Potential probiotic lactobacilli strains isolated from artisanal Mexican Cocido cheese: evidence-based biosafety and probiotic action-related traits on in vitro tests.

The biosafety of four potentially probiotic lactobacilli strains, isolated from artisanal Mexican Cocido cheese, was assessed through in vitro tests aimed to determine (1) the antibiotic susceptibility profile by broth microdilution, (2) the transferability of antibiotic resistance determinants by filter-mating, and (3) the phenotypic and genotypic stability during serial batch sub-culture (100-day period) by evaluating physiological and probiotic features and RAPD-PCR fingerprinting. Lactobacilli strains exhibited multidrug-resistance; however, resistance determinants were not transferred in the filter-mating assay. Significant (p < 0.05) differences were observed in bacterial morphology and some functional and technological properties when strains were serially sub-cultured over 50 generations (G50), compared to the initial cultures (G0). Conversely, the strains did not show mucinolytic and hemolytic activities either at G0 or after 100 generations (G100). Genetic polymorphism and genomic template instability on selected strains were detected, which suggest possible evolutionary arrangements that may occur when these bacteria are largely cultured. Our findings suggest that the assessed strains did not raise in vitro biosafety concerns; however, complementary studies are still needed to establish the safe potential applications in humans and animals.

Fermented milks with specific Lactobacillus spp. with potential cardioprotective effects.

In vitro and in vivo studies have reported the potential cardioprotective effects of fermented milks (FM). The aim of the present study was to evaluate the inhibitory activities of angiotensin converting enzyme (ACE), thrombin enzyme (TI) and micellar solubility of cholesterol of FM after 24 and 48 h of fermentation with Limosilactobacillus fermentum (J20, J23, J28 and J38), Lactiplantibacillus plantarum (J25) or Lactiplantibacillus pentosus (J34 and J37) exposed to simulated gastrointestinal digestion. Results showed that FM with J20 and J23 at 48 h of fermentation presented significantly (p < 0.05) higher degree of hydrolysis than other FM, and were not significantly different (p > 0.05) between them. Conversely, peptide relative abundance was significantly (p < 0.05) higher in FM with J20 than FM with J23. Moreover, IC50 (protein concentration necessary to inhibit enzyme activity by 50%) for ACE inhibition were 0.33 and 0.5 mg/mL for FM with J20 and J23, respectively. For TI inhibition, the IC50 were 0.3 and 0.24 mg/mL for FM with J20 and J23, respectively. Results exhibited 51 and 74% inhibition of micellar solubility cholesterol for FM with J20 and J23, respectively. Therefore, these results showed that not only peptide abundance, but also specific peptides might be responsible for these potential cardioprotective effects.

Key Stress Response Mechanisms of Probiotics During Their Journey Through the Digestive System: A Review.

The survival of probiotic microorganisms during their exposure to harsh environments plays a critical role in the fulfillment of their functional properties. In particular, transit through the human gastrointestinal tract (GIT) is considered one of the most challenging habitats that probiotics must endure, because of the particularly stressful conditions (e.g., oxygen level, pH variations, nutrient limitations, high osmolarity, oxidation, peristalsis) prevailing in the different sections of the GIT, which in turn can affect the growth, viability, physiological status, and functionality of microbial cells. Consequently, probiotics have developed a series of strategies, called "mechanisms of stress response," to protect themselves from these adverse conditions. Such mechanisms may include but are not limited to the induction of new metabolic pathways, formation/production of particular metabolites, and changes of transcription rates. It should be highlighted that some of such mechanisms can be conserved across several different strains or can be unique for specific genera. Hence, this review attempts to review the state-of-the-art knowledge of mechanisms of stress response displayed by potential probiotic strains during their transit through the GIT. In addition, evidence whether stress responses can compromise the biosafety of such strains is also discussed.

Safety Assessment of the Potential Probiotic Bacterium Limosilactobacillus fermentum J23 Using the Mexican Fruit Fly (Anastrepha ludens Loew, Diptera: Tephritidae) as a Novel In Vivo Model.

