D-Lactic Acid (D-Lactate) (Rapid) Assay Kit

Many of the enzymatic test kits are official methods of prestigious organisations such as the Association of Official Analytical Chemicals (AOAC) and the American Association of Cereal Chemists (AACC) in response to the interest from oenologists. Megazyme decided to use its long history of enzymatic bio-analysis to make a significant contribution to the wine industry, by the development of a range of advanced enzymatic test kits. This task has now been successfully completed through the strategic and comprehensive process of identifying limitations of existing enzymatic bio-analysis test kits where they occurred, and then using advanced techniques, such as molecular biology (photo 1), to rapidly overcome them. Novel test kits have also been developed for analytes of emerging interest to the oenologist, such as yeast available nitrogen (YAN; see pages 2-3 of issue 117 article), or where previously enzymes were simply either not available, or were too expensive to employ, such as for D-mannitol analysis.

It is without doubt that testing plays a pivotal role throughout the whole of the vinification process. To produce the best possible quality wine and to minimise process problems such as “stuck” fermentation or troublesome infections, it is now recognised that if possible testing should begin prior to harvesting of the grapes and continue through to bottling. Traditional methods of wine analysis are often expensive, time consuming, require either elaborate equipment or specialist expertise and frequently lack accuracy. However, enzymatic bio-analysis enables the accurate measurement of the vast majority of analytes of interest to the wine maker, using just one piece of apparatus, the spectrophotometer (see previous issue No. 116 for a detailed technical review). Grape juice and wine are amenable to enzymatic testing as being liquids they are homogenous, easy to manipulate, and can generally be analysed without any sample preparation.

Probiotics have been shown to provide health benefits for several metabolic diseases, including obesity, type 2 diabetes, and metabolic dysfunction-associated steatotic liver disease (MASLD), by modulating the gut microbiota. In this study, we evaluated the safety and efficacy of Lactobacillus delbrueckii subsp. lactis CKDB001 as a potential therapeutic candidate for the treatment of MASLD. We evaluated antibiotic resistance, hemolytic, gelatinase, and bile salt hydrolase activities, and the production of biogenic amines and D-lactate using in vitro analyses. We found that L. lactis CKDB001 treatment resulted in significant anti-adipogenic properties in the HepG2 cell line, reducing lipid accumulation and improving lipid profiles through mechanisms involving the upregulation of SIRT1 and PPARα, and downregulation of CD36 and ELOVL6. These results suggest that L. lactis CKDB001 is a safe and effective probiotic for managing MASLD. Further in vivo studies and clinical trials are required to validate these effects and fully elucidate their therapeutic potential and safety profiles.

Probiotics, as defined by the World Health Organization, are live microorganisms that, when consumed in sufficient quantities, provide health benefits to the host. Although some countries have approved specific probiotic species for use in food, safety concerns may still arise with individual strains. Lacticaseibacillus paracasei subsp. paracasei NTU 101 (NTU 101), isolated from the gut of healthy infants, has demonstrated various probiotic effects and shown safety in a prior 28-day animal feeding study. To further verify its safety and mitigate potential risks, we performed a comprehensive genotypic and phenotypic safety evaluation in accordance with the European Food Safety Authority guidelines for safety assessment through whole genome sequencing and related literature. In this research, minimum inhibitory concentration testing identified NTU 101’s resistance to chloramphenicol; however, subsequent gene analysis confirmed no associated risk of resistance. Assessments of safety, including biogenic amine content, hemolytic activity, mucin degradation, and D-lactic acid production, indicated a low level of risk. Additionally, a repeated-dose 90-day oral toxicity study in Sprague-Dawley rats revealed no toxicity at a dose of 2000 mg/kg body weight, further supporting the strain’s safety for consumption. Based on these comprehensive analyses, NTU 101 is considered safe for regular consumption as a health supplement.

