Lemon flavonoids nutraceutical (Eriomin®) attenuates prediabetes intestinal dysbiosis: A double-blind randomized controlled trial.

Eriocitrin (eriodictyol 7-O-ß-rutinoside), a citrus flavonoid from lemon juice and peel, reduces hyperglycemia and improves diabetes-related biomarkers in prediabetes patients. Eriocitrin is first metabolized by gut microbiota, producing energy for gut cells and short chain fatty acids that play a relevant role in glycemic control. The aim of this study was to assess the effect of Eriomin®, a nutraceutical composed of 70% eriocitrin, 5% hesperidin, and 4% naringin, on the microbiota of prediabetic patients. Patients were randomly divided into two groups and received unlabeled capsules of Eriomin® (200 mg/day) or placebo during 12 weeks. After treatment with the nutraceutical, it was a 6% decrease of hyperglycemia and 22% increase of GLP-1 blood levels of (p < .05). The profile of intestinal microorganisms, obtained by 16S rRNA sequencing of the patients' feces extract, showed changes in microbiota composition, such as lower growth of Firmicutes and less abundance of the Lachnospiraceae family. The family Ruminococcaceae increased and Blautia genus reduced with Eriomin® supplementation. In additional, Blautia was positively correlated with hyperglycemia reduction. In conclusion, the nutraceutical Eriomin® moderately reduced the growth of microorganisms associated with intestinal dysbiosis and increased the abundance of beneficial bacteria. Changes promoted mainly by the flavonoid eriocitrin in the microbiota were related to a lower glycemic level and increased production of GLP-1 in patients with prediabetes.

Revealing the beneficial effects of a dairy infant formula on the gut microbiota of early childhood children with autistic spectrum disorder using static and SHIME® fermentation models.

This study evaluated the impact of the Milnutri Profutura® (MNP) dairy infant formula on the gut microbiota of early childhood children (three to five years) with Autistic Spectrum Disorder (ASD) using static fermentation (time zero, 24, and 48 h) and the Simulator of the Human Intestinal Microbiol Ecosystem (SHIME®) (time zero, 72 h, and 7 days). The relative abundance of selected intestinal bacterial groups, pH values, organic acids, and sugars were verified at time zero, 24, and 48 h using flow cytometry and measurements. In addition, the diversity and changes in the gut microbiota, and the amounts of acetic, butyric, and propionic acids and ammonium ions (NH4+) in fermentation using the SHIME® were measured at time zero, 72 h, and 7 days. MNP increased Lactobacillus/Enterococcus and Bifidobacterium populations and decreased Bacteroides/Prevotella, Clostridium histolyticum and Eubacterium rectale/Clostridium coccoides populations (p < 0.05) at 24 and 48 h of static fermentation, showing a positive prebiotic activity score (65.18 ± 0.07). The pH, fructose and glucose decreased, while lactic, butyric, and propionic acids increased (p < 0.05) at 48 h of static fermentation. MNP increased (p < 0.05) the Firmicutes phylum during the fermentation in SHIME®. MNP decreased the diversity at 72 h of fermentation, mostly by the increase (p < 0.05) in the Lactobacillus genus. Microbial groups considered harmful such as Lachnospiraceae, Negativicoccus, and Lachnoclostridium were inhibited after administration with MNP. Propionic and butyric acids increased at 72 h and NH4+ decreased (p < 0.05) at the end of fermentation with MNP. The results indicate MNP as an infant formula which may benefit the gut microbiota of children with ASD.

An improved folate stable isotope dilution assay of unexploited food sources from Brazil.

Brazil has a diverse plant community, including underutilized non-conventional food crops (PANCs), which have the potential to be a rich source of food and contribute to food security. For assessing the folate content in a range of Brazilian PANCs, we extended the validation of an existing stable isotope dilution assay (SIDA) for the stably 13C-labelled 10-formyl-Pte[13C5]Glu (10-CHO-Pte[13C5]Glu). The SIDA method with an enzymatic treatment, purification step, and an LC-MS/MS measurement was validated regarding linearity, precision, LoD/LoQ, and recovery for 10-CHO-PteGlu. After successful validation, the study of some underutilized Brazilian non-conventional fruits and leaves from the São Paulo State University campus revealed them as an important source of folates. It provided the first insights into the folate content of unexploited food sources from Brazil. Pequi had the highest folate content among the fruits studied, with mean values of 333 µg/100 g based on fresh weight (FW). The analysis also shows that different cultivars of fruit or fruits from different growing locations have a high variability in folate content or other nutritional factors. In most fruits, the main vitamer was 5-CH3-H4folate, but jenipapo and taioba showed the highest content of 10-CHO-PteGlu with 28.22 µg/100 g (FW) in jenipapo peel and 75.64 µg/100 g (FW) in the taioba leaves. Thus, this study also provides results on the importance of the folate vitamer 10-CHO-PteGlu contributing to the total folate content.

