菊粉制备和乳酸菌微胶囊化包被的研究
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摘要
为了开发饲用微生态制剂,本研究采用热水浸提法从新鲜菊芋中提取菊粉,作为部分试验材料,并对提取的菊粉进行了含量测定,研究了菊粉对2株乳酸菌生长的影响。然后采用平板活菌计数技术测定了乳酸菌的耐酸性、耐胆盐性、耐热性和贮存稳定性,通过牛津杯法观察乳酸菌的抑菌效果。并在实验室条件下,筛选了乳酸菌微胶囊化的包被方法、材料、工艺及参数,以提高其抗逆性。最后将菊粉和微胶囊化乳酸菌混合用于仔猪饲料中,测定其对仔猪生产性能和免疫功能的影响。结果表明:
1.菊粉含量的测定采用还原糖的含量×0.9。还原糖的测定以果糖为标准物,按照3,5-二硝基水杨酸(DNS)比色法制作标准曲线,确定y=0.7564x-0.1107(R2=0.9998)为还原糖测定的回归方程。测得菊芋中菊粉含量为86.3%。
2.在MRS液体培养基中添加不同比例的菊粉,37℃下密闭培养,测定乳酸菌生长的OD600nm值和pH值。结果显示1%菊粉添加量能促进植物乳杆菌和保加利亚乳杆菌的生长,但是随着菊粉添加量的增加,菊粉对乳酸菌的生长几乎没有影响。
3.试验用植物乳杆菌的抗逆性不强,在pH值为1.5的人工胃液中不能存活,在pH值为2.5的人工胃液中处理2.5h后活菌数下降2个数量级以上;乳酸菌对温度的耐受性差,达到70℃时基本不生长;而对猪胆盐具有较好的耐受性,在胆盐质量分数为0.3%的MRS培养基中能够生长;常温下,菌液直接贮存存活率不高,15d后活菌含量下降2个数量级。该菌株对致病性大肠杆菌、沙门氏菌、金黄色葡萄球菌均有较强的抑制作用。
4.对几种实验室常用的微胶囊化方法进行筛选,证实挤压法包被乳酸菌效果较好。海藻酸钠形成的凝胶具有肠溶胃不溶的特性,因此可作为微胶囊化的主要壁材。通过正交试验确定挤压法最佳的包被配方为:3%海藻酸钠、4%脱脂奶粉、6%乳糖、2%氯化钙,制备的微胶囊外形颗粒圆整,大小均匀,产品活菌数最高达7.76×1010cfu/g。经微胶囊化的乳酸菌其耐酸性和贮存稳定性均有较大提高。
5.将菊粉和微胶囊化乳酸菌混合制成合生素,研究其对断奶仔猪的影响。结果表明,与对照组相比,乳酸菌组和合生素组日增重分别提高了19.63%(P<0.01)、24.85%(P<0.01),CD4+/CD8+分别提高了3.70%(P<0.05)、5.56%(P<<0.05),单核-巨噬细胞分别提高了12.21%(P<0.05)、13.17%(P<0.05)。
In order to study animal microecological preparation, a production extraction of Jerusalem artichoke, Inulin Fructan(GFn) ,was extracted through hot water extracting. And the study showed that the effect of inulin to Lactobacillus and the antimicrobial activity of Lactobacillus to E.coli, S.typhimurium, Sta.aureus.Then resistance to stress/bad conditions of Lactobacillus including thermoresistance,acid resistance,bile salt resistance and stability during storage was detected by plate count technique.And microencapsulation technique was adopted to immobilize Lactobacillus to improve its survival in detrimental conditions. Finally, The experiment studied the cooperation effect of inulin and microcapsules Lactobacillus on productive performance and immunity of weaned piglets.The results showed:
(1) The inulin content was measured with the 90 percent of reducing sugar.Reducing sugar was determined by the dinitrosalicylic acid method using D(一)Fructose as standard.The mathematical model was expressed according to following equation:y=0.7564x-0.1107(R2=0.9998). The inulin content was 86.3%.
