In vitro models as a tool to study the role of gut microbiota in obesity.

Obesity, a highly prevalent condition worldwide that leads to the development of multiple metabolic diseases, has been related to gut microbial dysbiosis. To understand this correlation, in vivo models have been extremely useful. However, its use is limited by associated ethical concerns, high costs, low representativeness, and low reproducibility. Therefore, new and improved in vitro models have been developed in recent years, representing a promising tool in the study of the role of gut microbiota modulation in weight management and metabolic health. This review aims to provide an update on the main findings obtained in vitro regarding gut microbiota modulation with probiotics, and food compounds, and its interaction with the host metabolism, associated with obesity. Available in vitro colon models currently used to study obesity are discussed, including batch and dynamic fermentation systems, and models that allow the study of microbiota-host interactions using cell cultures. In vitro models have demonstrated that homeostatic microbiota may help overcome obesity by producing satiety-related neurotransmitters and metabolites that protect the gut barrier and improve the metabolic activity of adipose tissue. In vitro models may be the key to finding new treatments for obesity-related disorders.

Novel Bio-Functional Aloe vera Beverages Fermented by Probiotic Enterococcus faecium and Lactobacillus lactis.

Aloe vera has been medicinally used for centuries. Its bioactive compounds have been shown to be very effective in the treatment of numerous diseases. In this work, a novel functional beverage was developed and characterized to combine the health benefits of probiotic bacteria with the Aloe vera plant itself. Two Aloe vera juices were obtained by fermentation either by a novel isolated Enterococcus faecium or a commercial Lactococcus lactis. The extraction of Aloe vera biocompounds for further fermentation was optimized. Extraction with water plus cellulase enhanced the carbohydrates and phenolic compounds in the obtained extracts. The biotransformation of the bioactive compounds from the extracts during fermentation was assessed. Both probiotic bacteria were able to grow on the Aloe vera extract. Lactic acid and short-chain fatty acids (SCFA) together with fourteen individual phenolic compounds were quantified in the produced Aloe vera juice, mainly epicatechin, aloin, ellagic acid, and hesperidin. The amount of total phenolic compounds was maintained through fermentation. The antioxidant activity was significantly increased in the produced juice by the ABTS method. The novel produced Aloe vera juice showed great potential as a functional beverage containing probiotics, prebiotics, SCFA, and phenolic compounds in its final composition.

Spontaneously fermented traditional beverages as a source of bioactive compounds: an overview.

Fermented food has been present throughout history, since fermentation not only helps preserving food, but also provides specific organoleptic characteristics typically associated to these foods. Most of the traditional fermented foods and artisanal beverages are produced by spontaneous generation, meaning no control of the microbiota, or the substrate used. Nevertheless, even not being standardized, they are an important source of bioactive compounds, such as antioxidant compounds, bioactive beeps, short chain fatty acids, amino acids, vitamins, and minerals. This review compiles a list of relevant traditional fermented beverages around the world, aiming to detail the fermentation process itself-including source of microorganisms, substrates, produced metabolites and the operational conditions involved. As well as to list the bioactive compounds present in each fermented food, together with their impact in the human health. Traditional fermented beverages from Mexico will be highlighted. These compounds are of high interest for the food, pharmaceutical and cosmetics industry. To scale-up the home fermentation processes, it is necessary to fully understand the microbiology and biochemistry behind these traditional products. The use of good quality raw materials with standardized methodologies and defined microorganisms, may improve and increase the production of the desirable bioactive compounds and open a market for novel functional products.

New Trends and Technological Challenges in the Industrial Production and Purification of Fructo-oligosaccharides.

An increased commercial interest in fructo-oligosaccharides (FOS) has emerged in the last decade due to their prebiotic activity. At large scale, the FOS are produced by microbial enzymes from sucrose. A mixture of FOS and other saccharides is obtained in this process. The presence of such saccharides reduces the prebiotic, caloric, and cariogenic value of the final product. Therefore, many efforts have been conducted to obtain a product with increased FOS purity. This review comprises the most important technological and physicochemical aspects including FOS production and recovery processes; safety, dose and health claims concerning its intake; and commercially available FOS.