Cashew By-Product as a Functional Substrate for the Development of Probiotic Fermented Milk.

Cashew (Anacardium occidentale) processing generates a by-product (CB) with potential for health benefits and that could be a favorable ingredient to be added to a probiotic food matrix. This study aimed to assess the functional attributes of CB in fermented milk with a probiotic and a starter culture using in vitro gastrointestinal conditions. Two formulations were tested, without CB (Control Formulation-CF) and with CB (Test Formulation-TF), and the two strains most adapted to CB, the probiotic Lacticaseibacillus paracasei subsp. paracasei F19® and the starter Streptococcus thermophilus ST-M6®, were chosen to be fermented in the CF and the TF. During a 28-day period of refrigeration (4 °C), both strains used in the CF and TF maintained a population above 8.0 log CFU/mL. Strains cultured in the TF had a significant increase in total phenolic compounds and greater antioxidant potential during their shelf life, along with improved survival of F19® after in vitro-simulated gastrointestinal conditions. Our study revealed the promising potential of CB in the probiotic beverage. The CB-containing formulation (TF) also exhibited higher phenolic content and antioxidant activity. Furthermore, it acted as a protector for bacteria during gastrointestinal simulation, highlighting its potential as a healthy and sustainable product.

Exploring the Association between Citrus Nutraceutical Eriocitrin and Metformin for Improving Pre-Diabetes in a Dynamic Microbiome Model.

Pre-diabetes is recognized as an altered metabolic state, which precedes type 2 diabetes, and it is associated with great dysfunction of the intestinal microbiota, known as dysbiosis. Natural compounds, capable of reducing blood glucose without side effects and with a beneficial effect on the microbiota, have been studied as substitutes or adjuvants to conventional hypoglycemic agents, such as metformin. In this work, the effect of the nutraceutical Eriomin®, a mixture of citrus flavonoids (eriocitrin, hesperidin, naringin, and didymin), which reduces glycemia and increases glucagon-like peptide-1 (GLP-1) in pre-diabetic patients, was tested in the Simulator of Human Intestinal Microbial Ecosystem (SHIME®), inoculated with pre-diabetic microbiota. After treatment with Eriomin® plus metformin, a significant increase in acetate and butyrate production was observed. Furthermore, sequencing of the 16S rRNA gene of the microorganisms showed that Eriomin® plus metformin stimulated the growth of Bacteroides and Subdoligranulum genera. Bacteroides are the largest fraction of the intestinal microbiota and are potential colonizers of the colon, with some species producing acetic and propionic fatty acids. In addition, Subdoligranulum species are associated with better host glycemic metabolism. In conclusion, Eriomin® associated with metformin improved the composition and metabolism of the intestinal microbiota, suggesting a potential use in pre-diabetes therapy.

Exploring the Potential of Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 as Promising Psychobiotics Using SHIME.

