5L立瓶发酵制备益生菌直投式发酵剂及应用试验

对长双歧杆菌、保加利亚乳杆菌、嗜热乳酸链球菌在5 L立瓶中分别进行培养,确定了各自的发酵最适收获期,菌体收集后经制备所获得冷冻浓缩直投式发酵剂活菌数大于1010mL-1,冷冻干燥直投式发酵剂活菌数大于1011g-1。以制备的冷冻浓缩、冷冻干燥直投式发酵剂,进行牛乳发酵应用试验,生产的益生菌酸奶风味纯正、品质良好。     

双歧杆菌豆奶粉的研制

本文对双歧杆菌的分离、鉴定，双歧杆菌菌粉的制备工艺，双歧杆菌豆奶粉的制备及其稳定性进行了研究。采用发酵工艺、冷冻干燥工艺及喷雾干燥工艺等生产的双歧杆菌豆奶粉的活菌稳定期在6个月以上。     

双歧杆菌豆奶粉的研制

对双歧杆菌的分离、鉴定、双歧杆菌菌粉的制备工艺，双歧杆菌豆奶粉的制备及其稳定性进行了研究。采用发酵工艺，冷冻干燥工艺及喷雾干燥工艺等生产的双歧杆菌豆奶粉的活菌稳定期在６个月以上。     

冻干乳酸双歧杆菌微胶囊的制备及存活性能研究

为了提高双歧杆菌在贮存和消费过程中的菌体存活率,利用冷冻干燥与微囊化结合的方法,将双歧杆菌和高效活菌保护剂一起作为核心物质,采用乳化法制备双歧杆菌微胶囊。通过正交试验,对双歧杆菌冻干过程中加入的保护剂种类及其最佳配比进行了研究。并探讨了双歧杆菌在模拟胃肠道环境以及高渗透压等极端环境条件下的存活能力。结果表明：加入10%菊糖、6%大豆蛋白、12%海藻糖、10%甘露醇可显著提高微囊化双歧杆菌冻干活菌数,使其存活率达到69.2%。较之冻干菌粉,双歧杆菌微囊在模拟胃液、肠液、高胆汁盐以及高渗透压溶液中均具有较好的耐受性。     

双歧杆菌和嗜酸乳杆菌二联活菌微胶囊的研制

目的制备长双歧杆菌和嗜酸乳杆菌二联活菌微胶囊,以期提高口服时的存活率和常温下的保存期。方法根据微胶囊在人工胃液中的耐酸性和在人工肠液中的崩解性,确定空气悬浮法制备微胶囊的最佳工艺条件。扫描电镜观察微胶囊形态与大小。根据恒温37℃、相对湿度60%~65%条件下贮存3个月后菌体存活率,考察其贮存稳定性。结果制备的微胶囊具有良好的耐酸性和肠溶性。微胶囊包囊产率56.49%,包囊效率87.45%。微胶囊表面覆盖着一层光滑平整的囊膜,粒径大小基本在400~500μm。加保护剂的微胶囊菌体存活率高于10%,且远远高于冻干菌粉和无保护剂微胶囊组。结论确定了空气悬浮法制备长双歧杆菌和嗜酸乳杆菌二联活菌微胶囊的最佳工艺条件,微胶囊化有利于提高活菌的稳定性。     

双歧杆菌和嗜酸乳杆菌二联活菌微胶囊的研制

目的制备长双歧杆菌和嗜酸乳杆菌二联活菌微胶囊,以期提高口服时的存活率和常温下的保存期。方法根据微胶囊在人工胃液中的耐酸性和在人工肠液中的崩解性,确定空气悬浮法制备微胶囊的最佳工艺条件。扫描电镜观察微胶囊形态与大小。根据恒温37℃、相对湿度60％～65％条件下贮存3个月后菌体存活率,考察其贮存稳定性。结果制备的微胶囊具有良好的耐酸性和肠溶性。微胶囊包囊产率56．49％,包囊效率87．45％。微胶囊表面覆盖着一层光滑平整的囊膜,粒径大小基本在400～500μm。加保护剂的微胶囊菌体存活率高于10％,且远远高于冻干菌粉和无保护剂微胶囊组。结论确定了空气悬浮法制备长双歧杆菌和嗜酸乳杆菌二联活菌微胶囊的最佳工艺条件,微胶囊化有利于提高活菌的稳定性。     

