高抗逆性双歧杆菌和嗜酸乳杆菌的筛选及应用研究
发布时间：2018-08-10 20:26
【摘要】： 双歧杆菌和嗜酸乳杆菌是人体的微生态调节剂。研究发现,要想获得预期的保健效果,一般来说,食品中活菌体的数量不少于10~6cfu/mL左右或日摄入量在10~8cfu左右,只有这样才能补偿益生菌在人的胃、肠中数量的下降。然而,由于益生菌的自身生长特性及周围环境因素的影响,导致进入市场的产品中活菌含量较低。因此,如何提高产品中益生菌活菌数已成为商业的重点研究课题。通过实验,筛选出黏附性高、对人体胃肠环境耐受性强的双歧杆菌和嗜酸乳杆菌各一株,对其冻干保护剂进行了优化,并对冻干粉储存稳定性进行测试。将两种菌的冻干粉等量混合后独立包装,与酶解法制得的低乳糖乳粉共同饮用,起到缓解乳糖不耐症和微生态调节的双重保护作用。 供试双歧杆菌5株,分别来源于国内三种益生菌药品及健康婴儿粪便;供试嗜酸乳杆菌5株,2株来源于国内二种益生菌药品,2株来源于本实验室保藏,1株购于黑龙江应用微生物研究所。分别对筛选到的不同菌株进行个体形态,菌落形态及其生理生化鉴定,确认分别属于双歧杆菌属和乳酸菌属。通过用激光共聚焦显微镜观察供试菌对结肠癌细胞COCA-Ⅱ的黏附性、分别通过耐酸和耐胆汁盐试验,筛选出指标较高的双歧杆菌3株和嗜酸乳杆菌2株。通过模拟胃液和模拟肠液进行复筛,分别筛选出高耐受性菌各1株,分别命名为双歧杆菌L-1、嗜酸乳杆菌La-5。L-1对COCA-Ⅱ的黏附数为:16.6±2.1黏附菌数/细胞;在pH3.0模拟胃液中作用120min后的存活率为72%;在胆汁盐浓度为0.2%的模拟肠液中,L-1 24h后其活菌数仍大于10~6cfu/mL。La-5对COCA-Ⅱ数为18.8±2.4黏附菌数/细胞;在pH2.5模拟胃液中作用3h后,其活菌数仍大于10~7cfu/mL;胆汁盐浓度为0.2%的模拟肠液时,La-5 3h后其活菌数仍大于10~6cfu/mL。 应用单因素试验,析因试验和响应面分析法等数学统计方法,分别对L-1和La-5冻干保护剂进行优化,试验得出L-1冻干保护剂的最佳配方为:海藻糖18%、甘油6%、维生素C 0.1%、明胶0.1%,双歧杆菌的冻干粉冻干保护率可达86.7%;嗜酸乳杆菌La-5冻干保护剂的最佳配方为:海藻糖19%,甘油6%,MnSO_40.1%,明胶0.1%,嗜酸乳杆菌菌的冻干粉冻干保护率可达96.8%,经验证均与模型预测基本相符。 经过储藏稳定性试验得出,L-1与La-5冻干粉经过密封包装,储存60天后,在20℃(即常温)储存条件下,L-1冻干粉活菌数仅下降0.68个对数点,为10~(9.32)cfu/g。La-5冻干粉活菌数只0.15个对数点,为10~(10.85)cfu/g;在37℃储存条件下,L-1冻干粉活菌数下降1.31个对数点,为10~(8.69)cfu/ g。La-5冻干粉活菌数下降0.25个对数点,为10~(10.75)cfu/g。 以新鲜牛奶为原料,采用中温乳糖酶水解制得低乳糖奶粉。应用单因素试验和均匀试验,确定各因素对奶粉褐变程度的影响。结果表明,影响褐变程度的因素依次为:水解的温度㧐水解的pH值㧐褐变抑制剂添加时间㧐褐变抑制剂的用量。最佳工艺条件为:乳糖酶添加量为0.15U/kg,水解的温度39℃,水解的pH值为6.6,复合褐变抑制剂(BHA:VE=1:1)添加量为0.1%,添加时间为水解前20min。该条件下制备的低乳糖奶粉乳糖水解率能够达70%,白度达到87.2。将两种菌的冻干粉等量混合用4层复合材料包装成2g独立包装。添加到低乳糖乳粉,冻干粉的添加量为1袋/10g。
[Abstract]:Bifidobacterium and Lactobacillus acidophilus are microecological regulators of the human body. Studies have found that to achieve the desired health effects, generally speaking, the number of living organisms in food is not less than 10-6 cfu/mL or the daily intake is about 10-8 cfu. Only in this way can the decrease of probiotics in the stomach and intestines be compensated. How to increase the viability of probiotic bacteria in products has become an important research topic in commerce. Bifidobacterium and Lactobacillus acidophilus strains with high adhesion and strong tolerance to human gastrointestinal environment were screened out through experiments. The freeze-drying protectant was optimized and the storage stability of the freeze-drying powder was tested. The freeze-drying powder of the two bacteria was packaged independently and drinked together with the low-lactose milk powder produced by enzymatic hydrolysis to alleviate lactose intolerance and regulate microecology.
