A comprehensive review of the characterization, host interactions, and stabilization advancements on probiotics: Addressing the challenges in functional food diversification.

The demand for functional food is rising in tandem with the prevalence of chronic diseases. Probiotics play a crucial role in functional food development, yet their ability to confer health benefits to the host remains a topic of debate according to Food and Agriculture Organization/World Health Organization requirements. The application of culturomics, innovative isolation techniques, within the realm of probiotics is increasingly deemed essential for fully harnessing the latent potential of microbial reservoirs. Nevertheless, its application remains confined predominantly to human fecal sources. Following the integration of probiogenomics, significant advancements have been made in the safety assessment of probiotics. However, the adoption of novel probiotic microorganisms has yet to match the requisite pace. Progress in research concerning host-probiotic interactions by employing omics technologies, particularly in animal models, is notable. Nonetheless, the comprehensive elucidation of mechanisms of action and human trial studies are lagging behind. Additionally, the viability of probiotics, spanning from their production as functional foods to their transit to the human colon, has markedly improved through encapsulation techniques. Nevertheless, opportunities for exploration persist regarding alternative coating materials and diverse encapsulation methodologies. Furthermore, there is a discernible transition in the domain of probiotic-based functional foods, shifting away from a primarily dairy-centric focus toward inclusion in a broader array of food categories. This comprehensive review addresses critical issues ranging from isolation sources and novel techniques to the final functional food developments. while doing so, it explores probiogenomics applications for probiotic characterization, investigations into host-probiotic interactions, and strategies for probiotic stabilization under harsh environmental conditions.

Microencapsulation of Lactobacillus plantarum by spray drying: Protective effects during simulated food processing, gastrointestinal conditions, and in kefir.

Probiotics are incorporated into food products because of numerous favorable effects on human health. The viability of probiotics is often affected by unfavorable interference during processing. The encapsulation can provide protection to probiotics during mechanical processing, storage, and gastrointestinal digestion. This study aimed to evaluate the protective effect of whey protein isolate (WPI) and dextran (DX) conjugates for Lactobacillus plantarum. The WPI-DX conjugate was prepared by Maillard-based glycation and confirmed by gel electrophoresis. Extending the heating time from 1 to 5 h decreased the content of tryptophan residues and increased the amide I and amide II bands. The enhanced protective ability of Maillard reaction products (MRPs) for L. plantarum was observed under conditions of stress (pH, heat, and salt) and in vitro digestion. In situ viability tests showed that encapsulation improved the survival of bacteria in kefir during 15 days of storage at 4 °C. Overall, our results provide valuable information for the development of functional probiotic food products.

Chickpea-Derived Prebiotic Substances Trigger Biofilm Formation by Bacillus subtilis.

Chickpea-based foods are known for their low allergenicity and rich nutritional package. As an essential dietary legume, chickpea is often processed into milk or hummus or as an industrial source of protein and starch. The current study explores the feasibility of using the chickpea-derived prebiotic substances as a scaffold for growing Bacillus subtilis (a prospective probiotic bacterium) to develop synbiotic chickpea-based functional food. We report that the chickpea-derived fibers enhance the formation of the B. subtilis biofilms and the production of the antimicrobial pigment pulcherrimin. Furthermore, electron micrograph imaging confirms the bacterial embedding onto the chickpea fibers, which may provide a survival tactic to shield and protect the bacterial population from environmental insults. Overall, it is believed that chickpea-derived prebiotic substances provide a staple basis for developing functional probiotics and synbiotic food.

Encapsulation and dispersion of Lactobacillus acidophilus in a chocolate coating as a strategy for maintaining cell viability in cereal bars.