Safety assessment of probiotics is difficult but essential. In this work, the Mexican fruit fly, Anastrepha ludens (Loew) (Diptera: Tephritidae), was used as in vivo model to assess the biosafety of Limosilactobacillus fermentum J23. In the first set of experiments, the strain was orally administered to adult flies through direct feeding, whereas in the second set of experiments, it was supplemented through the larval rearing medium. Data showed that L. fermentum J23 did not lead to increased mortality or treatment-related toxicity signs in adult female and male flies. Ingestion of L. fermentum J23 by adult female flies led to a statistically significant improvement in locomotor activity compared to the control groups (ca. 59% decrease in climbing time, p < 0.0001). A positive trend in lifespan extension under stress (maximum lifespan = 144 h) was also observed. When L. fermentum J23 was administered to the larvae, the adult emergence (p = 0.0099), sex ratio (p = 0.0043), and flight ability (p = 0.0009) increased significantly by 7%, 31%, and 8%, respectively, compared to the control diet. No statistical effect between the control diet and the L. fermentum J23-based diet for the number of pupae recovered, pupal weight, duration of the pupal stage, lifespan under stress, and morphological development was observed. We conclude that feeding L. fermentum J23 to the novel experimental model A. ludens had no toxic effects and could be safely considered a potential probiotic for food supplements; however, further studies are still needed to establish its biosafety in humans.

Current trends and perspectives on bioaccessibility and bioavailability of food bioactive peptides: in vitro and ex vivo studies.

The bioaccessibility and bioavailability of food-derived bioactive compounds are important issues when assessing their in vivo physiological health-promoting effects. Food components such as proteins and peptides are exposed to different proteases and peptidases during gastrointestinal digestion and absorption. Different in vitro approaches have therefore been developed to evaluate the bioaccessibility and stability of bioactive peptides. The static simulated gastrointestinal digestion model (SGD) was widely reported to assess the bioaccessibility of bioactive peptides. On the other hand, although the dynamic SGD model may better simulate human digestion, it has rarely been explored in bioaccessibility studies of food bioactive peptides due to its high cost and lack of standardization. For bioavailability studies, the Caco-2 cell monolayer model has been used extensively for the assessment of food bioactive peptides. In fact, very few reports using alternative methods for determining transepithelial transport of bioactive peptides have been employed. In this sense, ex vivo tissue-based models such as the Ussing chamber and the everted sac gut have been used. Current evidence supports the fact that using SGD with cell-based models for evaluating the bioaccessibility, absorption, and bioavailability of food-derived bioactive peptides, is the most commonly used approach. Nevertheless, SGD with ex vivo tissue-based models such as the everted sac, remains to be further explored because it seems to be the model that better mimics the physiological process - it is also fast and inexpensive, and several compounds may be tested simultaneously. In the present review, we discuss information available on the different in vitro approaches for the determination of bioaccessibility and bioavailability of food-derived bioactive peptides with special emphasis on ex vivo tissue-based models such as the everted sac and the Ussing chamber models. © 2022 Society of Chemical Industry.

Conventional and in silico approaches to select promising food-derived bioactive peptides: A review.

The interest for food-derived bioactive peptides, either from common or unconventional sources, has increased due to their potential therapeutic effect against a wide range of diseases. The study of such bioactive peptides using conventional methods is a long journey, expensive and time-consuming. Hence, bioinformatic approaches, which can not only help to predict the formation of bioactive peptides from any known protein source, but also to analyze the protein structure/function relationship, have gained a new meaning in this scientific field. Therefore, this review aims to provides an overview of conventional characterization methods and the most recent advances in the field of in silico approaches for predicting and screening promising food-derived bioactive peptides.

Peptides, Exopolysaccharides, and Short-Chain Fatty Acids from Fermented Milk and Perspectives on Inflammatory Bowel Diseases.

