Unlocking Nature's Toolbox: glutamate-inducible recombinant protein production from the Komagatella phaffii PEPCK promoter.

BACKGROUND: Komagataella phaffii (a.k.a. Pichia pastoris) harbors a glutamate utilization pathway in which synthesis of glutamate dehydrogenase 2 and phosphoenolpyruvate carboxykinase (PEPCK) is induced by glutamate. Glutamate-inducible synthesis of these enzymes is regulated by Rtg1p, a cytosolic, basic helix-loop-helix protein. Here, we report food-grade monosodium glutamate (MSG)-inducible recombinant protein production from K. phaffii PEPCK promoter (PPEPCK) using green fluorescent protein (GFP) and receptor binding domain of SARS-CoV-2 virus (RBD) as model proteins. RESULTS: PPEPCK-RBD/GFP expression cassette was integrated at two different sites in the genome to improve recombinant protein yield from PPEPCK. The traditional, methanol-inducible alcohol oxidase 1 promoter (PAOX1) was used as the benchmark. Initial studies carried out with MSG as the inducer resulted in low recombinant protein yield. A new strategy employing MSG/ethanol mixed feeding improved biomass generation as well as recombinant protein yield. Cell density of 100-120 A600 units/ml was achieved after 72 h of induction in shake flask cultivations, resulting in recombinant protein yield from PPEPCK that is comparable or even higher than that from PAOX1. CONCLUSIONS: We have designed an induction medium for recombinant protein production from K. phaffii PPEPCK in shake flask cultivations. It consists of 1.0% yeast extract, 2.0% peptone, 0.17% yeast nitrogen base with ammonium sulfate, 100 mM potassium phosphate (pH 6.0), 0.4 mg/L biotin, 2.0% MSG, and 2% ethanol. Substitution of ammonium sulphate with 0.5% urea is optional. Carbon source was replenished every 24 h during 72 h induction period. Under these conditions, GFP and RBD yields from PPEPCK equaled and even surpassed those from PAOX1. Compared to the traditional methanol-inducible expression system, the inducers of glutamate-inducible expression system are non-toxic and their metabolism does not generate toxic metabolites such as formaldehyde and hydrogen peroxide. This study sets the stage for MSG-inducible, industrial scale recombinant protein production from K. phaffii PPEPCK in bioreactors.

The investigation of the effects of monosodium glutamate on healthy rats and rats with STZ-induced diabetes.

Monosodium glutamate (MSG, E621) is a flavor-enhancing food additive used widely in the food preparation industry and consumed regularly. It is considered that long-term consumption of MSG causes metabolic syndrome and obesity. Diabetes mellitus (DM) is a chronic metabolic disease characterized by high blood sugar, polyuria, polydipsia, and polyphagia, in which insulin secreted from pancreatic ß cells is inadequate for maintaining blood glucose homeostasis. Rats were application 65 mg/kg streptozotocin (STZ) solution intraperitoneally and a diabetes model was created. For this purpose, freshly prepared STZ was injected into the peritoneum. Tumor necrosis factor-α, interleukin (IL)-10, IL-6, and IL-1ß levels in STZ, MSG, and STZ + MSG groups were found to be significantly increased in inflammation parameters measured on the 28th day of administration when compared to the Control Group (p < 0.001). Also, although malondialdehyde (MDA) levels increased significantly in the STZ + MSG group when compared to the control group (p < 0.001), glutathione (GSH), and superoxide dismutase (SOD) levels were significantly decreased in the STZ, MSG, and STZ + MSG groups when compared to the control group (p < 0.001). Also, although glucose levels increased significantly in STZ and STZ + MSG at the end of the 28th day (p < 0.01), insulin levels decreased in STZ, MSG, and STZ + MSG groups when compared to the control groups (p < 0.01). As a result, it was found that STZ and MSG application significantly increased cytokine production, increased MDA, which is an oxidant parameter in pancreatic tissue, and decreased antioxidants (GSH and SOD) when compared to the control groups. It was also found that MSG disrupted the normal histological structure in pancreatic cells, and the damage was much more in both exocrine and endocrine pancreatic areas in the STZ + MSG group when compared to the STZ and MSG groups. It was considered that with the increased use of MSG, the susceptibility to DM might increase along with tissue damage significantly in diabetic groups, therefore, MSG must be used in a limited and controlled manner.