(2) The experiment got the growth OD600nm and pH of Lactobacillus in sealed bottles with different levels of inulin. The results showed that the best percent of inulin was 1% to L.planticola and L.bulgaricus.But with the increase of inulin, the growth and reproduction of Lactobacillus had almost no change.
(3) L.planticola was sensitive to rigrous conditions was studied. Incubated in simulated gastric juice(pH=1.5)for 0.5h, no flora could be detected.And incubated (pH=2.5)for 2.5h,the number of viable L.planticola was decreased about 2 log count. When the culture was heated from 40℃to 70℃and kept for 60s,the number of viable L.planticola decreased from 6.6×10~9 to 0. Higher temperature,longer time,less culture would make less viable bacteria survive. When the culture was stored in liquid at ambient temperature for 15d,the number of viable L.planticola was decreased about 2 log count.The L.planticola had better tolerance in 3% bile salt and antimicrobial activity to E.coli, S.typhimurium and Sta.aureus.
(4) The extrusion method was selected for microencapsulating L.planticola.Sodium alginate has good bio-compatibility and the gel is soluble in intestinal juice but not in gastric juice.It was used as the wall material of Lactobacillus microencapsulation in this study.We had found out the optimum process of making microcapsule and addtive magnitude of supporters though the orthogonal experiment .The results showed that the optimum conditions for microencapsulation were established as follows:the alginate concentration as 3%, skim milk as 4%, milk sugar as 6%, calcium chloride as 2%,and its livability reached 7.67×10~(10)cfu/g. The resistance to stress/bad conditions of Lactobacillus microencapsulation including acid resistance and stability during storage had been greatly improved.
(5) The cooperation effect of inulin and microcapsules Lactobacillus on productive performance and immunity of weaned piglets was studied.Compared with the control, the average daily gain(ADG) of L.lactis group and sybiotics group increased by19.63%(P<0.01)、24.85%(P<0.01), CD4+/CD8+ increased by 3.70%(P<0.05)、5.56%(P<0.05), mononuclear phagocyte increased by 12.21%(P<0.05)、13.17%(P<0.05); The sybiotics group had better effect than the L.lactis group, but which was not remarkable.
1.菊粉含量的测定采用还原糖的含量×0.9。还原糖的测定以果糖为标准物,按照3,5-二硝基水杨酸(DNS)比色法制作标准曲线,确定y=0.7564x-0.1107(R2=0.9998)为还原糖测定的回归方程。测得菊芋中菊粉含量为86.3%。
2.在MRS液体培养基中添加不同比例的菊粉,37℃下密闭培养,测定乳酸菌生长的OD600nm值和pH值。结果显示1%菊粉添加量能促进植物乳杆菌和保加利亚乳杆菌的生长,但是随着菊粉添加量的增加,菊粉对乳酸菌的生长几乎没有影响。
3.试验用植物乳杆菌的抗逆性不强,在pH值为1.5的人工胃液中不能存活,在pH值为2.5的人工胃液中处理2.5h后活菌数下降2个数量级以上;乳酸菌对温度的耐受性差,达到70℃时基本不生长;而对猪胆盐具有较好的耐受性,在胆盐质量分数为0.3%的MRS培养基中能够生长;常温下,菌液直接贮存存活率不高,15d后活菌含量下降2个数量级。该菌株对致病性大肠杆菌、沙门氏菌、金黄色葡萄球菌均有较强的抑制作用。
4.对几种实验室常用的微胶囊化方法进行筛选,证实挤压法包被乳酸菌效果较好。海藻酸钠形成的凝胶具有肠溶胃不溶的特性,因此可作为微胶囊化的主要壁材。通过正交试验确定挤压法最佳的包被配方为:3%海藻酸钠、4%脱脂奶粉、6%乳糖、2%氯化钙,制备的微胶囊外形颗粒圆整,大小均匀,产品活菌数最高达7.76×1010cfu/g。经微胶囊化的乳酸菌其耐酸性和贮存稳定性均有较大提高。
5.将菊粉和微胶囊化乳酸菌混合制成合生素,研究其对断奶仔猪的影响。结果表明,与对照组相比,乳酸菌组和合生素组日增重分别提高了19.63%(P<0.01)、24.85%(P<0.01),CD4+/CD8+分别提高了3.70%(P<0.05)、5.56%(P<<0.05),单核-巨噬细胞分别提高了12.21%(P<0.05)、13.17%(P<0.05)。