Flour from Pereskia aculeata leaf and green banana were used as ingredients in the formulation of a cereal bar with added Lactobacillus acidophilus LA02-ID-1688. Encapsulation in a calcium-alginate hydrogel reinforced with magnesium hydroxide was used as a strategy to protect the probiotic cells under gastrointestinal conditions and to prolong shelf-life. The results are relevant especially for maintaining cell viability during shelf-life; a challenge for the food industry in relation to dry probiotic products. Encapsulation promoted the protection of probiotic cells in simulated gastric and intestinal conditions, allowing the maintenance of high viable cell counts (> 10 log CFU, colony forming unit). Encapsulation also contributed to cellular protection under extreme temperature conditions, with reductions of cell viability of < 1 logarithmic cycle when the capsules were subjected to 55ºC/10 min. Even at 75ºC/10 min, encapsulation protected the probiotic cells 3-times greater than the free-cells. The food bar proved to be rich in dietary fiber (19 g 100 g-1), lipids (12.63 g 100 g-1) and showed an appreciable protein content (5.44 g 100 g-1). A high viable probiotic cell count on storage over 120 days (12.54 log CFU) was observed, maintaining a probiotic survival rate > 90% and viability levels sufficient to promote health benefits.

Evaluation of the Cholesterol-Lowering Mechanism of Enterococcus faecium Strain 132 and Lactobacillus paracasei Strain 201 in Hypercholesterolemia Rats.

Hypercholesterolemia can cause many diseases, but it can effectively regulated by Lactobacillus. This study aimed to evaluate the cholesterol-lowering mechanism of Enterococcus faecium strain 132 and Lactobacillusparacasei strain 201. These results showed that both the strains decreased serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), liver TC and TG and increased fecal TC, TG and total bile acid (TBA) levels. Additionally, both strains also reduced glutamic-pyruvic transaminase (ALT), glutamic oxaloacetic transaminase (AST) and levels of tissue inflammation levels to improve the lipid profile, and they reduced fat accumulation partially by alleviating inflammatory responses. Furthermore, both strains regulated the expression of the CYP8B1, CYP7A1, SREBP-1, SCD1 and LDL-R gene to promote cholesterol metabolism and reduce TG accumulation. Interventions with both strains also altered the gut microbiota, and decreasing the abundance of Veillonellaceae, Erysipelotrichaceae and Prevotella. Furthermore, fecal acetic acid and propionic acid were increased by this intervention. Overall, the results suggested that E. faecium strain 132 and L. paracasei strain 201 can alleviate hypercholesterolemia in rats and might be applied as a new type of hypercholesterolemia agent in functional foods.

Production of L-carnitine-enriched edible filamentous fungal biomass through submerged cultivation.

The edible filamentous fungi are hot candidate for future supply of functional food and feed with e.g. protein, essential amino acids, and compounds with immunostimulant activity. L-carnitine that plays a crucial role in energy metabolism represents a functional compound normally produced by Zygomycetes filamentous fungus Rhizopus oligosporus in solid-state fermentation. The present study provides the first insights on production of L-carnitine-enriched edible fungal biomass through submerged cultivation of several Ascomycetes and Zygomycetes including Aspergillus oryzae, Neurospora intermedia, Rhizopus oryzae, and Rhizopus oligosporus. A. oryzae with 3 mg L-carnitine yield per gram of fungal biomass, indicates great potential on production of this bioactive compound which is remarkably higher than the other tested fungi in this work and also previous studies. In addition to fungal strain, other factors such as cultivation time and presence of yeast extract were found to play a role. Further studies on submerged growth optimization of A. oryzae in both high-quality recipes and in medium based on low-value substrates are proposed in order to clarify its potential for production of L-carnitine-enriched fungal biomass.

Postbiotics-parabiotics: the new horizons in microbial biotherapy and functional foods.

Probiotics have several health benefits by modulating gut microbiome; however, techno-functional limitations such as viability controls have hampered their full potential applications in the food and pharmaceutical sectors. Therefore, the focus is gradually shifting from viable probiotic bacteria towards non-viable paraprobiotics and/or probiotics derived biomolecules, so-called postbiotics. Paraprobiotics and postbiotics are the emerging concepts in the functional foods field because they impart an array of health-promoting properties. Although, these terms are not well defined, however, for time being these terms have been defined as here. The postbiotics are the complex mixture of metabolic products secreted by probiotics in cell-free supernatants such as enzymes, secreted proteins, short chain fatty acids, vitamins, secreted biosurfactants, amino acids, peptides, organic acids, etc. While, the paraprobiotics are the inactivated microbial cells of probiotics (intact or ruptured containing cell components such as peptidoglycans, teichoic acids, surface proteins, etc.) or crude cell extracts (i.e. with complex chemical composition)". However, in many instances postbiotics have been used for whole category of postbiotics and parabiotics. These elicit several advantages over probiotics like; (i) availability in their pure form, (ii) ease in production and storage, (iii) availability of production process for industrial-scale-up, (iv) specific mechanism of action, (v) better accessibility of Microbes Associated Molecular Pattern (MAMP) during recognition and interaction with Pattern Recognition Receptors (PRR) and (vi) more likely to trigger only the targeted responses by specific ligand-receptor interactions. The current review comprehensively summarizes and discussed various methodologies implied to extract, purify, and identification of paraprobiotic and postbiotic compounds and their potential health benefits.

