【作者】 李晓岩；

【导师】 陈西广；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2009， 博士

【摘要】 随着经济的发展,生活水平的不断提高,人们对具有保健功能的健康食品的需求量也越来越大,由此,在功能性食品、保健食品和营养滋补剂领域均占有一席之地的益生菌制剂也越来越受到人们的重视。益生菌是活的微生物,摄入充足的量后,能够对宿主产生一种或多种特殊且经论证的健康益处。乳酸菌作为益生菌的重要成员之一,具有改善肠道菌群结构、消除致癌因子、提高机体免疫力、降低胆固醇等重要生理功效。实际上,大多数的益生菌在进入消化道后对低pH的胃酸、胆汁酸等的抵抗能力较弱,难以有足够的活菌数量到达并定居于肠道发挥作用。微胶囊技术是提高益生菌活性的一种有效方法,将益生菌包埋于保护性结构中,能够增强它们对外界不良环境(干燥脱水、胃酸、胆汁、胰酶等)的抵抗能力,有利于使其顺利通过胃到达肠道发挥益生作用。挤压法是益生菌微胶囊化使用最广泛的一种方法,具有制备条件温和,不会严重影响被包埋菌体活性等优点。本文以海藻酸钠、明胶、壳聚糖、羧甲基壳聚糖为壁材,稻麸纤维、微孔淀粉为载体,利用挤压法制备出四类乳酸菌微胶囊,对其性质进行检测,考察微胶囊化对乳酸菌的保护作用,并对影响干态乳酸菌存活性的因素进行了探讨。以海藻酸钠为壁材、稻麸纤维为载体,利用挤压法成功的制备出乳酸菌-海藻酸钠微胶囊。结果表明,海藻酸钠的浓度(2%,3%,4%,5%,w/v)对干态乳酸菌的存活性及其在模拟胃肠液中(pH 1.2,pH 6.8)的释放情况影响不明显,以5%作为后续实验中的海藻酸钠浓度;4℃干燥的微胶囊样品有利于菌体活性的保持(相比于25℃);与裸菌相比,微胶囊化及载体(稻麸纤维)的添加有利于提高干态乳酸菌的存活能力;海藻糖作为一种非还原性二糖,能够被乳酸菌利用,但对菌体的生长代谢没有促进作用,作为生长组分和保护剂,海藻糖能够增强菌体对干燥环境的抵抗能力,4℃储存8周后样品中乳酸菌的活菌数量为8.1×10~7 cfu/g;乳酸菌的微胶囊化有利于提高菌体对胃酸的耐受性,在模拟胃液(pH 1.2)中处理2 h后菌体的存活率可达89%;随着储存环境相对湿度的增加(由33%升高到97%),微胶囊样品中乳酸菌的活菌数量减少。以海藻酸钠、明胶为壁材,利用挤压法制备乳酸菌-海藻酸钠/明胶微胶囊。微胶囊的粒径大小为1.1±0.2 mm。4℃储存一周后,微胶囊样品中干态乳酸菌的活菌数量最低为107 cfu/g。柠檬酸钠处理样品组与非处理组的表面结构相差较大,前者呈现片状结构,而后者则分布有许多皱褶;随着储存时间的延长,微胶囊样品中的活菌数量呈现下降趋势,柠檬酸钠处理样品组活菌数的下降程度相对较小;随着相对湿度从33%升高到97%,乳酸菌微胶囊的吸湿率明显增大;溶胀介质中的pH和离子强度对不同乳酸菌微胶囊的溶胀性具有基本相同的影响规律。当pH较低(2.4)时,微胶囊很快达到溶胀平衡,随着pH的升高(大于5.2),微球出现先溶胀后溶蚀的现象。随着离子强度从0.01升高到1 mol/l,微胶囊的溶胀率降低;微胶囊中的乳酸菌能够在模拟胃肠液中连续释放,且乳酸菌在模拟肠液中的释放速度和释放量明显大于在模拟胃液中。用酸降解法制备不同分子量的壳聚糖(120 kDa,338 kDa,540 kDa和1360 kDa);将脱乙酰化与乙酰化相结合,可以制备得到具有不同脱乙酰度的壳聚糖样品(91.26%,83.34%和65.41%);以海藻酸钠及壳聚糖为壁材、微孔淀粉为载体,利用三种方法制备乳酸菌-海藻酸钠/壳聚糖微胶囊。所有微胶囊均呈现圆形、无粘连的球状结构,粒径为2.1±0.2 mm;所有微胶囊样品的含水量均小于10%;随着相对湿度的增大,各样品的吸湿率明显增大;制备方法对乳酸菌微胶囊在模拟胃肠液中的溶胀性没有明显影响;由于外源交联法制备的微胶囊样品中乳酸菌的活菌数量高且对胃酸的耐受能力强,以其为后续试验中制备乳酸菌-海藻酸钠/壳聚糖微胶囊的方法;随着微孔淀粉添加量的增大(从0.2升高到0.4 g/ml),乳酸菌的存活量呈现增加趋势。在储存过程中,添加量为0.3和0.4 g/ml的样品中乳酸菌的存活量相差不大,确定后续实验中的微孔淀粉用量为0.3 g/ml;利用外源交联法制备乳酸菌-海藻酸钠/壳聚糖微胶囊,考察壳聚糖的分子量、浓度及脱乙酰度对微胶囊在不同pH、离子强度介质中的溶胀性及对乳酸菌存活性的影响,得出壳聚糖的最佳指标为:分子量为120 kDa,浓度为1%(w/v),脱乙酰度为91.26%。通过壳聚糖在浓碱液中与氯乙酸反应,制备得到不同分子量的羧甲基壳聚糖(172.7 kDa,490.2 kDa,720.9 kDa和1771.5 kDa);所制备的羧甲基壳聚糖为淡黄色粉末,易溶于水,水溶液的透明度良好。以海藻酸钠、壳聚糖及羧甲基壳聚糖为壁材、微孔淀粉为载体,运用挤压法制备出乳酸菌-海藻酸钠/壳聚糖/羧甲基壳聚糖微胶囊。所有微胶囊的圆整性良好,粒径的大小为2.2±0.1 mm,羧甲基壳聚糖的引入使得微胶囊表面变得光滑、平整;所有微胶囊样品的含水量均小于10%;随着相对湿度的增大(从33%增加到97%),微胶囊的吸湿率明显增大;微胶囊膜通透性实验表明,乳酸菌-海藻酸钠/壳聚糖/羧甲基壳聚糖微胶囊膜为一种选择透过性膜,羧甲基壳聚糖的分子量对膜通透性的影响没有明显的规律性。随着羧甲基壳聚糖浓度的增大,膜的通透能力下降;羧甲基壳聚糖的分子量及浓度对乳酸菌-海藻酸钠/壳聚糖/羧甲基壳聚糖微胶囊在不同pH及离子强度中的溶胀行为影响不明显,且对乳酸菌存活性的影响没有明显的规律性;综上所述,得出制备微胶囊的羧甲基壳聚糖的最佳条件为:分子量为721 kDa,浓度为1%,(w/v);乳酸菌在海藻酸钠/壳聚糖/羧甲基壳聚糖微胶囊内的生长曲线与游离菌体类似,均呈现“S”型;随着相对湿度的升高(从33%升高到97%),乳酸菌-海藻酸钠/壳聚糖/羧甲基壳聚糖微胶囊样品中乳酸菌的活菌数量减少;在固定化乳酸菌的耐受性实验中发现,以海藻酸钠、壳聚糖及羧甲基壳聚糖为壁材制备的三层微胶囊,对乳酸菌有明显的保护作用,大大提高了菌体对胃酸、胆盐的耐受能力。当益生元(菊粉和低聚果糖)的添加浓度为0.5,1.5,2.0%(w/v)时,菊粉对乳酸菌的增殖有促进作用,但影响不明显。低聚果糖对乳酸菌的生长没有促进作用。浓度为0.5%(w/v)的菊粉对乳酸菌在干燥过程中有明显的保护作用,且能提高菌体在储存过程中的稳定性;壳寡糖具有良好的微生态学效应,可作为微生态调节剂开发利用。实验发现,壳寡糖作为生长组分,有利于改善干燥菌体的存活情况,在储存第4周,壳寡糖添加组中乳酸菌的最低活菌数为5.6×10~7 cfu/g。作为干燥保护剂,壳寡糖浓度为0.4%时的保护效果最好,在储存第6周,菌体的活菌数量为3.6×10~8 cfu/g;在培养基中加入醋酸钠、柠檬酸钠和磷酸氢二钠等缓冲盐,能够调节培养液的酸碱度,促进菌体的生长,增加菌液中还原糖的产量,有利于提高菌体对渗透压的耐受性;另外,缓冲盐对菌体在干燥过程中的保护效果因菌体的干燥温度而异,当干燥温度为25℃时,保护效果较好;当菌体的干燥温度为25℃时,海藻糖(5%,w/v)和脱脂奶粉(10%,w/v)对菌体在复水过程中的保护效果较为明显。随着复水时间的延长,复水介质(海藻糖和脱脂奶粉)的浓度对菌体活性的恢复影响不大。总之,利用生物大分子材料(海藻酸钠、明胶、壳聚糖及羧甲基壳聚糖)对乳酸菌进行微胶囊化,有利于改善乳酸菌在干燥及储存过程中的存活情况,能够提高菌体对低pH的胃酸、胆汁的耐受性,为益生菌作用的发挥提供了前提,具有广阔的应用前景。更多还原

