内蒙古传统酸奶乳酸菌的筛选及体外益生效果评价

对内蒙古传统酸奶中分离出的8株乳酸菌菌株进行形态学及16S rDNA鉴定,确定其中包括鼠李糖乳杆菌、植物乳杆菌、干酪乳杆菌、瑞士乳杆菌和嗜热链球菌。然后进一步对菌株的酸、胆盐、人工胃肠液耐受性、胆固醇降解能力及自由基清除能力进行体外益生效果评价。结果表明,鼠李糖乳杆菌217-3对pH=1.5的强酸和人工胃肠液有较高的耐受性,同时还能耐受1.5%的胆盐。此外,其胆固醇降解和自由基清除能力也具有较高的水平。综上所述,鼠李糖乳杆菌217-3具有显著的益生性能,为高活性乳酸菌菌剂及相关发酵产品的开发奠定了一定的理论基础。     

芽孢杆菌35家族β-半乳糖苷酶的性质及其在合成低聚半乳糖中的应用

低聚半乳糖是一种重要的益生元,芽孢杆菌(Bacillales sp.)来源的β-半乳糖苷酶具有转半乳糖苷活性,能合成低聚半乳糖。研究了一个新的芽孢杆菌来源的β-半乳糖苷酶的表达、酶学性质及其在合成低聚半乳糖中的应用。从芽孢杆菌中克隆了一个糖苷水解酶35家族的β-半乳糖苷酶基因(BABgal35A),并在大肠杆菌BL21(DE3)中表达。多重序列比对结果表明:BABgal35A与环状芽孢杆菌(Bacillus circulans)来源的β-半乳糖苷酶同源性最高,为79.9%,是一个新型的β-半乳糖苷酶(命名为BABgal35A)。粗酶液经Ni-IDA亲和层析纯化得到电泳级纯酶,蛋白电泳分析表明其分子质量为60kDa左右,最适pH值和温度分别为5.0和55℃,并且该酶在pH 值为4.5~8.0,温度为20~45℃时稳定性好。BABgal35A对oNP-β-galactopyranoside具有较高的催化活性。该酶以乳糖为底物合成低聚半乳糖的产率达34%。研究结果表明,芽孢杆菌35家族β-半乳糖苷酶在低聚半乳糖的制备中具有潜在的应用价值。     

真菌多糖对鼠李糖乳杆菌性质的影响

研究了不同质量浓度的真菌多糖对鼠李糖乳杆菌的影响。结果表明,质量浓度为15g/L的真菌多糖对鼠李糖乳杆菌的增殖效果最显著,其活菌数最大可达到3.08×1010mL-1,能够使发酵周期提前2～3h。其产酸能力得到明显的提高,并且此真菌多糖益生元较好地提高了鼠李糖乳杆菌的耐酸、耐胆盐的能力。     

不同益生元对青春双歧杆菌耐胆盐能力的影响

为了解水苏糖、低聚异麦芽糖、低聚果糖3种益生元对青春双歧杆菌耐胆盐能力的影响情况,该研究在PTYG液体培养基中分别添加不同浓度的3种益生元.并在不同的胆盐浓度梯度下进行青春双歧杆菌耐胆盐能力的研究.结果表明,3种益生元均可提高青春双歧杆菌耐胆盐能力,低聚异麦芽糖的效果最好;在猪胆盐浓度为0.3%的PTYG液体培养基中,水苏糖、低聚异麦芽糖、低聚果糖提高青春双歧杆菌耐胆盐能力的最适添加量分别为1.0%、1.5%、O.2%,其中低聚异麦芽糖的添加量(1.5%)提高青春双歧杆菌耐胆盐能力的效果最好.     