Akkermansia muciniphila and Parabacteroides distasonis synergistically protect from colitis by promoting ILC3 in the gut.

Inflammatory bowel disease (IBD) is a group of inflammatory conditions of the gastrointestinal tract. The etiology of IBD remains elusive, but the disease is suggested to arise from the interaction of environmental and genetic factors that trigger inadequate immune responses and inflammation in the intestine. The gut microbiome majorly contributes to disease as an environmental variable, and although some causative bacteria are identified, little is known about which specific members of the microbiome aid in the intestinal epithelial barrier function to protect from disease. While chemically inducing colitis in mice from two distinct animal facilities, we serendipitously found that mice in one facility showed remarkable resistance to disease development, which was associated with increased markers of epithelial barrier integrity. Importantly, we show that Akkermansia muciniphila and Parabacteroides distasonis were significantly increased in the microbiota of resistant mice. To causally connect these microbes to protection against disease, we colonized susceptible mice with the two bacterial species. Our results demonstrate that A. muciniphila and P. distasonis synergistically drive a protective effect in both acute and chronic models of colitis by boosting the frequency of type 3 innate lymphoid cells in the colon and by improving gut epithelial integrity. Altogether, our work reveals a combined effort of commensal microbes in offering protection against severe intestinal inflammation by shaping gut immunity and by enhancing intestinal epithelial barrier stability. Our study highlights the beneficial role of gut bacteria in dictating intestinal homeostasis, which is an important step toward employing microbiome-driven therapeutic approaches for IBD clinical management. IMPORTANCE: The contribution of the gut microbiome to the balance between homeostasis and inflammation is widely known. Nevertheless, the etiology of inflammatory bowel disease, which is known to be influenced by genetics, immune response, and environmental cues, remains unclear. Unlocking novel players involved in the dictation of a protective gut, namely, in the microbiota component, is therefore crucial to develop novel strategies to tackle IBD. Herein, we revealed a synergistic interaction between two commensal bacterial strains, Akkermansia muciniphila and Parabacteroides distasonis, which induce protection against both acute and chronic models of colitis induction, by enhancing epithelial barrier integrity and promoting group 3 innate lymphoid cells in the colonic mucosa. This study provides a novel insight on how commensal bacteria can beneficially act to promote intestinal homeostasis, which may open new avenues toward the use of microbiome-derived strategies to tackle IBD.

Development of a functional prebiotic strawberry preparation by in situ enzymatic conversion of sucrose into fructo-oligosaccharides.

Food industry has been pressed to develop products with reduced sugar and low caloric value, while maintaining unchanged their rheological and physicochemical properties. The development of a strawberry preparation for the dairy industry, with prebiotic functionality, was herein investigated by in situ conversion of its intrinsic sucrose content into prebiotic fructo-oligosaccharides (FOS). Two commercial enzymatic complexes, Viscozyme® L and Pectinex® Ultra SP-L, were evaluated for the synthesis of FOS. Operational parameters such as temperature, pH, and enzyme:substrate ratio (E:S) were optimized to maximize FOS yield. The rheological and physicochemical properties of the obtained strawberry preparation were evaluated. For functional analysis, the resistance of FOS to the harsh conditions of the gastro-intestinal digestion was evaluated by applying the standardized INFOGEST static protocol. At optimal conditions (60 ℃, pH 5.0), Pectinex® produced 265 ± 3 g·L-1 FOS, yielding 0.57 ± 0.01 gFOS·ginitial sucrose-1 after 7 h reaction (E:S:1:40); and Viscozyme® produced 295 ± 1 g·L-1 FOS, yielding 0.66 ± 0.00 gFOS·ginitial sucrose-1 after 5 h (E:S:1:30). The obtained strawberry preparations contained more than 50 %(w/w) prebiotic FOS incorporated (DP 3-5), with 80 % reduction of its sucrose content. The caloric value was therefore reduced by 26-31 %. FOS showed resistance to gastrointestinal digestion being only slightly hydrolysed (<10 %). 1F-Fructofuranosylnystose was not digested at any phase of the digestion. Although the physicochemical properties of the prebiotic preparations were different from the original one, parameters such as the lower °Brix, water activity, consistency and viscosity, and its different color, may be easily adjusted. Results indicate that in situ synthesis strategies are efficient alternatives in the manufacture of reduced sugar and low-caloric food products with prebiotic potential.

