内源乳化凝胶法制备嗜热链球菌微胶囊及其性质的研究
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摘要
肠道益生菌群是构筑肠道粘膜屏障的主要组成部分,为机体抵御病原菌的侵袭提供强大的生物屏障,在维持肠道功能及机体的健康方面发挥着重要的生理作用。通过微胶囊化能将益生菌菌体与外界环境分开,避免其受损害,利于储存和运输;采用肠溶性壁材,能防止胃液破坏,使其到达肠道遇肠液后溶解,从而使尽可能多的菌体到达肠道并定植于肠粘膜上,真正起到保健和治疗的作用。本文以嗜热链球菌为模式菌株,以内源乳化凝胶法对其进行包埋,得到微米级壳聚糖/海藻酸微胶囊,并研究了其在模拟胃肠道环境下的释放性和耐受性,从而可以得到以下结论:
(1)内源乳化凝胶法制备微胶囊的最佳条件:海藻酸钠浓度为15g·L-1,乳化剂Span80用量为0.7%,水与油的体积比为1:8,CaCO3与NaALG质量比为1:5。在此条件下得到目的粒径40～60μm范围内微胶囊的百分含量为80.2%。通过对酪蛋白的包埋及其释放特性研究,得出最佳的壳聚糖覆膜条件为壳聚糖浓度4mg/mL,壳聚糖溶液pH=6,成膜反应时间10min。
(2)优化嗜热链球菌(Streptoccus thermophilus ST1)高密度培养基,最适碳源为蔗糖,最佳浓度为0.5%;最适氮源为大豆蛋白胨,最佳浓度为2.0%;适量的酵母膏和吐温80能促进嗜热链球菌的增殖,最适添加浓度为0.5%和0.1%,过量反而会抑制乳酸菌的增殖;柠檬酸二铵、乙酸钠和硫酸镁等无机盐也能促进乳酸菌的增殖,最适添加浓度为0.2%,0.3%和0.05%,过量也会抑制乳酸菌的增殖。通过Plackett-Burman试验得出蔗糖、大豆蛋白胨和酵母膏对菌体生长影响最为显著。通过响应面优化试验得出最适添加浓度为0.56%,0.54%和2.12%,菌体密度达到1.223。同时研究了温度、初始pH、接种量对乳酸菌高密度培养的影响。优化嗜热链球菌(Streptoccus thermophilus ST1)高密度培养条件,最适培养温度39℃,最适初始pH7.7,最适接种量5%。优化后大大缩短了生长延滞期,菌体密度达1.412。
在5L发酵罐中进行嗜热链球菌的增殖培养, 6h菌体浓度达到最大,可达到OD600为1.71。
(3)嗜热链球菌海藻酸微胶囊的最佳制备工艺为菌体:胶量=1:1,Span80浓度为0.7%,乳化时间为15min,海藻酸钠浓度为1.5%,包埋率为75.6%。
壳聚糖/海藻酸微胶囊较海藻酸微胶囊提高了被包埋嗜热链球菌的存活率,提高了65.74%;壳聚糖/海藻酸微胶囊在人工肠液中较海藻酸微胶囊难释放,释放时间长,释放缓慢;壳聚糖覆盖后,微胶囊在胃液作用下有更高的胃酸耐受性;同时耐胆汁酸性加强,在人工胃液中(pH=1.2)放置2h,活菌数仍能达到8.2×107c fu/mL,在胆汁酸溶液中处理6h,活菌数能达到1.99×10 9cfu/mL。
壳聚糖/海藻酸微胶囊耐胃酸后仍有一定的发酵活力。放于冰箱中4℃保存2个月,菌体存活率仍然达到94.8%,根据国家对保健产品的要求,经计算该微胶囊有效期为94.544天,延长了保藏时间。
综上所述,内源乳化凝胶法制备嗜热链球菌壳聚糖/海藻酸微胶囊是可行的,能增强菌体对外界环境因素的抵抗能力,显著提高菌体在低温保存期和到达肠道后的存活率,使乳酸菌更好地起到有益于健康的作用。微胶囊的粒径范围控制在40～60μm,便于将微胶囊加入到饮料、食品等中去,为新型微生态制剂的开发奠定了基础。
The intestinal flora plays an important role in human health, the main function of which is to prevent colonization of potential pathogenic microorganisms, exert health benefits of inherent basic nutrition and improve intestinal microbial balance of the host. Microencapsulation can be used to detach probiotics from outside environment and prevent the damage, which will advantage the storage and conveyance. The damage of gastric acid can be avoided with the enteric dissolved materials so that a larger number of live cells can get to intestines and make a true function in health care.
