Microencapsulation of probiotic bacteria: technology and potential applications

In the recent past, there has been an explosion of probiotic health-based products. Many reports indicated that there is poor survival of probiotic bacteria in these products. Further, the survival of these bacteria in the human gastro-intestinal system is questionable. Providing probiotic living cells with a physical barrier against adverse environmental conditions is therefore an approach currently receiving considerable interest. The technology of micro-encapsulation of probiotic bacterial cells evolved from the immobilised cell culture technology used in the biotechnological industry. Several methods of micro-encapsulation of probiotic bacteria have been reported and include spray drying, extrusion, emulsion and phase separation. None of these reported methods however, has resulted in the large numbers of shelf-stable, viable probiotic bacterial cells necessary for use in industry for development of new probiotic products. The most commonly reported micro-encapsulation procedure is based on the calcium-alginate gel capsule formation. Kappa-carrageenan, gellan gum, gelatin and starch are also used as excipients for the micro-encapsulation of probiotic bacteria. The currently available equipment for micro-encapsulation is not able to generate large quantities of uniform sized micro or nano capsules. There is a need to design and develop equipment that will be able to generate precise and uniform micro or nano capsules in large quantities for industrial applications. The reported food vehicles for delivery of encapsulated probiotic bacteria are yoghurt, cheese, ice cream and mayonnaise. Studies need to be done on the application of micro-encapsulation of probiotic bacteria in other food systems. The number of probiotic supplements will increase in the future. More studies, however, need to be conducted on the efficacy of micro-encapsulation to deliver probiotic bacteria and their controlled or targeted release in the gastrointestinal tract.     

Development of Yoghurt with Juçara Pulp (<Emphasis Type="Italic">Euterpe edulis</Emphasis> M.) and the Probiotic <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> La5

Yoghurts are dairy products consumed worldwide and can be supplemented with substances that provide extra health benefits as well as probiotic strains. In this context, the present study aimed to prepare a yoghurt added of juçara (Euterpe edulis M.) pulp and the commercial probiotic strain Lactobacillus acidophilus La5. Moreover, the probiotic survival during storage and after in vitro exposure to simulated gastric and enteric conditions was evaluated. Four formulations of yoghurt were prepared: (a) natural yoghurt, (b) yoghurt added of probiotic, (c) yoghurt added of juçara pulp, and (d) yoghurt added of probiotic culture and juçara pulp. The preparations were evaluated for survival of probiotic strain during storage and its tolerance to gastric and enteric conditions in vitro. The probiotic population in yoghurt remained unchanged during 28 days of storage. In addition, juçara pulp increased the probiotic resistance to simulated gastric and enteric conditions in the first day of storage. These data indicate that juçara pulp is a potential ingredient for the production of probiotic yoghurts.     

Immunomodulatory effects of probiotic bacteria DNA: IL-1 and IL-10 response in human peripheral blood mononuclear cells

A new therapeutic approach for inflammatory bowel diseases is based on the administration of probiotic bacteria. Prokaryotic DNA contains unmethylated CpG motifs which can activate immune responses, but it is unknown whether bacterial DNA is involved in the beneficial effects obtained by probiotic treatment. Peripheral blood mononuclear cells (PBMC) from healthy donors were incubated with pure DNA of eight probiotic strains and with total bacterial DNA from human feces collected before and after probiotic ingestion. Cytokine production was analyzed in culture supernatants. Modification of human microflora after probiotic administration was proven by polymerase chain reaction analysis. Here we show that Bifidobacterium genomic DNA induced secretion of the antiinflammatory interleukin-10 by PBMC. Total bacterial DNA from feces collected after probiotic administration modulated the immune response by a decrease of interleukin-1 beta and an increase of interleukin-10.     

Differential protein expression in early Atlantic cod larvae (Gadus morhua) in response to treatment with probiotic bacteria

Proteome analysis was used to study the effects of probiotic bacteria treatment on the protein expression in early Atlantic cod (Gadus morhua) larvae. The main focus was on proteins connected to immune function, growth and development. The results demonstrated that none of the identified cod larvae proteins showing up-regulation after administration of probiotic bacteria are known to have a function in immunity. In contrast, several proteins exhibiting down-regulation after exposure to the probiotic bacteria may be related to stress and immune responses. This indicates that the probiotic treated larvae were experiencing a lower level of immune-stimulation than the control group. It is therefore tempting to postulate that the probiotic bacteria mixture used in the present study reduced the environmental stress by inhibiting the growth of pathogenic bacteria. Most of the identified up-regulated proteins in the probiotic group relative to the control may be linked to growth and development. The most pronounced up-regulation of proteins was found in several muscle α-actin isoforms indicating improved growth upon probiotic bacteria treatment.     