Psychobiotics are probiotics that have the characteristics of modulating central nervous system (CNS) functions or reconciled actions by the gut-brain axis (GBA) through neural, humoral and metabolic pathways to improve gastrointestinal activity as well as anxiolytic and even antidepressant abilities. The aim of this work was to evaluate the effect of Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 on the gut microbiota of mildly anxious adults using SHIME®. The protocol included a one-week control period and two weeks of treatment with L. helveticus R0052 and B. longum R0175. Ammonia (NH4+), short chain fatty acids (SCFAs), gamma-aminobutyric acid (GABA), cytokines and microbiota composition were determined. Probiotic strains decreased significantly throughout the gastric phase. The highest survival rates were exhibited by L. helveticus R0052 (81.58%; 77.22%) after the gastric and intestinal phase when compared to B. longum (68.80%; 64.64%). At the genus level, a taxonomic assignment performed in the ascending colon in the SHIME® model showed that probiotics (7 and 14 days) significantly (p < 0.005) increased the abundance of Lactobacillus and Olsenella and significantly decreased Lachnospira and Escheria-Shigella. The probiotic treatment (7 and 14 days) decreased (p < 0.001) NH4+ production when compared to the control period. For SCFAs, we observed after probiotic treatment (14 days) an increase (p < 0.001) in acetic acid production and total SCFAs when compared to the control period. Probiotic treatment increased (p < 0.001) the secretion of anti-inflammatory (IL-6 and IL-10) and decreased (p < 0.001) pro-inflammatory cytokines (TNF-alpha) when compared to the control period. The gut-brain axis plays an important role in the gut microbiota, producing SCFAs and GABA, stimulating the production of anti-anxiety homeostasis. The signature of the microbiota in anxiety disorders provides a promising direction for the prevention of mental illness and opens a new perspective for using the psychobiotic as a main actor of therapeutic targets.

QseC sensor kinase modulates the human microbiota during enterohemorrhagic Escherichia coli O157:H7 infection in the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®).

Enterohemorrhagic Escherichia coli (EHEC) is an important gastrointestinal pathogen known for its ability to cause hemorrhagic colitis and induce hemolytic-uremic syndrome. The inner membrane QseC histidine kinase sensor has shown to be an important regulator of the locus of enterocyte effacement (LEE) island, where important EHEC key virulence genes are located. However, the QseC role during EHEC infection in human microbiota remains unknown. Herein, using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®), we investigated whether the QseC sensor has a role in human microbiota modulation by EHEC in a dynamic model. Our data demonstrated that the QseC sensor modulates human microbiota during EHEC infection, and its absence leads to an increase in Lactobacillaceae and Bifidobacterium genus predominance, although non-effect on Bacteroides genus by EHEC strains was observed. In co-culture, the Lactobacillus acidophilus has affected EHEC growth and impaired the EHEC growth under space-niche competition, although no growth difference was observed in the QseC sensor presence. Also, differences in EHEC growth were not detected in competition with Bacteroides thetaiotaomicron and EHEC strains did not affect B. thetaiotaomicron growth either. When investigating the mechanisms behind the SHIME results, we found that hcp-2 expression for the type 6 secretion system, known to be involved in bacterial competition, is under QseC sensor regulation beneath different environmental signals, such as glucose and butyrate. Our findings broaden the knowledge about the QseC sensor in modulating the human microbiota and its importance for EHEC pathogenesis.

Functional Fermented Milk with Fruit Pulp Modulates the In Vitro Intestinal Microbiota.

The effect of putative probiotic fermented milk (FM) with buriti pulp (FMB) or passion fruit pulp (FMPF) or without fruit pulp (FMC) on the microbiota of healthy humans was evaluated. FM formulations were administered into a simulator of the human intestinal microbial ecosystem (SHIME®) to evaluate the viability of lactic acid bacteria (LAB), microbiota composition, presence of short-chain fatty acids (SCFA), and ammonium ions. The probiotic LAB viability in FM was affected by the addition of the fruit pulp. Phocaeicola was dominant in the FMPF and FMB samples; Bifidobacterium was related to FM formulations, while Alistipes was associated with FMPF and FMB, and Lactobacillus and Lacticaseibacillus were predominant in FMC. Trabulsiella was the central element in the FMC, while Mediterraneibacter was the central one in the FMPF and FMB networks. The FM formulations increased the acetic acid, and a remarkably high amount of propionic and butyric acids were detected in the FMB treatment. All FM formulations decreased the ammonium ions compared to the control; FMPF samples stood out for having lower amounts of ammonia. The probiotic FM with fruit pulp boosted the beneficial effects on the intestinal microbiota of healthy humans in addition to increasing SCFA in SHIME® and decreasing ammonium ions, which could be related to the presence of bioactive compounds.

Spirulina platensis biomass enhances the proliferation rate of Lactobacillus acidophilus 5 (La-5) and combined with La-5 impact the gut microbiota of medium-age healthy individuals through an in vitro gut microbiome model.

