Simulated Gastrointestinal Passage Research Articles

Can a functional cheese spread incorporating Akkermansia muciniphila deliver beneficial physicochemical and biological properties while enhancing probiotic stability and viability during aerobic storage and in vitro digestion?

In the present study, next generation probiotic Akkermansia muciniphila was incorporated into a dairy matrix containing Portuguese whey cheese and Greek-style yoghurt in a proportion of 3.5:1, respectively. Subsequently, this innovative food was characterized in terms of microbiological and physicochemical parameters, total phenolic content and antioxidant, antidiabetic, and antihypertensive activities, as well as its protective effect on A. muciniphila viability during 21 d of refrigerated aerobic storage and when subjected to simulated gastrointestinal passage. The probiotic cheese spread displayed high microbiological quality, low total phenolic content (0.36 mg gallic acid equivalents/g of dried cheese) and interesting biological activities, including antidiabetic (98.10% α-glucosidase inhibition) and antihypertensive (49.18% angiotensin converting enzyme inhibition). Simultaneously, this food ensured a high A. muciniphila viability (>108 CFU/g) during 21 d of refrigerated aerobic storage with subsequent in vitro digestion. Additionally, this probiotic cheese presented a similar profile in terms of texture, color, water activity and pH when compared to the cheese control (without A. muciniphila), suggesting a potentially high acceptance among consumers. In conclusion, the developed cheese spread seems to be a promising and suitable food vector to safeguard A. muciniphila viability during refrigerated aerobic storage for at least 21 d with subsequent gastrointestinal passage.

Production of GABA in milk fermented by Bifidobacterium adolescentis strains selected on the bases of their technological and gastrointestinal performance

There is an increasing interest in producing foods enriched in gamma-aminobutyric acid (GABA), due to their purported health promoting attributes. GABA is the main inhibitor neurotransmitter of the central nervous system, and several microbial species are capable to produce it through decarboxylation of glutamate. Among them, several lactic acid bacteria species have been previously investigated as an appealing alternative to produce GABA enriched foods via microbial fermentation. In this work we report for the first time an investigation into the possibility of utilizing high GABA-producing Bifidobacterium adolescentis strains as a mean to produce fermented probiotic milks naturally enriched in GABA. To this end, in silico and in vitro analyses were conducted in a collection of GABA-producing B. adolescentis strains, with the main goal to scrutinize their metabolic and safety traits, including antibiotic resistance patterns, as well as their technological robustness and performance to survive a simulated gastrointestinal passage. One of the strains, IPLA60004, exhibited better survival to lyophilization and cold storage (for up to 4 weeks at 4 °C), as well as survival to gastrointestinal passage, as compared to the other strains under investigation. Besides, the elaboration of milk drinks fermented with this strain, yielded products with the highest GABA concentration and viable bifidobacterial cell counts, achieving conversion rates of the precursor, monosodium glutamate (GMS), up to 70 %. To our knowledge, this is the first report on the elaboration of GABA enriched milks through fermentation with B. adolescentis.

Effect of emulsification/internal gelation-based microencapsulation on the viability of Akkermansia muciniphila upon prolonged storage and simulated gastrointestinal passage

Akkermansia muciniphila is a common human intestinal commensal with a mucin-degrading nature. Its immunomodulatory characteristics and regulatory role of mucus layer and gut barrier integrity highlight the potential benefits of using this bacterium as an interventional player against inflammatory/cardio-metabolic disorders. In this work, we evaluate the effect of microencapsulation by the emulsification/internal gelation method on A. muciniphila survival during aerobic storage (0, 15, 30 and 95 days) and subsequent exposure to simulated gastrointestinal passage, in comparison with that of free cells. The present results show that microencapsulation by internal gelation promotes a 64.4 % entrapment efficacy of A. muciniphila cells (maintaining a 108 order of magnitude for cell viability). Moreover, physical characterization showed that microparticles mean size was 53,5 ± 12,1 µm and, as observed by electron scanning microscopy, microcapsules were spherical in shape. More importantly, as storage time increased, encapsulated A. muciniphila demonstrated higher stability in GI conditions, when compared to its free counterpart. In conclusion, microencapsulation by internal gelation seems to be an appropriate strategy in protecting A. muciniphila against the detrimental gastrointestinal transit after long periods of aerobic refrigerated storage.