Using Streptoccus thermophilus as the mode bacteria, the paper studied the preparation of chitosan/alginate microcapsules by emulsification/internal gelation, and further investigated the release and survival properties of the bacteria in the gastrointestinal tract. Based on the experimental results, main conclusions can be drawn as follows:
(1) The optimal conditions for the preparation of alginate microcapsules are: alginate concentration 1.5%(w/v), emulsifier Span80 concentration 0.7%(v/v), the ratio of water to oil 1:8 (v/v) and the ratio of CaCO3 to NaALG 1:5(w/w) , under the optimal conditions, the content of the microcapsules with the aimed diameter of 40～60μm is 80.2%. Microcapsules will improve their chemical and mechanical stability with a chitosan membrance by the encapsulation and releasing of casein, and the optimal conditions of the chitosan are observed when chitosan concentration is 4mg/ml, chitosan initial pH value is 6, and reacting time is 10min.
(2)The enrichment culture formula of Streptoccus thermophilus is determined. Experiments shows that the best addition of carbon source is sucrose 0.56%, and the best addition of nitrogen source is soyer peptone 2.0%. The equal amount of yeast extract, Tween80, ammonium citrate sodium, sodium acetate and MgSO 4·7H2O can promote the growth of Streptoccus thermophilus, the best additions of which are 0.5%, 0.1%, 0.2%, 0.3%and 0.02% respectively, but excessive additions can restrain the growth.Further research is done by SAS software combined with the Plackett-Burman design and response surface analysis method, which provides the most prominent influential factors for the growth of thalli and the best additions with sucrose 0.56%, soyer peptone 2.12% and yeast extract 0.54%. Moreover, the OD value can reach up to 1.223.
The optimal enrichment culture conditions of Streptoccus thermophilus can also obtained, namely, initial pH value at 7.7, suitable temperature 39℃, and inoculate amounts 5%. By adopting the optimal conditions, the arrearage phrase of Streptoccus thermophilus is shortened and OD value is enhanced to 1.412. Furthermore, Streptoccus thermophilus incubated in 5L fermentor is analyzed giving the best culture time of 6h. Results show that the density of the bacteria gets to the best at this time, and OD600 could reach up to 1.71.
(3) The optimal conditions for the preparation of alginate microcapsules containing Streptoccus thermophilus are: the ratio of bacteria and alginate 1:1, Span80 concentration 0.7%, emulsification time 10min, and alginate concentration 1.5%. The trapping efficiency will reach up to 75.6%.
The survival rate of Streptoccus thermophilus is advanced by 65.74% when the alginate microcapsules are coated with chitosan. The experiment shows that the release of chitosan/alginate microcapsule is slower than before in the artifical intestinal juice. In order to exert positive health effects, Streptoccus thermophilus should resist
the stressful conditions of stomach upper intestine that contain bile.The chitosan/alginate microcapsule has a high survival rate in the gastic juice and bile solution when it is in the gastric juice for about 2h and in the bile acid for 6h with its viability reaching up to 8.2×107cfu/mL and 1.99×109cfu/mL respectively.
The chitosan/alginate microcapsule proves to have strong fermentation activity. Its survival rate still keeps in 94.8% when it has been storaged for 2 months under 4℃in the refrigeratory. And its storage period of validity is 94.54 days calculated according to the demand of our country for the health care production.