This study first evaluated the stimulatory effect of S. platensis biomass on the growth of L. acidophilus and the metabolic activity during fermentation (37 °C, 72 h) in a culture medium. The results demonstrated a higher impact of S. platensis biomass than fructooligosaccharide (FOS), an established prebiotic. Higher L. acidophilus proliferation rates and metabolic activity were observed (lower pH values and higher concentrations of acetic, lactic, and propionic acids) in the presence of S. platensis. Then, we evaluated the effects of the S. platensis biomass (1.5 g, twice a day, 5 days) in association with L. acidophilus (106 CFU/g) on the gut microbiota composition of medium-age healthy individuals through the Simulator of Human Intestinal Microbial Ecosystem (SHIME®) and measurement of metabolites. L. acidophilus (La5) and L. acidophilus + S. platensis (Spi-La5) could positively modulate the intestinal microbiota. The administration of La5 resulted in increases in Bacteroides, Megasphaera, Lactobacillus, and Parabacteroides genus abundance, with a consequent decrease in ammonium ions. The administration of Spi-La5 increased the abundance of the genus Erysipelatoclostridium, Roseburia, Enterococcus, Bifidobacterium, Coriobacteriaceae UCG-003, Enterobacter, and Paraclostridium. The results demonstrate that the intestinal microbiota was differently modified by administrating La5 and Spi-La5 and indicate the latter as an alternative for microbiota positive modulation in healthy individuals.

Corrigendum to 'Evidences and perspectives of the use of probiotics, prebiotics, synbiotics, and postbiotics as adjuvants for prevention and treatment of COVID-19: A bibliometric analysis and integrative review' [Trends in Food Science & Technology 120 (2022) 174-192].

[This corrects the article DOI: 10.1016/j.tifs.2021.12.033.].

Agro-industrial by-products: Valuable sources of bioactive compounds.

In a world with eminent scarcity of natural resources and increasing incidence of chronic diseases related to unhealthy eating habits, the search for biologically active and environmentally friendly food products is raising among customers. Agro-industrial by-products have caught special attention from the scientific community for being an available, cost-effective and sustainable source of a wide array of bioactive compounds. Review papers frequently restrain their research to by-products derived from the production of most worldwide consumed crops. Therefore, the aim of this review is to summarize the latest overall research, which focus on the biological potential of agro-industrial by-products and their bioactive compound profile, targeting their application as food ingredients, including not only researches with worldwide consumed crops, but also local foodstuff. A total of 152 research papers, browsed in 2 databases, and involving more than 30 countries were gathered. The richness of bioactive compounds of food by-products from different industries, from fruits to marine products, is ascertained throughout this review. The diversity of food residue being investigated for their nutritional and biological capabilities and the content of specific molecules in each food group are remarkable points. Higher literature reports about fruits by-products may be explained by its wide range of bioactive compounds, especially in Latin American fruits, which includes all flavonoids subclasses, besides betaxanthins, carotenoids and phytosterols. Researchers mainly focus on the quantification of fiber, polyphenols and antioxidant capacity of the investigated by-products, obstructing the investigation of specific biological activities, which are precisely related to the main phytochemicals of the residue matrix, as each molecule has an individual mechanism of action that should be considered when evaluating its biological capabilities. Furthermore, the addition of food by-products has also been advantageous in the production of fortified or enriched bakery, dairy and meat products and functional beverages. All along this literature review, it becomes clearer the high nutritional and nutraceutical value that many by-products possess, besides their attested biological activities, such as antioxidant, anticarcinogenic, antimicrobial, among others.

Evidences and perspectives of the use of probiotics, prebiotics, synbiotics, and postbiotics as adjuvants for prevention and treatment of COVID-19: A bibliometric analysis and systematic review.

