- Digestion Of Short Chain Fatty Acids
- Effects Of Bifidobacterium Animalis Subsp Lactis Bb
- The Athlete And Gut Microbiome: Short
- Signs Of Pancreatic Cancer You Should Never Ignore
- Assessment Of Study Quality
- Mechanisms Mediating The Regulation Of Peroxisomal Fatty Acid Beta
- Final Thoughts On Essential Fatty Acids
- Improving Gut Epithelial Function
- Benefits Of Short Chain Fatty Acids
- Essential Fatty Acids And Skin Health
- Research Review: How Do Omega
- What Are Short Chain Fatty Acids?
If you’re currently trying to break a weight loss plateau, have high blood sugars, digestive problems or if you’re simply in the mood for some self-experimentation, then trying out resistant starch seems like a good idea. That being said, you can add resistant starch to your diet without adding any digestible carbohydrates. For this purpose, many people have recommended supplements, such as raw potato starch. A few studies in animals show that resistant starch can cause weight loss, but this effect hasn’t been studied properly in people. Several studies show that soluble fiber supplements can contribute to weight loss, primarily by increasing feelings of fullness and reducing appetite . Some studies have found a 33–50% improvement in insulin sensitivity after four weeks of consuming 15–30 grams per day .
Butyrate also increases intestinal epithelial cell production of antimicrobial peptides indicating a role in host defence . SCFAs increase the production of cytokines (TNF-a, IL-6) and chemokines in vitro . Activation of GPCRs induces membrane hyper-polarization, an increase in potassium ion influx and activation of the inflammasome NLPR3, increasing production of IL-18 a key cytokine that regulates repair and maintenance of epithelial barrier integrity .
- Adult CD patients naïve to active treatment showed reductions in Firmicutes and Clostridia, and increases in Bacteroidetes .
- Pectin, in which less than 50% of the carboxyl acid units occur as the methyl ester, is normally referred to as low ester or LM-pectin.
- In one trial, 20 healthy men and women ingested either placebo or fish oil capsules (2.8 g DHA and 1.2 g EPA per day) for four weeks .
- By comparing the GFD patients with the ND patients, we found no statistically significant differences between the measured bacteria.
- Alternatively, the SCFAs can be repackaged as enteric coated capsules that deliver time released product for maternal use, or added as a supplement to baby formula or food for at least the first 6-12 or months of life.
- In this system there are short-chain fatty acids , medium-chain fatty acids , and long-chain fatty acids .
- A recent work shows opposite patterns upon mucin usage as substrate by Lactobacillus and Akkermansia , thereby suggesting that trophic interactions may underlie, at least in part, these opposite trends of both microorganisms.
- Pharmaceutically acceptable carriers for liquid formulations are aqueous or non-aqueous solutions, suspensions, emulsions or oils, Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate.
- Ketone bodies have been suggested to produce larger amounts of energy compared to glucose.
- Then, it is feasible that decreased Akkermansia levels may lead to a compromised barrier function and increased gut permeability, hence promoting metabolic endotoxemia, which has been related to the development of obesity and associated disorders .
- Your gut bacteria convert resistant starch and pectin to short-chain fatty acids, and eating them has been shown to reduce diarrhea in children.
- The amounts of BB-12® in the PRE and POST were not different and the ingredients in the yogurt smoothie were identical .
- Butyrate has also been shown to decrease inflammation and diarrhea and protect against the growth of cancer causing cells in the colon.
- In addition, GPR43 is expressed in adipose tissues, where it controls fat accumulation.
The gut microbiota is an important factor that can regulate host energy metabolism and behavior (Tremaroli & Backhed, 2012). Additionally, the gut microbiota converts dietary fiber to SCFA, which increases the abundance of health-promoting bacteria and reduces the abundance of potentially pathogenic bacteria (Zentek et al., 2003). The ratio of Firmicutes to Bacteroides is higher in animals with genetically or diet-induced obesity, although this remains controversial (Ley et al., 2005; Moschen, Wieser & Tilg, 2012). We previously found that various dietary supplements such as fiber-like materials reduce this ratio in experimental animal models of obesity (Nakatani et al., 2018; Watanabe et al., 2018). Our findings of the 16S rRNA gene amplicon sequences revealed that the ratio of Firmicutes to Bacteroides was reduced more by Fuji FF than by cellulose. Our results at the family level showed an elevated relative abundance of Bacteroidales S24-7 groups, which are thought to be anti-diabetic, in the phylum Bacteroides (Krych et al., 2015).