黑果腺肋花楸多糖对3 种益生菌生长的影响

为考察不同浓度黑果腺肋花楸多糖(1%、2%、4%、6%、8%、10%、12%)对嗜酸乳杆菌、鼠李糖乳杆菌和乳双歧杆菌的生长作用及在模拟人体胃肠液环境下的耐受性影响,通过在含有不同浓度黑果腺肋花楸多糖及不同pH值的模拟胃肠液中培养益生菌,考察其存活率。研究结果表明,黑果腺肋花楸多糖可显著促进嗜酸乳杆菌、鼠李糖乳杆菌和乳双歧杆菌的生长,当添加黑果腺肋花楸多糖质量分数达到8%时,益生菌生长达到最佳。黑果腺肋花楸多糖可显著提高嗜酸乳杆菌、鼠李糖乳杆菌和乳双歧杆菌在pH1.5胃液中的耐受性(p<0.05),其存活率可达到64.45%～95.88%,作用效果优于低聚果糖;同时黑果腺肋花楸多糖可增强嗜酸乳杆菌和乳双歧杆菌对肠液的耐受性,其存活率可达到130.86%～132.51%,作用效果优于低聚果糖,但对鼠李糖乳杆菌肠液耐受性影响较弱,其存活率为51.88%,与低聚果糖作用效果相近。     

发酵蔬菜源食品用益生乳酸菌的筛选及降胆固醇能力评价

为了从发酵蔬菜中筛选出具有降解胆固醇能力的食品用益生乳酸菌,作者以融合魏斯氏菌（Weissella confuse) RW作为对照,采用定向分离法结合16S rDNA分析不同来源的发酵蔬菜样品中得到5株具有降胆固醇能力的益生乳酸菌。结果表明,5株菌对pH 2.0、质量浓度为3 g/L的胆盐以及模拟胃肠液均具有耐受性,降胆固醇能力强（54.79%~62.54%）、能够抑制病原菌且黏附于Caco-2细胞上,表现出优良的益生特性。此外,5株菌均无溶血现象,不产生物胺,对多数抗生素敏感,安全性高。通过16S rDNA鉴定5株菌分别为植物乳植杆菌（Lactiplantibacillus plantarum）JCSMC-1、植物乳杆菌（Lactobacillus plantarum）SYB和JCSMC6-2、副干酪乳酸菌（Lacticaseibacillus paracasei）SYD和XSMC-1。本研究获得的1株植物乳植杆菌、2株植物乳杆菌、2株副干酪乳酸菌均具有食品用益生菌的基本特性,为开发食品用益生菌提供了新资源。     

降胆固醇乳酸菌的筛选及其益生元干预生长作用分析

本研究从传统自然发酵的酵素饮品中分离筛选出一株高效降胆固醇菌株,该菌株具有较好的胆盐耐受性和耐酸能力。在pH3的酸性环境下,耐酸率为71.53%,该菌株能够长时间耐受胆盐环境,培养16h的胆盐耐受率为42.09%。以二甲苯为吸附剂,疏水率为35.91%,有较好的黏附特性;益生元不仅能够促进菌株生长,而且可显著提高降胆固醇能力。4种益生元中乳果糖促进生物量增加幅度最大,培养至16 h时生物量达到最高。乳果糖对菌株产酸性能影响最显著,pH值可降至3.56,胆固醇去除率达41.10%。该菌株对菊粉的利用率低,说明菌株对不同益生元具有选择性代谢。菌株经形态学观察和16S rDNA鉴定为副干酪乳杆菌,并命名为Lactobacillus paracasei S0940。本研究为进一步研究体内降脂能力以及开发功能性乳酸菌食品提供理论依据。     

内蒙古奶豆腐中潜在益生性乳酸菌的筛选

对从内蒙古奶豆腐中分离的16 株乳酸菌进行16S rDNA分子鉴定,其中9 株鉴定为乳酸片球菌、5 株为屎肠球菌、1 株为植物乳杆菌、1 株为鼠李糖乳杆菌。进一步对分离得到的菌株进行模拟人工胃液和胆盐耐受性、疏水性、体外降胆固醇和抗氧化特性研究。结果表明：植物乳杆菌M1-2和鼠李糖乳杆菌M6-1对模拟人工胃液有较高的耐受性;屎肠球菌M7-1、M8-1和M8-2具有较高的胆盐耐受性;植物乳杆菌M1-2和鼠李糖乳杆菌M6-1对甲苯和十六烷的疏水性显著高于其他菌株。体外功能性实验结果表明,屎肠球菌M1-1、植物乳杆菌M1-2、乳酸片球菌M1-3和M2-1的体外降胆固醇能力较高,胆固醇降解率均超过30%;鼠李糖乳杆菌M6-1对过氧化氢的耐受性、羟自由基（•OH）和1,1-二苯基-2-三硝基苯肼（1,1-diphenyl-2-picrylhydrazyl,DPPH）自由基的清除能力最高。综上所述,植物乳杆菌M1-2和鼠李糖乳杆菌M6-1具有较好的益生特性,可作为今后开发功能性产品的潜在益生菌株。     