Cancer immune therapy with engineered immune cells is standard of care in certain hematologic malignancies. However, solid tumor outcomes in clinical trials are lagging significantly behind. Engineered T cells face a myriad of challenges within the tumor microenvironment, one of which is competition for metabolites and a carbon source for adenosine triphosphate generation. Glucose is the main source of carbon for cellular metabolism, and it is critically limited in the microenvironment of solid tumors due to poor vascularization and competition with tumor cells for uptake. Here, we aimed to overcome this limitation by equipping T cells with the capability to use an alternative carbon source. We demonstrate that fructose can act as an alternative carbon source for T cells modified with chimeric antigen receptors or T cell receptors and the expression of glucose transporter 5. This simple manipulation is highly compatible with clinical development approaches to optimize cell therapy outcomes.

Background: Timely diagnosis of acute intestinal necrosis (AIN) is lifesaving, but challenging due to unclear clinical presentation. D-lactate has been proposed as an AIN biomarker. Objectives: We aimed to test the diagnostic performance in a clinical setting. Methods: We performed a cross-sectional prospective study, including all adult patients with acute referral to a single tertiary gastrointestinal surgical department during 2015–2016 and supplemented by enrollment of high-risk in-hospital patients suspected of having AIN during 2016–2019. AIN was verified intraoperatively, and D-lactate was analyzed using an automatic spectrophotometric set-up. A D-lactate cut-off for AIN was estimated using the receiver operating characteristic curve. The performance according to patient subgroups was estimated using the area under the receiver operating characteristic curve (AUC). Given the exploratory nature of this study, a formal power calculation was not feasible. Results: Forty-four AIN patients and 2914 controls were enrolled. The D-lactate cut-off was found to be 0.0925 mM. Due to lipemic interference, D-lactate could not be quantified in half of the patients, leaving 23 AIN patients and 1456 controls for analysis. The AUC for the diagnosis of AIN by D-lactate was 0.588 (95% confidence interval 0.475–0.712), with a sensitivity of 0.261 and specificity of 0.892. Analysis of high-risk patients showed similar results (AUC 0.579; 95% confidence interval 0.422–0.736). Conclusion: D-lactate showed low sensitivity for AIN in both average-risk and high-risk patients. Moreover, lipemic interference precluded valid spectrophotometric assessment of D-lactate in half of the patients, further disqualifying the clinical utility of D-lactate as a diagnostic marker for AIN.

The microbial fuel cell (MFC) technology is emerging as an effective technology for wastewater treatment to remove and detect many pollutants and simultaneously generate power. The anode is an essential component for bacterial attachment and extracellular electron transfer (EET). Thus, anode performance is critical for improving an MFC system's overall performance. Materials that are electronically conductive and provide favorable conditions for creating biofilm are good candidates for the anode. Carbon paper and carbon felt are the most used anode materials. However, the characteristics of these materials can be modified to increase their performance. This paper presents a modification of the conventional carbon anode by electrolyzing the carbon felt in a phosphate buffer and an experimental evaluation of the impact of the treatment. The tests were performed using an air cathode MFC, electrogenic bacteria Shewanella Baltica 20 fed on lactose-rich L-B. The treatment with phosphate buffer generates functional groups on the surface of the carbon felt, which establishes robust biofilm on the anode and offers lower charge-transfer resistance compared to the untreated carbon felt. This facilitates electron transfer from exoelectrogens to the anode, increasing the current density and power density output. Three times higher power density was observed in the cell with a modified anode than in an untreated carbon-felt anode.

