Gut Microbial Dysbiosis Differs in Two Distinct Cachectic Tumor-Bearing Models Consuming the Same Diet.

The impact of cancer cachexia on the colonic microbiota is poorly characterized. This study assessed the effect of two cachectic-producing tumor types on the gut microbiota to determine if a similar dysbiosis could be found. In addition, it was determined if a diet containing an immunonutrient-rich food (walnuts) known to promote the growth of probiotic bacteria in the colon could alter the dysbiosis and slow cachexia. Male Fisher 344 rats were randomly assigned to a semi-purified diet with or without walnuts. Then, within each diet group, rats were further assigned randomly to a treatment group: tumor-bearing ad libitum fed (TB), non-tumor-bearing ad libitum fed (NTB-AL), and non-tumor-bearing group pair-fed to the TB (NTB-PF). The TB group was implanted either with the Ward colon carcinoma or MCA-induced sarcoma, both transplantable tumor lines. Fecal samples were collected after the development of cachexia, and bacteria species were identified using 16S rRNA gene analysis. Both TB groups developed cachexia but had a differently altered gut microbiome. Beta diversity was unaffected by treatment (NTB-AL, TB, and NTB-PF) regardless of tumor type but was affected by diet. Also, diet consistently changed the relative abundance of several bacteria taxa, while treatment and tumor type did not. The control diet increased the abundance of A. Anaeroplasma, while the walnut diet increased the genus Ruminococcus. There were no common fecal bacterial changes characteristic of cachexia found. Diet consistently changed the gut microbiota, but these changes were insufficient to slow the progression of cachexia, suggesting cancer cachexia is more complex than a few gut microbiota shifts.

Influence of Hesperidin on the Physico-Chemical, Microbiological and Sensory Characteristics of Frozen Yogurt.

Frozen yogurts contain yogurt culture bacteria, which might impart health benefits to their consumers. Global frozen yogurt market sales are expected to grow by 4.8% by 2028, which represents an important opportunity for the industry, consumers and researchers. Polyphenols are metabolites found in plants which have antioxidant and anti-inflammatory properties and might prevent chronic diseases such as cancer, diabetes and cardiovascular diseases. The objective of this study was to elucidate the effect of the polyphenol hesperidin on the physico-chemical, microbiological and sensory characteristics of frozen yogurts. Hesperidin was incorporated into frozen yogurt at three concentrations (125, 250 and 500 mg/90 g of product), while yogurt with no hesperidin was used as a control. The viscosity and overrun of the frozen yogurt were analyzed on day 0. The hardness, pH, color and Lactobacillus bulgaricus and Streptococcus thermophilus counts were determined after 0, 30 and 60 d. The melting rate was determined at 60 and 90 min after 0, 30 and 60 d. The bile and acid tolerances of both S. thermophilus and L. bulgaricus were measured after 7 and 60 d. A hedonic scale of nine points was used to measure sensory attributes. Data were analyzed at α = 0.05 with an ANOVA with Tukey's adjustment, and McNemar's test was used to analyze purchase intent. Hesperidin did not influence the pH, overrun or microbial characteristics. Polyphenol addition compared to the control decreased the melting rate but increased the hardness and bile tolerance of L. bulgaricus, as well as the L* and b* values. The sensory characteristics were not influenced by the lowest concentration of hesperidin, as it was not statistically different from the control. Moreover, consumers were interested in purchasing frozen yogurt with added hesperidin after learning about the health claim. This study can assist in the development of a healthier frozen yogurt in an increasingly competitive market.

Influences of Yogurt with Functional Ingredients from Various Sources That Help Treat Leaky Gut on Intestinal Barrier Dysfunction in Caco-2 Cells.

