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.     

乳酸菌群体感应的研究进展

乳酸菌的益生特性已引起公众的广泛关注。群体感应是细菌感受外界环境变化并做出反应的转导机制,对乳酸菌的存活及益生特性至关重要。因此,近些年来乳酸菌的群体感应成为研究热点。该文综述了乳酸菌群体感应的信号分子及其双组分系统,群体感应对乳酸菌环境适应的调控（生物膜、耐酸、耐胆盐）,群体感应对乳酸菌益生特性（抑制致病菌、与宿主相互作用）的影响以及实际应用,为乳酸菌群体感应今后的基础研究和工业化应用提供参考。     

A Review of Oxygen Toxicity in Probiotic Yogurts: Influence on the Survival of Probiotic Bacteria and Protective Techniques

Oxygen toxicity is considered a significant factor influencing the viability of probiotic bacteria such as Lactobacillus and Bifidobacterium spp. in yogurts. This review assesses the involvement of oxygen during the manufacture of yogurt and its diffusion into the final product. The oxygen sensitivity of Lactobacillus acidophilus and Bifidobacterium spp. is discussed. The impact of dissolved oxygen of the product on the survival of these probiotic bacteria is highlighted. Additionally, microbiological, chemical, and packaging techniques recommended to protect probiotic bacteria from oxygen toxicity in dairy products are reviewed.     

luxS基因介导乳酸菌益生特性研究进展

一些乳酸菌具有高肠道耐受性、高粘附肠上皮细胞和产具有抑菌活性的细菌素等益生特性。某些乳酸菌的细菌素合成量、耐受性及黏附特性可以被诱导物-2（autoinduction-2,AI-2）提高,AI-2是通过甲基循环合成的一种信号分子。luxS基因可以编码合成LuxS蛋白,而LuxS蛋白是AI-2合成的关键酶,因此展开对luxS在细菌素合成量和耐受性、黏附特性方面的作用研究具有重要意义。该文通过讨论luxS基因在乳酸菌益生特性的研究现状,提出该研究领域中存在的问题以及发展趋势,从而为提高乳酸菌的益生特性提供理论依据。     

Survival of Microencapsulated Probiotic Bacteria after Processing and during Storage: A Review

The use of live probiotic bacteria as food supplement has become popular. Capability of probiotic bacteria to be kept at room temperature becomes necessary for customer's convenience and manufacturer's cost reduction. Hence, production of dried form of probiotic bacteria is important. Two common drying methods commonly used for microencapsulation are freeze drying and spray drying. In spite of their benefits, both methods have adverse effects on cell membrane integrity and protein structures resulting in decrease in bacterial viability. Microencapsulation of probiotic bacteria has been a promising technology to ensure bacterial stability during the drying process and to preserve their viability during storage without significantly losing their functional properties such acid tolerance, bile tolerance, surface hydrophobicity, and enzyme activities. Storage at room temperatures instead of freezing or low temperature storage is preferable for minimizing costs of handling, transportation, and storage. Concepts of water activity and glass transition become important in terms of determination of bacterial survival during the storage. The effectiveness of microencapsulation is also affected by microcapsule materials. Carbohydrate- and protein-based microencapsulants and their combination are discussed in terms of their protecting effect on probiotic bacteria during dehydration, during exposure to harsh gastrointestinal transit and small intestine transit and during storage.     

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.     

乳酸菌的益生特性及应用研究进展

活性乳酸菌产品因其良好的益生功效越来越受到人们的青睐,市场需求量不断增大。然而,受乳酸菌厌氧和热敏感性的限制,以及加工中加热和氧气胁迫的影响,产品中活性乳酸菌数量大幅度下降。进入体内的活性乳酸菌在胃液高酸性和肠道高胆汁酸等复杂环境的胁迫下进一步降低,严重影响产品的益生功效。为此,人们在提高乳酸菌对热、氧气、胃肠环境耐受性方面进行了大量研究,并开发了系列新技术,但缺乏彼此间的综合对比与分析。本文围绕如何提高乳酸菌的加工存活率,以及胃肠耐受性和肠道递送问题,对静电纺丝、静电喷雾、乳滴技术、多酚纳米盔甲、热诱导预处理等新技术的作用效果进行了总结与对比,以期为相关研究和技术应用提供参考。     