Background: Intra-abdominal infections are the second most common cause of sepsis in the intensive care unit. Intestinal epithelial injury due to abdominal sepsis results in a variety of pathological changes, such as intestinal bacteria and toxins entering the blood, leading to persistent systemic inflammation and multiple organ dysfunction. The increased apoptosis of intestinal epithelial cells induced by sepsis further exacerbates the progression of sepsis. Although several studies have revealed that circRNAs are involved in intestinal epithelial injury in sepsis, few studies have identified the roles of circRNAs in intestinal epithelial apoptosis. Methods: We used laser capture microdissection to obtain purified epithelial cells located in intestinal crypts from four patients with abdominal sepsis induced by intestinal perforation and four samples from age and sex-matched non-septic patients. Microarray analysis of circRNAs was conducted to assess differentially expressed circRNAs between patients with and without sepsis. Lastly, in vitro and in vivo assays were performed to study the mechanism of circFLNA in intestinal epithelial apoptosis during sepsis. Results: circFLNA was upregulated in the intestinal epithelium after abdominal sepsis induced by intestinal perforation. Inhibition of miR-766-3p impaired si-circFLNA-mediated inhibition of apoptosis and inflammation factor levels in lipopolysaccharide (LPS)-treated HIEC-6 cells. circFLNA aggravated apoptosis and inflammation through the Fas-mediated apoptosis pathway in both LPS-treated HIEC-6 cells and a mouse cecal ligation and puncture model. Conclusion: Our findings showed that circFLNA promotes intestinal injury in abdominal sepsis through the Fas-mediated apoptosis pathway by sponging miR-766-3p. The circFLNA/miR-766-3p/Fas axis has potential as a novel therapeutic target for treating intestinal injury in sepsis.

Streptococcus mutans is known as a contributor to dental caries. In this work, Lactobacillus pentosus MJM60383 was selected for its strong antagonistic activity against S. mutans and was characterized by good oral probiotic properties including lysozyme tolerance, adhesive ability to oral cells, good aggregation (auto-aggregation, co-aggregation) ability, hydrogen peroxide production and inhibition of biofilm formation of S. mutans. L. pentosus MJM60383 also exhibited safety as a probiotic characterized by no hemolytic activity, no D-lactate production, no biogenic amine production, and susceptibility to antibiotics. Furthermore, the biofilm formation of S. mutans was also significantly inhibited by the supernatant of L. pentosus MJM60383. An anti-biofilm mechanism study revealed that sucrose decomposition and the production of water-insoluble exopolysaccharides by S. mutans were inhibited by the treatment with L. pentosus MJM60383 supernatant. Real-time PCR analysis indicated that the supernatant of L. pentosus MJM60383 significantly inhibited the mRNA expression of S. mutans glycosyltransferases, which synthesize glucan to construct biofilm architecture and mediate bacterial adherence. Our study demonstrated L. pentosus MJM60383 as a potential oral probiotic and revealed its anti-biofilm mechanism.

Non-alcoholic fatty liver disease (NALFD) is a disease characterized by liver steatosis. The liver is a key organ involved in the metabolism of fat, protein, and carbohydrate, enzyme activation, and storage of glycogen, which is closely related to the intestine by the bidirectional relation of the gut-liver axis. Abnormal intestinal microbiota composition can affect energy metabolism and lipogenesis. In this experiment, we investigated the beneficial effect of Lactobacillus reuteri MJM60668 on lipid metabolism and lipogenesis. C57BL/6 mice were fed a high-fat diet (HFD) and orally administrated with MJM60668. Our results showed that mice treated with MJM60668 significantly decreased liver weight and liver/body weight ratio, without affecting food intake. Serum levels of ALT, AST, TG, TCHO, and IL-1β in mice fed with MJM60668 were decreased compared to the HFD group. Investigation of gene and protein expression on the lipogenesis and lipid metabolism showed that the expression of ACC, FAS, and SREBP was decreased, and PPARα and CPT was increased. Furthermore, an increase of adiponectin in serum was shown in our experiment. Moreover, serum IL-1β level was also significantly decreased in the treated mice. These results suggested that MJM60668 can strongly inhibit lipogenesis, enhance fatty acid oxidation, and suppress inflammation. Additionally, supplementation of MJM60668 increased the proportion of Akkermansiaceae and Lachnospiracea, confirming a potential improvement of gut microbiota, which is related to mucus barrier and decrease of triglycerides levels.