Microencapsulating role of whey protein isolate and sucrose in protecting the cell membrane and enhancing survival of probiotic lactobacilli strains during spray drying, storage, and simulated gastrointestinal passage

Limosilactobacillus reuteriKUB-AC5 andLactobacillus johnsoniiKUNN19-2 were spray-dried using whey protein isolate, sucrose, and whey protein isolate combined with sucrose at 130 °C and 150 °C. Differences in membrane fatty acid compositions of probiotics led to differences in cell membrane fluidity, cell injury, and cell survival during spray drying. Combination of whey protein isolate and sucrose improved survival of L. reuteriKUB-AC5, whose cell membrane were more fluid and more sensitive to stress, while whey protein isolate provided the best protection for L. johnsoniiKUNN19-2 against changes in protein secondary structures. Moreover, a relationship was found between membrane damage and the shelf-life ofmicrocapsules. Both microencapsulated cells prepared at the inlet temperature of 130 °C showed an enhanced survival upon simulated gastric digestion compared to the free cells. A thorough understanding of injury sites during spray drying will contribute to establishing optimal conditions and encapsulant formulations that maintain the cells’ viability during spray drying, storage, and gastrointestinal passage.

Spray-Drying Encapsulation of the Live Biotherapeutic Candidate Akkermansia muciniphila DSM 22959 to Survive Aerobic Storage.

Akkermansia muciniphila is regarded as a promising next-generation probiotic or live biotherapeutic candidate. Effective delivery strategies must be developed to ensure high enough viability of the probiotic strain throughout its industrial formulation, distribution chain, shelf-life, and, ultimately, the host’s gastrointestinal tract, where it should exert its beneficial effect(s). Among the possible methodologies, spray-drying is considered industrially attractive regarding its costs, efficiency, and scalability, with the due parameter customization. In this study, spray-drying was explored as a one-step process to encapsulate A. muciniphila DSM 22959, testing the drying settings and three different dairy-based matrices. Microcapsule morphology and size was assessed, and viability throughout storage at 4 or 22 °C and simulated gastrointestinal passage was determined. Akkermansia muciniphila microencapsulation by spray-drying, using 10% skim milk and inlet/outlet temperatures of 150/65 °C, is effective in terms of viability stabilization, both during prolonged aerobic storage and exposure to simulated gastrointestinal passage. Akkermansia muciniphila viability was maintained at around 107 CFU/g up to 28 days at 4 °C under aerobic conditions with viability losses inferior to 1 log reduction. This methodology provides the necessary conditions to efficiently deliver the recommended dose of live A. muciniphila in the human gut as a live biotherapeutic product.

Dose-Response Recovery of Probiotic Strains in Simulated Gastro-Intestinal Passage

The probiotic definition stipulates “adequate amounts”. Here, we investigated the metabolic output and recovery rate of probiotic strains using a simulated upper gastro-intestinal passage and colonic fermentation. Two different doses, 7 × 109 colony forming units (CFU) and 7 × 1010 CFU, of a probiotic mixture (Bifidobacterium lactis Bl-04, Lactobacillus acidophilus La-14, Lactobacillus paracasei Lpc-37, and Lactobacillus plantarum Lp-115) or placebo were tested. The four strains were quantified by qPCR and the metabolites analyzed by gas chromatography. There was a dose-response in the detection of all four strains. There was a slightly larger increase between the two doses for L. paracasei Lpc-37 as compared with the other strains; this may suggest a greater robustness of this strain. Compared with the placebo, the high dose simulations generated more propionic acid and a higher total of short chain fatty acids (SCFA). Higher doses of a species are required to reach measurable increases above the baseline level of this species.