Impact of two probiotic Lactobacillus strains feeding on fecal lactobacilli and weight gains in chicken

Two probiotic strains, Lactobacillus agilis JCM 1048 and L. salivarius subsp. salicinius JCM 1230 isolated from chicken intestine, exhibited probiotic characteristics that can be applied for chicken production. After 7 days of probiotic feeding (FD7), the count of intestinal lactobacilli in the probiotic group (group P, n=10) was significantly (p<0.05) higher than that in the control group (group C, n=9). After 40 days of probiotic feeding (FD40), the lactobacilli and enterococci counts were stable but the Enterobacteriaceae number was significantly reduced (p<0.05). A total of 163 isolated lactobacilli were identified as the L. acidophilus/gallinarum group (49.7%), L. agilis (30.7%), L. salivarius (9.2%), L. reuteri (9.2%), and Lactobacillus spp. (1.2%). The probiotic lactobacilli positively affected the Lactobacillus biota in chickens at FD7, with a significant increase in the number (p<0.05) of L. agilis and group P. The viable counts of each Lactobacillus species at FD40, however, showed no differences between two groups. An increasing incidence of L. agilis was also noted with probiotic feeding. The probiotic effect of two strains resulted in significantly increased weight gains (10.7%) of group P in comparison with group C at FD40 (p<0.01).     

Bacterial biota of shrimp intestine is significantly modified by the use of a probiotic mixture: a high throughput sequencing approach

The use of probiotics is a common practice of current shrimp aquaculture. Despite the immunophysiological responses that have been measured in shrimp exposed to probiotics, no information is currently available on the effect of this practice on the intestinal microbiota. The objective of this work was to evaluate the effect of a probiotic mixture on the intestinal microbiota of shrimp cultured under farm conditions. A culture-independent method based on high-throughput-sequencing (16S rRNA) was used to examine intestinal bacterial communities. A traditional system (without probiotics) was used as the reference. Targeted metagenomics analysis revealed that the probiotic mixture was based on bacteria in the phyla Proteobacteria and Firmicutes. A total of 23 species of bacteria were detected in the probiotic mixture; of these, 11 were detected in the intestine of shrimp reared in both systems, and 12 were novel for the system. Eight of the novel species were detected in shrimp cultured with the probiotic mixture; however, none of these novel species were related to marine or inclusively aquacultural environments, and only one (Bacillus subtilis) was recognized as probiotic for shrimp. The use of the probiotic mixture modified the bacterial profile of the shrimp intestine; however, most of the bacteria incorporated into the intestine were nonindigenous to the marine environment with no previous evidence of probiotic effects on any marine organism. The use of this probiotic mixture may represent a risk of causing environmental imbalances, particularly because farms using these types of probiotic mixtures discharge their effluents directly into the ocean without prior treatment.     

Viability of human-derived probiotic lactobacilli in ice cream produced with sucrose and aspartame

A mixture of human-derived probiotic strains of Lactobacillus acidophilus, L. agilis and L. rhamnosus was used as a probiotic culture in ice cream manufacture. Viability and survival of these probiotic cultures were investigated in two different ice cream formulations. Ice cream with sucrose and ice cream with aspartame were prepared and each of these was divided into two subgroups: one with direct addition of the probiotic culture and one with milk fermented by the same probiotic culture. Ice cream samples were stored at −20°C for 6 months and the survival rate of cultures were determined monthly. Probiotic cultures underwent tests for resistance to bile salts, antibiotics, acidic conditions; they were found to be highly resistant to such challenges. Chemical analysis of ice cream samples, such as determination of acidity, pH and solid matter, was also performed. The probiotic cultures remained unchanged in ice cream stored for up to 6 months regardless of the sweeteners used. Using probiotic cultures in ice cream mixes did not alter the characteristics of the product.     