The impact of yogurts made with starter culture bacteria (L. bulgaricus and S. thermophilus) and supplemented with ingredients (maitake mushrooms, quercetin, L-glutamine, slippery elm bark, licorice root, N-acetyl-D-glucosamine, zinc orotate, and marshmallow root) that can help treat leaky gut were investigated using the Caco-2 cell monolayer as a measure of intestinal barrier dysfunction. Milk from the same source was equally dispersed into nine pails, and the eight ingredients were randomly allocated to the eight pails. The control had no ingredients. The Caco-2 cells were treated with isoflavone genistein (negative control) and growth media (positive control). Inflammation was stimulated using an inflammatory cocktail of cytokines (interferon-γ, tumor necrosis factor-α, and interleukin-1ß) and lipopolysaccharide. The yogurt without ingredients (control yogurt) was compared to the yogurt treatments (yogurts with ingredients) that help treat leaky gut. Transepithelial electrical resistance (TEER) and paracellular permeability were measured to evaluate the integrity of the Caco-2 monolayer. Transmission electron microscopy (TEM), immunofluorescence microscopy (IM), and real-time quantitative polymerase chain reaction (RTQPCR) were applied to measure the integrity of tight junction proteins. The yogurts were subjected to gastric and intestinal digestion, and TEER was recorded. Ferrous ion chelating activity, ferric reducing potential, and DPPH radical scavenging were also examined to determine the yogurts' antioxidant capacity. Yogurt with quercetin and marshmallow root improved the antioxidant activity and TEER and had the lowest permeability in fluorescein isothiocyanate (FITC)-dextran and Lucifer yellow flux among the yogurt samples. TEM, IM, and RTQPCR revealed that yogurt enhanced tight junction proteins' localization and gene expression. Intestinal digestion of the yogurt negatively impacted inflammation-induced Caco-2 barrier dysfunction, while yogurt with quercetin, marshmallow root, maitake mushroom, and licorice root had the highest TEER values compared to the control yogurt. Yogurt fortification with quercetin, marshmallow root, maitake mushroom, and licorice root may improve functionality when dealing with intestinal barrier dysfunction.

Physico-chemical, microbiological, and sensory characteristics of yogurt as affected by various ingredients.

l-Glutamine, quercetin, slippery elm bark, marshmallow root, N-acetyl-d-glucosamine, licorice root, maitake mushrooms, and zinc orotate have been reported to help treat leaky gut. The purpose of this research was to explore the impact of these functional ingredients on the physico-chemical, microbiological, and sensory properties of yogurt. The milk from same source was equally divided into 9 pails and the 8 ingredients were randomly assigned to the 8 pails. The control had no ingredient. Milk was fermented to yogurt. The pH, titratable acidity, syneresis, viscosity, color (L*, a*, b*, C*, and h*), Streptococcus thermophilus counts, and Lactobacillus delbrueckii spp. bulgaricus counts of yogurts were determined on d 1, 7, 14, 21, 28, 35, and 42, whereas coliform counts, yeast and mold counts, and rheological characteristics were determined on d 1 and 42. The sensory study was performed on d 3 and particle size of the functional ingredients (powder form) was also determined. When compared with control, the incorporation of slippery elm bark into yogurts led to less syneresis. l-Glutamine increased pH and n' values (relaxation exponent derived from G') and lowered titratable acidity values. N-Acetyl-d-glucosamine incorporation resulted in higher n' and lower titratable acidity values, whereas maitake mushroom led to lower n' values. Incorporating quercetin increased the growth of L. bulgaricus. Adding maitake mushrooms increased the growth of S. thermophilus but lowered apparent viscosity values, whereas quercetin decreased its S. thermophilus counts. Quercetin decreased L* and a* values but increased b* values, and maitake mushroom increased a* values. Thixotropic behavior increased with the addition of licorice root and quercetin. Adding slippery elm bark, N-acetyl-d-glucosamine, licorice root, maitake mushrooms, and zinc orotate into yogurt did not affect the sensory properties, whereas yogurts with quercetin had the lowest sensory scores. Overall, most of these ingredients did not cause major changes to yogurt properties.

Gut Microbiota Composition and Predicted Microbial Metabolic Pathways of Obesity Prone and Obesity Resistant Outbred Sprague-Dawley CD Rats May Account for Differences in Their Phenotype.