Optimization of fermentation conditions for the production of γ-aminobutyric acid by Lactobacillus hilgardii GZ2 from traditional Chinese fermented beverage system.

γ-Aminobutyric acid (GABA) is a crucial neurotransmitter with wide application prospects. In this study, we focused on a GABA-producing strain from a traditional Chinese fermented beverage system. Among the six isolates, Lactobacillus hilgardii GZ2 exhibited the greatest ability to produce GABA in the traditional Chinese fermented beverage system. To increase GABA production, we optimized carbon sources, nitrogen sources, temperature, pH, and monosodium glutamate and glucose concentrations and conducted fed-batch fermentation. The best carbon and nitrogen sources for GABA production and cell growth were glucose, yeast extract and tryptone. Gradual increases in GABA were observed as the glucose and monosodium glutamate concentrations increased from 10 g/L to 50 g/L. During fed-batch fermentation, lactic acid was used to maintain the pH at 5.56, and after feeding with 0.03 g/mL glucose and 0.4 g/mL sodium glutamate for 72 h, the GABA yield reached 239 g/L. This novel high-GABA-producing strain holds great potential for the industrial production of GABA, as well as the development of health-promoting functional foods and medical fields.

Enhancing menaquinone-7 biosynthesis through strengthening precursor supply and product secretion.

Menaquinone-7 (MK-7) is an important class of vitamin K2 that is essential in human health and can prevent osteoporosis and cardiovascular disease. However, due to the complex synthesis pathway, the synthesis efficiency is low. The main objective of this study was to explore the effect of enhanced supply of precursors in Bacillus natto. Three precursors of pyruvate, shikimic acid, and sodium glutamate were chosen to investigate the effect of enhanced supply of precursors on MK-7 synthesis. Then, the optimal concentrations, different combinations, and different adding times were systematically studied, respectively. Results showed that the combination of shikimic acid and sodium glutamate could boost MK-7 production by 2 times, reaching 50 mg/L of MK-7 titer and 0.52 mg/(L·h) of MK-7 productivity. Furthermore, adding shikimic acid and sodium glutamate initially and feeding pyruvate at 48 h and 72 h increased MK-7 production to 58 mg/L. At the same time, the expression of the three related genes was also significantly upregulated. Subsequently, a new fermentation strategy combining the precursors enhancement and product secretion was proposed to enhance MK-7 yield and MK-7 productivity to 63 mg/L and 0.45 mg/(L·h). This study proposed a new fermentation regulation strategy for the enhancement of vitamin K2 biosynthesis.

Hybrid substrate-based pH autobuffering GABA fermentation by Levilactobacillus brevis CD0817.

The probiotic fermentation of the bioactive substance gamma-aminobutyric acid (GABA) is an attractive research topic. There is still room for further improvement in reported GABA fermentation methods based on a single substrate (L-glutamic acid or L-monosodium glutamate). Here, we devised a pH auto-buffering strategy to facilitate the fermentation of GABA by Levilactobacillus brevis CD0817. This strategy features a mixture of neutral monosodium L-glutamate plus acidic L-glutamic acid as the substrate. This mixture provides a mild initial pH; moreover, the newly dissolved L-glutamic acid automatically offsets the pH increase caused by substrate decarboxylation, maintaining the acidity essential for GABA fermentation. In this study, a flask trial was first performed to optimize the GABA fermentation parameters of Levilactobacillus brevis CD0817. The optimized parameters were further validated in a 10 L fermenter. The flask trial results revealed that the appropriate fermentation medium was composed of powdery L-glutamic acid (750 g/L), monosodium L-glutamate (34 g/L [0.2 mol/L]), glucose (5 g/L), yeast extract (35 g/L), MnSO4·H2O (50 mg/L [0.3 mmol/L]), and Tween 80 (1.0 g/L). The appropriate fermentation temperature was 30 °C. The fermenter trial results revealed that GABA was slowly synthesized from 0-4 h, rapidly synthesized until 32 h, and finally reached 353.1 ± 8.3 g/L at 48 h, with the pH increasing from the initial value of 4.56 to the ultimate value of 6.10. The proposed pH auto-buffering strategy may be popular for other GABA fermentations.