Modulation of Designed Gut Bacterial Communities by Prebiotics and the Impact of Their Metabolites on Intestinal Cells.

The impact of prebiotics on human health is associated with their capacity to modulate microbiota, improving beneficial microbiota-host interactions. Herein, the prebiotic potential of microbial-fructo-oligosaccharides (microbial-FOSs) produced by a co-culture of Aspergillus ibericus plus Saccharomyces cerevisiae was evaluated on seven- and nine-strain bacterial consortia (7SC and 9SC, respectively), designed to represent the human gut microbiota. The 7SC was composed of Bacteroides dorei, Bacteroides vulgatus, Bifidobacterium adolescentis, Bifidobacterium longum, Escherichia coli, Lactobacillus acidophilus, and Lactobacillus rhamnosus. The 9SC also comprised the aforementioned bacteria, with the addition of Bacteroides thetaiotaomicron and Roseburia faecis. The effect of microbial-FOSs on the metabolic activity of intestinal Caco-2/HT29-MTX-E12 co-culture was also assessed. The results showed that microbial-FOS selectively promoted the growth of probiotic bacteria and completely suppressed the growth of E. coli. The microbial-FOSs promoted the highest production rates of lactate and total short-chain fatty acids (SCFA) as compared to the commercial prebiotic Frutalose® OFP. Butyrate was only produced in the 9SC consortium, which included the R. faecis-a butyrate-producing bacteria. The inclusion of this bacteria plus another Bacteroides in the 9SC promoted a greater metabolic activity in the Caco-2/HT29-MTX-E12 co-culture. The microbial-FOSs showed potential as promising prebiotics as they selectively promote the growth of probiotic bacteria, producing high concentrations of SCFA, and stimulating the metabolic activity of gut cells.

Detoxification of ochratoxin A and zearalenone by Pleurotus ostreatus during in vitro gastrointestinal digestion.

Powdered Pleurotusostreatus (PO) was tested for the detoxification of ochratoxin A (OTA) and zearalenone (ZEN) through in vitro gastrointestinal digestion in the absence and presence of ground feed and cornmeal. Ochratoxin α was detected in the chromatograms after OTA elimination, suggesting the presence of an OTA-hydrolytic enzyme in the PO, whereas ZEN was adsorbed by PO. OTA was totally eliminated at pH 5 and 7, while ZEN was better adsorbed at pH 3. In simulated gastrointestinal conditions conducted without feed matrices, PO eliminated OTA by 85% and ZEN by 23% at the end of the intestinal phase. When digestions were conducted with ground feed and cornmeal, PO eliminated OTA by 13 and 34%, and ZEN by 20 and 2%, respectively. PO demonstrated great potential for OTA detoxification, but the feed matrices adsorbed the mycotoxins, reducing their bioaccessibility considerably in the oral and gastric phases and negatively influencing PO detoxification capacity.

Effect of Gastrointestinal Digestion on the Bioaccessibility of Phenolic Compounds and Antioxidant Activity of Fermented Aloe vera Juices.