Food-grade expression of nattokinase in Lactobacillus delbrueckii subsp. bulgaricus and its thrombolytic activity in vitro.

OBJECTIVES: To produce nattokinase in a food-grade expression system and evaluate its thrombolytic activity in vitro. RESULTS: No nattokinase activity from reconstituted strains was observed in simulated gastric juice, but the enzyme was stable in intestinal fluid, the relative activity of which was found to be 60% after 4 h. Due to the nattokinase being produced intracellularly by recombinant bacterial strains, the persistence of the bacteria in gastric juice ensured transmission of the nattokinase into intestinal juice. Because of subsequent disintegration of the bacteria, the highest nattokinase activity was observed after 3 h at approximately 32%, following its carriage within the recombinant strains to the intestinal fluid. CONCLUSIONS: This study demonstrated that nattokinase from recombinant strains exhibited good thrombolytic activity in vitro and may be used by the dairy fermentation industry for the development of novel thrombolytic functional foods.

Riboflavin-overproducing lactobacilli for the enrichment of fermented soymilk: insights into improved nutritional and functional attributes.

The influence of riboflavin (B2)-overproducing lactobacilli on the antioxidant status, isoflavone conversion, off-flavor reduction, amino acid profile, and viscosity of B2-bio-enriched fermented soymilk was investigated. Results showed that B2 in fermented soymilk was notably increased from 0.2 to 3.8 µg/mL for Lactobacillus fermentum UFG169 and to 1.9 µg/mL for Lactobacillus plantarum UFG10. The apparent viscosity significantly changed with rising acidity and agglutination of protein. The off-flavor volatile substances (hexanal and nonanal) were significantly reduced in fermented soymilk. Furthermore, a large amount of glucoside form isoflavones was deglycosylated into bioactive aglycones after 4 h up to 32 h. B2 content and isoflavones significantly improved the antioxidant status of soymilk. Partial least squares regression analysis correlated the strain activity and fermentation time with the improved nutritional and functional soymilk qualities. This study demonstrated the strategy for strain development for B2-bio-enriched fermentation to extend the health-promoting benefits of soymilk and soy-related foods. KEY POINTS: • B2-enriched fermentation enhanced the nutrition and functional status of soymilk. • Fermentation time significantly affected the apparent viscosity of fermented soymilk. • Off-flavor volatile substances were significantly reduced or even diminished. • Increased B2and bioactive isoflavones contributed to improved antioxidant potential.

Potential Pharmaceutical and Food Applications of Postbiotics: A Review.

In recent decades, functional foods with ingredients comprising probiotics, prebiotics and postbiotics have been gaining a lot of attention from scientists. Probiotics and postbiotics are usually applied in pharmaceutical formulations and/or commercial food-based products. These bioactive agents can be associated with host eukaryotic cells and have a key role in maintaining and restoring host health. The review describes the concept of postbiotics, their quality control and potential applications in pharmaceutical formulations and commercial food-based products for health promotion, prevention of disease and complementary treatment. Despite the effectiveness of probiotic products, researchers have introduced the concept of postbiotic to optimize their beneficial effects as well as to meet the needs of consumers to provide a safe product. The finding of recent studies suggests that postbiotics might be appropriate alternative agents for live probiotic cells and can be applied in medical, veterinary and food practice to prevent and to treat some diseases, promote animal health status and develop functional foods. Presently scientific literature confirms that postbiotics, as potential alternative agents, may have superiority in terms of safety relative to their parent live cells, and due to their unique characteristics in terms of clinical, technological and economical aspects, can be applied as promising tools in the drug and food industry for developing health benefits, and therapeutic aims.