The M 1 -muscarinic acetylcholine receptor subtype may play a role in learning and memory performance in the hippocampus of neonatal monosodium glutamate-obese rats.

Here, we investigate the effects of obesity induced by monosodium glutamate (MSG) on cognitive impairment and whether this model induces any alteration in the affinity, density, and subtypes of muscarinic acetylcholine receptors (mAChRs) in rat hippocampus. Healthy rats were used as controls, and MSG-obese rats were selected via the Lee index > 0.300. The effects of MSG-induced obesity on hippocampal spatial learning and memory processes were evaluated by using the working memory versions of the Morris' water maze task and the evaluation of mAChRs by binding assay and their subtypes by immunoprecipitation assays. [ 3 H]Quinuclidinyl benzilate specific binding analysis showed that the equilibrium dissociation constant (K D ) did not differ between control and MSG, indicating that affinity is not affected by obesity induced by MSG. The maximum number of binding sites (B max ) obtained in MSG subjects was lower than that obtained from control rats, indicating a decrease in the expression of total mAChRs. Immunoprecipitation assays reveal a decrease in the expression of M 1 subtype of MSG when compared with control rats (M 2 to M 5 subtypes did not differ between control and MSG). We also observed that MSG promotes a disruption of the spatial working memory which was accompanied by a decrease in the M 1 mAChR subtype in rat hippocampus, thus suggesting deleterious long-term effects besides the obesity. In conclusion, these findings provide new insights into how obesity can influence spatial learning and memory that is hippocampal-dependent. The data suggest that the M 1 mAChR subtype protein expression is a potential therapeutic target.

Dietary supplementation with monosodium glutamate enhances milk production by lactating sows and the growth of suckling piglets.

This study was conducted to test the hypothesis that increasing dietary content of glutamate through addition of monosodium glutamate (MSG) enhances milk production by lactating sows and the growth of their offspring. Thirty multiparous sows (Landrace × Large White) were assigned randomly into one of three dietary groups: control (a corn- and soybean meal-based diet), the basal diet + 1% MSG, and the basal diet + 2% MSG. Diets were made isonitrogenous by the addition of appropriate amounts of L-alanine. Lactating sows had free access to drinking water and were fed twice daily their respective diets. The number of live-born piglets was standardized to 9 per sow at day 0 of lactation (the day of farrowing). On days 3, 15, and 29 of lactation, body weight and milk consumption of piglets were measured, and blood samples obtained from sows and piglets at 2 h and 1 h after feeding, respectively. Feed intake of sows did not differ (P > 0.05) among the three groups of sows. Concentrations of aspartate, glutamine, citrulline, arginine, tryptophan, proline, branched-chain amino acids, and glutamate were greater (P < 0.05) in the plasma of MSG-supplemented sows and their piglets than for controls. When compared with the control, dietary supplementation with 1-2% MSG increased (P < 0.05): concentrations of many free amino acids (including glutamate plus glutamine) and all protein-bound amino acids in milk; the milk intake of piglets by 14-25%; and daily weight gains of piglets by 23-44%. These results indicate that dietary supplementation with 1-2% MSG to lactating sows enhances milk production to support the growth of sow-reared piglets.

Role of cysteine in the improvement of γ-aminobutyric acid production by nonproteolytic Levilactobacillus brevis in coculture with Streptococcus thermophilus.

Previous research has showed that nonproteolytic Levilactobacillus brevis 145 (L) in coculture with Streptococcus thermophilus 1275 (S), not Lactobacillus delbrueckii ssp. bulgaricus (Lbu), was able to produce γ-aminobutyric acid (GABA) during milk fermentation in the presence of monosodium glutamate (MSG). It was assumed that differences of casein hydrolysis patterns between Strep. thermophilus 1275 and L. bulgaricus caused the phenomenon. Moreover, the GABA content was low and residual MSG was high in SL-fermented milk. In our research, comparison of peptide profiles determined by liquid chromatography/tandem mass spectrometry showed that αS2-casein, ß-casein, and κ-casein degradation by L. bulgaricus and Strep. thermophilus varied. Importantly, the peptide number in the L and Lbu coculture group increased compared with the Lbu monoculture group, whereas the peptide number in the SL coculture group decreased in comparison with S monoculture group, suggesting that L. bulgaricus was not able to provide peptides for the growth of Lb. brevis 145. Furthermore, we found that after supplementation with cysteine (50 mg/L) during milk fermentation by SL, 10 g/L MSG was converted into 4.8 g/L GABA with a minimum level of residual MSG, viable cell counts of Lb. brevis and lactic acid production were increased, and the casein hydrolysis pattern was not influenced. Moreover, sulfhydryl group-containing chemicals including cystine, reduced glutathione, and oxidized glutathione showed effects similar to that of cysteine in improving GABA production. Finally, when L. bulgaricus YIB2 was combined with SL, supplementation of cysteine was also able to significantly improve GABA production.