Probiotic bacteria: selective enumeration and survival in dairy foods

A number of health benefits have been claimed for probiotic bacteria such as Lactobacillus acidophilus, Bifidobacterium spp., and Lactobacillus casei. Because of the potential health benefits, these organisms are increasingly incorporated into dairy foods. However, studies have shown low viability of probiotics in market preparations. In order to assess viability of probiotic bacteria, it is important to have a working method for selective enumeration of these probiotic bacteria. Viability of probiotic bacteria is important in order to provide health benefits. Viability of probiotic bacteria can be improved by appropriate selection of acid and bile resistant strains, use of oxygen impermeable containers, two-step fermentation, micro-encapsulation, stress adaptation, incorporation of micronutrients such as peptides and amino acids and by sonication of yogurt bacteria. This review will cover selective enumeration and survival of probiotic bacteria in dairy foods.     

Probiotics,prebiotics, and microencapsulation: A review

The development of a suitable technology for the production of probiotics is a key research for industrial production, which should take into account the viability and the stability of the organisms involved. Microbial criteria, stress tolerance during processing, and storage of the product constitute the basis for the production of probiotics. Generally, the bacteria belonging to the genera Lactobacillus and Bifidobacterium have been used as probiotics. Based on their positive qualities, probiotic bacteria are widely used in the production of food. Interest in the incorporation of the probiotic bacteria into other products apart from dairy products has been increasing and represents a great challenge. The recognition of dose delivery systems for probiotic bacteria has also resulted in research efforts aimed at developing probiotic food outside the dairy sector. Producing probiotic juices has been considered more in the recent years, due to an increased concern in personal health of consumers. This review focuses on probiotics, prebiotics, and the microencapsulation of living cells.     

德氏乳杆菌保加利亚亚种耐酸机制

德氏乳杆菌保加利亚亚种是最具经济价值的乳酸菌之一,其耐酸能力是影响其发酵性能和发挥益生功效的关键因素。本文就该菌的耐酸能力调节进行阐述。     

Processing optimization of probiotic yogurt containing glucose oxidase using response surface methodology

Exposure to oxygen may induce a lack of functionality of probiotic dairy foods because the anaerobic metabolism of probiotic bacteria compromises during storage the maintenance of their viability to provide benefits to consumer health. Glucose oxidase can constitute a potential alternative to increase the survival of probiotic bacteria in yogurt because it consumes the oxygen permeating to the inside of the pot during storage, thus making it possible to avoid the use of chemical additives. This research aimed to optimize the processing of probiotic yogurt supplemented with glucose oxidase using response surface methodology and to determine the levels of glucose and glucose oxidase that minimize the concentration of dissolved oxygen and maximize the Bifidobacterium longum count by the desirability function. Response surface methodology mathematical models adequately described the process, with adjusted determination coefficients of 83% for the oxygen and 94% for the B. longum. Linear and quadratic effects of the glucose oxidase were reported for the oxygen model, whereas for the B. longum count model an influence of the glucose oxidase at the linear level was observed followed by the quadratic influence of glucose and quadratic effect of glucose oxidase. The desirability function indicated that 62.32 ppm of glucose oxidase and 4.35 ppm of glucose was the best combination of these components for optimization of probiotic yogurt processing. An additional validation experiment was performed and results showed acceptable error between the predicted and experimental results.     

