Effect of Various Encapsulating Materials on the Stability of Probiotic Bacteria

ABSTRACT:  Ten probiotic bacteria, including Lactobacillus rhamnosus , Bifidobacterium longum , L. salivarius , L. plantarum , L. acidophilus , L. paracasei , B. lactis type Bl-04, B. lactis type Bi-07, HOWARU L. rhamnosus , and HOWARU B. bifidum , were encapsulated in various coating materials, namely alginate, guar gum, xanthan gum, locust bean gum, and carrageenan gum. The various encapsulated probiotic bacteria were studied for their acid and bile tolerance. Free probiotic organisms were used as a control. The acid tolerance of probiotic organisms was tested at pH 2 over a 2-h incubation period. Bile tolerance was tested with taurocholic acid over an 8-h incubation period. The permeability of the capsules was also examined using a water-soluble dye, 6-carboxyflourescin (6-CF). The permeability was monitored by measuring the amount of 6-CF released from the capsules during a 2-w storage period. Results indicated that probiotic bacteria encapsulated in alginate, xanthan gum, and carrageenan gum survived better ( P < 0.05) than free probiotic bacteria, under acidic conditions. When free probiotic bacteria were exposed to taurocholic acid, viability was reduced by 6.36 log CFU/mL, whereas only 3.63, 3.27, and 4.12 log CFU/mL was lost in probiotic organisms encapsulated in alginate, xanthan gum, and carrageenan gum, respectively. All encapsulating materials tested released small amounts of 6-CF; however, alginate and xanthan gum retained 22.1% and 18.6% more fluorescent dye than guar gum. In general, microcapsules made of alginate, xanthan gum, and carrageenan gum greatly improved the survival of probiotic bacteria when exposed to acidic conditions and bile salts.     

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。     

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。     

结冷胶发酵液微滤除菌工艺

采用有机微滤膜对结冷胶发酵液进行过滤除菌实验。考察了不同膜材料的过滤性能,以及结冷胶浓度、温度、压力等操作参数对除菌效果的影响。将结冷胶发酵液脱酰基处理后稀释成浓度为3 g/L的料液,再将酸碱度调至pH10,在0.1 MPa、65℃下用孔径为0.45μm的聚醚砜膜过滤,菌体去除率和结冷胶回收率分别为97%和84%。滤膜污染后采用0.75%HCl和1%NaOH交替清洗,可使膜通量得到较好地恢复。     

具有抑制肠道致病菌和黏附Caco-2细胞作用的益生性乳酸菌的筛选及鉴定

为了筛选具有肠道益生特性的乳酸菌,进一步开发益生菌资源,本文研究采用牛津杯、人工模拟胃肠液及体外黏附Caco-2细胞等方法,以鼠李糖乳杆菌GG(Lactobacillus rhamnosus GG,LGG)为对照菌株,对实验室保藏15株乳酸菌的益生特性进行筛选与评估。实验结果表明:15株乳酸菌对两株沙门氏菌都具有一定抑制效果,抑菌圈直径为10.95~22.06 mm;乳酸菌C174、D24、D599、C37、D512具有较好的耐酸耐胆盐能力,在人工胃液3 h内存活率达到60%以上,人工肠液4 h内存活率达到80%以上;在黏附试验中,C174对Caco-2细胞具有较强黏附能力,黏附数量为740 CFU/100细胞,略高于对照菌株鼠李糖乳杆菌(543 CFU/100细胞);通过对具有高黏附性的菌株C174进行生理生化和16S rRNA分子测序鉴定,结果表明菌株C174为植物乳杆菌(Lactobacillus plantarum),命名为Lactobacillus plantarum ZJ-174。Lactobacillus plantarum ZJ-174的抑菌能力强、能够耐酸耐胆盐,并具有很强的黏附能力,是一株潜在的益生菌,具有治疗或预防人和动物肠道疾病等应用价值。     