Digestion Of Short Chain Fatty Acids
The pH was found to be a major factor during cultivation, influencing the growth performance of the separate strains and caproate toxicity. We have completed separate pilot studies of 3.4 g/day EPA + DHA ethyl esters (4 g Lovaza™) administered for 6 months to explore effects on benign breast tissue risk biomarkers for breast cancer in premenopausal and postmenopausal women at increased risk for breast cancer. Favorable modulation of several tissue risk biomarkers for breast cancer was observed . Additional preclinical studies suggest that EPA and DHA increase expression of BRCA1/2, phosphatase and tensin homolog , and other proteins associated with cell cycle control and DNA repair . In general, the action of COX and LOX enzymes on the omega-3 fatty acids EPA and DHA is to produce eicosanoids with less affinity for the corresponding receptors as well as resolvins that block inflammatory cell recruitment and promote phagocytosis.
In combination with the overexpression of the ilvIHCD genes for 2-ketoisovalerate biosynthesis, isobutanol was produced at a titer of 22 g/L. Subtilis by engineering an Ehrlich pathway together with the overexpression of the acetolactate synthase genes (alsS, ilvC, and ilvD as shown in Fig.1) responsible for the synthesis of 2-ketoisovalerate . Glutamicum, the most widely used branched-chain amino acid producer in fermentation industry, has a natural ability to accumulate 2-ketoisovalerate and 2-keto-3-methylvalerate at high intracellular concentrations .
Effects Of Bifidobacterium Animalis Subsp Lactis Bb
These engineering efforts have largely expanded the capability to bioproduce advanced biofuels in microbial hosts that have more attractive features than the native host . Subtilis fabH2 and its bkd operon, a terminally branched LCFA biosynthetic pathway was first constructed in E. However, the initially engineered strain only produced 2.5 mg/L of branched-chain LCFAs. Instead, a high proportion of straight-chain fatty acids were co-produced . To increase the proportion of terminally branched LCFAs in total free fatty acids, Jiang et al. replaced the acetyl-CoA-specific E.
In one embodiment, the method of the invention may comprise administration of the composition of the invention in combination with one or more chemotherapeutic agent. In some embodiments, a composition of the invention is administered before, during, or after administration of at least one antiproliferative agent for the treatment of cancer. Antiproliferative agents are compounds that decrease the proliferation of cells.
Many factors affect the amount of short-chain fatty acids in your colon, including how many microorganisms are present, the food source and the time it takes food to travel through your digestive system. Whereas small quantities of short-chain fatty acids are absorbed through the portal venous circulation, 80% to 90% of all fat absorbed from the gut is transported by way of the thoracic duct in the form of chylomicrons. Thus, feedings restricted to medium- or short-chain triglycerides theoretically result in reduced lymph flow in the thoracic duct and may enhance spontaneous healing of a thoracic duct fistula. Short-chain fatty acids are produced by the friendly bacteria in your gut.
The Athlete And Gut Microbiome: Short
The addition of beta-acids decreased acetate, propionate and total SCFA. The fermentation of oat β (1 ® 3) glucan and glucose were not altered by hop beta-acids. Of particular importance is the role of fatty acids in the formation of eicosanoids, which are a group of signaling molecules involved in the immune response.
Lower Ehhadh expression was also noted in murine hepatocellular carcinomas and in subsets of certain human cancers, supporting the likely generality of these results. Our results demonstrate the feasibility of C12 or DDDA dietary supplementation that is non-toxic, inexpensive and likely compatible with more standard chemotherapies. Hypertrophy is one of the pathological processes where increased acetylation is implicated in disease progression. In response to various hypertrophic stimuli, SIRT3-deficient mice appear to be more sensitive to injuries and manifested various abnormalities compared to their wild-type counterparts . On the other hand, SIRT3 transgenic mice are protected from hypertrophic injuries . Activation of forkhead box O3a-dependent and manganese superoxide dismutase and catalase are also seen in response to SIRT3 activation.