Effects of probiotics on growth,survival, and intestinal and liver morphometry of Gangetic mystus (Mystus cavasius)

The usage of probiotics proved advantageous in aquaculture due to its positive impact on fish growth, immune response and environment. This study was aimed to assess the effects of probiotics on growth, survival and histometry of intestine and liver in Gangetic mystus (Mystus cavasius) using two separate experiments for a period of 8 weeks (in aquaria) and 16 weeks (in earthen ponds). Three different probiotic treatments were incorporated i.e. commercial probiotic one; CP-1 (T1), commercial probiotic two; CP-2 (T2), Lab developed (Lab dev.) probiotic (T3) including a control. The results indicated that the probiotics usage especially Lab dev. probiotic (T3) significantly improved the growth parameters such as weight gain (g) and specific growth rate (SGR, %/day) as well as ensured better feed conversion efficiency. Zero mortality was observed in aquaria whereas probiotic application enhanced survivability in earthen ponds. Moreover, all probiotic treatment exhibited positive results for different histo-morphometric features of intestine and liver. Mucus secreting goblet cells and fattening of mucosal fold increased significantly with probiotic usage. The amount of regular shaped nucleus was maximum in T3 with least intra cellular distance between liver tissues in earthen ponds. The greatest value for hemoglobin with lowest glucose level was observed in T3 as well. Furthermore, probiotic ensured low concentration of ammonia during culture. Overall, it was anticipated that the application of probiotics in Gangetic mystus culture resulted positive effect on its growth, feed utilization, survivability, histo-morphometry, immunity and hematological parameters.     

4种市售抗生素对发酵床垫料中益生微生物生长的影响

目的研究不同剂量土霉素、盐酸环丙沙星、克拉霉素、乙酰甲喹对垫料中益生微生物生长的影响。方法以饲料中抗生素添加量、抗生素在动物体内转化率为依据,观察0.1×国标、1×国标和10×国标对应的抗生素残留对垫料中主要益生微生物生长的影响。结果与空白组相比,0.1×国标的土霉素和克拉霉素残留对3种益生微生物影响小,盐酸环丙沙星和乙酰甲喹残留使枯草芽胞杆菌和酵母菌数量不可检测;1×国标量的土霉素残留对3种益生微生物影响较小,而盐酸环丙沙星、克拉霉素、乙酰甲喹残留造成了益生微生物的不可检测;除植物乳杆菌在10×国标的土霉素残留中生长变化小外,所有检测菌在该浓度其他抗生素残留中均不可检测。结论土霉素、盐酸环丙沙星、克拉霉素、乙酰甲喹残留对垫料中主要益生微生物生长均有不同程度的影响。     

Application of genotypic and phenotypic analyses to commercial probiotic strain identity and relatedness

AIMS: The objective of this study was to generate strain-specific genomic patterns of a bank of 67 commercial and reference probiotic strains, with a focus on probiotic lactobacilli. METHODS AND RESULTS: Pulsed-field gel electrophoresis (PFGE) was used as the primary method for strain differentiation. This method was compared with carbohydrate fermentation analysis. To supplement visual comparison, PFGE patterns were analysed quantitatively by cluster analysis using unweighted pair group method with arithmetic averages. SmaI, NotI and XbaI were found to effectively generate clear and easy-to-interpret PFGE patterns of a range of probiotic strains. Some probiotic strains from different sources shared highly similar PFGE patterns. CONCLUSIONS: Results document the value of genotypic strain identification methods, combined with phenotypic methods, for determining probiotic strain identity and relatedness. No correlation was found between relatedness determined by carbohydrate fermentation profiles alone compared with PFGE analysis alone. Some commercial strains are probably derived from similar sources. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach is valuable to the probiotic industry to develop commercial strain identification patterns, to provide quality control of strain manufacturing production runs, to track use of protected strains and to determine the relatedness among different research and commercial probiotic strains.     