降胆固醇乳酸菌的益生特性及其作用机理

考察了具有降胆固醇乳酸菌(乳酸乳球菌L14-3、鼠李糖乳杆菌118-1、粪肠球菌M53-2、植物乳杆菌117-1、植物乳杆菌23-1)的益生特性,且初探乳酸菌降胆固醇的作用机理。通过模拟胃液、胰液进行乳酸菌的耐酸耐胆盐测定,并评价了乳酸菌对抗生素的抗性。通过胆固醇、胆盐的共沉淀作用和菌体细胞吸收两种方式去除发酵液中的胆固醇,利用邻苯二甲醛法测定胆固醇的含量,确定胆固醇的去除率。结果表明:通过共沉淀作用和菌体吸收,乳酸乳球菌L14-3和鼠李糖乳杆菌118-1分别去除胆固醇的比例最高。在pH=2.0的人工胃液中,粪肠球菌M53-2和乳酸乳球菌L14-3的活菌数显著高于其他3株菌(P0.05)。粪肠球菌M53-2对pH 3.0的人工胃液有较好的耐受性,存活率显著高于其他4株菌(P0.05)。在pH=8.0人工胰液中,鼠李糖乳杆菌118-1和植物乳杆菌117-1的存活率显著高于其他菌株(P0.05)。植物乳杆菌117-1耐药性最好。因此,研究具有降胆固醇的益生菌具有广阔应用前景。     

红参对乳酸菌体外增菌的影响

采用比浊法和活菌计数法研究在基础培养基/菊芋汁基础培养基中添加不同量低聚木糖、低聚果糖、红参提取液及其酸水解物对鼠李糖乳杆菌AS 1.2466T/保加利亚乳杆菌ATCC 11842生长情况的影响。结果表明:不同添加物对鼠李糖乳杆菌AS 1.2466T的增菌效果为红参提取液>红参酸水解物>低聚木糖>低聚果糖。当红参提取液添加量为12.50%时,鼠李糖乳杆菌AS 1.2466T的最大OD600值为1.172,活菌数为2.72×10^8 CFU/mL,比基础培养基中最大活菌数7.75×10^7 CFU/mL增加1个数量级。对保加利亚乳杆菌ATCC 11842的增菌效果为红参提取液>低聚木糖>红参酸水解物>低聚果糖。当红参提取液添加量为6.25%时,保加利亚乳杆菌ATCC 11842的最大OD600值为0.627,活菌数为4.43×10^8 CFU/mL,比菊芋汁基础培养基中最大活菌数1.48×10^7 CFU/mL增加1个数量级。红参提取液比低聚木糖、低聚果糖具有较好的增菌效果,将红参作为益生元研究其益生功能,为研究红参对肠道微生物的影响提供试验依据。     

耐酸耐胆盐乳酸菌的鉴定及筛选

从自然发酵的酸奶中分离出2 株细菌,经16S rDNA分子鉴定为Lactobacillus plantarum SN1和Lactobacillusrhamnosus SN6,并对其生长曲线、产酸速率、耐酸耐胆盐能力进行了研究。L. plantarum SN1和L. rhamnosus SN6在2 h后进入对数期,16 h后达到稳定期,其OD600 nm值分别为8.47、7.43。L. plantarum SN1和L. rhamnosus SN6的产酸速率较快,pH值在8 h后就降到了4.2以下,48 h后降到3.3左右。L. plantarum SN1和L. rhamnosus SN6在pH 4的培养基培养16 h后,其相对OD600 nm值分别为49.29%、47.14%,具有较强的耐酸能力。L. plantarum SN1和L. rhamnosus SN6在0.3 g/L胆盐质量浓度下培养16 h后,相对OD600 nm值分别为57.7%、69.48%;在0.6 g/L胆盐质量浓度下的相对OD600 nm值分别为48.22%、29.56%,具有较强的耐胆盐能力。结果表明：L. plantarum SN1和L. rhamnosus SN6是生长性能好、产酸能力强、耐酸耐胆盐的益生菌株。     