Signs Of Pancreatic Cancer You Should Never Ignore
The pharmaceutical compositions provided herein may also be administered as controlled-release compositions, i.e. compositions in which the active ingredient is released over a period of time after administration. Controlled- or sustained-release compositions include formulation in lipophilic depots (e.g. fatty acids, waxes, oils). In another embodiment, the composition is an immediate-release composition, i.e. a composition in which all the active ingredient is released immediately after administration. Samples that may be used for measuring such an index include tissues and fluids obtained from an individual, such as blood, a biopsy, or a fecal sample. A key Th1-type cytokine involved in the pathogenesis of inflammatory disease is IFN-γ which inhibits apoptosis of keratinocytes by stimulating expression of the anti-apoptotic protein Bcl-x. Therefore, in one embodiment, the method of the invention may comprise administration of the composition of the invention in combination with one or more inhibitors of IFN-γ.
In contrast, the VLCFAs were excluded from membrane lipids in the developing seeds of transgenic Arabidopsis. The inefficient catabolism of VLCFAs in nonseed tissues in this instance may have resulted in their accumulation in these tissues. It is speculated that VLCFAs may play a role in membrane bilayer structure/function so that they are not catabolized, by analogy with the presence of C24 to C26 VLCFAs in sphingolipids. One possibility is that VLCFAs may be required for the formation of highly curved membrane structures, which may mimic the structural role of sphingolipids. The yeast Slc mutant, which lacks sphingolipids synthesis, can survive without sphingolipids by making novel glycerolipids containing C26 fatty acids, strongly supporting this hypothesis. Expression of a nasturtium acyl-CoA-dependent diacylglycerol acyltransferase 1 gene in high-erucic acid rapeseed or canola did not increase the erucic acid levels but significantly increased oil content.
Assessment Of Study Quality
Oris MG1 live cells together with dead cells than was induced by 60 mM butyric acid. The ionized form present at low pH causes cytoplasmic membrane damage to a major barrier to proton influx . Proteins embedded in cell membranes are exposed to the detrimental effect of the acidic pH at their cellular location and inactivated . Fimbrillin-dependent and fimbrillin-independent INAC in live cells is considered to rely on the effects of nonionized organic acids on the membrane surface of the fimbriae in dental plaques.
Fibre may be, in part, so important for human health because it is the main food source for bacteria which then produce short chain fatty acids. To be fair not all fibre can be fermented by these bacteria and fibre does have other benefits including bulking of the stool . So a nice little summary here is that dietary fiber can be used as a food source for different bacteria which then produce short chain fatty acids.
Mechanisms Mediating The Regulation Of Peroxisomal Fatty Acid Beta
Consistent with this mechanism, we demonstrated that perinatal deficits in DHA accrual were associated with a significant reduction in membrane-bound MARCKS, and an associated increase in cytosolic MARCKS, in the rat hippocampus. These findings suggest that lower membrane phosphatidylserine content in response to reduced DHA levels are associated with a dysregulation in the signal transduction processes that regulate F-actin cytoskeletal structural plasticity. The strength of this study is in finding a verified association between stool patterns and plasma and fecal fatty acids. The multifaceted, self-reported questionnaires used for stool status assessment had previously been validated against objective fecal characteristics as well .
Final Thoughts On Essential Fatty Acids
We found that FFAR2, FFAR3 mRNA were present at very low levels and that no changes were observed after IL-1β stimulation in H4 cells. However, the GPR 109A mRNA was detectable and up-regulated to varying degrees was noted upon IL-1β stimulation with all three SCFAs pretreatment of H4 cells. Necrotizing enterocolitis is an inflammatory necrosis of the distal small intestine and colon that commonly affects very premature infants (10% incidence) less than 1500grams in birthweight . We have reported that its pathogenesis is in part due to an inappropriate reaction of colonizing bacteria with an immature intestine . For example, colonizing bacteria can create an excessive inflammatory response in the immature intestine rather than developing immune homeostasis . Follow-up testing will involve checking your baby’s urine and blood samples for harmful amounts of acids and toxins.