Synbiotic fermented soymilk with Weissella cibaria FB069 and xylooligosaccharides prevents proliferation in human colon cancer cells.

AIM: Studies on the anticancer effects of synbiotic fermented soymilk are rare. The aim of the present study was to evaluate the effect of synbiotic fermented soymilk supplemented with xylooligosaccharides and inoculated with Weissella cibaria FB069 (FSMXW) in the proliferation of colon cancer cell and compare it with the effect of soymilk inoculated with Lactobacillus rhamnosus GG (LGG). METHODS AND RESULTS: Both FB069 and LGG were able to grow in soy-based products and rapidly reduce their pH as a result of fermentation. The addition of XOS significantly enhanced the acidification rate, viscosity and total cell concentration in fermented soymilk inoculated with W. cibaria FB069. However, the same effect was not observed following inoculation with LGG. Moreover, the synbiotic FSMXW showed higher dextran, folate, GABA and aglycone content. FSMXW inhibited the proliferation of Caco-2 and HCT116 cell lines, by reducing the transcription of MD2, TLR4, MyD88, and NF-κb. CONCLUSIONS: The synbiotic soymilk containing XOS and W. cibaria FB069 increase nutrient and functional compounds through fermentation process. SIGNIFICANCE AND IMPACT OF THE STUDY: Our finding suggests that W. cibaria and XOS can be potentially employed in developing functional foods and health-related products.

How probiotics face food stress: They get by with a little help.

The dietary consumption of probiotics in the form of pharmaceuticals or functional food can improve human health and contribute to disease prevention. However, the biological activity and health potential of food-delivered probiotics can be severely compromised by the stress conditions encountered by the microorganisms throughout the manufacture process, from probiotic preparation to their inclusion into food, subsequent storage and ingestion. Here, we give an account of the stress factors that can have major negative impacts on probiotic viability and functionality, with a focus on food-related environmental adverse conditions. We also describe some of the mechanisms elicited by the microbial cells to counteract these stresses and summarize a few relevant approaches proposed in literature to develop more robust and competitive probiotics by enhancing their stress tolerance, with the aim to improve the efficacy and health value of probiotic functional food.

Milk with Juçara (Euterpe edulis Martius) Pulp: Fermentation by L. reuteri LR92 and Reuterin Production in Situ

Abstract This study aimed to develop and evaluate fermented milk by Lactobacillus reuteri LR92 with addition of juçara pulp (FMJ) and reuterin production in situ. The fermentation process was analyzed for 24 hours and the storage of FMJ for 30 days at 4 °C. During the fermentation, there was consumption of 25% (w / v) of lactose and increase of 0.01 to 0.85% (w / v) of lactic acid. The FMJ presented 0.43 ± 0.01 mM of reuterin, inhibiting Staphylococcus aureus strains under in vitro test. For the carbohydrates, the percentages (g.100g-1) found were 7.31 ± 1.07; 9.19 ± 0.82; 1.60 ± 0.50 and 0.08 ± 0.00 for sucrose, lactose, galactose and fructose respectively. The survival of L. reuteri, present in FMJ, was 2.47 log CFU / mL after 6 hours of gastrointestinal simulation. In sensory analysis FMJ received a grade 7 for global acceptance indicating good acceptance of the product.

Cereal bars functionalized through Bifidobacterium animalis subsp. lactis BB-12 and inulin incorporated in edible coatings of whey protein isolate or alginate.

Currently, cereal bars are gaining interest globally because of their nutritionally balanced and convenient nature. One healthy strategy is to add probiotics to cereal bars, to make them a functional food product. So, in this study a cereal bar functionalized with edible coatings of whey protein isolate (WPI) and alginate (ALG) incorporated with Bifidobacterium animalis subsp. lactis BB-12 and inulin was developed and evaluated for its consumer acceptability and physicochemical and microbiological properties, throughout 90 days of storage. WPI-coated cereal bars were shown to be the solution that better maintained the level of the incorporated probiotic strain when compared to the ones coated with ALG, throughout storage and throughout in vitro gastrointestinal digestion. The physicochemical properties of the bars, namely aw, moisture content, color and texture, were not altered during the storage period. The sensory evaluation showed that coated bars were accepted as well as control bars. Moreover, the consumers appreciated better the odor and flavor of WPI-coated bars than those of ALG-coated bars.