Plant-based beverages are enriched by the fermentation process. However, their biocompounds are transformed during gastrointestinal digestion, improving their bioaccessibility, which is of primary importance when considering the associated health benefits. This study aimed to evaluate the effect of in vitro gastrointestinal digestion on phenolic compound bioaccessibility and antioxidant activity of novel Aloe vera juices fermented by probiotic Enterococcus faecium and Lactococcus lactis. Aloe vera juices were digested using the standardized static INFOGEST protocol. During digestion, phenolic compounds and antioxidant activity (DPPH, ABTS, and FRAP) were accessed. The digestion process was seen to significantly increase the total phenolic content of the fermented Aloe vera juices. The fermentation of Aloe vera increased the bioaccessibility of juice biocompounds, particularly for kaempferol, ellagic acid, resveratrol, hesperidin, ferulic acid, and aloin. The phenolics released during digestion were able to reduce the oxidative radicals assessed by ABTS and FRAP tests, increasing the antioxidant action in the intestine, where they are absorbed. The fermentation of Aloe vera by probiotics is an excellent process to increase the bioavailability of beverages, resulting in natural added-value functional products.

Successive Fermentation of Aguamiel and Molasses by Aspergillus oryzae and Saccharomyces cerevisiae to Obtain High Purity Fructooligosaccharides.

Fructooligosaccharides (FOS) are usually synthesized with pure enzymes using highly concentrated sucrose solutions. In this work, low-cost aguamiel and molasses were explored as sucrose alternatives to produce FOS, via whole-cell fermentation, with an Aspergillus oryzae DIA-MF strain. FOS production process was optimized through a central composite experimental design, with two independent variables: initial sucrose concentration in a medium composed of aguamiel and molasses (AgMe), and inoculum concentration. The optimized process-165 g/L initial sucrose in AgMe (adjusted with concentrated molasses) and 1 × 107 spores/mL inoculum concentration-resulted in an FOS production of 119 ± 12 g/L and a yield of 0.64 ± 0.05 g FOS/g GFi. Among the FOSs produced were kestose, nystose, 1-fructofuranosyl-nystose, and potentially a novel trisaccharide produced by this strain. To reduce the content of mono- and disaccharides in the mixture, run a successive fermentation was run with two Saccharomyces cerevisiae strains. Fermentations run with S. cerevisiae S227 improved FOS purity in the mixture from 39 ± 3% to 61.0 ± 0.6% (w/w) after 16 h of fermentation. This study showed that agro-industrial wastes such as molasses with aguamiel are excellent alternatives as substrate sources for the production of prebiotic FOS, resulting in a lower-cost process.

Evaluation of Microbial-Fructo-Oligosaccharides Metabolism by Human Gut Microbiota Fermentation as Compared to Commercial Inulin-Derived Oligosaccharides.

The prebiotic potential of fructo-oligosaccharides (microbial-FOS) produced by a newly isolated Aspergillus ibericus, and purified by Saccharomyces cerevisiae YIL162 W, was evaluated. Their chemical structure and functionality were compared to a non-microbial commercial FOS sample. Prebiotics were fermented in vitro by fecal microbiota of five healthy volunteers. Microbial-FOS significantly stimulated the growth of Bifidobacterium probiotic strains, triggering a beneficial effect on gut microbiota composition. A higher amount of total short-chain fatty acids (SCFA) was produced by microbial-FOS fermentation as compared to commercial-FOS, particularly propionate and butyrate. Inulin neoseries oligosaccharides, with a degree of polymerization (DP) up to 5 (e.g., neokestose and neonystose), were identified only in the microbial-FOS mixture. More than 10% of the microbial-oligosaccharides showed a DP higher than 5. Differences identified in the structures of the FOS samples may explain their different functionalities. Results indicate that microbial-FOS exhibit promising potential as nutraceutical ingredients for positive gut microbiota modulation.

Mesalazine and inflammatory bowel disease - From well-established therapies to progress beyond the state of the art.