BACKGROUND: Coronavirus disease-19 (COVID-19) is an infectious disease transmitted by the virus responsible for the severe acute respiratory syndrome 2 (SARS-CoV-2), which exhibit several clinical manifestations including gastrointestinal symptoms. SCOPE AND APPROACH: This review aimed to provide insights and perspectives for the use of probiotics, prebiotics, synbiotics, and postbiotics as adjuvants for prevention/treatment and/or modulation of the microbiota in COVID-19 patients. Eighty-four studies published in the Scopus database from the onset of the pandemic until December 2021 were assessed and submitted to a bibliometric analysis adapted from VOSviewer software. KEY FINDINGS AND CONCLUSIONS: Through bibliometric analysis, it might be suggested that the modulation of the gut/lung microbiome is promising as an adjuvant for the prevention/treatment of COVID-19 patients, due to immunomodulation properties related to probiotics and prebiotics. So far, few clinical studies involving the application of probiotics in COVID-19 patients have been completed, but reduction in the duration of the disease and the severity of symptoms as fatigue, olfactory dysfunction and breathlessness, nausea and vomiting and other gastrointestinal symptoms were some of the main findings. However, probiotics are not recommended to immunocompromised patients in corticosteroid therapy. The future perspectives point to the modulation of the intestinal microbiota by probiotics, prebiotics, synbiotics, and postbiotics represent a promising adjuvant approach for improving the health of patients with COVID-19.

Effect of probiotic, prebiotic, and synbiotic on the gut microbiota of autistic children using an in vitro gut microbiome model.

Imbalances in gut microbiota composition occur in individuals with autism spectrum disorder (ASD). The administration of probiotics, prebiotics, and synbiotics is emerging as a potential and promising strategy for regulating the gut microbiota and improving ASD-related symptoms. We first investigated the survival of the probiotics Limosilactobacillus (L.) reuteri and Bifidobacterium (B.) longum alone, mixed and combined with a galacto-oligosaccharide (GOS) under simulated gastrointestinal conditions. Next, we evaluated the impact of probiotics (L. reuteri + B. longum), prebiotic (GOS), and synbiotic (L. reuteri + B. longum + GOS) on gut microbiota composition and metabolism of children with ASD using an in vitro fermentation model (SHIME®). The combination of L. reuteri, B. longum, and GOS showed elevated gastrointestinal resistance. The probiotic, prebiotic, and synbiotic treatments resulted in a positive modulation of the gut microbiota and metabolic activity of children with ASD. More specifically, the probiotic treatment increased the relative abundance of Lactobacillus, while the prebiotic treatment increased the relative abundance of Bifidobacterium and decreased the relative abundance of Lachnoclostridium. Changes in microbial metabolism were associated with increased short-chain fatty acid concentrations and reduced ammonium levels, particularly in the prebiotic and synbiotic treatments.

Human microbiota modulation via QseC sensor kinase mediated in the Escherichia coli O104:H4 outbreak strain infection in microbiome model.

BACKGROUND: The intestinal microbiota plays a crucial role in human health, adjusting its composition and the microbial metabolites protects the gut against invading microorganisms. Enteroaggregative E. coli (EAEC) is an important diarrheagenic pathogen, which may cause acute or persistent diarrhea (≥14 days). The outbreak strain has the potent Shiga toxin, forms a dense biofilm and communicate via QseBC two-component system regulating the expression of many important virulence factors. RESULTS: Herein, we investigated the QseC histidine sensor kinase role in the microbiota shift during O104:H4 C227-11 infection in the colonic model SHIME® (Simulator of the Human Intestinal Microbial Ecosystem) and in vivo mice model. The microbiota imbalance caused by C227-11 infection affected ỿ-Proteobacteria and Lactobacillus spp. predominance, with direct alteration in intestinal metabolites driven by microbiota change, such as Short-chain fatty acids (SCFA). However, in the absence of QseC sensor kinase, the microbiota recovery was delayed on day 3 p.i., with change in the intestinal production of SCFA, like an increase in acetate production. The higher predominance of Lactobacillus spp. in the microbiota and significant augmented qseC gene expression levels were also observed during C227-11 mice infection upon intestinal depletion. Novel insights during pathogenic bacteria infection with the intestinal microbiota were observed. The QseC kinase sensor seems to have a role in the microbiota shift during the infectious process by Shiga toxin-producing EAEC C227-11. CONCLUSIONS: The QseC role in C227-11 infection helps to unravel the intestine microbiota modulation and its metabolites during SHIME® and in vivo models, besides they contribute to elucidate bacterial intestinal pathogenesis and the microbiota relationships.

Probiotic infant cereal improves children's gut microbiota: Insights using the Simulator of Human Intestinal Microbial Ecosystem (SHIME®).