Crohn's disease and ulcerative colitis are characterized by chronic inflammatory processes and an imbalanced immune response along the gastrointestinal (GI) tract. Pharmacological treatments have been widely used, although their long-term application has adverse side effects. On the other hand, milks fermented with specific lactic acid bacteria (LAB) have been shown to be useful as alternative or complementary aids. Many metabolites such as peptides, exopolysaccharides, and short-chain fatty acids are produced during milk fermentation. These components have been shown to change the pH of the gastrointestinal lumen, aid intestine mucosal recovery, modulate the microbiota, and reduce the inflammatory response (innate and adaptive immune system), both in vitro and in vivo. Therefore, the objective of the present review is to describe how these bioactive compounds from fermented milk by specific LAB can decrease the deleterious symptoms of inflammatory bowel disease.

Antidepressant and Anxiolytic Effects of Fermented Huauzontle, a Prehispanic Mexican Pseudocereal.

This study aimed to assess the potential antidepressant- and anxiolytic-like effects of huauzontle fermented by Lactiplantibacillus plantarum Lp22. The possible association between oxidative stress/inflammation biomarkers and unconditional behavioural tests was also evaluated. Red light-induced stress mice C57Bl/6 (n = 5 per group) received orally either fermented or unfermented huauzontle, diazepam or fluoxetine. A non-stressed group which received saline solution was also included. Then, anxiety-related and depression-related behaviour tests were performed; after that, blood and tissues samples were collected to determine oxidative stress/inflammation biomarkers. The mice receiving both fermented and unfermented huauzontle spent more time (94 s) in open arms in the elevated plus maze test p < 0.05; besides, travelled longer distance (p < 0.05) and increased by more than 50% the exploration time for the open field, as well as the time spent in the illuminated zone (197 s) in the light/dark test. Furthermore, reduced immobility time in the tail suspension and forced swim tests (23.1 and 15.85, respectively), and anhedonia was no detected in the sucrose preference test. The oxidative stress index was lower in the liver of fermented huauzontle-treated mice, while enhanced levels of IL-10, MCP-1 and BDNF in plasma, and lipoxygenase (LOX) activity in the hippocampus were found. Finally, PCA revealed a positive correlation among LOX and BDNF and parameters determined in the anxiety tests, as between catalase activity and immobility time in the depression test. These findings indicate the novel potential therapeutic applications of fermented huauzontle on depression and anxiety-like behaviours possibly mediated by antioxidant and anti-inflammatory mechanisms.

Insects as an Alternative Protein Source.

The recent COVID-19 pandemic drastically affected food supply chains worldwide, showing the vulnerability of food security. Efforts to develop alternative protein sources that are sustainable and can help alleviate global food shortage problems should be prioritized. Insects have been part of our diet for thousands of years and still are today, and market trends show a global increase in the number of food-grade insect producers. The global market for edible insects has been forecasted to reach US$8 billion by the year 2030. Insects are highly nutritious and have bioactive peptides with potential therapeutic effects. This review provides an overview of the consumption of insects from ancient to modern times, discusses the rationale for using insects as alternative protein sources, and presents a summary of the major insects consumed worldwide as well as a brief description of the traditional and novel technologies currently used to process insects and/or extract their nutritional components.

Tejuino, a Traditional Fermented Beverage: Composition, Safety Quality, and Microbial Identification.

This study aims to analyze the chemical and microbial composition and characterize volatile compounds from the artisanal and commercial Tejuino beverage. For this, eight samples are analyzed (four artisanal and four commercial). The chemical and microbiological quality is determined by standard methods, and volatile compounds are determined by solid-phase microextraction. Overall, the physicochemical composition and microbiological quality are higher for artisanal Tejuino (p < 0.05). The pH values were 3.20 and 3.62, and 0.76 and 0.46 meq of lactic acid for artisanal and commercial Tejuino, respectively. With volatile compounds analyzed, esters, benzenes, and aldehydes were predominant; meanwhile, ethanol was a volatile compound with the highest concentration for all samples. Saccharomyces cerevisiae and Limosilactobacillus fermentum were identified in artisanal Tejuino; yeasts of the Pichia genera and Lactiplantibacillus plantarum, for commercial Tejuino, and Enterococcus genus were identified in both samples. The characterization of both types of Tejuino allows us to update the information available on this important Mexican beverage. In addition, the isolation of lactic acid bacteria, as representative bacteria of both drinks, offers an area of opportunity to know the potential functionality of these bacteria in traditional fermented products.