降胆固醇和耐酸耐胆盐益生菌的筛选研究

本研究通过含有胆固醇胶束的MRS-胆固醇液体培养基从五株益生菌中筛选出降胆固醇能力较强的益生菌,接着对筛选出的益生菌进行耐酸、耐胆盐试验。结果表明,五株益生菌都有降胆固醇的功能,其中干酪乳杆菌LCA-1、鼠李糖乳杆菌LR-D和植物乳杆菌LP-C的胆固醇去除率较高,分别为25.26%、22.18%和20.03%;LR-D能够短时耐受低p H值环境,在p H 2.5的发酵液中培养2h的存活率能够达到14.39%,但是耐胆盐能力较差;LP-C能够长时间耐受低p H值的环境,具有较强的胆盐耐受能力,在高胆盐环境(0.5%)中培养2 h的存活率高达133.75%,培养24 h的活菌数仍保持在1.13×105 cfu/m L,此时其余两株菌的活菌未检出,说明LP-C能够耐受高胆盐环境。植物乳杆菌LP-C具有较高的胆固醇去除率,能够耐受低p H值和高胆盐环境。因此,可用于进一步研究与开发。     

Acid, bile, and heat tolerance of free and microencapsulated probiotic bacteria

ABSTRACT:  Eight strains of probiotic bacteria, including Lactobacillus rhamnosus , Bifidobacterium longum, L. salivarius, L. plantarum , L. acidophilus , L. paracasei , B. lactis type Bl-O4, and B. lactis type Bi-07, were studied for their acid, bile, and heat tolerance. Microencapsulation in alginate matrix was used to enhance survival of the bacteria in acid and bile as well as a brief exposure to heat. Free probiotic organisms were used as a control. The acid tolerance of probiotic organisms was tested using HCl in MRS broth over a 2-h incubation period. Bile tolerance was tested using 2 types of bile salts, oxgall and taurocholic acid, over an 8-h incubation period. Heat tolerance was tested by exposing the probiotic organisms to 65 °C for up to 1 h. Results indicated microencapsulated probiotic bacteria survived better ( P < 0.05) than free probiotic bacteria in MRS containing HCl. When free probiotic bacteria were exposed to oxgall, viability was reduced by 6.51-log CFU/mL, whereas only 3.36-log CFU/mL was lost in microencapsulated strains. At 30 min of heat treatment, microencapsulated probiotic bacteria survived with an average loss of only 4.17-log CFU/mL, compared to 6.74-log CFU/mL loss with free probiotic bacteria. However, after 1 h of heating both free and microencapsulated probiotic strains showed similar losses in viability. Overall microencapsulation improved the survival of probiotic bacteria when exposed to acidic conditions, bile salts, and mild heat treatment.     

母乳婴儿源乳杆菌的筛选及其免疫调节能力研究

以分离自母乳婴儿源的乳杆菌——鼠李糖乳杆菌BF-1、植物乳杆菌BF-15、唾液乳杆菌BF-29、干酪乳杆菌BF-55为对象,研究这些菌株对体外模拟人工胃、肠液及胆盐的耐受性,对Caco-2细胞的黏附能力、安全性和对小鼠脾淋巴细胞增殖活性的影响,探讨菌株的免疫调节活性。试验结果表明:植物乳杆菌BF-15对人工消化液和胆盐有较强的耐受性,对Caco-2细胞黏附能力[(7.10±0.30)CFU/cell]显著高于阳性对照菌株LGG[(3.90±0.30)CFU/cell](P<0.05);BF-15除对氨基糖苷类、糖肽类抗生素的固有耐药性外,对苯唑西林、头孢噻吩也有耐药性,无抗性质粒;BF-15和LGG的活性和热致死菌在一定的菌浓范围(1×10^6~10^7CFU/mL)均促进体外小鼠淋巴细胞增殖,表现出剂量依赖关系,同时活性菌株作用效果明显优于热致死菌株。在相同菌浓条件下,两株菌体外免疫调节能力没有显著性差异(P>0.05)。母乳婴儿源乳杆菌——植物乳杆菌BF-15具有较强的抗逆性,较高的黏附性和一定的免疫调节能力。     