基于MTT比色和响应面法优化侧孢短芽孢杆菌最大活菌数培养条件

目的：探索获得侧孢短芽孢杆菌（Brevibacillus laterosporus）最大活菌数的最佳培养基成分及培养条件。方法：在建立MTT比色法与平板计数法的相关回归方程基础上,对获得最大活菌数的侧孢短芽孢杆菌最适培养基成分（碳源、氮源、无机盐）和培养条件（初始pH、温度、接种量、磷酸二氢钾）进行优化。结果：MTT比色法与平板计数法对活菌数测定结果表现出显著的线性关联（R²>0.999）;麦芽糖、氯化钙、初始pH、磷酸二氢钾为显著影响因子,最佳发酵条件为麦芽糖8.75 g/L,氯化钙0.17 g/L,初始pH 7.07、磷酸二氢钾3.73 g/L,此条件下活菌数为8.12×10⁸ CFU/mL,与理论活菌数（8.25×10⁸ CFU/mL）无显著差异。结论：基于MTT比色和响应面法优化侧孢短芽孢杆菌最大活菌数培养条件,优化后的活菌数较优化前提高了3.02倍。     

Inhibition of Salmonella Enteritidis by cerein 8A, EDTA and sodium lactate

The ability of the bacteriocin cerein 8A to inhibit Salmonella Enteritidis in combination with EDTA and sodium lactate was investigated. Salmonella Enteritidis was incubated with combinations of cerein 8A (3200 AU/mL) and EDTA (20, 50, 100 mmol/L) or sodium lactate (200 mmol/L). All treatments caused a significant reduction in the OD₆₀₀ values of Salmonella Enteritidis cultures. The addition of cerein 8A plus EDTA resulted in higher inhibition in comparison with the bacteriocin alone; the greater the concentration of EDTA, the greater the inhibitory effect. The combination of cerein 8A plus 100 mmol/L EDTA results in a more efficient treatment to reduce the number of viable cells of Salmonella Enteritidis. The combination of cerein 8A plus sodium lactate also showed significant inhibition of the indicator organism. Transmission electron microscopy showed damaged cell walls and loss of protoplasmic material in treated cells. The cells of Salmonella Enteritidis treated with cerein 8A plus EDTA appeared more injured. The bacteriocin cerein 8A may be useful to inhibit Gram-negative bacteria, with enhanced effect in combination with chelating agents. Control of Salmonella Enteritidis, a Gram-negative bacterium constantly linked to food outbreaks, addresses an important aspect of food safety.     

SigB plays a major role in Listeria monocytogenes tolerance to bile stress

The ability of Listeria monocytogenes to tolerate high levels of bile stress is critical to its successful infection and colonization in the human gastrointestinal tract. L. monocytogenes encodes bile salt hydrolase by a bsh gene which plays a significant role in hydrolyzing high concentrations of bile salt when L. monocytogenes grows under hypoxemic condition. As the bsh promoter contains consensus SigB and PrfA binding sites, we investigated the role of SigB (σ^B) and PrfA in L. monocytogenes tolerance against bile stress by comparing the survival of isogenic deletion mutants of L. monocytogenes EGD_ΔsigB, EGD_ΔprfA and EGD_ΔprfAΔsigB with their parent strain EGD at high levels of bile salt. Our results show that the sigB deletion significantly reduced the MICs of bile salt for EGD_ΔsigB and EGD_ΔprfAΔsigB (2.6% and 2.2% vs 3.5% in wild type strain EGD), while the growth rates of these two sigB deletion mutants (EGD_ΔsigB and EGD_ΔprfAΔsigB) were affected the most in the presence of 3% bile salt. Pre-exposure to alkali (pH 9.0) and osmotic (0.3 M NaCl) stresses for a short period of time (30 min) resulted in improved growth of L. monocytogenes as well as its prfA-sigB isogenic mutants even under sublethal concentrations of bile salt, while pre-exposure to acid pH (pH 4.5) failed to provide cross-protection against subsequent bile stress. Furthermore, the sigB gene had more remarkable influence than that of prfA on bsh expression, as much lower levels of bsh transciption were observed in EGD_ΔsigB and EGD_ΔprfAΔsigB. Meanwhile, bsh expression in the deletion mutants did not respond to elevated levels of bile salt. These data indicate that σ^B might play a crucial role in Listeria survival under bile salt environment in the gastrointestinal tract before its successful colonization, invasion and intracellular propagation.     