Simulated gastrointestinal conditions increase adhesion ability of Lactobacillus paracasei strains isolated from kefir to Caco-2 cells and mucin

Gastrointestinal conditions along the digestive tract are the main stress to which probiotics administrated orally are exposed because they must survive these adverse conditions and arrive alive to the intestine. Adhesion to epithelium has been considered one of the key criteria for the characterization of probiotics because it extends their residence time in the intestine and as a consequence, can influence the health of the host by modifying the local microbiota or modulating the immune response. Nevertheless, there are very few reports on the adhesion properties to epithelium and mucus of microorganisms after passing through the gastrointestinal tract. In the present work, we evaluate the adhesion ability in vitro of L. paracasei strains isolated from kefir grains after acid and bile stress and we observed that they survive simulated gastrointestinal passage in different levels depending on the strain. L. paracasei CIDCA 8339, 83120 and 83123 were more resistant than L. paracasei CIDCA 83121 and 83124, with a higher susceptibility to simulated gastric conditions. Proteomic analysis of L. paracasei subjected to acid and bile stress revealed that most of the proteins that were positively regulated correspond to the glycolytic pathway enzymes, with an overall effect of stress on the activation of the energy source. Moreover, it is worth to remark that after gastrointestinal passage, L. paracasei strains have increased their ability to adhere to mucin and epithelial cells in vitro being this factor of relevance for maintenance of the strain in the gut environment to exert its probiotic action.

Microencapsulated Starter Culture During Yoghurt Manufacturing, Effect on Technological Features

The potential of living cell microencapsulation in sustaining cells’ viability, functionality and targeted release in gastrointestinal tract is relatively well documented. Differently, the effects exerted by the capsules on cell metabolic activities during fermentation of a food matrix as well as on cell physiology are poorly addressed. This paper aimed at studying the effects of chitosan-alginate capsules (matrix and core-shell) on metabolic activities of Streptococcus thermophilus and probiotic Lactobacillus delbrueckii during milk fermentation for yoghurt production. This food system has been used to monitor growth, acidification kinetics and strain proteolytic activity. Bacterial viability has been monitored during yoghurt storage at 4 °C for 28 days and an in vitro digestion to evaluate the protective effect exerted by the capsules. Furthermore, production of volatile metabolites associated with starter culture activity was monitored by headspace solid-phase microextraction-GC/MS to explore possible influence of microenvironment on cell metabolism. Results indicate that both kinds of capsules influenced at different extent cell functionalities (growth, acidification and proteolysis), while they improve cell viability during yoghurt storage and simulated gastrointestinal passage. The volatile pattern revealed that capsules influenced their production in yoghurt: 12 out of 28 volatiles recovered in yoghurt fermented by free and encapsulated starters had significantly different concentration. However, concentration of the main aroma constituents (e.g. acetaldehyde, diacetyl, acetoin) was not significantly affected. Due to the leakage of bacteria from microcapsules during fermentation, the final product resulted in co-existing of free and still encapsulated cells, with the main advantage of an increased viability during yoghurt storage and simulated digestion of the encapsulated counterpart.

Effect of double alginate microencapsulation on in vitro digestibility and thermal tolerance of Lactobacillus plantarum NCDC201 and L. casei NCDC297

This study was undertaken for the microencapsulation of Lactobacillus plantarum NCDC201 and L. casei NCDC297 into double alginate coatings. Microencapsulated probiotics showed significant improvement in their survivability after simulated gastrointestinal passage and exposure to heat treatments. The resistance in simulated gastric juice (SGJ) (120 min) was 47.50% and 45.82% higher as compared to free cells of L. plantarum NCDC201 and L. casei NCDC297, respectively. After incubation in simulated intestinal juice (SIJ) (120 min), the viable probiotic population was 6.34 log CFU/ml and 6.92 log CFU/ml for microencapsulated L. plantarum NCDC201 and L. casei NCDC297, respectively. Similarly, microencapsulated probiotics showed relevant counts at higher heat exposure (75 °C for 1 and 10 min). SEM results indicated the absence of free bacteria confirming the formation of microcapsules, with spherical morphology, continuous and compact surfaces. ATR-FTIR analysis confirmed the cross linking of the microcapsules by calcium chloride and successful immobilization of the probiotics into the polymer microcapsules. DSC suggested the formation of cross-linking and structure of “egg box” and increase in the melting temperature of microcapsules. This study has concluded that double alginate coating technique enhanced the stability of probiotics at high temperature (75 ± 1 °C) and in simulated gastric and intestinal conditions.