Like humans, outbred Sprague-Dawley CD rats exhibit a polygenic pattern of inheritance of the obese phenotype and not all individuals exposed to a high calorie intake develop obesity. We hypothesized that differences in gut microbiota composition account for phenotype differences between obese prone (OP) and obese resistant (OR) rats. We studied the gut microbiota composition of OPand OR rats after a high fat (HF) diet and how they respond to fermentation of resistant starch (RS). In phase 1 of the study 28 OP and 28 OR rats were fed a HF diet. In order to determine causal role of microbiota on phenotypes, In phase 2, a microbiota transplant between the two phenotypes was performed before switching all rats to a HF diet supplemented with 20% RS. We determined microbiota composition by 16S sequencing and predicted microbiota function by PICRUSt2. Despite a similar calorie intake, in phase 2 OP rats gained more weight and accumulated more abdominal fat in both phase 1 and 2 compared to OR rats (P < 0.001; n = 6). The OP rats fermented RS more robustly compared with OR rats with an increase in total bacteria, short chain fatty acids, and increased weight of the cecum, but microbiota of OP rats had much lower alpha diversity and evenness. The microbiota of OP rats, had higher amounts of bacteria from order Bacteroidales, specifically family Muribaculaceae (S24-7), which is known to possess several starch degrading enzymes and was reported in previous studies to increase with fermentation of RS. The OR rats fermented RS less but had higher bacterial diversity and evenness and had significantly higher bacterial counts from phylum Firmicutes particularly order Clostridiales, genus Clostridium and an uncultured bacterium of the genus Akkermansia. The microbiota of OR rats had enhanced bacterial chemotaxis, phosphotransferase system (PTS), and fatty acid biosynthesis compared to OP rats whose microbiota had higher glycan degradation and LPS biosynthesis pathways. The microbiota transplant did not change obesity phenotype or microbiota composition. In conclusion, a higher alpha-diversity and evenness of the microbiota and higher proportions of Clostridiales and Akkermansia in OR rats were associated with a better metabolic phenotype with lower body fat. However, robust RS fermentation caused a lower diversity and evenness and did not result in a leaner phenotype.

Metagenomic insights into the effects of Urtica dioica vegetable on the gut microbiota of C57BL/6J obese mice, particularly the composition of Clostridia.

Urtica dioica (UT) vegetable attenuates diet induced weight gain and insulin resistance. We hypothesized that UT imparts metabolic health by impacting the gut microbiota composition. We examined effects of UT on the cecal bacterial taxonomic signature of C57BL/6J mice fed isocaloric diets: a low-fat diet (LFD) with 10% fat, a high fat diet (HFD) with 45% fat or the HFD supplemented with 9% UT (HFUT). Among Firmicutes, the HFD had no significant impact on Clostridia, but increased Bacilli particularly genus Lactococcus and Lactobacillus. HFUT lowered Lactococcus but not Lactobacillus to levels of the LFD (P<.01; n=9). Further examination of Clostridia showed that HFUT increased genus Clostridium by over 2-fold particularly the species C. vincentii and C. disporicum and increased genus Turicibacter by three-fold (P<.05; n=9). Abundance of Clostridium and Turicibacter negatively correlated with body weight (P<.05; R2=0.42) and HOMA-IR (P<.05; R2=0.45). Turicibacter and Clostridium have been shown to be more abundant in lean phenotypes compared to obese. Clostridium impacts host phenotype by inducing intestinal T cell responses. The HFUT diet had no effect on members of Actinobacteria. Among Bacteroidetes, HFUT mainly increased proliferation of Bacteroides thetaiotaomicron (P<.05; n=9) with no significant impact on other groups. Functional analysis showed that HFUT enhanced bacterial beta-alanine and D-arginine metabolism both of which are associated with a lean phenotype and enhanced insulin sensitivity. We conclude that increasing the proliferation of Clostridium and Turicibacter and altering amino acid metabolism may be contributing mechanism(s) by which Urtica dioica impacts metabolic health.

Differences in Capacity of High-Amylose Resistant Starch, Whole-Grain Flour, and a Combination of Both to Modify Intestinal Responses of Male Sprague Dawley Rats Fed Moderate and High Fat Diets.

Gastrointestinal tract (GIT) responses to a high-amylose resistant starch (RS) product were compared to those observed when RS was combined with whole grain (WG) and to controls with low RS intake in rats fed moderate or high fat diets. Regardless of fat intake, rats fed RS or WG + RS diets had higher cecum weights, higher intestinal quantities of short chain fatty acids, and lower intestinal content pH, and their GIT cells had increased gene expression for gluconeogenesis and barrier function compared to controls. Whereas RS resulted in greater GIT content acetate and propionate and lowest pH, the WG + RS diets yielded higher butyrate. Rats fed the RS diet with MF had higher cecum weights than those fed either the RS diet with HF or the WG + RS diet with either MF or HF. Diets containing combinations of RS and other dietary fibers should be considered for RS-mediated GIT benefits.