Five strains of Bifidobacterium were isolated from three domestic probiotics and healthy infant feces, five strains of Lactobacillus acidophilus, two from two domestic probiotics, two from our laboratory and one from Heilongjiang Institute of Applied Microbiology. The adherence of the tested bacteria to COCA-II was observed by laser confocal microscopy. Three strains of Bifidobacterium and two strains of Lactobacillus acidophilus were screened out by acid-and bile-salt-tolerant tests. The two strains were fed into simulated gastric juice and simulated intestinal juice. After re-screening, one strain of highly tolerant bacteria was screened and named as Bifidobacterium L-1, Lactobacillus acidophilus L-5.L-1 adhering to COCA-II was 16.6+2.1 adherent bacteria/cells, the survival rate was 72% after 120 minutes in simulated gastric juice at pH 3.0, and the number of viable bacteria was still greater than 10-6 cfu/m after 24 hours in simulated intestinal juice with bile salt concentration of 0.2%. L.La-5 still had more viable bacteria than 10-7 cfu/mL after 3 hours of exposure to simulated gastric juice at pH 2.5, and more viable bacteria than 10-6 cfu/mL after 53 hours of exposure to simulated intestinal juice with bile salt concentration of 0.2%.
L-1 and La-5 freeze-drying protectants were optimized by single factor test, factorial test and response surface analysis. The optimum formula of L-1 freeze-drying protectant was obtained as follows: trehalose 18%, glycerol 6%, vitamin C 0.1%, gelatin 0.1%, Bifidobacterium freeze-drying protectant rate 86.7%; Lactobacillus acidophilus La-5 freeze-drying protectant. The optimum formulation of the protectant was trehalose 19%, glycerol 6%, MnSO_40.1%, gelatin 0.1%, and the freeze-drying protective rate of Lactobacillus acidophilus powder was 96.8%. The experimental results showed that the protective rate was basically consistent with the model prediction.
The results of storage stability test showed that the viable bacteria count of L-1 and La-5 freeze-dried powder decreased by 0.68 logarithm points after 60 days storage at 20 C (normal temperature), and the viable bacteria count of L-1 freeze-dried powder was only 0.15 logarithm points of 10_ (9.32) cfu/g. La-5 freeze-dried powder and 10_ (10.85) cfu/g under 37 C storage condition. 1.31 logarithmic points, 10 ~ (8.69) CFU / g.La-5 lyophilized powder viable bacteria count decreased by 0.25 logarithmic points, 10 ~ (10.75) CFU / g.
Low lactose milk powder was prepared from fresh milk by hydrolysis of lactase at medium temperature.The effects of various factors on browning degree of milk powder were determined by single factor test and uniform test.The results showed that the factors influencing browning degree were: hydrolysis temperature? PH value of hydrolysis? Adding time of browning inhibitor? Dosage of browning inhibitor. The technological conditions were as follows: the addition of lactase was 0.15U/kg, the hydrolysis temperature was 39 C, the hydrolysis pH was 6.6, the addition of compound browning inhibitor (BHA:VE=1:1) was 0.1%, and the addition time was 20 minutes before hydrolysis. The material was packaged into 2G independent packaging. The addition of low lactose milk powder to freeze-dried powder was 1 bags of /10g..