Comparison of different methods for microencapsulation of probiotics

At the Department of Food Technology at Fulda University of Applied Sciences different methods for microencapsulation of Lactobacillus reuteri DSM 20016 were investigated. The aim of these studies was to develop a process to stabilize the probiotic bacteria for storage and to prevent them from the gastric conditions, to ensure that a satisfactory amount of the probiotics could reach their target location, the human intestine. Drying processes like spray drying and freeze drying were tested as well as fluidized bed granulation with optional Wurster coating using different auxiliary materials. As encapsulation material maltodextrine, sweet whey powder or gummi arabicum were used. The coating experiments were performed with an aqueous shellac solution. In the performed studies the fluidized bed bottom spray granulation with an additional Wurster coating turned out to be an encouraging procedure to keep the probiotics in a stable form resistant against gastric conditions. The survival rate in the simulated gastro-intestinal passage could be increased up to the sevenfold amount of the untreated bacteria.

In-vitro probiotic characteristics assessment of feruloyl esterase and glutamate decarboxylase producing Lactobacillus spp. isolated from traditional fermented millet porridge (kambu koozh)

Abstract Feruloyl esterase producing lactic acid bacteria was isolated from pearl millet porridge (kambu koozh) and characterized for its probiotic properties with respect to their biological barrier resistance. Among the five feruloyl esterase hyperproducers, CFR5 ( Lactobacillus fermentum ) was found to tolerate phenol and bile acids and survive in simulated gastro-intestinal passage conditions. Ability of the strain CFR5 to produce β-galactosidase and glutamate decarboxylase enzyme as well as invitro cholesterol reducing properties makes the strain suitable for its utilization in the formulation of functional foods.

Effect of peanut butter matrices on the fate of probiotics during simulated gastrointestinal passage

This study observed the extent to which peanut butter could enhance the viability of selected probiotics during simulated gastrointestinal passage. Full fat and reduced fat peanut butter were inoculated with commercial probiotic product U, N or C at 107 CFU/g. Inoculated products were homogenized in 0.5% NaCl solution. Probiotic products (107 CFU/g) suspended directly in 0.5% NaCl solution served as controls. Digestion in the stomach, upper and lower small intestines was mimicked (each stage for 120 min) with the appropriate enzymes at 37 °C with agitation. Aliquots collected at 30–360 min were enumerated for probiotics. Lower cell populations were observed in samples inoculated with probiotic culture C, followed by U and N (p < 0.05). After 360 min, in peanut butter homogenates, bacterial population from N (5.67 log CFU) was higher than those from U (3.48 log CFU) and C (3.00 log CFU). On average, Streptococcus/Lactococcus species had the highest survivability followed by Bifidobacterium and Lactobacillus species. The fat content of peanut butter did not significantly impact probiotic survivability. Results suggest that peanut butter matrices could protect probiotic bacteria during simulated gastrointestinal passage and could be used as a vehicle for probiotic delivery.