Improving Gut Epithelial Function
Specifically Studying unique populations as they span the epidemiologic transition, allows us to investigate several risk factors simultaneously, including environmental co-variates, (e.g. local diet/PA), which have been shown to impact both gut microbiota and SCFAs. In addition, continuing follow-up in a previously established, longitudinal cohort of African-origin adults, and considered high-risk for the development of obesity and metabolic disorders will allow us to capitalize on identifying causal factors. Notably, we can leverage up to 10 years of detailed phenotype information already collected in the parent study, METS, including yearly weight change, to model the contribution of these on the gut microbiota and SCFAs. Geng, C.; Meng, Q.; Zhang, M.; Liu, X. Effects of medium chain fatty acid on animal growth performance and the possible mechanism. Dierick, N.A.; Decuypere, J.A.; Degeyter, I. The combined use of whole Cuphea seeds containing medium chain fatty acids and an exogenous lipase in piglet nutrition. Butyrate can act as an energy source for normal colon epithelial cells, promoting their proliferation, but can also inhibit proliferation and induce apoptosis .
Benefits Of Short Chain Fatty Acids
Short-chain fatty acids help battle inflammation and promote gut health, but it’s important not to go overboard. Most of us don’t get the dietary fiber we need in the first place, and fresh food should be the first place you turn because getting too much fiber has the opposite effect. To account for this, the list below focuses on the most powerful types of fiber for short chain fatty acid levels , along with various foods that contain the fiber. For the past decade, we have all witnessed the confusing marketing war over how many billions of bacteria or how many strains of probiotics could fit into a capsule.
Essential Fatty Acids And Skin Health
As discussed in the preceding section, the increased acetylation of proteins following enhanced fatty acid ß-oxidation and increased acetyl-CoA generation may contribute to the metabolic phenotype observed in I/R. In addition, as NAD+ availability is a critical determinant for Sirtuin activity, ischemia-induced decreases in the NAD+/NADH redox couple during I/R may inactivate SIRT3 and lead to the hyperacetylation of mitochondrial proteins . Furthermore, the mRNA and protein levels of NAMPT, the rate-limiting enzyme that converts NAM to NMN in the NAD+ salvage synthesis pathway is downregulated in the heart during I/R injury, further reducing NAD+ levels and thus Sirtuin activity . Several studies have demonstrated the cardioprotective role of NAD+ in I/R injury either by exogenous NAD+ supplementation or enzymatic manipulation . However, this mechanism has not yet been investigated in relation to changes in metabolic enzymes activity or metabolic alterations as a result of acetylation suppression by NAD+ boosting.
In mouse epidermis, topical EPA (10 nmol/cm2, 30 min) protected against UVB-induced immunosuppression, but also increased lipid peroxidation and decreased vitamin C levels compared to oleic acid treated control mice . Similarly, dietary EPA supplementation (20% of total fatty acids consumed daily for Delta 8 Disposable Cartridges ten days) protected skin from UVB-induced immunosuppression but increased lipid peroxidation and reduced vitamin C and glutathione levels in mouse epidermis . The essential role of LA in barrier function was determined in several animal experiments similar to those conducted by Burr and Burr .
Sphaeroides significantly decreased the aspartate aminotransferase and creatinine level. Sphaeroides treatment on the basic physiological health of mice was observed. Sphaeroides significantly increased the content of acetic acids in the gut of mice, while there were no significant changes in the concentration of butyric and propionic acid. High-throughput sequencing analysis indicated that there was a significant difference in α-diversity between these 2 groups, while there was no significant difference in β-diversity between them. Sphaeroides 8513 could modulate the microbial ecology in the gut of mice. This study, METS-Microbiome, proposes to examine the influence of the gut microbiota, and its metabolites of carbohydrate digestion, short chain fatty acids , on obesity, and weight change.
Similarly, palmitic acid increases the phosphorylation of nuclear factor of kappa light peptide gene enhancer in B-cells inhibitor α (IκBα), which leads to phosphorylation and translocation of NF-κB p65 to the nucleus . Phosphorylation of p38, c-Jun , IκBα and NF-κB p65 , endoplasmic reticulum stress is also described after treatment of macrophages with stearic acid. Activation of both MAPK signaling and NF-κB pathway by SFAs leads to the production of cytokines and activation of cyclooxygenase (COX-2) and iNOS. In addition to binding to TLRs, palmitic and lauric acids enhances TLR4 expression in different macrophage cell lines as well as expression of other receptors involved in the immune response, such as CD36 and CD86 .