蜂粮中Lactobacillus kunkeei的益生特性研究

蜂粮是植物花粉、蜂蜜和蜜蜂唾液的发酵混合物,是营养丰富的天然食材。作者从蜂粮中分离到61株乳酸杆菌,经16S rRNA基因测序,鉴定为Lactobacillus kunkeei。首先,用邻苯二甲醛法对61株L.kunkeei进行胆固醇去除能力检测,其中5株菌胆固醇去除能力均大于15%,菌株B35对胆固醇的去除率达到(29.07±1.30)%。随后,对这5株L.kunkeei进行酸耐受性、胆盐耐受性、抗生素耐药性及抑菌等益生特性进行研究。耐酸实验和耐胆盐实验结果表明,5株L.kunkeei均具有良好的耐酸性和胆盐耐受性。药敏试验结果表明,5株L.kunkeei对4种临床常用抗生素(红霉素、克林霉素、庆大霉素、万古霉素)均敏感。通过琼脂扩散法抑菌实验,发现5株L.kunkeei对大肠埃希氏菌、金黄色葡萄球菌均有一定的抑菌作用。B35菌株的细胞黏附性能良好,对Caco-2细胞的黏附率达到3.32%。从蜂粮中分离到5株具有良好益生特性的菌株,其中菌株B35的益生特性最佳,为益生菌的开发与利用提供了新的菌种资源。     

4种多糖益生元对鼠李糖乳杆菌微胶囊稳定性的影响

为提高鼠李糖乳杆菌（Lactobacillus rhamnosus GG,LGG）的稳定性，采用复合凝聚法制备LGG微胶囊，探究菊糖、低聚果糖、普鲁兰多糖和水苏糖4种益生元对LGG微胶囊性能的影响，并结合扫描电镜和差示扫描量热仪分析微胶囊的微结构和热稳定性。结果表明：益生元对LGG微胶囊的性能均有积极影响。模拟胃肠液处理后，菊糖活菌数存活率最高，达9.5(lg(CFU/g))；水苏糖对LGG微胶囊在胆盐和高温下的保护能力最强，75℃、30 min后活菌数达8.7(lg(CFU/g))；水分活度0.75条件下低聚果糖的加入提高了LGG微胶囊的贮藏稳定性；差示扫描量热分析结果表明益生元提高了LGG微胶囊的热稳定性，水苏糖的微胶囊熔融温度达172℃，但低聚果糖降低了LGG微胶囊的熔融温度；扫描电镜分析表明添加益生元后微胶囊结构没有变化。本研究为后续添加益生元对LGG微胶囊性能的影响研究提供理论基础。     

益生菌的筛选及其发酵特性研究

为获得性能优良的益生菌生产菌株,通过乳酸菌的溶钙圈筛选,耐酸、耐胆盐检测及体外抗氧化和分解胆固醇能力测试进行了菌株的选育;对筛选菌株进行16S rRNA基因测序及生理生化特性鉴定;采用正交试验优化培养基配方和发酵条件。结果表明,从牛奶储罐中分离、筛选到一株各项性能优良的菌株,编号为SX68,经鉴定其为植物乳杆菌（Lactobacillus plantarum）。优化培养基配方为：蛋白胨10 g/L,牛肉膏10 g/L,酵母提取物5 g/L,磷酸氢二钾3 g/L,柠檬酸氢二胺4 g/L,乙酸钠5 g/L,葡萄糖30 g/L,吐温-80 1 mL/L,MgSO4·7H2O 0.58 g/L,MnSO4·4H2O 0.25 g/L,pH 6.4;最佳发酵条件为：起始pH 6.5,发酵温度35 ℃,接种量20 mL/L,溶氧量＜100 mL/L,搅拌转速50 r/min,罐压0.03 MPa。在此优化条件下,菌株SX68最高发酵生物量（OD600 nm值）为15.68,活菌数为9.6×1012 CFU/mL,可为其用于益生菌生产奠定基础。     