Invited review: Effect of antihypertensive fermented milks on gut microbiota.

Hypertension is a risk factor for the development of other cardiovascular diseases and remains one of the leading causes of death worldwide. Although genetic and environmental factors are associated with the development of hypertension, it has been recently recognized that gut microbiota (GM) may also have an effect on human health. In this sense, gut dysbiosis (a marked decrease in richness and diversity of GM) has been linked to different metabolic diseases, such as hypertension. Therefore, different studies have been pursued to reduce gut dysbiosis and diminish hypertension. Different strategies to maintain a balanced GM, particularly through diet and the use of probiotics, are being evaluated. Most recently, the effect of antihypertensive fermented milks on GM has been addressed. New evidence suggests that antihypertensive fermented milks may modulate GM. Thus, the aim of this review is to present available information related to the effect of antihypertensive fermented milks on gut microbiota.

Invited review: Potential antiobesity effect of fermented dairy products.

The growing prevalence of obesity affects millions of people around the world and has gained increased attention over the years because it is associated with the development of other chronic degenerative diseases. Different organizations recommend lifestyle changes to treat obesity; nevertheless, other strategies in addition to lifestyle changes have recently been suggested. One of these strategies is the use of probiotics in fermented dairy products; however, a need exists to review the different studies available related to the potential antiobesity effect of these products. Because probiotic fermented dairy products that support weight management are not available in the market, there is a great opportunity for the development of functional dairy products with new lactic acid bacteria that may present this added health benefit. Thus, the purpose of this overview is to highlight the importance of probiotic fermented dairy products as potential antiobesogenic functional foods and present in vitro and in vivo studies required before this kind of product may be introduced to the market. Overall, most studies attributed the antiobesity effect of fermented dairy foods to the probiotic strains present; however, bioactive peptides released during milk fermentation may also be responsible for this effect.

Novel bacteriocins produced by Lactobacillus fermentum strains with bacteriostatic effects in milk against selected indicator microorganisms.

The aim of this work was to isolate and characterize bacteriocins produced by 2 Lactobacillus fermentum strains isolated from artisanal Mexican Cocido cheese. Fractions (F ≤3 kDa) obtained from cell-free supernatants of Lb. fermentum strains J23 and J32 were further fractionated by reversed-phase HPLC on a C18 column. Antimicrobial activities of F ≤3 kDa and bacteriocin-containing fractions (BCF), obtained from fractionation of F ≤3 kDa against 4 indicator microorganisms, were determined by the disk diffusion method and growth inhibition in milk. Subsequently, isolated BCF were analyzed by reversed-phase HPLC tandem mass spectrometry. Results showed that BCF presented antimicrobial activity against the 4 indicator microorganisms tested. For J23, one of the fractions (F3) presented the highest activity against Escherichia coli and was also inhibitory against Staphylococcus aureus, Listeria innocua, Salmonella Typhimurium, and Salmonella Choleraesuis. Similarly, fractions F3 and F4 produced by J32 presented antimicrobial activity against all indicator microorganisms. Furthermore, generation time and growth rate showed that F3 from J23 presented significantly higher antimicrobial activity against the 4 indicator microorganisms (2 gram-positive and 2 gram-negative) when inoculated in milk compared with F3 from J32. Interestingly, this fraction presented a broader antimicrobial spectrum in milk than nisin (control). Reversed-phase HPLC tandem mass spectrometry analysis revealed the presence of several peptides in BCF; however, F3 from J23 that was the most active fraction of all presented only 1 bacteriocin. The chemical characterization of this bacteriocin suggested that it was a novel peptide with 10 hydrophobic AA residues in its sequence and a molecular weight of 2,056 Da. This bacteriocin and its producing strain, J23, may find application as a biopreservative against these indicator microorganisms in dairy products.