Bacteroidetes were more abundant in the stool samples of those who ate a predominantly plant based diet, while Firmicutes were more abundant in those who ate a predominantly animal products diet. Both these categories of organisms contain microbial species that can break down complex carbohydrates, resulting in the production of SCFAs. The Mediterranean diet is characterised by high intake of fruit, vegetables, legumes, nuts and cereals; moderately high intake of fish; regular but moderate alcohol consumption; and low intake of saturated fat, red meat, and dairy products. SCFAs serve as an energy source for the colonocyte and are rapidly absorbed and have been shown to play a role in gut motility, gut barrier maintenance, inflammation, glucose homeostasis, and energy harvesting.
Research Review: How Do Omega
In addition, systemic inflammation induced by inflammatory stimuli such as a single injection of LPS leads to injury of intestinal barrier function and down-regulated expression of occludin and claudin-1 in weaning pigs . However, Rakhshandeh et al. reported that repeated LPS injection did not alter transepithelial resistance in the ileum of growing pigs although it decreased nutrient digestibility and increased active glucose transport. The reasons for this discrepancy might be that the pigs develop a tolerance to the multiple, subsequent LPS challenges , and/or the growing pigs are more resistant to LPS challenge relative to weaning pigs. Moreover, early weaning also results in increased intestinal permeability and decreased expression of tight junction proteins in piglets .
The cells were then treated for 24 hr and harvested and assayed for luciferase and -galactosidase activities using the Luciferase Assay System and the -Galactosidase Enzyme Assay System . LRD, JPR, KBA, PB, EVL and TEL are the principal investigators at each of the five study sites. JAG is responsible for the microbiome, and bioinformatics analyses and interpretation. BTL is an endocrinologist with expertise in SCFAs metabolism, diabetes and metabolism. The investigative team, with its geographic diversity, existing data, established partnerships, and multidisciplinary expertise, is uniquely positioned to conduct this type of research. With this study, we have enriched the existing METS investigative team of epidemiologists, biostatisticians, nutritionists and exercise physiologists with expertise in endocrinology, microbiology, metabolomics and bioinformatics.
If you have a question about what makes a “short” fatty acid, check out this link. Then, we addressed in our group of adult subjects, whether an altered gut microbiota composition and/or serum inflammatory mediators may have a negative impact on the host metabolism and if a clinical relevance of these players could be expected. The steepest rise in SCFA concentration occurred during the first 6 hours for all substrates and calculation of SCFA production always yielded positive values. In both controls and patients with DP-IBS, lactulose fermentation resulted in the highest rates of SCFA production followed by pectin, starch, and hemicellulose . During the first 6 hours, incubations with lactulose, starch, and hemicellulose resulted in significantly increased production rates for total SCFA, acetate, and propionate in control fecal homogenates versus those from DP-IBS patients.
Martin RE, Bazan NG. Changing fatty acid content of growth cone lipids prior to synaptogenesis. Suzuki H, Manabe S, Wada O, Crawford MA. Rapid incorporation of docosahexaenoic acid from dietary sources into brain microsomal, synaptosomal and mitochondrial membranes in adult mice. Valentine CJ, Morrow G, Pennell M, Morrow AL, Hodge A, Haban-Bartz A, Collins K, Rogers LK. Randomized controlled trial of docosahexaenoic acid supplementation in midwestern U.S. human milk donors. Francois CA, Connor SL, Bolewicz LC, Connor WE. Supplementing lactating women with flaxseed oil does not increase docosahexaenoic acid in their milk. It is clear from the preclinical ‘prevention’ studies that combination EPA and DHA treatment has anti-CRC activity.
Essential Fatty Acids
It stabilizes the enzyme during the experiment, but it is relatively neutral so it doesn’t affect the properties of the enzyme. Each one of your ten trillion cells requires a constant supply of nourishment. But we don’t have to worry about this–we merely eat our dinner and our body does the rest. The food is digested and the useful pieces are delivered to cells throughout the body, using the bloodstream as the delivery system. They float in the watery bloodstream and are picked up by cells along the way. Other important nutrients, however, are not soluble in water, so special carriers must be made to chaperone them to hungry cells.
The increased lipid accumulation in the resident macrophages reflects the fatty acid composition of the adipocytes. The dietary fatty acid determines the fatty acid composition of the adipose tissue. Macrophages then accumulate fatty acids indirectly provided by the diet. The composition varies with the acyl chain length, e.g., short-, medium-, or long-chain, and saturated fatty acids. We described herein in detail the impact of the different dietary fatty acids on macrophage functions.