Abundance of the species Clostridium butyricum in the gut microbiota contributes to differences in obesity phenotype in outbred Sprague-Dawley CD rats.

OBJECTIVES: Gut microbiota profiles contribute to differences in obesity phenotype. We examined the abundance of the species Clostridium butyricum in relation to obesity phenotype. METHODS: In outbred Sprague -Dawley rats we examined effects of dietary fat, resistant starch (RS), and a microbiota transplant on obesity phenotype. Using targeted qPCR, we examined the abundance of total gut bacteria and C. butyricum in relation to the propensity of obesity prone and obesity resistant rats to accumulate abdominal fat. RESULTS: Before inclusion of dietary RS, obesity resistant (OR) rats had higher amounts of total bacteria, and C. butyricum compared to obesity prone (OP) rats (P < 0.005 in study I, P < 0.0001 in study II). A high fat diet (HF) lowered C. butyricum levels while RS had no effect. Dietary RS elicited robust fermentation and increased total bacteria only in OP rats. In preparation for the transplant, antibiotics were administered to recipient rats. Four weeks thereafter, total bacteria levels were restored but, C. butyricum levels were not. The transplant between the two phenotypes had no effect on abundance of C. butyricum and obesity phenotype. CONCLUSIONS: While C. butyricum is a known saccharolytic, its proliferation is not enhanced by fermentation of resistant starch. C. butyricum maybe one of the species that constitute a core microbiota involved in energy storage and metabolism through mechanisms that are not yet known.

Validation of a Concussion Screening Battery for Use in Medical Settings: Predicting Centers for Disease Control Concussion Symptoms in Children and Adolescents.

OBJECTIVE: Effective screening for concussion is increasingly important, and medical professionals play a critical role in diagnostic and return-to-play decisions. However, few well-validated measures are available to assist in those decisions. This study aims to determine whether previously validated measures assessing neurocognitive and neurobehavioral abilities can predict Centers for Disease Control (CDC) concussion symptom endorsement in a sample of child or youth athletes. METHOD: Participants were 113 individuals, aged 6-17, representing 29 consecutive cases undergoing a post-concussion evaluation by a pediatric neurologist and 84 consecutive cases completing standardized baseline assessments (i.e., not being evaluated as a follow-up to a concussion). All participants completed the same standardized battery of tests comprised of the Connors' Continuous Performance Test (CPT 3), the Balance Error Scoring System (BESS), and the NIH 4-Meter Gait Test as well as completing a checklist of CDC concussion symptoms. RESULTS: Regression analyses indicate that the screening battery explained 33% of the variance (d = 1.4) in concussion symptom endorsement, after controlling for age. The neurocognitive test alone (CPT 3) accounts for 21.5% of the variance (d = 1.05) in symptoms after controlling for age, and the neurobehavioral measures (BESS and NIH 4-Meter Gait) then account for an additional 11.5% variance (accounting for 18.6% variance, d = .96, when entered first). These effect sizes are considered large to very large and reflect a marked increase in predictive validity relative to existing measures commonly used in concussion assessments. CONCLUSIONS: A relatively brief screening battery can function in medical settings to predict significant and substantial variability in CDC concussion symptoms in a pediatric sample.

Novel Resistant Starch Type 4 Products of Different Starch Origins, Production Methods, and Amounts Are Not Equally Fermented when Fed to Sprague-Dawley Rats.

SCOPE: The possible mechanisms of production of four novel resistant starch type 4 (RS4) products for total cecal fermentation in an in vivo rodent model are evaluated. METHODS AND RESULTS: Forty weanling rats are randomly assigned to five groups (n = 8) for a 3-week study. Starches are the RS type 4 products, as 10% of weight of RS diets (RSA-RSD), and AMIOCA starch (100% amylopectin) comprises 53.6% weight of control (CON) and 43.6% weight of RS diets. The RS products vary by percent purity and origin (potato, corn, tapioca). At euthanasia, cecal contents, serum, GI tract, and abdominal fat are collected. RSB, RSC, and RSD fed rats have greater empty cecum weights, lower cecal content pH, higher cecal content wet weight, and higher total cecal content acetate and propionate than the CON and RSA fed rats. Two other indicators of fermentation, total cecal contents butyrate and glucagon-like peptide 1, do not have significant ANOVA F values, which require more subjects for 80% power. CONCLUSION: RS4 products that are produced from different starch origins with varying amounts of RS4 content and different methods of production are not uniformly fermented in an in vivo model.