Production and characterization of yeast extracts produced by Saccharomyces cerevisiae, Saccharomyces boulardii and Kluyveromyces marxianus.

In recent years, prejudice in society against monosodium glutamate (MSG) has directed food manufacturers to alternative sources. Yeast extracts are considered as "natural" due to the production process and stand out due to their nutritional properties as well as giving a flavor similar to MSG. In this study, chemical, functional and flavor properties of yeast extract powders produced from Saccharomyces cerevisiae TGM10, Saccharomyces boulardii S11 and Kluyveromyces marxianus TGM66 were evaluated. Results revealed that the most protein-rich sample was S. cerevisiae TGM10 extract (69.17%), followed by S. boulardii S11 (66.16%) and K. marxianus TGM66 (62.42%) extracts, respectively and S. cerevisiae TGM10 extract was also the richest yeast extract for essential amino acids. Additionally, flavor-enhancing amino acids such as glutamic acid, aspartic acid, alanine and glycine were dominant in S. cerevisiae TGM10 extract (47.41 g/100 g protein). Sensorial evaluation of yeast extracts demonstrated that salty taste, umami taste and meaty flavor scores of yeast extracts were lower than MSG whereas for fruity flavor, yeast extracts had the highest scores. These findings revealed the potential of three yeast strains to produce yeast extracts in order to increase the nutritional value and flavor of foods.

Cysteine protected cells from H2O2-induced damage and promoted long-chain fatty acids synthesis in vivo to improve γ-aminobutyric acid production in Levilactobacillus brevis.

Levilactobacillus brevis NPS-QW-145 isolated from kimchi is deficient in glutamate dehydrogenase-encoding gene (gdhA) to form glutamate, hence it required exogenous supplementation of glutamate/monosodium glutamate (MSG) for decarboxylation reaction to produce γ-aminobutyric acid (GABA). However, GABA conversion rate from MSG was relatively low. The individual effect of 20 amino acids on regulating GABA biosynthesis was investigated. Cysteine was selected to significantly improve GABA production from MSG. It was found that Lb. brevis was capable of producing H2O2, cysteine protected Lb. brevis against H2O2-induced oxidative damage to increase cell viability for the enhancement of GABA production. Moreover, cysteine promoted glucose consumption to produce acetyl-CoA for synthesizing long-chain fatty acids to significantly up-regulate GABA biosynthesis. These findings deciphered antioxidative capability of cysteine in Lb. brevis 145 and provided a theoretical basis for fatty acids synthesis-mediated GABA synthesis in Lb. brevis 145, and possibly in other lactic acid bacteria.

Enhancing the production of γ-aminobutyric acid in Escherichia coli BL21 by engineering the enzymes of the regeneration pathway of the coenzyme factor pyridoxal 5'-phosphate.

The compound γ-aminobutyric acid (GABA) was widely used in various fields. To enhance the production of GABA in Escherichia coli BL21(DE3), the enzymes of the regeneration pathway of the coenzyme factor pyridoxal 5'-phosphate (PLP) were engineered. The recombinant E. coli strain was screened and identified. The initial concentrations of L-monosodium glutamate (L-MSG) had an obvious influence on the production of GABA. The highest concentration of GABA in recombinant E. coli BL21/pET28a-gadA was 5.54 g/L when the initial L-MSG concentration was 10 g/L, whereas it was 8.45 g/L in recombinant E. coli BL21/pET28a-gadA-SNO1-SNZ1 at an initial L-MSG concentration of 15 g/L. The corresponding conversion yields of GABA in these two strains were 91.0% and 92.7%, respectively. When the initial concentrations of L-MSG were more than 15 g/L, the concentrations of GABA in E. coli BL21/pET28a-gadA-SNO1-SNZ1 were significantly higher as compared to those in recombinant E. coli BL21/pET28a-gadA, and it reached a maximum of 13.20 g/L at an initial L-MSG concentration of 25 g/L, demonstrating that the introduction of the enzymes of the regeneration pathway of PLP favored to enhance the production of GABA. This study provides new insight into producing GABA effectively in E. coli BL21(DE3).