Evaluation of acrylamide-removing properties of bacterial consortia under simulated gastrointestinal conditions.

BACKGROUND: Previous studies have demonstrated the acrylamide-removing properties of probiotic monocultures; however, potential advantages of consortia over monocultures in reducing the dietary exposure to acrylamide have not been proven. Hence this work aims to assess the acrylamide (AA)-binding properties of bacterial consortia, consisting of either probiotic strains and / or representative bacteria of duodenal microbiota, exposed to simulated gastrointestinal conditions (SGC). The AA binding capacity of ten probiotic strains (PS) and six duodenal strains (NDS) was evaluated under different conditions; then, three different consortia (PS, NDS, and PS + NDS) were assessed under SGC. RESULTS: Among individual PS, Bacillus coagulans GBI-30, Lactobacillus fermentum J23, L. pentosus J37 and J24, and L. casei Shirota, exhibited the highest AA-binding capacity (80-87%), while Bifidobacterium catenulatun ATCC27676, Streptococcus salivarius subsp. thermophilus ATCC19258, and S. gallolyticus ATCC9809 were the best (ca. 68%) NDS monocultures. Probiotic strain consortia showed higher (P < 0.05) AA binding capacity (> 90%) than monoculture bacteria. Conversely, individual NDS cultures displayed higher (P < 0.05) binding capacity than NDS consortia (60%). A significant reduction (P < 0.05) in AA removal capacity was observed when consortia were exposed to SGC, PS consortia being the most effective (> 60% removal). CONCLUSION: These results suggest that consortia of specific PS could play an important role in reducing the intestinal availability of acrylamide. © 2021 Society of Chemical Industry.

Protective Effect of Lacticaseibacillus casei CRL 431 Postbiotics on Mitochondrial Function and Oxidative Status in Rats with Aflatoxin B1-Induced Oxidative Stress.

Studies have shown that the intracellular content of probiotic (postbiotics) has antioxidant properties, which can improve the antioxidant status in vivo. However, its absorption and mechanisms underlying the protective effects are still unknown. The antioxidant capacity of Lacticaseibacillus casei CRL431 (IC-431) postbiotics was determined after an in vitro simulated digestive process. Permeability of antioxidant constituents of IC-431 was determined by an ex vivo everted duodenum assay. Aflatoxin B1-induced oxidative stress rat models were established and treated with IC-431; biomarkers of hepatic mitochondrial function and H2O2 levels, oxidative stress, and oxidative stress index (OSi) were examined. The antioxidant capacity of IC-431 (477 ± 45.25 µmol Trolox Equivalent/L) was reduced by exposure to the simulated digestive process. No difference (p > 0.05) was found among digested and the permeate fraction of IC-431. A protective effect was observed by significantly lower OSi and higher liver glutathione peroxidase and catalase activities. Lower H2O2 production, a higher degree of mitochondrial uncoupling, and lower mitochondrial respiration coefficient were also observed (p < 0.05). These results suggest that IC-431 antioxidant components permeate intestinal barriers to enter the bloodstream and regulate antioxidant status during AFB1-induced oxidative stress by reducing hepatic mitochondrial dysfunction, thus enhancing antioxidant enzyme response.

Exploring the Milk-Clotting and Proteolytic Activities in Different Tissues of Vallesia glabra: a New Source of Plant Proteolytic Enzymes.