双歧杆菌和嗜酸乳杆菌二联活菌微胶囊的研制

目的 制备长双歧杆菌和嗜酸乳杆菌二联活菌微胶囊,以期提高口服时的存活率和常温下的保存期.方法 根据微胶囊在人工胃液中的耐酸性和在人工肠液中的崩解性,确定空气悬浮法制备微胶囊的最佳工艺条件.扫描电镜观察微胶囊形态与大小.根据恒温37℃、相对湿度60%～65%条件下贮存3个月后菌体存活率,考察其贮存稳定性.结果 制备的微胶囊具有良好的耐酸性和肠溶性.微胶囊包囊产率56.49%,包囊效率87.45%.微胶囊表面覆盖着一层光滑平整的囊膜,粒径大小基本在400～500μm.加保护剂的微胶囊菌体存活率高于10%,且远远高于冻干菌粉和无保护剂微胶囊组.结论 确定了空气悬浮法制备长双歧杆菌和嗜酸乳杆菌二联活菌微胶囊的最佳工艺条件,微胶囊化有利于提高活菌的稳定性.     

高活性副干酪乳杆菌冻干菌粉的制备及工艺优化

目的:优化副干酪乳杆菌多种冻干保护剂的配比和真空冷冻干燥工艺参数,提高发酵剂的活菌含量,为冻干菌粉的制备提供指导。方法:以存活因子为指标,通过单因素实验筛选大分子保护剂、糖类、聚合物类、抗氧化剂类保护剂,应用正交试验优化保护剂配比,并通过单因素分析冷冻干燥的工艺参数,采用扫描电子显微镜观察冻干菌体形态。结果:副干酪乳杆菌冻干保护剂配方为:脱脂奶粉15%,蔗糖20%,聚乙烯吡咯烷酮(PVP K-30) 7%,谷胱甘肽(GSH)1.3%;真空冷冻干燥的工艺参数为在15℃下预处理2 h,保护剂pH为6.5,菌液和保护剂的比例为1∶1.5 (v/v),冻干厚度为0.5 cm,预冻方式为在液氮(-196℃)下处理15 min。在此条件下,副干酪乳杆菌经真空冷冻干燥后存活因子达(0.998±0.001),活菌数为(2.35±0.02)×1011CFU/g。经电镜观察发现菌体形态饱满,结构完整。结论:研究结果表明优化后的冻干保护剂及冻干工艺参数适用于副干酪乳杆菌菌体的保存,菌体活性保持良好,该工艺具有良好的应用推广前景。     

动物双歧杆菌冻干保护剂配方优化及酸对菌粉存活率的影响

目的 筛选动物双歧杆菌冻干菌粉保护剂,优化冻干保护剂配方,探究菌悬液制备过程中有机酸积累对菌粉存活率的影响。方法 以菌粉中动物双歧杆菌存活率为指标,通过发酵培养、离心收集菌泥、制备菌悬液、预冻和冷冻干燥的菌粉制备工艺,通过单因素实验和正交实验优化冻干保护剂。根据单因素实验探究冻干前菌悬液制备条件和最适pH。结果 最佳保护剂组合为5.00%麦芽糊精、6.00%海藻糖、0.15%抗坏血酸、1.50%谷氨酸钠、1.00%甘油。通过对菌悬液制备过程中菌粉活菌数的研究确定菌悬液制备和冻干条件,菌悬液pH 6.5,无菌水洗涤2次菌, 4℃菌悬液融合30 min,在-80℃预冻2 h,-40℃下干燥24 h,获得的冻干菌粉活菌数为1.38×10¹² CFU/g,菌粉最高存活率可达98.60%。结论 本研究优化后的保护剂组合可以制备高活性动物双歧杆菌菌粉,提高经济效益。     

耐氧性双歧杆菌的驯化及其培养条件的研究

通过对一株青春双歧杆菌的耐氧驯化,获得了具一定耐氧能力的双歧杆菌ad,该菌株可在含有一定空气容积的密闭容器中良好生长。并探讨了以大豆为主要原料发酵培养青春双歧杆菌ad的工艺条件,其最佳培养基配方为:豆浆浓度3BX,葡萄糖1%,自制乳肽0.5%,番茄汁2.5%,胡萝卜汁2.5%;pH7.0;接种量2%;培养温度37℃;厌氧培养24～48h。发酵液活菌数可达6.9×109cfu/mL。青春双歧杆菌ad发酵液原液直接冷冻干燥,无须添加其他保护剂;双歧杆菌ad冻干前增加温度前处理环节,可提高菌体成活率;双歧杆菌ad含水冻干粉活菌数达1.0×1010cfu/g;绝干样品活菌数达1.1×1010cfu/g。     