Inflammatory bowel disease incidence has been constantly rising for the past few decades. Current therapies attempt to mitigate its symptoms since no cure is established. The most commonly prescribed drug for these patients is 5-aminosalicylic acid (5-ASA). Due to the low rate and seriousness of side effects compared to other therapies, 5-ASA is still largely prescribed in many stages of inflammatory bowel disease, including scenarios where evidence suggests low effectiveness. Although commercialized formulations have come a long way in improving pharmacokinetics, it is still necessary to design and develop novel delivery systems capable of increasing effectiveness at different stages of the disease. In particular, micro- and nano-sized particles might be the key to its success in Crohn's disease and in more serious disease stages. This review provides an overview on the clinical significance of 5-ASA formulations, its limitations, challenges, and the most recent micro- and nanoparticle delivery systems being designed for its controlled release. Emergent alternatives for 5-ASA are also discussed, as well as the future prospects for its application in inflammatory bowel disease therapies.

Evaluation of functional and nutritional potential of a protein concentrate from Pleurotus ostreatus mushroom.

Edible mushrooms used as a protein-rich food may be an attractive alternative to conventional protein sources, while promoting its valorization. This work aimed to obtain a protein concentrate from a Pleurotus ostreatus mushroom flour, its characterization, and nutritional and functional properties evaluation. Methodologies applied for extraction and precipitation of protein were optimized - pH 4 and 12, respectively; and flour-solvent ratio of 1:20 w/v. The protein density was increased by 78%. P. ostreatus flour and concentrate were characterized by proximal composition. The content of total phenolic compounds in the protein concentrate decreased, leading to a positive effect on protein digestibility, while the DPPH radical scavenging activity was not significantly affected. Peptides with molecular weights from 12 to 35 kDa, with possible bioactivity, were identified by electrophoresis. Protein digestibility assessed by in vitro gastrointestinal digestion showed a 4.2-fold higher hydrolysis degree in the protein concentrate than the flour.

Valorisation of rejected unripe plantain fruits of Musa AAB Simmonds: from nutritional characterisation to the conceptual process design for prebiotic production.

The increasing consumption of plantain fruits with specific quality standards generates high agricultural waste. This work aimed at valorising the rejected unripe pulp of Dominico-Hartón plantain fruits (Musa AAB Simmonds). The pulp was characterised physico-chemically, thermally and functionally. The data gathered experimentally and collected from different databases were used to design a production process of isomalto-oligosaccharides (IMO) syrup. The plantain flour contains high levels of starch (87 ± 2%) and amylose (31.2 ± 0.8%). The flour showed stability at high temperatures (pasting temperature of 79.26 ± 0.02 °C), allowing its use in high temperature processes. In vitro gastrointestinal digestion of the plantain flour showed that when cooked, the glycemic index of the flour increased from 47.7 ± 2.2 to 84.2 ± 1.8, while its resistant starch content only slightly decreased from 71.7 ± 1% to 52.6 ± 2%, suggesting that this type of flour preserves high content of dietary fibre after digestion. The conceptual process design showed that 24.48 g of IMO are theoretically obtained from 53.24 g of plantain flour maltose. These results suggest that the rejected plantain pulp holds high potential as an ingredient for the production of prebiotic compounds such as IMO.

Edible mushrooms as a novel protein source for functional foods.

Fast demographic growth has led to increasing interest in low-cost alternative protein sources to meet population needs. Consequently, scientific researchers have been focused on finding under-exploited sources of protein, alternative to those of animal origin. Usually plant proteins have been used for this purpose; however, most of them are not considered to be high quality due to their lack of some essential amino acids. Mushroom proteins usually have a complete essential amino acid profile, which may cover the dietetic requirements, as well as may have certain economic advantages as compared to animal and plant sources. Many mushrooms also have the ability to grow in agro-industrial waste, as submerged cultures, reaching high yields in a short period of time. Edible mushrooms can be processed to obtain a wide variety of food products enriched with high quality protein, which may have as well improved functional properties, giving them an added value. This review discusses the use of mushrooms as sustainable functional food.

Microbial co-culturing strategies for fructo-oligosaccharide production.