Thus, these pieces of evidence point to a Lactobacillus within-group heterogeneity with relevance for the human health, as suggested by other authors . In agreement with this hypothesis our findings on the interactions between Lactobacillus abundance and nutrient parameters, evidenced different association patterns as depending on the metabolic “health” status of the subject. Controversy observed in mice studies and clinical interventions in humans is also consistent with this idea.
Adipocytes store fat derived from the diet and from liver metabolism. Under energy stress these cells may degrade their stored fat to supply fatty acids and also glycerol to the circulation. These metabolic activities are regulated by several hormones (e.g., insulin, glucagon and epinephrine).
In one embodiment, the method comprises administering to a subject in need thereof a composition comprising at least one SCFA, or a biologically-active derivative or precursor thereof, in combination with one or more immune checkpoint inhibitor. “Checkpoint inhibitor” as used herein includes inhibitors or molecules that block immune checkpoints as commonly understood in the field of cancer immunotherapy. Commonly the checkpoint inhibitors Do CBD GUMMIES Eliminate Pain? are antibodies that block the immune checkpoint proteins. Immune checkpoint proteins include, but are not limited to, PD1, PDL1, PDL2, CTLA-4, LAG3, TIM3, B7-H3, BTLA, VISTA, CD40, CEACAM1, CD80, CD86, OX40, CD27, GITR, DNAM-1, TIGIT, TMIGD2 and DC-SIGN. Some examples of known checkpoint inhibitors include, but are not limited to, ipilimumab, pembrolizumab, nivolumab, pidilizumab, atezolizumab, avelumab, druvbalumab and others.
They are also rich sources of energy, and may be broken down inside cells to form ATP. Thus, your body maintains a storehouse of fatty acids, stored as fat. When your body needs energy or needs building materials, fat cells release fatty acids into the blood. There, they are picked up by serum albumin and delivered to distant parts of the body. U. Axling, C. Olsson, J. Xu et al., “Green tea powder and Lactobacillus plantarum affect gut microbiota, lipid metabolism and inflammation in high-fat fed C57BL/6J mice,” Nutrition and Metabolism, vol.
What Are Short Chain Fatty Acids?
Akkermansia is known to reside within the mucus layer of the intestine, thus contributing to strengthen the intestinal wall . Then, it is feasible that decreased Akkermansia levels may lead to a compromised barrier function and increased gut permeability, hence promoting metabolic endotoxemia, which has been related to the development of obesity and associated disorders . Therefore, these results suggest that Akkermansia may be linked to the inflammatory milieu by modulating the FFA profile in the host. Hyper-oxygenated fatty acids are produced by a specific industrial processes for topical skin creams.
Sunburn, also referred to as erythema, is caused by excess exposure to ultraviolet radiation . Even at levels that may not cause sunburn, UVR causes cellular damage that induces inflammation and suppresses the immune system in the skin . Because both omega-6 and omega-3 EFAs are converted into compounds that participate in inflammatory and immune reactions, their levels in skin can influence the cellular response to UVR. Experiments using human keratinocyte cell cultures and isolated skin extracts demonstrate that UV exposure reduces 12-LOX expression but increases 15-LOX expression . Furthermore, 15-LOX metabolites inhibit 12-LOX expression and the formation of its proinflammatory product, 12-HETE . Notably, COX-2 is also induced by UVR, and there is an orchestrated expression pattern of COX- and LOX-derived eicosanoids that mediate the response to UVR .
A part of the same fresh fecal sample collected for SCFA analysis was stored in the refrigerator and processed within 2 hours of defecation. Samples were prepared by diluting with 18 times their weight of cooled phosphate buffer (pH 7.4) and saturated with nitrogen to preserve an anaerobic environment. Homogenization of samples was carried out at 4°C in sterile containers in the presence of CBD Gummies 3000mg Jar vs. CBD Gummies 3000mg Party Pack nitrogen. Then 25 mL of the homogenized fecal suspension was transferred into 120-mL sterile containers. Next, 100 mg of each substrate was added to each container along with a small magnet and the samples stirred under nitrogen to facilitate dispersion and homogenization. All containers were tightly closed, sealed, and incubated at 37°C in a rotating water bath for 6, 16, and 24 hours.