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R371
本文编号：2176102
[Abstract]:Bifidobacterium and Lactobacillus acidophilus are microecological regulators of the human body. Studies have found that to achieve the desired health effects, generally speaking, the number of living organisms in food is not less than 10-6 cfu/mL or the daily intake is about 10-8 cfu. Only in this way can the decrease of probiotics in the stomach and intestines be compensated. How to increase the viability of probiotic bacteria in products has become an important research topic in commerce. Bifidobacterium and Lactobacillus acidophilus strains with high adhesion and strong tolerance to human gastrointestinal environment were screened out through experiments. The freeze-drying protectant was optimized and the storage stability of the freeze-drying powder was tested. The freeze-drying powder of the two bacteria was packaged independently and drinked together with the low-lactose milk powder produced by enzymatic hydrolysis to alleviate lactose intolerance and regulate microecology.
Five strains of Bifidobacterium were isolated from three domestic probiotics and healthy infant feces, five strains of Lactobacillus acidophilus, two from two domestic probiotics, two from our laboratory and one from Heilongjiang Institute of Applied Microbiology. The adherence of the tested bacteria to COCA-II was observed by laser confocal microscopy. Three strains of Bifidobacterium and two strains of Lactobacillus acidophilus were screened out by acid-and bile-salt-tolerant tests. The two strains were fed into simulated gastric juice and simulated intestinal juice. After re-screening, one strain of highly tolerant bacteria was screened and named as Bifidobacterium L-1, Lactobacillus acidophilus L-5.L-1 adhering to COCA-II was 16.6+2.1 adherent bacteria/cells, the survival rate was 72% after 120 minutes in simulated gastric juice at pH 3.0, and the number of viable bacteria was still greater than 10-6 cfu/m after 24 hours in simulated intestinal juice with bile salt concentration of 0.2%. L.La-5 still had more viable bacteria than 10-7 cfu/mL after 3 hours of exposure to simulated gastric juice at pH 2.5, and more viable bacteria than 10-6 cfu/mL after 53 hours of exposure to simulated intestinal juice with bile salt concentration of 0.2%.
L-1 and La-5 freeze-drying protectants were optimized by single factor test, factorial test and response surface analysis. The optimum formula of L-1 freeze-drying protectant was obtained as follows: trehalose 18%, glycerol 6%, vitamin C 0.1%, gelatin 0.1%, Bifidobacterium freeze-drying protectant rate 86.7%; Lactobacillus acidophilus La-5 freeze-drying protectant. The optimum formulation of the protectant was trehalose 19%, glycerol 6%, MnSO_40.1%, gelatin 0.1%, and the freeze-drying protective rate of Lactobacillus acidophilus powder was 96.8%. The experimental results showed that the protective rate was basically consistent with the model prediction.
The results of storage stability test showed that the viable bacteria count of L-1 and La-5 freeze-dried powder decreased by 0.68 logarithm points after 60 days storage at 20 C (normal temperature), and the viable bacteria count of L-1 freeze-dried powder was only 0.15 logarithm points of 10_ (9.32) cfu/g. La-5 freeze-dried powder and 10_ (10.85) cfu/g under 37 C storage condition. 1.31 logarithmic points, 10 ~ (8.69) CFU / g.La-5 lyophilized powder viable bacteria count decreased by 0.25 logarithmic points, 10 ~ (10.75) CFU / g.
Low lactose milk powder was prepared from fresh milk by hydrolysis of lactase at medium temperature.The effects of various factors on browning degree of milk powder were determined by single factor test and uniform test.The results showed that the factors influencing browning degree were: hydrolysis temperature? PH value of hydrolysis? Adding time of browning inhibitor? Dosage of browning inhibitor. The technological conditions were as follows: the addition of lactase was 0.15U/kg, the hydrolysis temperature was 39 C, the hydrolysis pH was 6.6, the addition of compound browning inhibitor (BHA:VE=1:1) was 0.1%, and the addition time was 20 minutes before hydrolysis. The material was packaged into 2G independent packaging. The addition of low lactose milk powder to freeze-dried powder was 1 bags of /10g..
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R371
【引证文献】
相关期刊论文 前2条
1 刘延国;张琳;冯香安;李杰;;益生菌在消化道中的抑菌作用[J];中国饲料;2011年16期
2 陈琳;彭虹旎;孔垂斌;王增元;吕左航;甄丽;;几株饲用益生菌对体外模拟胃肠道环境的抗逆性评估[J];中国微生态学杂志;2010年08期
，本文编号：2176102
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