嗜热链球菌M5-5冻干保护剂配方的优化

为了研究不同冻干保护剂在真空冷冻干燥过程中对嗜热链球菌M5-5菌体细胞的保护效果,采用单因素试验和正交试验,以冻干后的菌体存活率为评价指标,考察9种冻干保护剂在冷冻干燥过程中对M5-5菌体的保护效果,以期获得最优保护剂配方。结果表明,海藻糖、甘油和脱脂乳作为单一冻干保护剂时,菌体细胞存活率分别为43.85%、47.51%和40.86%,显著高于其他6种保护剂（P＜0.05）;通过正交试验确定冻干保护剂的最优配方为海藻糖60 g/L、谷氨酸钠20 g/L、甘油20 g/L、脱脂乳150 g/L,此时嗜热链球菌M5-5的冻干存活率可达88.41%。扫描电镜结果显示,添加保护剂配方组菌体细胞完整,表面光滑,说明该保护剂配方能有效减少冷冻干燥过程对菌体细胞的损伤。     

Viability of lactic acid bacteria and bifidobacteria in fermented soymilk after drying, subsequent rehydration and storage

To develop a probiotic dietary adjunct, soymilk fermented with various combinations of lactic acid bacteria (Streptococcus thermophilus and Lactobacillus acidophilus) and bifidobacteria (Bifidobacterium longum and Bifidobacterium infantis) was subjected to freeze-drying and spray-drying. Survival of the starter organisms during the drying process, subsequent rehydration at different temperatures and during a 4-month period of storage under different storage conditions was examined. After freeze-drying, lactic acid bacteria and bifidobacteria exhibited a survival percent of 46.2-75.1% and 43.2-51.9%, respectively, higher than that noted after spray-drying. Regardless of the drying condition, S. thermophilus showed a higher percentage of survival than L. acidophilus, while B. longum survived better than B. infantis. Further study with soymilk fermented with S. thermophilus and B. longum revealed that the freeze-dried and spray-dried fermented soymilk rehydrated at 35-50 degrees C and 20 degrees C, respectively, was optimum for the recovery of the starter organisms. Both S. thermophilus and B. longum survived better in the freeze-dried than the spray-dried fermented soymilk during storage. A higher percent of survival was also noted for both the starter organisms when the dried fermented soymilk was stored at 4 degrees C than 25 degrees C. Holding the dried fermented soymilk in the laminated pouch enabled S. thermophilus and B. longum to exhibit a higher percentage of survival than in the deoxidant- and desiccant-containing glass or polyester (PET) bottle. Among all the packaging materials and storage temperatures tested, starter organisms were most stable in the dried fermented soymilk held in laminated pouch and stored at 4 degrees C. Under this storage condition, S. thermophilus and B. longum showed a survival percentage of 51.1% and 68.8%, respectively, in the freeze-dried fermented soymilk after 4 months of storage. Meanwhile, S. thermophilus and B. infantis in the spray-dried fermented soymilk showed a survival percent of 29.5% and 57.7%, respectively.     

冷冻保护剂对直投式木葡萄酸醋杆菌发酵剂菌体细胞特性的影响

本文研究了1%谷氨酸、5%海藻糖、10%脱脂乳及复合0.575%谷氨酸+0.075%海藻糖+6.4%脱脂乳作为冷冻保护剂对直投式木葡萄酸醋杆菌(GBX)发酵剂菌体细胞特性的影响,通过测定GBX发酵剂菌体细胞的DNA泄露、细胞液总抗氧化能力、SOD活性、纤维素合成酶活性和ATP酶活性变化等情况来衡量GBX菌体细胞的生理活性变化。结果发现:添加单一1%谷氨酸、5%海藻糖、10%脱脂乳以及复合0.575%谷氨酸+0.075%海藻糖+6.4%脱脂乳作为保护剂均能明显抑制冷冻干燥过程中菌体细胞的DNA泄露,提高细胞总抗氧化能力、SOD活性、纤维素合成酶和ATP酶活性,表明添加冷冻保护剂能够一定程度上保护菌体细胞,其中复合保护剂对菌体细胞的保护效果最佳,电子扫描显微镜也验证了冷冻保护剂能确实降低菌体细胞在冷冻干燥过程中损害程度,从而使菌体细胞保持良好的生理活性。     

黑曲霉孢子粉真空冷冻干燥保护剂配方优化

采用单因素试验和混合均匀试验设计,对黑曲霉孢子粉真空冷冻干燥保护剂配方进行筛选和优化。通过Design-Expert软件对实验数据进行回归分析,得到保护剂最佳配方为脱脂奶粉6.43 g/100 mL、蔗糖13.03 g/100 mL、甘油6.88 g/100 mL,孢子存活率预测值为88.82%,实际值为87.03%。     

hs-3、ws-3乳酸菌发酵剂冻干研究

通过研究冻干保护剂的冻干厚度、干燥时间与冻干发酵剂含水量的关系,确定hs-3、ws-3乳酸菌冷冻干燥时间为13h,冻干厚度为0.75cm。对不同复合冷冻保护剂进行筛选,确定10%胡萝卜汁 6%葡萄糖 1%琼脂 1%麦芽糊精 1%番茄汁为乳酸菌优良的冷冻复合保护剂。冻干混合菌种发酵剂经发酵活力检测性能优良。     