Fructo-oligosaccharide (FOS) mixtures produced by fermentation contain large amounts of non-prebiotic sugars. Here we propose a mixed culture of Aureobasidium pullulans and Saccharomyces cerevisiae cells to produce FOS and consume the small saccharides simultaneously, thereby increasing FOS purity in the mixture. The use of immobilised A. pullulans in co-culture with encapsulated S. cerevisiae, inoculated after 10 h fermentation, enhanced FOS production in a 5 L bioreactor. Using this strategy, a maximal FOS concentration of 119 g L-1, and yield of 0.59 gFOS gsucrose-1, were obtained after 20 h fermentation, increasing FOS productivity from about 4.9 to 5.9 gFOS L-1 h-1 compared to a control fermentation of immobilized A. pullulans in monoculture. In addition, the encapsulated S. cerevisiae cells were able to decrease the glucose in the medium to about 7.6% (w/w) after 63 h fermentation. This provided a final fermentation mixture with 2.0% (w/w) sucrose and a FOS purity of over 67.0% (w/w). Moreover, a concentration of up to 58.0 g L-1 of ethanol was obtained through the enzymatic transformation of glucose. The resulting pre-purified FOS mixture could improve the separation and purification of FOS in downstream treatments, such as simulated moving bed chromatography.

Process development for the production of prebiotic fructo-oligosaccharides by penicillium citreonigrum.

A new isolated P. citreonigrum URM 4459 was selected to produce fructooligosaccharides (FOS) in an efficient, economical and fast one-step fermentation. Optimal culture conditions were stablished by experimental design. Experiments run in bioreactor resulted in a high yield, content, productivity and purity of FOS (0.65 ±â€¯0.06 gFOS/ginitial Sucrose, 126.3 ±â€¯0.1 g/L, 2.28 ±â€¯0.08 g/L.h and 61 ±â€¯0%). The FOS mixture was purified up to 92% (w/w) with an activated charcoal column. FOS produced were able to promote lactobacilli and bifidobacteria growth. Higher bacteria cell density was obtained for microbial-FOS mixtures than commercial Raftilose® P95. Some strains grew even faster in the FOS mixture produced than in all other carbon sources. FOS were resistant to the simulated gastrointestinal conditions. A high amount of a reducing trisaccharide was identified in the FOS produced mixture, possibly neokestose, which may explain the great prebiotic potential of the FOS.

Trypsin purification using magnetic particles of azocasein-iron composite.

This work presents an inexpensive, simple and fast procedure to purify trypsin based on affinity binding with ferromagnetic particles of azocasein composite (mAzo). Crude extract was obtained from intestines of fish Nile tilapia (Oreochromis niloticus) homogenized in buffer (01g tissue/ml). This extract was exposed to 100mg of mAzo and washed to remove unbound proteins by magnetic field. Trypsin was leached off under high ionic strength (3M NaCl). Preparation was achieved containing specific activity about 60 times higher than that of the crude extract. SDS-PAGE showed that the purified protein had molecular weight (24kDa) in concordance with the literature for the Nile tilapia trypsin. The mAzo composite can be reused and applied to purify trypsin from other sources.

New improved method for fructooligosaccharides production by Aureobasidium pullulans.

Fructooligosaccharides are prebiotics with numerous health benefits within which the improvement of gut microbiota balance can be highlighted, playing a key role in individual health. In this study, an integrated one-stage method for FOS production via sucrose fermentation by Aureobasidium pullulans was developed and optimized using experimental design tools. Optimization of temperature and agitation speed for maximizing the FOS production was performed using response surface methodology. Temperature was found to be the most significant parameter. The optimum fermentation conditions were found to be 32 °C and 385 rpm. Under these conditions, the model predicted a total FOS production yield of 64.7 gFOS/gsucrose. The model was validated at optimal conditions in order to check its adequacy and accuracy and an experimental yield of 64.1 (±0.0) gFOS/gsucrose was obtained. A significant improvement of the total FOS production yields by A. pullulans using a one-stage process was obtained.