Microbial composition and activity in the gastrointestinal tract (GIT) of cattle has important implications for animal health and welfare, driving the focus of research toward ways to modify their function and abundance. However, our understanding of microbial adaption to nutritional changes remains limited. The aim of this study was to examine the progressive mechanisms of adaptation in the rumen and hindgut of cattle receiving increasing amounts of starch with or without dietary supplementation of a blended phytogenic feed additive (PFA; containing menthol, thymol and eugenol). We used 16S rRNA gene amplicon sequencing to assess the microbial composition and predicted metabolic pathways in ruminal solid and liquid digesta, and feces. Furthermore, we employed targeted liquid chromatography-mass spectrometry methods to evaluate rumen fluid metabolites. Results indicated a rapid microbial adaptation to diet change, starting on the second day of starch feeding for the particle associated rumen liquid (PARL) microbes. Solid rumen digesta- and feces-associated microbes started changing from the following day. The PARL niche was the most responsive to dietary changes, with the highest number of taxa and predicted pathways affected by the increase in starch intake, as well as by the phytogenic supplementation. Despite the differences in the microbial composition and metabolic potential of the different GIT niches, all showed similar changes toward carbohydrate metabolism. Metabolite measurement confirmed the high prevalence of glucose and volatile fatty acids (VFAs) in the rumen due to the increased substrate availability and metabolic activity of the microbiota. Families Prevotellaceae, Ruminococcaceae and Lachnospiraceae were found to be positively correlated with carbohydrate metabolism, with the latter two showing wide-ranging predicted metabolic capabilities. Phytogenic supplementation affected low abundant taxa and demonstrated the potential to prevent unwanted implications of feeding high-concentrate diet, such as reduction of microbial diversity. The inclusion of 50% concentrate in the diet caused a major shift in microbial composition and activity in the GIT of cattle. This study demonstrated the ability of microorganisms in various GIT niches to adjust differentially, yet rapidly, to changing dietary conditions, and revealed the potential beneficial effects of supplementation with a PFA during dietary adaptation.

Environmental conditions (pH and temperature) are expected to influence microbial community composition and product spectrum in mixed-culture food waste (FW) fermentation. However, some conditions may favour growth of multiple organisms that compete for common substrates or consume target metabolites. The inoculum plays an integral role in mixed-culture fermentation, but it is currently unknown how an adapted inoculum, known to selectively produce the target metabolite, would influence fermentation, and how environmental conditions could control fermentation outcomes. Therefore, this study assessed the effects of pH (uncontrolled vs. controlled pH 4.0-6.0) and temperature (35-60°C) on lactic acid (LA) from synthetic mixed FW batch fermentation (80 g_VS·L⁻¹) utilising an adapted fermentation inoculum known to produce significant LA (10% inoculum volume). Concentrations of LA and competing organic acids were measured. Uncontrolled pH encouraged Lactobacillus growth but resulted in a low LA yield due to inhibitory conditions. Controlled pH 6.0 improved LA production but introduced LA consumption and competitive butyrate production. Observed butyrate production was dependent on pH and temperature and correlated with the growth of Clostridium Sensu Stricto 12. At pH 6.0 and 50°C, observable LA consumption was eliminated, and the LA yield was maximised at 0.55 g_LA·g_VS⁻¹ (39 g_LA·L⁻¹) while Lactobacillus remained dominant. The adapted inoculum effectively promoted LA production, while pH and temperature regulation were effective control levers to target LA.

Uric acid is the final product of purine metabolism in human. The increase of serum uric acid is tightly related to the incidence of hyperuricemia and gout. Also, it has been reported that the intake of purine-rich foods like meat and seafood is associated with an increased risk of gout. Therefore, the reduction of purine absorption is one of therapeutic approaches to prevent hyperuricemia and gout. Currently, probiotics are being studied for the management of hyperuricemia and gout. In this study, we aimed to investigate the effect of Lactobacillus brevis MJM60390 on hyperuricemia induced by a high-purine diet and potassium oxonate in a mouse model. L. brevis MJM60390 among 24 lactic acid bacteria isolated from fermented foods showed the highest ability to assimilate inosine and guanosine in vitro and typical probiotic characteristics, like the absence of bioamine production, D-lactate production, hemolytic activity, as well as tolerance to simulated orogastrointestinal conditions and adherence to Caco-2 cells. In an in vivo animal study, the uric acid level in serum was significantly reduced to a normal level after oral administration of L. brevis MJM60390 for 2 weeks. The activity of xanthine oxidase catalyzing the formation of uric acid was also inhibited by 30%. Interestingly, damage to the glomerulus, Bowman's capsule, and tubules in the hyperuricemia model were reversed by supplementation with this strain. Fecal microbiome analysis revealed that L. brevis MJM60390 supplementation enhanced the relative abundance of the Rikenellaceae family, which produces the short-chain fatty acid butyrate and helps to maintain good gut condition. Therefore, these results demonstrated that L. brevis MJM60390 can be a probiotic candidate to prevent hyperuricemia.