Suppression of hTAS2R16 Signaling by Umami Substances.

Interaction between umami and bitter taste has long been observed in human sensory studies and in neural responses in animal models, however, the molecular mechanism for their action has not been delineated. Humans detect diverse bitter compounds using 25-30 members of the type 2 taste receptor (TAS2R) family of G protein-coupled receptor. In this study, we investigated the putative mechanism of antagonism by umami substances using HEK293T cells expressing hTAS2R16 and two known probenecid-insensitive mutant receptors, hTAS2R16 N96T and P44T. In wild type receptor, Glu-Glu, inosine monophosphate (IMP), and l-theanine behave as partial insurmountable antagonists, and monosodium glutamate (MSG) acts as a surmountable antagonist in comparison with probenecid as a full insurmountable antagonist. The synergism with IMP of umami substances still stands in the suppression of hTAS2R16 signaling. In mutagenesis analysis, we found that Glu-Glu, MSG, and l-theanine share at least one critical binding site on N96 and P44 with probenecid. These results provide the first evidence for a direct binding of umami substances to the hTAS2R16 through the probenecid binding pocket on the receptor, resulting in the suppression of bitterness.

Isolation of a novel poly-γ-glutamic acid-producing Bacillus licheniformis A14 strain and optimization of fermentation conditions for high-level production.

In the present study, bacteria producing poly-γ-glutamic acid were isolated from marine sands, and an efficient producer identified. γ-PGA was rapidly screened by thin-layer chromatography and UV spectrophotometer assay. Media optimization was carried out, and for the cost-effective production of γ-PGA, monosodium glutamate was used as the substrate for the synthesis of γ-PGA instead of glutamic acid. Lastly, Plackett-Buman design (PB) and Response surface methodology (RSM) were used to determine significant media components and their interaction effect to achieve maximum γ-PGA production. With this integrated method, a bacterial strain with a high yield of γ-PGA was obtained rapidly, and the production was increased up to 37.8 g/L after optimization.

Ectoine production with indigenous Marinococcus sp. MAR2 isolated from the marine environment.

Ectoine has fostered the development of products for skin care and cosmetics. In this study, we employed the marine bacterial strain Marinococcus sp. MAR2 to increase ectoine production by optimizing medium constituents using Response Surface Methodology (RSM) and a fed-batch strategy. The results from the steepest ascent and central composite design indicated that 54 g/L of yeast extract, 14.0 g/L of ammonium acetate, 74.4 g/L of sodium glutamate, and 6.2 g/L of sodium citrate constituted the optimal medium with maximum ectoine production (3.5 g/L). In addition, we performed fed-batch culture in the bioreactor, combining pH and dissolved oxygen to produce ectoine by Marinococcus sp. MAR2. The ectoine production, content, and productivity of 5.6 g/L, 10%, and 3.9 g/L/day were further reached by a fed-batch culture. Thus, the ectoine production by Marinococcus sp. MAR2 using RSM and fed-batch strategy shows its potential for industrial production.

Optimized cultural conditions of functional yogurt for γ-aminobutyric acid augmentation using response surface methodology.

Yogurt, a functional dairy food product, is an effective medium for delivering beneficial functional ingredients. One ingredient, γ-aminobutyric acid (GABA), has growing appeal in the development of functional foods for its potential in reducing the risk of diabetes, hypertension, and stress as a bioactive agent. However, the concentration of GABA in existing food products is remarkably low. We developed a functional yogurt rich in GABA using Streptococcus thermophilus fmb5. The GABA yield of yogurt was enhanced by optimization of culture conditions using single factor and response surface methods. The results showed that culture temperature, monosodium glutamate concentration, and culture time are the 3 main factors that affect GABA yield. The optimal culture conditions were determined as follows: 38.8°C for culture temperature, 20 g/L of monosodium glutamate, and 120 h of culture time. Under the above optimal conditions, the actual yield of GABA production was maximized at 9.66 g/L, which was 1.2 times or higher than that of from any single factor treatment. The GABA concentration, viable bacteria number, and water-holding capacity of GABA-rich yogurt were stable throughout the whole storage time. The results show that producing yogurt with Streptococcus thermophilus fmb5 and the optimized culture conditions will achieve high GABA concentrations that maximize health benefits to consumers.

