结果如下:
1.微囊化清酒乳杆菌的制备
分别采用液芯包囊法和传统的海藻酸钙凝胶包埋法对从发酵肉中分离的清酒乳杆菌LS10进行微囊化处理,并对两种微胶囊囊内细胞密度进行比较,同样的增殖条件液芯微胶囊囊内细胞密度要明显高于凝胶珠囊内细胞密度,依据实验目的,确定在接下来的实验中采用液芯包囊法制备微胶囊。
2.微囊化清酒乳杆菌高密度培养的研究
将乳清粉作为基础培养基,补充适当碳、氮源,促生长因子,pH缓冲剂,通过单因素试验和响应曲面试验,确定了微囊化清酒乳杆菌的增殖培养基配方(L~(-1)):乳清粉60g,葡萄糖18.87g,蛋白胨21.98g,番茄汁98.62mL,CaCO_3 4.39g,MgSO_40.28g,MnSO_4 0.18g。通过正交试验得出微囊化清酒乳杆菌的最佳培养条件为:温度37℃、初始pH 7.0、培养时间20h、接种量1%。在优化条件下,其囊内细胞密度达到10.59 log(cfu/g)。
3.微囊化清酒乳杆菌深层冷冻发酵剂活性保持技术研究
对微囊化清酒乳杆菌在不同冷冻温度(-20℃和-70℃)下的存活率进行比较,确定深低温-70℃作为微囊化清酒乳杆菌的冷冻温度;对不同解冻温度(4℃、22℃、42℃)下菌体的存活率进行比较,选择42℃作为微囊化清酒乳杆菌深层冷冻发酵剂的解冻温度。
采用Plackett-Burman设计法,对蔗糖、乳糖、葡萄糖、果糖、麦芽糊精、甘油、甘露醇、山梨醇、明胶、脱脂奶粉、L-半胱氨酸盐酸盐、精氨酸、抗坏血酸以及谷氨酸钠14种材料对微囊化清酒乳杆菌深低温冷冻的保护效果进行评价。结果表明:在冷冻中有显著保护作用的是蔗糖、甘油、谷氨酸钠以及脱脂奶粉。进而用响应曲面法来确定清酒乳杆菌LS10的冷冻保护剂的浓度范围,得到其最佳保护剂组成为:蔗糖45.75g/L,甘油63.95mL/L,谷氨酸钠64.70g/L,脱脂奶粉92.93g/L,使清酒乳杆菌LS10的冷冻存活率达到92.54%。
在研制出高活性肉品发酵剂的基础上,研究了该发酵剂在不同条件(-20℃和-70℃)下的保存特性,结果表明在-70℃条件下保存菌数下降较慢,效果较好,6个月后菌体存活率为63.59%。
Fermented meat in our country has a long history encompassing more than thousand years,but the use of microbial starter in meat processing has started only recently.People have now comprehended the role of lactic acid bacteria and other microorganism in overall quality and preservation of fermented meat.In European and American countries,starter is applied more and more extensively in meat industry.Our country is the major producer and consumer of fermented meat and there are a range of different fermented meats available all over the country.The demand for supreme quality and diversified meat products is sharply increasing with the improvement in people's living standards,while traditional processing technology no longer meet the requirement of modernized fermented meat. Therefore,in order to promote the development of the fermented meat industry,it is highly imperative to develop safty and high-activity meat starter to give a boost to meat processing industry.
Starting with the crux technology of preparing high-activity concentrated lactic acid bacteria as meat starter.The present research used liquid-core capsule immobilized technology on Lactobacillus sake LS10 which was isolated from fermented meat.The concentration of L.sake LS10 was studied using the single factor experiments,response surface methodology and orthogonal designs to optimize the enrichment media and culture conditions.Screening the optimal deep-frozen protective for encapsulated L.sake LS10 was done to improve the cell viability.The study provided a theoretical basis for preparing new immobilized lactic acid bacteria as meat starter.The main results were as follows:
1.Preparation of microencapsulated L.sake LS 10
Lactobacillus sake LS10 was immobilized in calcium alginate capsules by using liquid-core method and conventional embedding method,respectively.The capsulated cell density of liquid-core microcapsule was obviously higher than that of gel bead under the same culture condition.According to the test purpose,the liquid-core method was chosen.
2.Study on high-density cultivation of microencapsulated L.sake LS10
Based on whey culture medium,and supplemented with carbon source,nitrogen source,growth factors,pH-buffer,the optimum culture medium was determined by single factor experiments and response surface methodology.It was found that the strain LS10 required the following ingredients for the optimal growth(L~(-1)):whey powder 60g,glucose 18.87g,peptone 21.98g,tomato juice 98.62mL,CaCO_3 4.39g,MgSO_4 0.28g and MnSO_4 0.18g.The results of orthogonal designs showed that the optimal culturing conditions were temperature 37℃,pH 7.0,culture time 20h,inoculum 1%.At the optimized condition,the cell concentration in the liquid-core capsules was increased to 10.59 log(cfu/g).
3.Study on activity protection of microencapsulated deep-freezing starter
Cell viability of Lactobacillus sake LS10 was compared under different freezing temperature(-20℃and -70℃) and different thawing temperature(4℃,22℃and 42℃). The optimum freezing temperature was found to be -70℃while the optimum thawing temperature 42℃.
Plackett-Burman design was used to evaluate the deep-frozen protective effect of sucrose,lactose,glucose,fructose,maltodextrin,glycerol,mannitol,sorbitol,gelatin,skim milk,L-cysteine hydrochloride,arginine,ascorbic acid and sodium glutamate on encapsulated L.sake LS10.Results showed that sucrose,glycerol,sodium glutamate and skim milk were the effective deep-frozen protectants for L.sake LS10.Then the central composite design and response surface analysis were used to determine the optimal levels of these media.The results showed that the optimal protective medium for enhancing the viability of deep-frozen L.sake LS10 was sucrose 45.75g/L,glycerol 63.95 mL/L,sodium glutamate 64.70g/L,and skim milk 92.93 g/L,which allowed the cell viability of L.sake LS10 up to 92.54%after deep-freezing.
On the basis of preparation of deep-frozen concentrated meat starter,the change of bacterium count under different storage conditions(-20℃and -70℃) was studied.The results showed that frozen storage at -70℃contributed more to L.sake LS10 protection than that at -20℃,the cell viability was 63.59%six months later.
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