In order to study animal microecological preparation, a production extraction of Jerusalem artichoke, Inulin Fructan(GFn) ,was extracted through hot water extracting. And the study showed that the effect of inulin to Lactobacillus and the antimicrobial activity of Lactobacillus to E.coli, S.typhimurium, Sta.aureus.Then resistance to stress/bad conditions of Lactobacillus including thermoresistance,acid resistance,bile salt resistance and stability during storage was detected by plate count technique.And microencapsulation technique was adopted to immobilize Lactobacillus to improve its survival in detrimental conditions. Finally, The experiment studied the cooperation effect of inulin and microcapsules Lactobacillus on productive performance and immunity of weaned piglets.The results showed:
(1) The inulin content was measured with the 90 percent of reducing sugar.Reducing sugar was determined by the dinitrosalicylic acid method using D(一)Fructose as standard.The mathematical model was expressed according to following equation:y=0.7564x-0.1107(R2=0.9998). The inulin content was 86.3%.
(2) The experiment got the growth OD600nm and pH of Lactobacillus in sealed bottles with different levels of inulin. The results showed that the best percent of inulin was 1% to L.planticola and L.bulgaricus.But with the increase of inulin, the growth and reproduction of Lactobacillus had almost no change.
(3) L.planticola was sensitive to rigrous conditions was studied. Incubated in simulated gastric juice(pH=1.5)for 0.5h, no flora could be detected.And incubated (pH=2.5)for 2.5h,the number of viable L.planticola was decreased about 2 log count. When the culture was heated from 40℃to 70℃and kept for 60s,the number of viable L.planticola decreased from 6.6×10~9 to 0. Higher temperature,longer time,less culture would make less viable bacteria survive. When the culture was stored in liquid at ambient temperature for 15d,the number of viable L.planticola was decreased about 2 log count.The L.planticola had better tolerance in 3% bile salt and antimicrobial activity to E.coli, S.typhimurium and Sta.aureus.
(4) The extrusion method was selected for microencapsulating L.planticola.Sodium alginate has good bio-compatibility and the gel is soluble in intestinal juice but not in gastric juice.It was used as the wall material of Lactobacillus microencapsulation in this study.We had found out the optimum process of making microcapsule and addtive magnitude of supporters though the orthogonal experiment .The results showed that the optimum conditions for microencapsulation were established as follows:the alginate concentration as 3%, skim milk as 4%, milk sugar as 6%, calcium chloride as 2%,and its livability reached 7.67×10~(10)cfu/g. The resistance to stress/bad conditions of Lactobacillus microencapsulation including acid resistance and stability during storage had been greatly improved.
(5) The cooperation effect of inulin and microcapsules Lactobacillus on productive performance and immunity of weaned piglets was studied.Compared with the control, the average daily gain(ADG) of L.lactis group and sybiotics group increased by19.63%(P<0.01)、24.85%(P<0.01), CD4+/CD8+ increased by 3.70%(P<0.05)、5.56%(P<0.05), mononuclear phagocyte increased by 12.21%(P<0.05)、13.17%(P<0.05); The sybiotics group had better effect than the L.lactis group, but which was not remarkable.
引文
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76 张世凤,瞿建新.影响饲用微生物添加剂作用效果的原因浅析.河南畜牧兽医,1999,20(3):22-23.
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