Effect of osmotic dehydration of olives as pre-fermentation treatment and partial substitution of sodium chloride by monosodium glutamate in the fermentation profile of Kalamata natural black olives.

This study examined the effect of osmotic dehydration of Kalamata natural black olives as pre-fermentation treatment in combination with partial substitution of NaCl by monosodium glutamate (MSG) on the fermentation profile of olives. Osmotic dehydration was undertaken by immersing the olives in 70% (w/w) glucose syrup overnight at room temperature. Further on, three different mixtures of NaCl and MSG with/without prior osmotic dehydration of olives were investigated, namely (i) 6.65% NaCl - 0.35% MSG (5% substitution), (ii) 6.30% NaCl - 0.70% MSG (10% substitution), (iii) 5.95% NaCl - 1.05% MSG (15% substitution), and (iv) 7% NaCl without osmotic dehydration (control treatment). Changes in the microbial association (lactic acid bacteria [LAB], yeasts, Enterobacteriaceae), pH, titratable acidity, organic acids, sugars, and volatile compounds in the brine were analyzed for a period of 4 months. The final product was subjected to sensory analysis and the content of MSG in olives was determined. Results demonstrated that osmotic dehydration of olives prior to brining led to vigorous lactic acid processes as indicated by the obtained values of pH (3.7-4.1) and acidity (0.7-0.8%) regardless of the amount of MSG used. However, in non-osmotically dehydrated olives, the highest substitution level of MSG resulted in a final pH (4.5) that was beyond specification for this type of olives. MSG was degraded in the brines being almost completely converted to γ-aminobutyric acid (GABA) at the end of fermentation. Finally, the sensory assessment of fermented olives with/without osmotic dehydration and at all levels of MSG did not show any deviation compared to the control treatment.

Hydrogel Film-Immobilized Lactobacillus brevis RK03 for γ-Aminobutyric Acid Production.

Hydrogels of 2-hydroxyethyl methacrylate/polyethylene glycol diacrylate (HEMA/PEGDA) have been extensively studied for their use in biomedical and pharmaceutical applications owing to their nontoxic and highly hydrophilic characteristics. Recently, cells immobilized by HEMA/PEGDA hydrogels have also been studied for enhanced production in fermentation. Hydrogel films of HEMA/PEGDA copolymer were generated by Ultraviolet (UV)-initiated photopolymerization. The hydrogel films were used to immobilize viable Lactobacillus brevis RK03 cells for the bioconversion of monosodium glutamate (MSG) to γ-aminobutyric acid (GABA). The mechanical properties and fermentation yields of the L. brevis RK03 cells immobilized on polyacrylate hydrogel films with different monomeric formulations were investigated. Fermentation was carried out in 75 mL de Man, Rogosa and Sharpe (MRS) medium containing various concentrations of MSG. We found that HEMA (93%)/PEGDA (3%) hydrogels (sample H) maximized GABA production. The conversion rate of MSG to GABA reached a maximum value of 98.4% after 240 h. Bioconversion activity gradually declined after 420 h to 83.8% after five cycles of semi-continuous fermentation. Our results suggest that HEMA (93%)/PEGDA (3%) hydrogels have great potential for use in GABA production via semi-continuous fermentation.

Enhancement of gamma-aminobutyric acid (GABA) levels using an autochthonous Lactobacillus futsaii CS3 as starter culture in Thai fermented shrimp (Kung-Som).