Development of probiotic diets for the olive fly: evaluation of their effects on fly longevity and fecundity

One possible control strategy against the olive fly, Bactrocera oleae, the most serious olive crop pest, is the Sterile Insect Technique (SIT) application. However, a number of problems associated with this method remain that decrease the effectiveness of SIT, including the quality of reared insects. Taking the importance of the relationship between the olive fly and bacteria into consideration, the effects of probiotic diets enriched with Pseudomonas putida on B. oleae longevity and fecundity were evaluated. First, we found that the probiotic bacterium, P. putida, is conveyed from diets to the oesophageal bulb as well as to the fly midgut after feeding on the probiotic diet. Subsequently, B. oleae adults fed on either: (a) a standard full protein and sugar diet; (b) a sugar only diet; (c) a probiotic standard full protein and sugar diet; or (d) a probiotic sugar diet. Flies fed on probiotic diets were supplied with an inoculated gel containing P. putida; non‐inoculated gel was provided to the flies fed on non‐probiotic diets. B. oleae males and females that fed on sugar diets did not survive as long as those that fed on protein diets. A comparison of the longevity of adults fed on full diet and sugar with their respective probiotic diets revealed no significant difference. Males fed on the sugar only diet survived longer than males fed on probiotic sugar diet, and females fed on the full protein and sugar diet survived much longer than females fed on the full probiotic diet. As regarding fecundity, both full diets resulted in a higher number of eggs laid per female. Females fed on the probiotic sugar diet laid a higher number of eggs than females that fed on sugar only. The inoculated gel of the probiotic sugar diet contained a significantly higher quantity of leucine, isoleucine and proline than the non‐inoculated gel of the sugar only diet. The possible role of dietary bacteria in relation to functional aspects of olive fly physiology is discussed.     

Probiotic Supplement Use among Young Children in Taiwan: A Prospective Cohort Study

Objectives

The objective of this study is to provide details on probiotic supplement use among young children in Taiwan.

Participants and Methods

This study is based on the Taiwan Birth Cohort Study database. We used questionnaires to collect information on probiotic supplement use among young children from birth to 18 months of age, while also considering their demographic characteristics and other covariates. Low-birth-weight infants, preterm infants, those with birth defects, and those with caregivers who returned incomplete questionnaires were excluded. The final valid sample comprised 16,991 cases.

Results

Approximately half the children received probiotic supplements before the age of 18 months. Only 6.3% of the children received probiotic supplements during the two periods of birth to 6 months and 7 to 18 months. Firstborn children, native mothers, mothers with higher educational levels, higher family income, and parents who lead healthy lifestyles were positively related to probiotic supplement use among children. Young children who were breastfed, with eczema, or with gastrointestinal tract problems were significantly positively associated with probiotic supplement use.

Conclusion

The findings show that probiotic supplement usage among young children is associated with a more socially advantaged circumstance and certain child health factors, such as eczema, diarrhea, and constipation. Parents might use probiotic supplements for prevention or treatment of child diseases. The findings of this research could serve as a baseline for future studies, and provide insight into probiotic supplement use behavior for health professionals caring for infants and young children.     

Role of the probiotic strain Lactobacillus paracasei LMGP22043 carried by artichokes in influencing faecal bacteria and biochemical parameters in human subjects

Aims: To evaluate the positive influence of the probiotic strain Lactobacillus paracasei LMGP22043 carried by artichokes into the human gut with special reference to faecal bacterial balance, short‐chain fatty acid concentrations and enzyme activities in a randomized, double‐blind human trial in comparison with probiotic‐free artichokes (control). Methods: Twenty subjects were randomized into two groups, which consumed daily 180 g of the artichoke product (probiotic or control) during two 15‐day study periods (periods 1 and 2) separated by a 15‐day washout in a crossover manner. Faecal samples were subjected to microbiological and biochemical analyses, and a strain‐specific PCR was performed to monitor the probiotic strain. Results: The probiotic strain, transported by the vegetable matrix, transiently colonized the gut of 17/20 subjects (median 6·87 log CFU g^?1 faeces), antagonized Escherichia coli and Clostridium spp. and increased the genetic diversity of lactic population based on REP‐PCR profiles, mainly after period 1. Conclusions: The probiotic L. paracasei LMGP22043 successfully colonized the human gut and positively influenced faecal bacteria and biochemical parameters. Significance and Impact of the Study: The association of the probiotic L. paracasei with a food carrier rich in fibre can represent a new strategy for favouring a daily supply of probiotics and attracting more consumers to vegetable food fortified with probiotic strains.     