In summary, the production of microcapsules containing Streptoccus thermophilus by emulsification/internal gelation is feasible, which can resist the bad circumstance and improve the survival rate in the low temperature and intestinal tract so that the probiotics can exert health benefits more effectively. Under the aimed diameter of 40～60μm, the microcapsule can be appended to drinks and foods, which will provide the foundation for further research and development of new-type microbioecologics.
(1)内源乳化凝胶法制备微胶囊的最佳条件:海藻酸钠浓度为15g·L-1,乳化剂Span80用量为0.7%,水与油的体积比为1:8,CaCO3与NaALG质量比为1:5。在此条件下得到目的粒径40～60μm范围内微胶囊的百分含量为80.2%。通过对酪蛋白的包埋及其释放特性研究,得出最佳的壳聚糖覆膜条件为壳聚糖浓度4mg/mL,壳聚糖溶液pH=6,成膜反应时间10min。
(2)优化嗜热链球菌(Streptoccus thermophilus ST1)高密度培养基,最适碳源为蔗糖,最佳浓度为0.5%;最适氮源为大豆蛋白胨,最佳浓度为2.0%;适量的酵母膏和吐温80能促进嗜热链球菌的增殖,最适添加浓度为0.5%和0.1%,过量反而会抑制乳酸菌的增殖;柠檬酸二铵、乙酸钠和硫酸镁等无机盐也能促进乳酸菌的增殖,最适添加浓度为0.2%,0.3%和0.05%,过量也会抑制乳酸菌的增殖。通过Plackett-Burman试验得出蔗糖、大豆蛋白胨和酵母膏对菌体生长影响最为显著。通过响应面优化试验得出最适添加浓度为0.56%,0.54%和2.12%,菌体密度达到1.223。同时研究了温度、初始pH、接种量对乳酸菌高密度培养的影响。优化嗜热链球菌(Streptoccus thermophilus ST1)高密度培养条件,最适培养温度39℃,最适初始pH7.7,最适接种量5%。优化后大大缩短了生长延滞期,菌体密度达1.412。
在5L发酵罐中进行嗜热链球菌的增殖培养, 6h菌体浓度达到最大,可达到OD600为1.71。
(3)嗜热链球菌海藻酸微胶囊的最佳制备工艺为菌体:胶量=1:1,Span80浓度为0.7%,乳化时间为15min,海藻酸钠浓度为1.5%,包埋率为75.6%。
壳聚糖/海藻酸微胶囊较海藻酸微胶囊提高了被包埋嗜热链球菌的存活率,提高了65.74%;壳聚糖/海藻酸微胶囊在人工肠液中较海藻酸微胶囊难释放,释放时间长,释放缓慢;壳聚糖覆盖后,微胶囊在胃液作用下有更高的胃酸耐受性;同时耐胆汁酸性加强,在人工胃液中(pH=1.2)放置2h,活菌数仍能达到8.2×107c fu/mL,在胆汁酸溶液中处理6h,活菌数能达到1.99×10 9cfu/mL。
壳聚糖/海藻酸微胶囊耐胃酸后仍有一定的发酵活力。放于冰箱中4℃保存2个月,菌体存活率仍然达到94.8%,根据国家对保健产品的要求,经计算该微胶囊有效期为94.544天,延长了保藏时间。
综上所述,内源乳化凝胶法制备嗜热链球菌壳聚糖/海藻酸微胶囊是可行的,能增强菌体对外界环境因素的抵抗能力,显著提高菌体在低温保存期和到达肠道后的存活率,使乳酸菌更好地起到有益于健康的作用。微胶囊的粒径范围控制在40～60μm,便于将微胶囊加入到饮料、食品等中去,为新型微生态制剂的开发奠定了基础。
The intestinal flora plays an important role in human health, the main function of which is to prevent colonization of potential pathogenic microorganisms, exert health benefits of inherent basic nutrition and improve intestinal microbial balance of the host. Microencapsulation can be used to detach probiotics from outside environment and prevent the damage, which will advantage the storage and conveyance. The damage of gastric acid can be avoided with the enteric dissolved materials so that a larger number of live cells can get to intestines and make a true function in health care.