CD Obesity-Prone Rats, but not Obesity-Resistant Rats, Robustly Ferment Resistant Starch Without Increased Weight or Fat Accretion.

OBJECTIVE: This study used CD obesity-prone (OP) and obesity-resistant (OR) rats to examine how weight gain and fat accretion relate to fermentation levels and microbiota composition after feeding resistant starch (RS). METHODS: After feeding OP rats and OR rats a high-fat (HF) diet for 4 weeks, rats were stratified into three groups: they were fed either an HF diet (group 1: HF-HF) or were switched to a low-fat (LF) diet (group 2: HF-LF) or an LF diet supplemented with 20% RS by weight for 4 weeks (group 3: HF-LFRS). Energy intake, body weight, fermentation variables, and microbiota composition were determined. RESULTS: In OP rats, RS elicited robust fermentation (increased cecal contents, short-chain fatty acids, and serum glucagon-like peptide 1). Total bacteria, species of the Bacteroidales family S24-7, and the archaean Methanobrevibacter smithii increased. The robust fermentation did not elicit higher weight or fat accretion when compared with that of control rats fed the same isocaloric diets (HF-LF ± RS). In OR rats, body weight and fat accretion were also not different between HF-LF ± RS diets, but RS elicited minimal changes in fermentation and microbiota composition. CONCLUSIONS: Robust fermentation did not contribute to greater weight. Fermentation levels and changes in microbiota composition in response to dietary RS differed by obesity phenotype.

Obese ZDF rats fermented resistant starch with effects on gut microbiota but no reduction in abdominal fat.

SCOPE: To determine if whole-grain (WG) flour with resistant starch (RS) will produce greater fermentation than isolated RS in obese Zucker Diabetic Fatty (ZDF) rats, and whether greater fermentation results in different microbiota, reduced abdominal fat, and increased insulin sensitivity. METHODS AND RESULTS: This study utilized four groups fed diets made with either isolated digestible control starch, WG control flour (6.9% RS), isolated RS-rich corn starch (25% RS), or WG corn flour (25% RS). ZDF rats fermented RS and RS-rich WG flour to greatest extent among groups. High-RS groups had increased serum glucagon-like peptide 1 (GLP-1) active. Feeding isolated RS showed greater Bacteroidetes to Firmicutes phyla among groups, and rats consuming low RS diets possessed more bacteria in Lactobacillus genus. However, no differences in abdominal fat were observed, but rats with isolated RS had greatest insulin sensitivity among groups. CONCLUSIONS: Data demonstrated ZDF rats (i) possess a microbiota that fermented RS, and (ii) WG high-RS fermented better than purified RS. However, fermentation and microbiota changes did not translate into reduced abdominal fat. The defective leptin receptor may limit ZDF rats from responding to increased GLP-1 and different microbiota for reducing abdominal fat, but did not prevent improved insulin sensitivity.

Simultaneous delivery of antibiotics neomycin and ampicillin in drinking water inhibits fermentation of resistant starch in rats.

SCOPE: Antibiotics ampicillin 1 g/L and neomycin 0.5 g/L were added to drinking water before or during feeding of resistant starch (RS) to rats to inhibit fermentation. METHODS AND RESULTS: In a preliminary study, antibiotics and no RS were given prior to rats receiving a transplant of cecal contents via gavage from donor rats fed RS (without antibiotics) or a water gavage before feeding resistant starch to both groups. Antibiotics given prior to feeding RS did not prevent later fermentation of RS regardless of either type of gavage. In the second study, antibiotics were given simultaneously with feeding of RS. This resulted in inhibition of fermentation of RS with cecal contents pH >8 and low amounts of acetate and butyrate. Rats treated with antibiotics had reduced Bifidobacteria spp., but similar Bacteroides spp. to control groups to reduce acetate and butyrate and preserve the production of propionate. Despite reduced fermentation, rats given antibiotics had increased glucagon-like peptide 1 (GLP-1) and cecum size, measures that are usually associated with fermentation. CONCLUSIONS: A simultaneous delivery of antibiotics inhibited fermentation of RS. However, increased GLP-1 and cecum size would be confounding effects in assessing the mechanism for beneficial effects of dietary RS by knocking out fermentation.