Human Studies on Probiotics: What Is Scientifically Proven

ABSTRACT: New knowledge has revealed that probiotics have specific properties and targets in the human intestinal tract. It is also understood that each probiotic strain, independent of the genera and species, is unique and the properties of each strain have to be assessed in a case-by-case manner. Human intervention studies are required for verification of probiotic properties. The basis of probiotics lies in the normal intestinal microbiota. The concept of healthy gut microbiota can be used in terms of preventing, correcting, and dietary manipulation of potential microbiota aberrancies. This is reflected in the search of probiotics with disease-specific properties. The effects of GI tract conditions, such as pH, bile, and digestive enzymes, on the survival and adhesion properties of probiotic bacteria have been documented. Various bacteria show different levels of tolerance to the GI conditions. Adhesion on intestinal surface lengthens the retention time of a probiotic, and it is particularly important for the small intestine. The residence time of material in the small intestine is relatively short. Such properties need to be clearly defined for probiotic microbes discovered in the future. An effective probiotic should reside at desirable target sites sufficiently long at sufficient concentrations to elicit probiotic effects. More research on the assessment of desirable dosages is urgently needed.     

Usefulness of a set of simple in vitro tests for the screening and identification of probiotic candidate strains for dairy use

A set of simple in vitro tests (identification by species-specific PCR, genetic diversity, phage sensitivity, growth and viability in milk, resistance to salts and flavor compounds, bacterial interactions, tolerance to simulated gastric juice and bile, bile salts deconjugation, hydrophobicity and β-galactosidase and antibacterial activities), that can be carried out in almost every laboratory of microbiology, mainly in developing countries where there is often limited access to sophisticated techniques, allowed us to identify, among 19 intestinal human isolates, a potential candidate for new probiotic dairy foods for the local market. Lactobacillus gasseri LgF_37/1 performed well in the culture media used for the enumeration of probiotic bacteria in argentinian dairy products. This strain showed also high tolerance to the technological challenges assessed, bile salts resistance, the capacity to produce bacteriocin-like metabolites, to inhibit pathogenic bacteria, to deconjugate bile salts and high hydrophobicity. Further in vivo research should be carried out with this strain before claiming probiotic properties for it. However, the use of a set of simple in vitro techniques proved to be important to determine which strains should undergo future and more complex studies.     

发酵乳杆菌及其益生特性研究进展

发酵乳杆菌作为传统发酵食品中的优势乳酸菌具有安全性及遗传稳定的特点。作者着重阐述了发酵乳杆菌在传统食品中的分布以及应用,及其作为益生菌在耐受动物肠道不利条件、胆固醇降解、对腐败及病原菌的抑制、对人体的免疫增强作用等方面的益生特性及其机制的研究现状,并对菌株安全性方面的研究进行了综述,以期对发酵乳杆菌作为高品质的益生食品发酵剂奠定一定的理论基础。     

Protective approaches and mechanisms of microencapsulation to the survival of probiotic bacteria during processing,storage and gastrointestinal digestion: A review

Abstract

In recent years, there is a rising interest in the number of food products containing probiotic bacteria with favorable health benefit effects. However, the viability of probiotic bacteria is always questionable when they exposure to the harsh environment during processing, storage, and gastrointestinal digestion. To overcome these problems, microencapsulation of cells is currently receiving considerable attention and has obtained valuable effects. According to the drying temperature, the commonly used technologies can be divided into two patterns: high temperature drying (spray drying and fluid bed drying) and low temperature drying (ultrasonic vacuum spray drying, spray chilling, electrospinning, supercritical technique, freeze drying, extrusion, emulsion, enzyme gelation, and impinging aerosol technique). Furthermore, not only should the probiotic bacteria maintain high viability during processing but they also need to keep alive during storage and gastrointestinal digestion, where they additionally suffer from water, oxygen, heat as well as strong acid and bile conditions. This review focuses on demonstrating the effects of different microencapsulation techniques on the survival of bacteria during processing as well as protective approaches and mechanisms to the encapsulated probiotic bacteria during storage and gastrointestinal digestion that currently reported in the literature.     