Proteolytic enzymes are widely distributed in nature, playing essential roles in important biological functions. Recently, the use of plant proteases at the industrial level has mainly increased in the food industry (e.g., cheesemaking, meat tenderizing, and protein hydrolysate production). Current technological and scientific advances in the detection and characterization of proteolytic enzymes have encouraged the search for new natural sources. Thus, this work aimed to explore the milk-clotting and proteolytic properties of different tissues of Vallesia glabra. Aqueous extracts from the leaves, fruits, and seeds of V. glabra presented different protein profiles, proteolytic activity, and milk-clotting activity. The milk-clotting activity increased with temperature (30-65 °C), but this activity was higher in leaf (0.20 MCU/mL) compared with that in fruit and seed extracts (0.12 and 0.11 MCU/mL, respectively) at 50 °C. Proteolytic activity in the extracts assayed at different pH (2.5-12.0) suggested the presence of different types of active proteases, with maximum activity at acidic conditions (4.0-4.5). Inhibitory studies indicated that major activity in V. glabra extracts is related to cysteine proteases; however, the presence of serine, aspartic, and metalloproteases was also evident. The hydrolytic profile of caseins indicated that V. glabra leaves could be used as a rennet substitute in cheesemaking, representing a new and promising source of proteolytic enzymes.

An insight to fermented edible insects: A global perspective and prospective.

The practice of eating insects is not a new phenomenon; however, the interest for their consumption has increased in recent years due to their recognized nutritional value (high content of micro- and macronutrient), potential health benefits (presence of bioactive substances), and low-environmental impact (use of less resources and reduced pollution levels). Currently, research on insects has focused on the promotion of various processing technologies for their use as either ingredients (in a non-recognizable form) to the development of innovative products, or as sources of novel bioactive compounds. In this context, evidence has suggested that alternative technologies, particularly fermentation, could be used the obtain diverse insect-based ingredients/products with unique properties. Therefore, the purpose of this narrative review was to provide an overview of the available literature on fermentation applied to obtain new insect-based products, to summarize the patents and patent-applications to protect fermented edible insect products and processes, as well as to enlist examples of current available products in the market.

Postbiotics and paraprobiotics: A review of current evidence and emerging trends.

In recent years, new probiotic-related concepts such as postbiotics and paraprobiotics have been coined to indicate that non-viable microorganisms or bacterial-free extracts may provide benefits to the host by offering additional bioactivities to probiotics, including but not limited to anti-inflammatory, immunomodulatory, anti-proliferative and antioxidant activities. Despite in vitro and in vivo studies that support the promising use of postbiotics and paraprobiotics as health promoters, the mechanism of action and the signaling pathway involved have not yet been fully elucidated. Therefore, the aim of this chapter is to provide an overview of novel probiotic-related concepts and the scientific evidence that supports their bioactivities as well as the possible mechanisms underlying their health-promoting effects. Additionally, current trends in food, feed, and pharmaceutical applications are discussed.

Poro de Tabasco cheese: Chemical composition and microbiological quality during its artisanal manufacturing process.

Poro de Tabasco cheese (PTC) is one of the most popular cheeses in southern Mexico. It has been made by traditional, nonstandardized artisanal techniques dating back more than 50 yr. These techniques result in the cheese having a heterogeneous chemical and microbiological composition and, consequently, distinct organoleptic characteristics. Scientific interest in artisanal cheese is growing because it represents a source of bacteria with potential health benefits. However, the quality of raw-milk cheeses often does not comply with official sanitary standards. The objective of the present study was to explore the chemical composition and microbiological quality of PTC and to describe its production process. Based on chemical composition, this cheese can be classified as a hard, full-fat, fresh cheese, with moisture on a fat-free basis and fat in dry matter ranging from 41 to 55% and from 49 to 57%, respectively. The chemical and microbiological composition of PTC varied among the evaluated dairies due to the lack of standardization in the production process. Microbial populations decreased during production, which may be associated with high acidity and high salt and low moisture contents, the presence of lactic acid bacteria or antimicrobial substances, and the drainage of whey. However, despite the absence of Escherichia coli, Salmonella spp., and Listeria monocytogenes in final cheeses for all dairies, Staphylococcus aureus and its toxins were found in some samples from one dairy. Therefore, heat treatment for milk and good manufacturing practices should be implemented throughout the entire production process to ensure a safe product.