In vitro testing of commercial and potential probiotic lactic acid bacteria

Probiotics are defined as live microorganisms which when administered in adequate amounts confer a health benefit on the host. The objective of this study was to investigate the diversity of selected commercial and potential probiotic lactic acid bacteria using common in vitro screening assays such as transit tolerance in the upper human gastrointestinal tract, adhesion capacity to human intestinal cell lines and effect on epithelial barrier function. The selected bacteria include strains of Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus farciminis, Lactobacillus sakei, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus reuteri and Pediococcus pentosaceus. Viable counts after simulated gastric transit tolerance showed that L. reuteri strains and P. pentosaceus tolerate gastric juice well, with no reduction of viability, whereas L. pentosus, L. farciminis and L. sakei strains lost viability over 180min. All strains tested tolerate the simulated small intestinal juice well. The bacterial adhesion capacity to human intestinal cells revealed major species and strain differences. Overall, L. plantarum MF1298 and three L. reuteri strains had a significant higher adhesion capacity compared to the other strains tested. All strains, both living and UV-inactivated, had little effect on the epithelial barrier function. However, living L. reuteri strains revealed a tendency to increase the transepithelial electrical resistance (TER) from 6 to 24h. This work demonstrates the diversity of 18 potential probiotic bacteria, with major species and strain specific effects in the in vitro screening assays applied. Overall, L. reuteri strains reveal some interesting characteristics compared to the other strains investigated.     

Effect of addition of sucrose and aspartame on the compression resistance of hydrocolloids gels

The effect of adding sucrose (5–25% w/w) and aspartame (0.04–0.16% w/w) on the compression resistance of three hydrocolloid gelled systems: κ‐carrageenan, gellan gum and κ‐carrageenan/locust bean gum at three different concentrations (0.3, 0.75 and 1.2% w/w) was studied. Sucrose addition increased true rupture stress in the three‐gelled systems, this effect being stronger in gellan gels. The deformability modulus increased with sucrose concentration in gellan gels, but not in the other systems. Rupture stress and deformability modulus increased with the addition of sucrose only in the harder gels (0.75 and 1.2% w/w). The effect of sucrose addition on the true rupture strain was significant but, in general, not important, mainly for lower gum concentrations. Aspartame addition did not affect the compression parameters.     

结冷胶生物合成及应用

结冷胶是由少动鞘脂单胞菌产生的一种新型微生物多糖,具有用量低、透明度高、耐酸、耐高温、耐酶、兼容性好等优良特性,在食品、制药、化工等行业具有广泛的用途。通过介绍结冷胶理化方面的特性,总结了结冷胶发酵生产工艺,以及提高产量和质量的关键因素,进一步展望了未来结冷胶的发展趋势和应用前景。     

结冷胶在非膨化冷食品中的应用研究

从结冷胶的特点和冷食品新产品开发的要求出发，重点研究了结冷胶在冷食品中的应用，以适应冷食品发展的需要。选取适宜的胶体与其复配，达到最佳凝胶效果。然后将复配胶体用于非膨化冷食品中，得到较理想的酥脆冷食品配方。配方为：葡萄糖15％，甜蜜素0．10％，柠檬酸0．15％，柠檬酸钠0．03％，结冷胶0．02％，魔芋胶0．08％，卡拉胶0．05％，氯化钙0．1％，水84．47％。     

Survival of bifidobacteria in yogurt and simulated gastric juice following immobilization in gellan-xanthan beads

A novel acid-stable bead made of gellan gum and xanthan gum was used to immobilize Bifidobacteria. The beads (0.75% gellan and 1% xanthan gum) had an average diameter of 3 mm and did not shrink in 25% lactic acid solution, pH 1.5, or 20% acetic acid solution, pH 1.5, after storage at 4 degrees C for 4 weeks. Bifidobacterium infantis ATCC 15697, the most acid-tolerant strain tested, was immobilized in gellan-xanthan beads and its survival in peptone water, pH 4, pasteurized yogurt, and simulated gastric juice was monitored. In peptone water, pH 4, the reduction in cell count of immobilized cells of B. infantis ATCC 15697 was not significantly different from that obtained with free cells during 6 weeks of storage at 4 degrees C. However, counts of immobilized cells of B. infantis ATCC 15697 remained significantly higher than free cells (P < 0.0001) when both were exposed to simulated gastric juices at pH 2.5, 2.0 and 1.5. At pH 2.5, the viable count of free cells dropped from 1.23 x 10(9) CFU/ml to an undetectable level (< 10 CFU/ml) in 30 min, while the viable count of immobilized cells decreased by only 0.67 log cycle in the same time period. Immobilized cells also survived significantly better than free cells (P <0.05) in pasteurized yogurt after refrigerated storage for 5 weeks.     