【Abstract】 With the development of economy and the improving of people’s living standard, great interest has been focused on developing functional foods that exert a beneficial effect on the host health. Probiotic agents have gained great attention in the food technology field. Probiotics are defined as“live microorganisms which when administered in adequate amounts confer a health benefit on the host”. Lactic acid bacteria, which are among the most important probiotic microorganisms, have many beneficial effects on the human including maintenance of a healthy gut microflora, reducing risk factors for colon cancer, improving the organism immunity, and lowering serum cholesterol. However, studies indicate that the bacteria may not survive in sufficient numbers when incorporated into dairy products as well as during their passage through the gastro-intestinal tract. The low pH of the stomach and bile salts commonly encountered in the GIT are detrimental to their survival. Microencapsulation techniques have been widely utilized to protect probiotics against adverse environmental conditions (drying, gastric acid, bile salts, pancreatic enzyme, etc.). Among the available techniques for immobilizing living cells, the extrusion method has been frequently used, because the preparation process of microcapsules is under mild conditions compared to freeze drying and spray drying.In this paper, four kinds of Lactobacillus sp. loaded microcapsules are prepared with the extrusion method, using alginate, gelatin, chitosan and carboxymethyl chitosan as wall material, fibre and microporous starch as carrier. The characteristics of microcapsules are detected. Factors affecting the survival of dried Lactobacillus sp. are also studied.Lactobacillus sp. loaded microcapsules are prepared by extrusion technology with fibre and alginate. The survival condition of Lactobacillus sp. and the release profile of encapsulated cells in gastro-intestinal tract exhibit slight difference when the alginate concentration altered from 2% to 5% (w/v) and 5% is used in the succeeding experiments. Drying at 4℃is beneficial to the survival of Lactobacillus sp. compared with 25℃. The immobilization technique and the addition of fibre are much beneficial to the survival of encapsulated Lactobacillus sp.. Trehalose, as a reducing disaccharide, can slightly reduce the acid production of Lactobacillus sp. and has a negative effect on its proliferation. Trehalose can improve the survival ability of Lactobacillus sp. after drying and maintain the live cell numbers over 107 cfu/g after 8 weeks storage at 4℃. The survival rate of microencapsulated Lactobacillus sp. reaches 89% after they are exposed to simulated gastric fluids (pH 1.2) for 2 h. The numbers of viable Lactobacillus sp. decrease with the relative humidity increased from 33% to 97%.Lactobacillus sp. loaded