Manufacture of Cheddar cheese using probiotic Lactobacillus plantarum K25 and its cholesterol-lowering effects in a mice model

The probiotic adjunct Lactobacillus plantarum K25 was inoculated into milk to produce probiotic cheese. The effect of Lb. plantarum K25 on cheese composition, microbiological growth and survival during the manufacturing and ripening period, primary and secondary proteolysis during cheese ripening, and the in vivo cholesterol-lowering ability of the probiotic cheese were investigated. The results showed that the use of adjunct Lb. plantarum K25 in Cheddar cheese did not affect the cheese components including moisture, protein, fat, salt content and the pH value of cheese. During the whole ripening period, the probiotic adjunct maintained its viability, suggesting the effectiveness of Cheddar cheese as a vehicle for delivery of probiotic bacteria. No significant differences were observed in water-soluble nitrogen, 70?% ethanol-soluble nitrogen, 5?% phosphotungstic acid-soluble nitrogen, free amino acids and urea-PAGE patterns between the control and probiotic cheeses. Assessment of the in vivo cholesterol-lowering property of cheese with Lb. plantarum K25 showed that the levels of serum total cholesterol, low-density lipoprotein cholesterol and triglycerides decreased significantly, and the level of serum high-density lipoprotein cholesterol increased in mice fed with the probiotic cheese. The results indicated the potential function as a dietary item of the probiotic cheese with Lb. plantarum K25 to reduce the risk of cardiovascular diseases.     

Comparative Analysis of Antigiardial Potential of Heat Inactivated and Probiotic Protein of Probiotic Lactobacillus rhamnosus GG in Murine Giardiasis

The present study was designed to envisage the antigiardial efficacy of killed probiotic and probiotic protein (PP) of Lactobacillus rhamnosus GG in murine giardiasis. Experimentally, it was observed that animal administered either with probiotic protein emulsified with adjuvant (PP_(E) + Giardia) or killed probiotic (killed probiotic (i/p) + Giardia) had significantly reduced Giardia cycle with respect to observed severity and duration of giardiasis compared with Giardia-infected mice. Further, it was found that animals belonging to PP_(E) + Giardia and killed probiotic (i/p) + Giardia had significantly high levels of antigiardial IgA antibody and nitric oxide both in serum and in intestinal fluid compared with Giardia-infected and counter control mice. Histopathologyically, also animals belonging to PP_(E) + Giardia and killed probiotic (i/p) + Giardia animals had intact mucosal epithelium lining, basal crypts, and normal villi along with increased goblet cells compared with severe microvillus atrophy, vacuolated epithelial cells, and ileitis in Giardia-infected mice. This is the first-ever study to demonstrate that prior administration of either killed probiotics or probiotic protein of effective probiotic reduced both the severity and the duration of giardiasis mainly by modulating the gut microbiome and morphology along with mucosal immunity, but animals belonging to PP_(E) + Giardia had better response than killed probiotic (i/p) + Giardia suggesting that probiotic components do have adjuvant potential and may be used as the vaccine candidate for gastrointestinal diseases.

     

脆弱拟杆菌的研究进展

脆弱拟杆菌是定殖于哺乳动物肠道中的共生菌,同时也是临床感染病例中常见的条件致病菌。本文从致病及益生特性两大方面综述脆弱拟杆菌的研究现状,着重讨论了脆弱拟杆菌作为潜在益生菌在预防和治疗糖尿病及免疫性疾病中所起的重要作用,从而为筛选及应用益生脆弱拟杆菌菌株提供一定的参考。     

Lack of Heterogeneity in Bacteriocin Production Across a Selection of Commercial Probiotic Products

Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit to the host. Bacteriocin production has often been mooted as a desirable probiotic trait and, in specific cases, has been shown to promote probiotic survival within the gastrointestinal tract, contribute to the control of pathogens and even influence host gene expression in the gut. However, it is not clear what proportion of probiotic strains routinely found in commercial products produces bacteriocins, and additionally, it is not known which bacteriocins are produced most frequently. To address this, we conducted a culture-based assessment of the bacteriocinogenic ability of bacterial strains found in a variety of commercially available probiotic products. We detected eight bacteriocin-producing isolates from 16 tested products. Interestingly, in all cases, the isolates were Lactobacillus acidophilus, and the bacteriocin produced was identified as the narrow spectrum class II bacteriocin, lactacin B. The apparent absence of other bacteriocin-producing strains from across these products suggests a lack of heterogeneity in bacteriocin production within probiotic products and suggests that bacteriocin production is not being optimally harnessed as a probiotic trait.     