Comparative survival and evaluation of functional probiotic properties of spray-dried lactic acid bacteria

The effect of spray drying on the viability and retention of key probiotic properties like acid and bile tolerance and cholesterol assimilation of three probiotic lactic acid bacteria (LAB) ( Lactobacillus plantarum CFR 2191, Lactobacillus salivarius CFR 2158 and Pediococcus acidilactici CFR 2193) has been studied. More than 97% survival was exhibited by the three LAB (1% cell suspension) spray dried with maltodextrin and nonfat skimmed (NFSM) as carriers. LAB cultures spray dried with maltodextrin showed significantly greater ( P ≤  0.05) retention of functional properties than those with NFSM. The results highlight a cost-effective way of producing large quantities of selected probiotic cultures with functional properties for neutraceutical application.     

低酰基结冷胶酸性凝胶的凝胶特性

以葡萄糖酸-δ-内酯(glucono-δ-lactone,GDL)作为酸诱导剂,制备低酰基结冷胶(low acyl gellan gum,LA)酸性凝胶,考察基体质量浓度、GDL/LA复配比例以及酸液浸泡对酸性凝胶凝胶特性的影响。研究结果表明,GDL酸化为缓慢酸化,GDL/LA复配比例越高、体系的pH值越低,酸化速率越快。基体质量浓度和GDL/LA复配比例对酸性凝胶结构影响显著,断裂应力和保水性随着GDL/LA复配比例的增大先升高后降低。基体质量浓度越高,断裂应力和不透明性越大。GDL/LA复配比例增大,断裂应变减小,不透明性增大。当酸液pH值为1时,酸液浸泡对GDL/LA复配比例为2∶1和4∶1的酸性凝胶强度无影响,但GDL/LA复配比例为1∶4、1∶2和1∶1时,凝胶强度随浸泡时间的增加而增强,酸液浸泡可以促使酸性凝胶进行结构重建。     

Viability and Resistance of Lactobacilli Isolated from Cocoa Fermentation to Simulated Gastrointestinal Digestive Steps in Soy Yogurt

To study the potential probiotic characteristics such as decrease of pH, microbial viability, and tolerance to simulated digestive steps of fermented soy beverage (“soy yogurt”) produced with lactobacilli isolated from cocoa fermentation (Lactobacillus fermentum TcUESC01 and Lactobacillus plantarum TcUESC02) during fermentation and refrigerated storage. The sensory acceptance of the yogurts was also tested. Samples of soy yogurt produced with L. fermentum TcUESC01 or L. plantarum TcUESC02 were collected during fermentation (0, 4, 8, and 12 h) and refrigerated storage (1, 9, 18, and 27 d), and submitted to pH and bacterial viability determinations. Tolerance to simulated digestion steps was done with refrigerated storage samples at 9 °C. Simulated digestion was performed in 3 successive steps: exposure to pepsin‐HCl solution, bile shock, and simulated small intestinal juice. During storage, a decrease in pH and lactobacillus viability was observed. L. fermentum TcUESC01 showed to be more resistant than L. plantarum TcUESC02 to simulated gastrointestinal digestion. All soy yogurts showed acceptable hedonic scores (greater than 5 in a 9‐point hedonic scale ranging from “like extremely” to “dislike extremely”) in sensory evaluation for flavor, aroma, color, consistency, and overall impression. L. plantarum TcUESC02 and, especially, L. fermentum TcUESC01 showed potential probiotic characteristics when considering pH, cell viability, and tolerance to simulated digestive steps and did not affect the sensory characteristics when supplemented to soy yogurt during storage.     

Complex Coacervation as a Novel Microencapsulation Technique to Improve Viability of Probiotics Under Different Stresses

A physicochemical approach has been undertaken to develop polymeric microcapsules for delivering probiotic bacteria with improved viability in functional food products. Two probiotic strains of Lactobacillus paracasei subsp. paracasei (E6) and Lactobacillus paraplantarum (B1), isolated from traditional Greek dairy products, were microencapsulated by complex coacervation using whey protein isolate (WPI, 3 %?w/v) and gum arabic (GA, 3 %?w/v) solutions mixed at 2:1 weight ratio. The viability of the bacterial cells during processing (heat treatment and high salt concentrations), under simulated gut conditions (low pH and high bile concentrations) and upon storage, was evaluated. Entrapment of lactobacilli in the complex coacervate structure enhanced the viability of the microorganisms when exposed to a low pH environment (pH 2.0). Both encapsulated strains retained high viability in simulated gastric juice (>73 %; log scale), especially in comparison with non-encapsulated (free) cells (<19 %). Moreover, after 60 days of refrigerated storage at pH 4.0, the viability of microencapsulated cells was more than 86 %, implying improved protection in comparison with the free cells (<59 %). Complex coacervation with WPI/GA has the potential to deliver live probiotics in low pH foods or fermented products; it is also important to note that the complexes can dissolve at pH 7.0 (gut environment) releasing the microbial cells (desired feature of target delivery systems).     