Infant́s gut microbiota can be modulated by many factors, including mode of delivery, feeding regime, maternal diet/weight and probiotic and prebiotic consumption. The gut microbiota in dysbiosis has been associated with innumerous diseases. In this sense, early childhood intestinal microbiome modulation can be a strategy for disease prevention. This study had the purpose to evaluate the effect of an infant cereal with probiotic (Bifidobacterium animalis ssp. lactis BB-12®) on infant́s intestinal microbiota using SHIME®, which simulates human gastrointestinal conditions. The ascending colon was inoculated with fecal microbiota from three children (2-3 years old). NH4+, short chain fatty acids (SCFASs) and microbiota composition were determined by selective ion electrode, GC/MS and 16S sequencing, respectively. After treatment, butyric acid production increased (p < 0.05) 52% and a decrease in NH4+ production was observed (p < 0.01). The treatment stimulated an increase (p < 0.01) of Lactobacillaceae families, more precisely L. gasseri and L. kefiri. L. gasseri has been associated with the prevention of allergic rhinitis in children and L. kefiri in the prevention of obesity. Thus, infant cereal with BB-12® is able to stimulate the growth of L. gasseri and L. kefiri in a beneficial way, reducing NH4+ and increasing the production of SCFAs, especially butyric acid, in SHIME®.

Microbiota da pele: novos desafios / Skin microbiota: new challenges

Comunidades complexas de bactérias, fungos e vírus prosperam em nossa pele. A composição dessas comunidades depende das características da pele, como concentração das glândulas sebáceas, teor de umidade e temperatura, bem como da genética do hospedeiro e fatores ambientais exógenos. Estudos metagenômicos recentes descobriram uma diversidade surpreendente dentro desses ecossistemas e promoveram uma nova visão dos organismos comensais durantes as diferentes fases da vida humana. Portanto nesta revisão de literatura buscamos compreender as interações micróbio-hospedeiro e descobrir os fatores que impulsionam a colonização microbiana nos ajudará a entender a patogênese das doenças de pele e a desenvolver novas terapêuticas pro-microbianas e antimicrobianas.

Complex communities of bacteria, fungi and viruses thrive on our skin. The composition of these communities depends on the characteristics of the skin, such as concentration of sebaceous glands, moisture and temperature content, as well as the host's genetics and exogenous environmental factors. Recent metagenomic studies have discovered a surprising diversity within these ecosystems and have promoted a new view of commensal organisms during the different stages of human life. Thus, this review explores microbe-host interactions and discovering the factors that drive microbial colonization will help us understand the pathogenesis of skin diseases and develop new promicrobial and antimicrobial therapies

Immune status, well-being and gut microbiota in military supplemented with synbiotic ice cream and submitted to field training: a randomised clinical trial.

Strenuous physical activity, sleep deprivation and psychological stress are common features of military field training. The present study aimed to evaluate the effects of supplementation with a synbiotic ice cream on salivary IgA, gastrointestinal symptoms, well-being indicators and gut microbiota in young military participants undergoing field training. Sixty-five military completed the study: one group was supplemented for 30 d with synbiotic ice cream containing: 2·1 × 108 CFU/g for Lactobacillus acidophilus LA-5 and 2·7 × 109 CFU/g for Bifidobacterium animalis BB-12 and 2·3 g of inulin in the 60 g of ice cream at manufacture, and the other with a placebo ice cream. Volunteers were evaluated at pre-supplementation (baseline), post-supplementation and after a 5-d military training. Bifidobacterium and Lactobacillus genera were measured in stool samples and both showed a higher differential abundance post-supplementation and training. Salivary IgA and gastrointestinal symptoms decreased at post-training in both groups (P < 0·05; main effect of time); however, supplementation with synbiotic did not mitigate this effect. Tenseness and sleepiness were decreased in the synbiotic-treated group, but not in the placebo group at post-military training (P = 0·01 and 0·009, respectively; group × time effect). The other well-being indicators were not affected by the synbiotic supplementation. In conclusion, 30 d of synbiotic ice cream supplementation containing inulin, L. acidophilus LA-5 and B. animalis BB-12 favourably modulated gut microbiota and improved tenseness and sleepiness in healthy young military undergoing a 5-d field training. These improvements may be relevant to this population as they may influence the decision-making process in an environment of high physical and psychological stress.