microcapsules based on alginate and gelatin have been prepared by extrusion technology. The size of microcapsules is 1.1±0.2 mm and the product can increase the live cell number of Lactobacillus sp. to 107 cfu/g in the dry state after storage at 4℃for 1 week. The surface of microcapsules treated with sodium citrate presents as the form of sheets, while the one without treatment exhibits some rumple. The live cell number of Lactobacillus sp. decreases with the storage time prolonged and microcapsules treated sodium citrate is much beneficial to the survival of Lactobacillus sp.. The moisture absorption rate of microcapsules increases significantly with the relative humidity altered from 33% to 97%. The pH values and ion intensity of solution affect the swelling behavior of alginate/gelatin microcapsules in the same way. The microcapsules swell rapidly and reach its equilibrium at pH 2.4. When the pH is higher than 5.2, the microcapsules become unstable and disintegrate much rapidly. The swelling ratio of microcapsules decreases with the ion intensity increased from 0.01 to 1 mol/l. Cells of Lactobacillus sp. can be continuously released from the microcapsules during gastro-intestinal tract, and the release amounts and speeds of Lactobacillus sp. in simulated intestinal fluid are much higher and faster than that in simulated gastric fluid.Chitosans with different molecular weight (120 kDa, 338 kDa, 540 kDa and 1360 kDa) and degree of deacetylation (91.26%,83.34% and 65.41%) are prepared using acid degradation and acetylation methods. Lactobacillus sp. loaded microcapsules based on alginate, chitosan and microporous starch have been prepared by three different methods. All the microcapsules appear as a spherical structure about 2.1±0.2 mm in diameter. The water content of microcapsules is lower than 10%. The moisture absorption rate of microcapsules increases significantly with the relative humidity altered from 33% to 97%. The swelling profile of microcapsules in simulated gastro-intestinal fluid is not affected by preparation methods. The external crosslinking method is chosen for the following experiment because it can protect Lactobacillus sp. against the gastric acidity efficiently and provide the best protection of Lactobacillus sp. against desiccation. The survival condition of microencapsulated Lactobacillus sp. improves with the level of microporous starch increased from 0.2 to 0.4 g/ml. In the process of storage the survival of bacteria has no obvious difference when the microporous starch is added between 0.3 and 0.4 g/ml, the concentration of 0.3 g/ml is selected to use in the succeeding experiments. The chitosan molecular weight of 120 kDa, deacetylation degree of 91.26% and concentration of 1% (w/v) are the optimal condition to prepare Lactobacillus sp. microcapsules.Carboxymethyl chitosan with different molecular weight (172.7 kDa,490.2 kDa,720.9 kDa and 1771.5 kDa) are prepared with chitosan and chloroacetic acid in alkaline condition and all the samples are light yellow powder