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.

Inhibition of the CYP Enzymatic System Responsible of Heterocyclic Amines Bioactivation by an Asclepias subulata Extract.

Asclepias subulata plant extract has previously demonstrated antiproliferative activity and antimutagenicity against heterocyclic aromatic amines (HAAs) commonly found in cooked meat. The objective of this work was to evaluate the in vitro ability of an ethanolic extract from the medicinal plant Asclepias subulata extract (ASE), non-heated and heated (180 °C), to inhibit the activity of CYP1A1 and CYP1A2, which are largely responsible for HAAs bioactivation. Ethoxyresorufin and methoxyresorufin O-dealkylation assays were performed in rat liver microsomes exposed to ASE (0.002-960 µg/mL). ASE exerted an inhibitory effect in a dose-dependent manner. The half inhibitory concentration (IC50) for unheated ASE was 353.6 µg/mL and 75.9 µg/mL for heated ASE in EROD assay. An IC40 value of 288.4 ± 5.8 µg/mL was calculated for non-heated ASE in MROD assay. However, after heat treatment, the IC50 value was 232.1 ± 7.4 µg/mL. Molecular docking of corotoxigenin-3-O-glucopyranoside, one of the main components of ASE, with CYP1A1/2 structure, was performed. Results show that the interaction of corotoxigenin-3-O-glucopyranoside with CYP1A1/2s' α-helices, which are related with the active site and the heme cofactor, may explain the plant extract's inhibitory properties. Results showed that ASE inhibits CYP1A enzymatic subfamily and may potentially act as a chemopreventive agent by inhibiting bioactivation of promutagenic dietary HAAs.

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.

Bioactive peptides produced by engineered probiotics and other food-grade bacteria: A review.

Synthetic biology is employed for the study and design of engineered microbes with new and improved therapeutic functions. The main advantage of synthetic biology is the selective genetic manipulation of living organisms with desirable beneficial effects such as probiotics. Engineering technologies have contributed to the edition of metabolic processes involved in the mechanisms of action of probiotics, such as the generation of bioactive peptides. Hence, current information related to bioactive peptides, produced by different engineering probiotics, with antimicrobial, antiviral, antidiabetic, and antihypertensive activities, as well as their potential use as functional ingredients, is discussed here. Besides, the effectiveness and safety aspects of these bioactive peptides were also described.

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.

Hydrolysates and peptide fractions from pork and chicken skin collagen as pancreatic lipase inhibitors.

The objective of this work was to obtain hydrolysates and peptide fractions from pork (PSC) and chicken (CSC) skin collagen extracts and to evaluate their ability as pancreatic lipase inhibitors. Collagen extracts were hydrolyzed with collagenase or a protease from Bacillus licheniformis (MPRO NX®) at 6, 12, and 24 h. After 24 h incubation, the highest degree of hydrolysis of PSC (p < 0.05) was obtained with collagenase (72.58%), while in CSC was obtained with MPRO NX® (64.45%). Hydrolysates obtained at 24 h had the highest inhibitory activity of lipase (p < 0.05). CSC/collagenase hydrolysates (10 mg/mL) presented the highest inhibitory activity (75.53%) (p < 0.05). Ultrafiltrated fractions >5 kDa from CSC/collagenase and PSC/MPRO NX® hydrolysates were the most bioactive fractions (IC50: 4.33 mg/mL). The highest were obtained by CSC peptides (IC50s: 6.30 and 6.08 mg/mL). These results may be considered as a novel approach to use collagen hydrolysates, or their peptide fractions, as promising natural inhibitors of pancreatic lipase.

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.

Immunomodulation by Bifidobacterium animalis subsp. lactis Bb12: Integrative Analysis of miRNA Expression and TLR2 Pathway-Related Target Proteins in Swine Monocytes.

Bifidobacterium animalis subsp. lactis Bb12 is a widely used probiotic that provides numerous health benefits to its host, many due to its immunomodulatory properties. Although the precise mechanism of modulation is still under investigation, several reports associate the interaction of TLR2 with components of the bacterial cell wall inducing a signaling cascade that culminates with the production of cytokines and co-stimulatory molecules. MicroRNAs (miRNAs) have emerged as important post-transcriptional regulators of immune responses, including those toward probiotics. In this study, we analyzed the miRNA expression profile in swine monocytes exposed to Bb12 by using an anti-TLR2 blocking strategy and Bb12 involvement in the regulation of the TLR2 pathway. As a result, the expression of 40 miRNAs was influenced by the treatments (p < 0.01), and 15 differentially expressed miRNAs with validated miRNA-mRNA interactions with around 26 proteins related to the TLR2 pathway were identified. The miRNAs upregulated in response to Bb12 included miR-15a-5p, miR-16-5p, miR-26a-5p, miR-29b-3p, and miR-30d-5p, and the following showed downregulation: miR-181a-5p, miR-19b-3p, miR-21-5p, miR-23a-5p, and miR-221-3p. The expression of let-7c-5p, let-7f-5p, miR-146b-5p, miR-150-5p, and miR-155-5p was increased by Bb12 only when TLR2 was blocked. The identified miRNA common targets were downstream proteins from bacterial recognition via TLR2, such as MyD88, TRAF6, and MAPK members; transcription factors such as NF-κB and AP-1; and cytokines such as IL-6, IL-10, and TNF-α. TLR2 participation was abrogated by anti-TLR2 antibody and suggests that bacterial recognition is complemented by other receptors since there were still changes in the microtranscriptome.

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.

Are Bacteriocins a Feasible Solution for Current Diverse Global Problems?

The development of effective technologies to cope with persistent and progressive global problems in human health and sustainable development has become an imperative worldwide challenge. The search for natural alternatives has led to the discovery of bacteriocins, which are potent protein antimicrobial compounds produced by most bacteria. The relevance of these molecules is evidenced by more than 4,500 papers published in the last decade in Scopus indexed journals highlighting their versatility and potential to impact various aspects of daily life, including the food industry, medicine, and agriculture. Bacteriocins have demonstrated antibacterial, antifungal, antiviral, and anticancer activities, and they also act as microbiota regulators and plant growth promoters. This mini-review aims to provide insights into the current state and emerging roles of bacteriocins, as well as their potential and limitations as feasible solutions against current diverse global problems.

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.