Microencapsulation by vibrating technology of the probiotic strain Lactobacillus reuteri DSM 17938 to enhance its survival in foods and in gastrointestinal environment

The probiotic strain Lactobacillus reuteri DSM 17938 was microencapsulated in alginate (MC) and in chitosan-alginate (CMC) matrix by vibrating technology with the main purpose to improve its tolerance to adverse environments. Microencapsulation procedure proved to not affect cell viability as indicated by a high encapsulation yield (97%) as well as further chitosan coating that minimally affected cell population. The viability of microencapsulated probiotic was evaluated under several stress conditions such as 28 days storage in different solutions at 4 and 20 °C, simulated gastrointestinal passage and osmotic stress conditions. Also reuterin production and diffusion through microcapsules were assessed. Results of survival assays showed that in most of tested conditions microencapsulation significantly (P < 0.05) improved L. reuteri DSM 17938 resistance. Furthermore, microcapsules were freeze dried in skimmed milk and no significant variations in cell viability and microcapsules morphology were observed. Morphological analysis revealed some differences between MC and CMC although both microcapsules had a diameter of 110 ± 5 μm, a spherical shape and a uniform and continuous surface. This study proved that microencapsulation by n chitosan-alginate matrix could improve tolerance of L. reuteri DSM 17938 towards stress conditions encountered in food processing as well as preserve its functional properties.

Evaluation of the functional potential of Weissella and Lactobacillus isolates obtained from Nigerian traditional fermented foods and cow's intestine

The characterisation of 24 lactic acid bacteria (LAB) isolates from Nigerian traditional fermented dairy foods, including some cow's intestine isolates, was conducted in order to select isolates for potential use as probiotics. LAB isolates were identified by partial sequencing the 16S rRNA gene as belonging to the species Lactobacillus paracasei, Lactobacillus brevis and mainly Weissella confusa. At the end of a characterisation process, 2 L. paracasei and 2 W. confusa isolates were selected, and their resistance to a simulated gastrointestinal digestion and their ability to adhere to eukaryotic cell lines were assessed. The survival to the simulated gastrointestinal passage was higher when bacterial suspensions were made in skimmed milk (2.0±0.8 log units reduction) or at the simulated gastric juice pH 3 (2.7±0.9 log units reduction) than at pH 2.0 (5.5±0.7 log units reduction). Adhesion of LAB to both intestinal and vaginal epithelial models was comparable or higher than that of the reference Lactobacillus rhamnosus GG. However, some of the isolates increased the adhesion of the pathogen Escherichia coli LMG2092 to HT-29 and HeLa monolayers. Overall, isolates L. paracasei UI14 and W. confusa UI7 are good candidates for further studying potential benefits that support their use as probiotics. This is one of the few articles reporting the characterisation and the probiotic potential of Weissella, although more studies are needed in order to establish their safety for potential probiotic applications.

Ein enteritis-oralimpfstoff aus zwölf hitzeinaktivierten enterobakteriazeen 2. Mitteilung: Die immunogenität nach behandlung mit simuliertem magen- und darmsaft geprüft im aktiven mäuseschutzversuch

In the first communication (3), we reported on the conception, the composition, and the efficacy of the polyvalent oral vaccine from 6 strains of salmonellae, 2 strains of shigellae, and 4 strains of dyspepsia coli. The inactivation took place at 100°C/3 min. The question going to be answered in this communication was as follows: Does the immunogenicity of the vaccine decreased during the gastrointestinal passage under influence of acid and enzymes? We allowed the vaccine to react with simulated gastric juice and/or pepsin at pH = 3 and 37 C/60 min on the one hand and with simulated intestinal juice and/or pancreatin at pH = 7 and 37°C/180 min on the other hand either individually or in combination. The vaccinal preparations produced this way were examined for their immunogenicity in the mouse protection test. The mice were orally immunized with the aid of a probang for ten times (total dosis = 3.75 × 1010 germs) and intraperitoneally infected with the virulent enteropathogenic strain of E. coli 2,380 being contained in the twelvefold vaccine on the 10. day after the last oral vaccination. In the main test, 70.4 % of 351 non-vaccinated control animals died. 277 mice were immunized with the vaccine having been treated in the strongest way (gastric juice + pepsin + intestinal juice + pancreatin); 4.0 % of those died which is an index of efficacy of 94.3. The mice immunized with untreated vaccine served as positive controls and were protected in the same way; 3.1% of 225 mice died (index of efficacy = 95.6). The results show that the simulated gastro-intestinal passage did not have a negative influence upon the immunogenicity of the polyvalent vaccine.