Postbiotics produced at laboratory and industrial level as potential functional food ingredients with the capacity to protect mice against Salmonella infection.

AIM: To determine the protective capacity against Salmonella infection in mice of the cell-free fraction (postbiotic) of fermented milk, produced at laboratory and industrial level. METHODS AND RESULTS: The proteolytic activity (PA) of 5 commercial cultures and 11 autochthonous Lactobacillus strains was evaluated. The DSM-100H culture displayed the highest PA and it was selected for further studies. The capacity of the postbiotics produced by pH-controlled fermentation to stimulate the production of secretory IgA in faeces and to protect mice against Salmonella infection was evaluated. A significant increase in secretory IgA in faeces of mice fed 14 days the postbiotic obtained at the laboratory (F36) was detected compared to control animals. A significantly higher survival was observed in mice fed the F36 and the FiSD (industrial product) compared to controls. CONCLUSION: The postbiotics obtained showed immunomodulatory and protective capacity against Salmonella infection in mice. SIGNIFICANCE AND IMPACT OF THE STUDY: The pH-controlled milk fermentation by the proteolytic DSM-100H culture could be a suitable strategy to obtain a food ingredient to be added to a given food matrix, not adequate to host viable cells of probiotics, to confer it enhanced functionality and thus expand the functional food market.

Modeling of lactic acid fermentation of soy formulation with Lactobacillus plantarum HM1.

Lactic acid bacteria alone or with special adjunct probiotic strains are inevitable for the preparation of various specific functional foods. Moreover, because of their growth and metabolism, the final products are preserved for a certain time. Thus, growth dynamics of the lactic acid bacteria of the Fresco DVS 1010 culture ( Lactococcus lactis spp. lactis, Lactococcus lactis spp. cremoris, Streptococcus salivarius spp. thermophilus) during liquid-state fermentation of soya mashes and pH values within the process were analyzed in this study. Although milk is the most typical growth medium for the lactic acid bacteria, presumable viable counts of Fresco culture reached levels 109 CFU ml-1 after 8 h, representing 2-3 log increase in comparison to initial state (specific growth rates ranged from 1.06 to 1.64 h-1). After 21 days of storage period, the pH levels in the products were reduced to 4.50-4.70, representing a decrease of about 1.5-1.7 units. All prepared soybean products contained detectable amounts of raffinose-series oligosaccharides (0.25-0.68 g per 100 g) that were reduced in average by about 30.5% during period of 21 days.

Effects of Lactobacillus acidophilus LA-3 on physicochemical and sensory parameters of açaí and mango based smoothies and its survival following simulated gastrointestinal conditions.

The effects of the incorporation of Lactobacillus acidophilus LA3 (inoculated at 9 log CFU/mL) on physicochemical parameters, contents of sugars and organic acids, phenolic profile and sensory aspects of açaí (AS) and mango based smoothies (MS) during 28 days of refrigerate storage were assessed. The survival of L. acidophilus in AS and MS under simulated human gastrointestinal conditions during storage was also monitored. Counts of L. acidophilus LA-3 were approximately 8.5 log CFU/mL in AS and 7.5 log CFU/mL in MS after 1 day of storage and no changes (p ≥ 0.05) were observed in later evaluated storage time periods. Fruit smoothies (AS or MS) with L. acidophilus LA-3 presented lower (p < 0.05) contents of glucose, fructose and maltose and higher (p < 0.05) contents of lactic and succinic acids compared to those without L. acidophilus LA-3. Phenolic contents increased or decreased (p < 0.05) after 1 day of storage in AS and MS with L. acidophilus LA-3 and did not change (p ≥ 0.05) in AS or MS without L. acidophilus LA-3. The incorporation of L. acidophilus LA-3 increased the acidic flavor and fermented aroma in AS and MS. Viable counts of L. acidophilus LA-3 were approximately 7.5 and 8.5 log CFU/mL in AS and MS, respectively, at the end of in vitro digestion just after the incorporation, as well as on days 14 and 28 of storage. These results show the feasibility of incorporating L. acidophilus LA-3 into AS or MS and suggest these fruit smoothies as matrices for formulation of non-dairy beverages containing potentially probiotic lactobacilli.