自然发酵酸笋中乳酸菌的筛选鉴定及益生特性研究

为筛选自然发酵酸笋中的益生乳酸菌,采用MRS培养基分离,经生理生化实验及16S rDNA序列分析鉴定,通过溶血实验评价安全性以及耐酸耐胆盐实验评价耐受性,并测定乳酸菌体外抗氧化性。结果表明,共分离到69株乳酸菌,隶属于2个科3个属,分别为乳杆菌属（Lactobacillus）、片球菌属（Pediococcus）、链球菌属（Streptococcus）,均为γ-溶血的安全菌株,且均对0.3%胆盐具有良好耐受性,其中41株乳酸菌耐pH 2.0酸性条件,从中优选出的17株乳酸菌对人工胃肠液均有良好耐受性。4株短乳杆菌（Z1-02、ZR2-14、ZR2-12、ZR2-22）和2株消化乳杆菌（ZR1-02、ZR1-05）的1,1-二苯基-2-三硝基苯肼（DPPH）自由基清除率、2,2'-联氮-双-3-乙基苯并噻唑啉-6-磺酸（ABTS）自由基清除率、还原能力均分别＞62%、79%、0.393,其中乳杆菌Z1-02的综合抗氧化能力最强,这6株乳酸菌具备优良的益生性能和体外抗氧化活性。     

酢辣椒中益生乳酸菌的筛选及其功能特性

为从酢辣椒筛选出具有益生特性的菌株,开发辣椒益生菌产品,本实验从地方特色发酵食品酢辣椒中分离得到16 株乳酸菌,以产酸速度、耐人工胃液能力为指标进行初筛,通过生化实验和16S rDNA序列分析方法鉴定;并通过研究菌株对工艺逆境的耐受能力,表面疏水性和自聚合能力以及抑菌活性、抗氧化能力、降解亚硝盐能力和安全特性来评价乳酸菌的益生特性。结果表明：发酵乳杆菌17-1、发酵乳杆菌18-2和短乳杆菌L3-5能耐受0.4%苯酚、溶酶菌和热处理;具有较高的表面疏水性和自聚合能力;能产生具有广谱抑菌效果的细菌素来抑制病原菌的生长;菌株L3-5和17-1具有较高的抗氧化能力;3 株菌都具有胆盐水解酶活性和产γ-氨基丁酸能力,并且都属于安全性菌株。综上所述,传统酢辣椒是分离益生乳酸菌新的天然来源,从中分离得到的发酵乳杆菌17-1、发酵乳杆菌18-2和短乳杆菌L3-5可以作为潜在的益生性菌株。     

Metabolic and biochemical responses of probiotic bacteria to oxygen

The interaction between oxygen and probiotic bacteria was studied by growing Lactobacillus acidophilus and Bifidobacterium spp. in 0, 5, 10, 15, and 21% oxygen in a hypoxic glove box. The metabolic responses of each probiotic strain in the different oxygen environments were monitored by measuring the levels of lactic acid and determining the lactate-to-acetate ratio. Biochemical changes induced by oxygen were examined by monitoring the specific activities of NADH oxidase, NADH peroxidase, and superoxide dismutase. In addition, the ability to decompose hydrogen peroxide and the sensitivity of each strain to hydrogen peroxide was also determined. With an increase in oxygen percentage, levels of lactic acid in L. acidophilus strains decreased, whereas the lactate-to-acetate ratio reduced in all the bifidobacteria tested. At 21% oxygen, the specific activities of NADH oxidase and NADH peroxidase, and the hydrogen peroxide decomposing ability of five probiotic strains was significantly higher than at 0% oxygen. The sensitivity of the probiotic strains to hydrogen peroxide however, remained unaffected in all the different oxygen percentages. Superoxide dismutase levels did not reveal any conclusive trend. In both L. acidophilus and Bifidobacterium spp., NADH oxidase and NADH peroxidase functioned optimally at pH 5. Growth in the various oxygen environments did not change this optimum pH.     

乳酸菌作为粘膜重组疫苗呈递载体的研究进展

乳酸菌是对人和动物具有多种益生功能的食品级微生物,被广泛地应用于食品、医药、生物技术等领域。乳酸菌的安全益生特性及乳酸菌基因表达系统研究取得的重大进展,使得重组乳酸菌黏膜疫苗成为研究的热点。乳酸菌作为黏膜免疫的蛋白呈递载体,可诱导机体产生有效的免疫反应和免疫耐受,具有巨大的发展潜力。