Gamma-aminobutyric acid (GABA) is a non-proteinogenic amino acid, which has a variety of well-characterized beneficial physiological functions. In order to improve GABA levels and the fermentation process of Thai fermented shrimp (Kung-Som), autochthonous Lactobacillus futsaii CS3 was inoculated as a starter culture into Kung-Som, and its effects on the quality of Kung-Som were studied. The optimal conditions for GABA production in Kung-Som as obtained by response surface methodology (RSM) using a central composite design (CCD) were an inoculum size of roughly 107 CFU/g (X1) of L. futsaii cells together with the addition of 0.5% (w/w) monosodium glutamate (MSG) (X2), resulting in maximum GABA levels of 10,500 mg per kg fresh product. Under these optimized conditions, the experimental GABA content of Kung-Som with an added starter culture was up to four times higher than that of the control (without starter culture) or commercial Kung-Som products (10,120 mg/kg product). Kung-Som produced by inoculation with L. futsaii CS3 but without addition of MSG showed a considerably increased GABA content of 7790 mg/kg compared to the control. Fermentation time was reduced to less than 1 week for these samples compared to the control batches, which took up to 19 days. Polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) revealed that L. futsaii CS3 remained prominently throughout the Kung-Som fermentation, and that lactic acid bacteria (LAB) rapidly dominated the total microflora because of this inoculation with L. futsaii CS3. Kung-Som samples with starter culture were accepted as well as commercial ones by 30 panelists (p > 0.05). In conclusion, L. futsaii CS3 is a good starter culture for GABA production, resulting in, improved microbiological safety as well as reduced fermentation time.

MSG-Evoked c-Fos Activity in the Nucleus of the Solitary Tract Is Dependent upon Fluid Delivery and Stimulation Parameters.

The marker of neuronal activation, c-Fos, can be used to visualize spatial patterns of neural activity in response to taste stimulation. Because animals will not voluntarily consume aversive tastes, these stimuli are infused directly into the oral cavity via intraoral cannulae, whereas appetitive stimuli are given in drinking bottles. Differences in these 2 methods make comparison of taste-evoked brain activity between results that utilize these methods problematic. Surprisingly, the intraoral cannulae experimental conditions that produce a similar pattern of c-Fos activity in response to taste stimulation remain unexplored. Stimulation pattern (e.g., constant/intermittent) and hydration state (e.g., water-restricted/hydrated) are the 2 primary differences between delivering tastes via bottles versus intraoral cannulae. Thus, we quantified monosodium glutamate (MSG)-evoked brain activity, as measured by c-Fos, in the nucleus of the solitary tract (nTS; primary taste nucleus) across several conditions. The number and pattern of c-Fos neurons in the nTS of animals that were water-restricted and received a constant infusion of MSG via intraoral cannula most closely mimicked animals that consumed MSG from a bottle. Therefore, in order to compare c-Fos activity between cannulae-stimulated and bottle-stimulated animals, cannulated animals should be water restricted prior to stimulation, and receive taste stimuli at a constant flow.

Homeostatic effect of p-chloro-diphenyl diselenide on glucose metabolism and mitochondrial function alterations induced by monosodium glutamate administration to rats.

The metabolic syndrome is a group of metabolic alterations considered a worldwide public health problem. Organic selenium compounds have been reported to have many different pharmacological actions, such as anti-hypercholesterolemic and anti-hyperglycemic. The aim of this study was to evaluate the effect of p-chloro-diphenyl diselenide (p-ClPhSe)2, an organic selenium compound, in a model of obesity induced by monosodium glutamate (MSG) administration in rats. The rats were treated during the first ten postnatal days with MSG and received (p-ClPhSe)2 (10 mg/kg, intragastrically) from 45th to 51 th postnatal day. Glucose, lipid and lactate levels were determined in plasma of rats. Glycogen levels and activities of tyrosine aminotransferase, hexokinase, citrate synthase and glucose-6-phosphatase (G-6-Pase) were determined in livers of rats. Renal G-6-Pase activity was also determined. The purine content [Adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate] and mitochondrial functionality in the liver were also investigated. p-(ClPhSe)2 did not alter the reduction in growth performance and in the body weight caused by MSG but reduced epididymal fat deposition of rats. p-(ClPhSe)2 restored glycemia, triglycerides, cholesterol and lactate levels as well as the glucose metabolism altered in rats treated with MSG. p-(ClPhSe)2 restored hepatic mitochondrial dysfunction and the decrease in citrate synthase activity and ATP and ADP levels caused by MSG in rats. In summary, (p-ClPhSe)2 had homeostatic effects on glucose metabolism and mitochondrial function alterations induced by MSG administration to rats.