How to select a probiotic? A review and update of methods and criteria.

International competition within the dairy market and increasing public awareness about the importance of functional food consumption are providing new challenges for innovation in the probiotic sector. In this context, countless references are currently dedicated to the selection and characterization of new species and more specific strains of probiotic bacteria. In general, these studies adopt basic selection criteria established by the World Health Organization (WHO), including host-associated stress resistance, epithelium adhesion ability, and antimicrobial activity. These aspects are applied to ensure that the candidate probiotic could withstand the stressful conditions of the human digestive system and exert functional proprieties. However, it cannot be assumed that these novel microbial strains are capable of offering several biological benefits attributed to probiotics. Additionally, safety-associated selection criteria, such as plasmid-associated antibiotic resistance spreading and enterotoxin production, are often neglected. This article reviews the recent developments in the processes, strategies, and methods, such as anticarcinogenic, antidepression, antianxiety, antiobesity, antidiabetic, immunostimulatory, and cholesterol-lowering assessments, to select probiotic strains with the ultimate objective of assisting future probiotic microbe evaluation studies.

Fermented Dessert with Whey, Ingredients from the Peel of Jabuticaba (Myrciaria cauliflora) and an Indigenous Culture of Lactobacillus plantarum: Composition, Microbial Viability, Antioxidant Capacity and Sensory Features.

The use of agro-industrial wastes in combination with indigenous lactic acid bacteria is an interesting option to confer functional potential to food products. The microbial viability, chemical composition, antioxidant capacity, texture and sensory acceptability of a fermented dairy dessert containing the indigenous culture Lactobacillus plantarum CNPC003, whey and ingredients obtained from the jabuticaba (Myrciaria cauliflora) peel were compared with formulations without lactobacilli (control) or containing a commercial probiotic culture (Lactobacillus rhamnosus LR32). L. plantarum presented viability higher than 7 log CFU g-1 in the dessert, as did the commercial probiotic, for 21 days at 4 ± 1 °C. Total phenolic contents (45⁻60 mg gallic acid equivalents, GAE, 100 g-1) were comparable to those of other studies evaluating dairy products containing plant sources. The formulations were low in fat, presenting as acceptable for overall consumption, with attractive color and appreciable texture. Considering the total antioxidant capacity, 200⁻250 g of dessert would be necessary to capture 1 g of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. The dessert with Lactobacillus plantarum CNPC003 is seen as a viable alternative for the use of whey and jabuticaba peel, as well as a potential functional food due to the concentration of lactobacilli reached, besides the presence of antioxidant phenolic compounds.

Probiotic Properties of Exopolysaccharide-Producing Lactobacillus Strains Isolated from Tempoyak.

Tempoyak is a functional Malaysian food (an acid-fermented condiment) which is produced from the pulp of the durian (Durio zibethinus) fruit. The current study aimed to isolate and identify potential exopolysaccharide (EPS)-producing Lactobacillus strains from tempoyak for potential use as probiotics. Seven isolates (DUR2, DUR4, DUR5, DUR8, DUR12, DUR18, and DUR20) out of 44 were able to produce EPS, and exhibited resistance to acid and bile salt compared to the reference strains Lactobacillus rhmnosus (ATCC53103) and L. plantarum (ATCC8014). The seven isolated strains belonged to five different species-L. plantarum, L. fermentum, L. crispatus, L. reuteri, and L. pentosus-which were identified using API 50 CHL and 16S rRNA gene sequences (Polymerase chain reaction, PCR - based). The seven strains displayed different ability to produce EPS (100-850 mg/L). Isolates exhibited a high survivability to acid (pH 3.0), bile salts (0.3%), and gastrointestinal tract model (<70%). Results showed that the auto-aggregation and cell surface hydrophobicity ranged from 39.98% to 60.09% and 50.80% to 80.53%, respectively, whereas, the highest co-aggregation value (66.44%) was observed by L. fermentum (DUR8) with Pseudomonas aeruginosa. The isolates showed good inhibitory activity against tested pathogens, high antioxidant activity (32.29% to 73.36%), and good ability to reduce cholesterol (22.55% to 75.15%). Thus, the seven tested strains have value as probiotics.