植物乳杆菌HCS03-001安全性评价及其益生特性分析

为了对植物乳杆菌HCS03-001(CGMCC No.16258)进行安全性评价以及益生特性的研究,通过溶血实验、耐药实验及急性经口毒性实验评价其安全性,通过对酸环境及胆盐环境的耐受性实验、抑菌功能特性实验及通便功效试验评价该菌株的益生特性.结果表明,植物乳杆菌HCS03-001在哥伦比亚血琼脂培养基上无透明圈形成,即...     

Screening of potential lactobacilli antigenotoxicity by microbial and mammalian cell-based tests

Antigenotoxicity is considered an important property for probiotic lactobacilli. The ability of non probiotic lactobacilli from dairy products and starters to inhibit two reference genotoxins: 4-nitroquinoline-1-oxide and N-methyl-N'-nitro-N-nitrosoguanidine was evaluated. The study was carried out using short-term assays with different targets, such as procaryotic cells (SOS-Chromotest for genotoxicity in Escherichia coli and Ames test for mutagenicity in Salmonella typhimurium) and eucaryotic cells (Comet assay for genotoxicity in Caco-2 enterocytes). A high proportion of strains inhibiting 4-nitroquinoline-1-oxide activity was found in Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus plantarum. Inhibition of N-methyl-N'-nitro-N-nitrosoguanidine activity occurred in only one L. acidophilus strain. All the strains with antigenotoxic properties also demonstrated antimutagenic activity and produced modifications in genotoxin spectroscopic profiles. Strain viability during and after genotoxin exposure was confirmed. Concordance of the results obtained with microbial and mammalian cell-based tests is underlined.     

驼乳制品中抑制α-淀粉酶和α-葡萄糖苷酶活性乳酸菌的筛选及益生特性研究

目的:本研究以新疆阿勒泰地区的驼乳制品为研究对象,筛选对α-淀粉酶和α-葡萄糖苷酶有抑制活性的优良乳酸菌。方法:采用稀释涂布法分离纯化菌株,DNS和pNPG法筛选对α-淀粉酶和α-葡萄糖苷酶有抑制活性的菌株,通过耐酸性、耐胆盐、模拟胃肠道环境耐受性、抑菌性、抗氧化活性实验评价菌株的益生特性。结果:从驼乳制品中共计获得34株菌株,其中6株对α-淀粉酶和α-葡萄糖苷酶有抑制活性,经过形态学和16S rRNA分子鉴定,确定有4株为植物乳杆菌Lactiplantibacillus plantarum,2株为副干酪乳杆菌Lactiplantibacillus paracasei。6株乳酸菌对α-淀粉酶的抑制活性都达到50%以上,其中X34对α-淀粉酶的抑制率最高达到88.59%。本实验中筛选的乳酸菌对α-葡萄糖苷酶的抑制率在11%~16%之间,X31抑制率最高为15.43%。6株乳酸菌在不同pH（1.0、2.0、3.0）和不同浓度胆盐（1、2、3 g/L）的培养基中均可存活。6株乳酸菌在模拟胃液和肠液中的存活率分别达到83%和90%以上。对羟自由基和超氧阴离子自由基的清除率超过90%以上,其中X29对羟自由基的清除率最高为92.43%,对超氧阴离子自由基清除率最强的是X33清除率达到94.04%。6株乳酸菌对大肠杆菌、沙门氏菌和金黄色葡萄球菌有一定的抑制作用,其中X31对大肠杆菌的最大抑菌圈直径为19.63 mm,X23对沙门氏菌抑菌圈最大达到19.85 mm,菌株X33对金黄色葡萄球菌的抑菌圈直径最大为19.17 mm。结论:从驼乳制品中筛选到6株具有潜在降糖活性的乳酸菌,对强酸、胆盐和胃肠液有一定的耐受性,抗氧化能力较强,具有抑制致病菌的效果,为后期研发功能性降糖饮品提供益生菌株。