Inhibition of 1,2-dimethylhydrazine induced colon genotoxicity in rats by the administration of probiotic curd

Epidemiologic and experimental studies suggest that the probiotic organisms are effective in preventing colon carcinogenesis, which is the major cause of mortality and morbidity in western countries. Keeping this in view, a curd (a common Indian fermented milk product) was prepared by the addition of probiotic cultures Lactobacillus acidophilus, Lactobacillus casei and curd culture Lactococcus lactis biovar. diacetylactis. In present study, we have evaluated the anti tumor effect of probiotic curd by monitoring the DNA damage through comet assay. The rats were allocated to four groups, first group was DMH control group, second group was probiotic curd group in which probiotic curd was given along with DMH (1,2-dimethylhydrazine) injection, third group was normal curd group in which normal curd was given along with DMH injection and fourth group was normal control group. Animals received subcutaneous injection of DMH dissolved in normal saline at a dose rate of 20 mg/kg body weight, once weekly for 15 weeks. The rats were dissected at 40th week of experiment and comet assay was done in colonic cells to assess the DNA damage. A significant reduction in DNA damage (54.7%) was observed in probiotic curd group as compared to DMH control group (88.1%). The probiotic curd was effective to significantly reduce the L:W ratio in comparison to DMH control group and normal curd. The results of present study show the protective effects of probiotic curd against DMH induced genotoxicity in colonic cells.     

Intraspecific genotypic characterization of Lactobacillus rhamnosus strains intended for probiotic use and isolates of human origin

A set of 118 strains of the species Lactobacillus rhamnosus was collected, including probiotic strains, research strains with potential probiotic properties, food starter cultures, and human isolates. The majority of the strains were collected from companies, hospitals, or culture collections or were obtained after contacting authors who reported clinical case studies in the literature. The present work aimed to reveal the genotypic relationships between strains of these diverse sources. All strains were initially investigated using fluorescent amplified fragment length polymorphism (FAFLP) with three different primer combinations. Numerical analysis of FAFLP data allowed (i) confirmation of the identification of all strains as members of L. rhamnosus and (ii) delineation of seven stable intraspecific FAFLP clusters. Most of these clusters contained both (potentially) probiotic strains and isolates of human origin. For each of the clusters, strains of different sources were selected for pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments obtained with the enzymes NotI and AscI. Analysis of PFGE data indicated that (i) some (potentially) probiotic strains were indistinguishable from other probiotic strains, suggesting that several companies may use duplicate cultures of the same probiotic strain, and (ii) in a number of cases human isolates from sterile body sites were indistinguishable from a particular probiotic strain, suggesting that some of these isolates may be reisolations of commercial strains.     

A Review of the Advancements in Probiotic Delivery: Conventional vs. Non-conventional Formulations for Intestinal Flora Supplementation

Probiotic delivery systems are widely used nutraceutical products for the supplementation of natural intestinal flora. These delivery systems vary greatly in effectiveness to exert health benefits for a patient. Probiotic delivery systems can be categorized into conventional, pharmaceutical formulations, and non-conventional, mainly commercial food-based, products. The degree of health benefits provided by these probiotic formulations varies in their ability to deliver viable, functional bacteria in large enough numbers (effectiveness), to provide protection against the harsh effects of the gastric environment and intestinal bile (in vivo protection), and to survive formulation processes (viability). This review discusses the effectiveness of these probiotic delivery systems to deliver viable functional bacteria focusing on the ability to protect the encapsulated probiotics during formulation process as well as against harsh physiological conditions through formulation enhancements using coatings and polymer enhancements. A brief overview on the health benefits of probiotics, current formulation, patient and legal issues facing probiotic delivery, and possible recommendations for the enhanced delivery of probiotic bacteria are also provided. Newer advanced in vitro analyses that can accurately determine the effectiveness of a probiotic formulation are also discussed with an ideal probiotic delivery system hypothesized through a combination of the two probiotic delivery systems described.