Encapsulation of probiotic Lactobacillus bulgaricus in alginate–milk microspheres and evaluation of the survival in simulated gastrointestinal conditions

In this paper, a new encapsulation carrier was applied in order to improve the survival of Lactobacillus bulgaricus (L. bulgaricus). L. bulgaricus was encapsulated in alginate–milk microspheres prepared by extrusion method. Around 100% encapsulation yield was achieved. The tolerance of encapsulated L. bulgaricus to adverse environments such as low pH (pH 2.0 and 2.5), high concentration of bile salt (1.0% and 2.0%) and long time storage (1 month), was investigated. Release characteristic of encapsulated L. bulgaricus in Simulated Intestine Fluid (SIF) was also studied. The results showed that encapsulation could improve the tolerance of L. bulgaricus to adverse environments. The viability of encapsulated L. bulgaricus did not change after 120 min incubation in Simulated Gastric Fluid (SGF) pH 2.5. The viability of encapsulated L. bulgaricus could be kept more than 8 log CFU/g after 120 min incubation in SGF pH 2.0. The viability of encapsulated L. bulgaricus in 1% bile salt solution was reduced from 9.98 log CFU/g microspheres to 9.24 and 8.48 log CFU/g microspheres after 1 and 2 h incubation, respectively. Around 1.3 and 2.1 log CFU/g microspheres were reduced after 1 and 2 h exposure in 2% bile salt solution, respectively. Full viability of encapsulated L. bulgaricus could be preserved after 1 month’s storage at 4 °C. L. bulgaricus encapsulated in alginate–milk microspheres could be completely released in 1 h. These studies demonstrated encapsulation of L. bulgaricus in alginate–milk microspheres is an effective protection technique against extreme simulated gastrointestinal environment.     

Efficient expression of myo-inositol oxygenase in Escherichia coli and application for conversion of myo-inositol to glucuronic acid

Glucuronic acid is an important biochemical with wide applications in the food and medical industries. The myo-inositol oxygenase (MIOX) gene was synthesized and expressed in Escherichia coli BL 21(DE3). After optimization of induction conditions and the culture temperature, the highest MIOX activity (45.46 kU/L) was achieved when 0.1 mM isopropyl-thio-β-d-galactoside (IPTG) was added to cell cultures with an OD₆₀₀ value 1.0 followed by induction at 26°C for 8 hours. The purified MIOX enzyme exerted characteristics similar to the native form. Conversion of myo-inositol to glucuronic acid was performed using whole cells in a pH 8.5 buffer system. Whole cells harboring myo-inositol oxygenase were used as a biocatalyst to produce 2.13 g/L of glucuronic acid with a conversion rate of 99%. A promising novel process for glucuronic acid production from abundant agricultural byproducts is presented.     

Viability of the Lactobacillus rhamnosus HN001 Probiotic Strain in Swiss‐ and Dutch‐Type Cheese and Cheese‐Like Products

The objective of this study was to determine the viability of the probiotic Lactobacillus rhamnosus HN001 in Swiss‐type and Dutch‐type cheese and cheese‐like products (milk fat is substituted by stearin fraction of palm fat) during manufacture, ripening, and storage. The use of the probiotic L. rhamnosus HN001 in Dutch‐type cheese and cheese‐like products significantly (P = 0.1) changed their chemical composition (protein and fat content) and an insignificant increase (approximately 1.6% in cheese‐like products and approximately 0.3% in cheese) in yield. L. rhamnosus HN001 did not affect the rate of changes in the pH of ripened cheese and cheese‐like products. A minor increase in probiotic counts was observed in initial stages of production and were partially removed with whey. Ripened cheese and cheese‐like products were characterized by high survival rates of probiotic bacteria which exceeded 8 log CFU/g after ripening. An insignificant reduction in the number of viable probiotic cells was noted during storage of Swiss‐type and Dutch‐type cheese, whereas a significant increase in probiotic cell counts was observed in cheese‐like products during storage.