and water soluble. Microcapsules containing Lactobacillus sp. are prepared by extrusion technology with microporous starch, alginate, chitosan and carboxymethyl chitosan. All the microcapsules appear as a spherical structure about 2.2±0.1 mm in diameter. The water content of microcapsules is lower than 10%. With the relative humidity altered from 33% to 97%, the moisture absorption rate of microcapsules increases significantly while the live cell number decreases. The membrane of microcapsules shows permselectivity. The effect of carboxymethyl chitosan molecular weight on the permeability of microcapsules membrane has no regularity. The permeability of microcapsule membrane decreases with the increase of carboxymethyl chitosan concentration. The molecular weight and concentration of carboxymethyl chitosan has little effect on the swelling behavior of microcapsules in different pH and ion intensity condition. The carboxymethyl chitosan molecular weight of 721 kDa and concentration of 1% (w/v) are the optimal condition to prepare microcapsules. Lactobacillus sp. loaded in the microcapsules has the same growth profile (S-type) with the free cells. Results indicate that Lactobacillus sp. immobilized in the microcapsules survive much better under low pH and bile conditions compared with the free cells.Factors that influencing the survival of dried Lactobacillus sp. are studied. With the concentration of 0.5, 1.5, 2.0% (w/v), inulin is beneficial to the growth of Lactobacillus sp. while fructo oligosaccharides has no positive effect. Inulin with concentration of 0.5% is found to be the most effective of the prebiotic in retaining the viability of immobilized Lactobacillus sp. under drying conditions. Chitosan oligosaccharide can be developed and utilized as a micro-ecological regulator. As a component of growth medium, chitosan oligosaccharide can improve the live cell number to 5.6×10~7 cfu/g after storage at 4℃for 4 weeks. As the protective additive, chitosan oligosaccharide (0.4%, w/v) can improve the live cell number to 3.6×10~8 cfu/g after storage at 4℃for 6 weeks. The presence of buffer salts (disodium hydrogen phosphate, sodium acetate and sodium citrate) in the growth medium improves the viability of Lactobacillus sp. after dried at 25℃and its resistance to osmolarity. Trehalose and skimmed milk are found to be the most suitable rehydration medium in retaining the viability of microencapsulated Lactobacillus sp. in dry state. With the rehydration time increasing, the concentration of the rehydration medium (trehalose and skimmed milk) has no obvious effect on the recovery of dried Lactobacillus sp..In summary, immobilized Lactobacillus sp. with alginate, gelatin, chitosan and carboxymethyl chitosan proves to be very efficient in improving the viability of Lactobacillus sp. in dry state. The microencapsulation method reported in this paper also improves the resistance of Lactobacillus sp. to gastric acid and bile salts and it provides a basis for the broad application of probiotics.更多还原