金黄色葡萄球菌标准物质的研制

将金黄色葡萄球菌添加到复合保护剂中,经冻干制备成干粉状标准物质,通过比较冻干前后活菌数来验证保护剂保护效果,并研究标准物质的均匀性和稳定性。结果表明复合保护剂的保护率达到51.74%,可以在冻干过程中有效保护菌体,制备的金黄色葡萄球菌标准物质均匀性良好,可以稳定保存半年。该标准物质可用于食品检测实验室质量控制和食品中金黄色葡萄球菌检测结果的评价。     

Effect of β-cyclodextrin on physical properties of nanocapsules manufactured by emulsion–diffusion method

This study was carried out to investigate the effect of β-cyclodextrin (β-CD) as a cryoprotectant on the eugenol encapsulated nanocapsules. Nanocapsules (NCs) were prepared by emulsion–diffusion method and frozen at various temperatures (−30 °C to −10 °C). Physical stability of the NCs was determined by particle size analysis and morphological observation using scanning or transmission electron microscopy before and after freezing or freeze-drying process. Freeze-thawed and freeze-dried NCs without β-CD were remarkably aggregated. The particle size of NCs without β-CD was significantly increased as decreasing freezing temperature. On the contrary, NCs with β-CD were slightly increased in the range of 370–400 nm. Significant particle aggregation was not shown at NCs with β-CD at all freezing temperatures. In this study, β-CD could was effective as a cryoprotectant to maintain physical stability of NCs after freezing or freeze-drying. β-CD could be useful to protect NCs from freezing stress.     

Effect of oleic acid on the viability of different freeze-dried Lactiplantibacillus plantarum strains

Freeze drying is one of the most convenient ways to preserve microorganisms, but in the freeze-drying process, strains will inevitably suffer varying degrees of damage under different conditions. The deterioration of cell membrane integrity is one of the main forms of damage. The type and ratio of fatty acids in the cell membrane affect its characteristics. Therefore, it is worth investigating whether certain fatty acids can increase freeze-drying resistance. In this study, we found that adding a low concentration of oleic acid to a cryoprotectant could increase survival rate of strains of Lactiplantibacillus plantarum following freeze drying, and the optimal concentration of oleic acid was determined to be 0.001%. When 0.001% oleic acid was added to phosphate-buffered saline, the freeze-drying survival rate of L. plantarum increased by up to 6.63 times. Adding 0.001% oleic acid to sorbitol, the survival rate of L. plantarum increased by as much as 3.65 times. The 0.001% oleic acid–sucrose cryoprotectant resulted in a freeze-drying survival rate of L. plantarum of about 90%, a 2.26-fold improvement compared with sucrose alone. Although the effect of oleic acid depends on the cryoprotectants used and the strain treated, addition of oleic acid showed significant improvement overall. Further experiments showed that adding a low concentration of oleic acid to the cryoprotectants improved the freeze-drying survival rate of L. plantarum by maintaining cell membrane integrity and lactate dehydrogenase activity. Our findings provide a new strategy for safeguarding bacterial viability in commonly used cryoprotectants by the addition of a common food ingredient, which may be extensively applied in the food industry.     

真空冷冻干燥与喷雾干燥长双歧杆菌的工艺比较研究

为有效延长双歧杆菌的保存期和耐受性,利用真空冷冻干燥及喷雾干燥法制备活性菌粉。研究两种干燥方法的工艺参数,并对干燥得到的活性菌粉性能进行比较。真空冷冻干燥法制备的长双歧杆菌活性菌粉的活菌数为7.98×109CFU/g、存活率90.9%、含水量3.8%,4℃保藏90d后活菌数为2.5×108CFU/g,菌粉的水溶性好;喷雾干燥法制备的活性菌粉的活菌数为4.4×109CFU/g、存活率82.2%、包埋率71.9%、含水量5.6%,4℃保藏90d后活菌数为4×108CFU/g,菌粉在水中的溶解时间较长。真空冷冻干燥成本较高、操作时间长、菌粉的保藏期较短;而喷雾干燥工艺简单、生产成本低、工作效率高,包埋的菌粉保藏期较长。