Intestinal infarction is the fast-evolving endpoint of impaired blood perfusion to an intestinal segment which may have fatal outcome. Early diagnosis and treatment within 6 h reduce mortality. Currently, d-lactate is a promising biomarker, however, not available in the acute clinical setting. The aim of this study is implementation of d-lactate analysis in a routine clinical setting. We used a spectrophotometric method, based on enzymatic oxidation of d-lactate by d-lactate dehydrogenase (D-LDH) coupled to the reduction of nicotinamide-adenine dinucleotide (NAD⁺). The amount of NADH formed in this reaction is equivalent to d-lactate. The primary concern in this method is interfering NADH formed by oxidation of l-lactate by l-lactate dehydrogenase (L-LDH). A commercially available kit for d-lactate measurement was implemented on our existing automated routine laboratory equipment including pH-inactivation of L-LDH. Our setup fulfilled clinical quality goals. We were able to measure d-lactate with an acceptable performance of the analysis and a short turn-around time. The method can be used to distinguish between the expected cut-off for intestinal ischemia around 0.3 mM and the upper reference limit of 0.05 mM. With a turnaround time of just 9 min, the analysis has potential as a readily available detection of circulating d-lactate for early diagnosis of intestinal ischemia.

Introduction: We sought to quantify targeted metabolites (d-lactate, pyruvate, urea, ammonia) and the cytokine IL-8 produced by human cervicovaginal epithelial cells co-cultured with Ureaplasma urealyticum (a preterm birth-associated bacterium) or Lactobacillus crispatus (a healthy vaginal commensal associated with term birth). Methods: Concentrations of D-lactate, pyruvate, urea and ammonia measured by enzyme-based spectrophotometry and IL-8 by ELISA were determined and compared between monolayer-cultured HeLa cells (ATCC 35241) infected with strains of U. urealyticum (ATCC 27618, 0.5 mL = 3640 CFU/mL, U. urealyticum) or L. crispatus (ATCC 33820, MOI = 10,000, 1000 and 100, L. crispatus) and incubated in 5% CO₂ at 37°C for 24 h. Uninfected HeLa cells (Hc) were used as controls and cytotoxicity was determined by the amount (optical density) of lactate dehydrogenase (LDH) released by the dead HeLa cells. Results: The amount of LDH released by untreated Hc (P = 0.002) and U. urealyticum-infected cells (P < 0.0001) was higher than those of L. crispatus-infected cells, with U. urealyticum-infected cells recording the highest % cytotoxicity and L. crispatus-infected cells MOI 10,000 (Lc10,000) the least (P < 0.0001). Though there was no significant difference in the concentration of urea between the samples, U. urealyticum-infected cells showed higher ammonia compared to other samples (p = 0.03). In contrast, all L. crispatus samples had higher D-lactate than untreated Hc (p = 0.01) and U. urealyticum-infected cells (P = 0.01). Also, Lc10,000 had the highest D-lactate (p = 0.001) and lowest pyruvate (P = 0.04, excluding UU) compared to other samples. Furthermore, U. urealyticum-infected cells produced the highest IL-8 (P = 0.01) compared to other samples, with Lc10,000 producing undetectable levels. Conclusion: Infection of cervicovaginal epithelial cells by U. urealyticum stimulates production of ammonia from urea and induces elevated IL-8 production possibly leading to significantly higher cytotoxicity. In contrast, L. crispatus appeared protective against HeLa cell inflammation and death, producing more D-lactate and less IL-8, consistent with a role for L. crispatus in promoting vaginal floral health and reducing infection/inflammation-associated preterm birth.