Using Streptoccus thermophilus as the mode bacteria, the paper studied the preparation of chitosan/alginate microcapsules by emulsification/internal gelation, and further investigated the release and survival properties of the bacteria in the gastrointestinal tract. Based on the experimental results, main conclusions can be drawn as follows:
(1) The optimal conditions for the preparation of alginate microcapsules are: alginate concentration 1.5%(w/v), emulsifier Span80 concentration 0.7%(v/v), the ratio of water to oil 1:8 (v/v) and the ratio of CaCO3 to NaALG 1:5(w/w) , under the optimal conditions, the content of the microcapsules with the aimed diameter of 40～60μm is 80.2%. Microcapsules will improve their chemical and mechanical stability with a chitosan membrance by the encapsulation and releasing of casein, and the optimal conditions of the chitosan are observed when chitosan concentration is 4mg/ml, chitosan initial pH value is 6, and reacting time is 10min.
(2)The enrichment culture formula of Streptoccus thermophilus is determined. Experiments shows that the best addition of carbon source is sucrose 0.56%, and the best addition of nitrogen source is soyer peptone 2.0%. The equal amount of yeast extract, Tween80, ammonium citrate sodium, sodium acetate and MgSO 4·7H2O can promote the growth of Streptoccus thermophilus, the best additions of which are 0.5%, 0.1%, 0.2%, 0.3%and 0.02% respectively, but excessive additions can restrain the growth.Further research is done by SAS software combined with the Plackett-Burman design and response surface analysis method, which provides the most prominent influential factors for the growth of thalli and the best additions with sucrose 0.56%, soyer peptone 2.12% and yeast extract 0.54%. Moreover, the OD value can reach up to 1.223.
The optimal enrichment culture conditions of Streptoccus thermophilus can also obtained, namely, initial pH value at 7.7, suitable temperature 39℃, and inoculate amounts 5%. By adopting the optimal conditions, the arrearage phrase of Streptoccus thermophilus is shortened and OD value is enhanced to 1.412. Furthermore, Streptoccus thermophilus incubated in 5L fermentor is analyzed giving the best culture time of 6h. Results show that the density of the bacteria gets to the best at this time, and OD600 could reach up to 1.71.
(3) The optimal conditions for the preparation of alginate microcapsules containing Streptoccus thermophilus are: the ratio of bacteria and alginate 1:1, Span80 concentration 0.7%, emulsification time 10min, and alginate concentration 1.5%. The trapping efficiency will reach up to 75.6%.
The survival rate of Streptoccus thermophilus is advanced by 65.74% when the alginate microcapsules are coated with chitosan. The experiment shows that the release of chitosan/alginate microcapsule is slower than before in the artifical intestinal juice. In order to exert positive health effects, Streptoccus thermophilus should resist
the stressful conditions of stomach upper intestine that contain bile.The chitosan/alginate microcapsule has a high survival rate in the gastic juice and bile solution when it is in the gastric juice for about 2h and in the bile acid for 6h with its viability reaching up to 8.2×107cfu/mL and 1.99×109cfu/mL respectively.
The chitosan/alginate microcapsule proves to have strong fermentation activity. Its survival rate still keeps in 94.8% when it has been storaged for 2 months under 4℃in the refrigeratory. And its storage period of validity is 94.54 days calculated according to the demand of our country for the health care production.
In summary, the production of microcapsules containing Streptoccus thermophilus by emulsification/internal gelation is feasible, which can resist the bad circumstance and improve the survival rate in the low temperature and intestinal tract so that the probiotics can exert health benefits more effectively. Under the aimed diameter of 40～60μm, the microcapsule can be appended to drinks and foods, which will provide the foundation for further research and development of new-type microbioecologics.
引文
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