L-Lactic Acid (L-Lactate) 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.

Kefir is an increasingly popular dairy- or sugar-based fermented food product. The aim of our study was to investigate the bacterial and fungal communities in 28 retail kefirs including 21 milk kefirs, including 3 thick kefir yogurt and 4 water kefir products. Full-length amplicon nanopore sequencing of both 16S rRNA (for bacteria) and intergenic spacer (for fungi) was undertaken. The diversity within and between groups was analyzed (α and β diversity) and linear discriminant analysis effect size analysis was undertaken to identify biomarkers that differentially characterize the microbial communities associated with different kefir types. The pH, lactic acid concentration, total viable counts (TVC), lactic acid bacteria (LAB), total coliform counts (TCC), and yeast counts were also investigated. The main bacterial genera (and species) were Lactococcus (cremoris) and Streptococcus (thermophilus), and other bacteria such as Lactobacillus (delbrueckii) and Lentilactobacillus (kefiri) were also detected. The fungal populations were mainly composed of Brettanomyces (anomalus), Zygotorulaspora (florentina) and Kazachstania (unispora), but with many different fungal genera/species detected. The pH ranged from 3.1 to 4.7 with a mean of 4.2 ± 0.07 and the lactic acid content ranged from 0.1 to 9 g/L with a mean of 5.6 ± 0.53. In milk kefirs the TVC, LAB, TCC, and yeast counts ranged from 3.1 to 9.1, 3.4 to 9.0, not detected (ND) to 1.6 and ND to 6.5 log₁₀ cfu/mL or cfu/g, respectively. The corresponding counts in water kefirs were 4.1 to 7.3 (TVC), 4.1 to 7.0 (LAB), ND to 1.1 (TCC), and 3.9 to 7.0 (yeast) log₁₀ cfu/mL or cfu/g, respectively. It was concluded that although the 28 retail kefirs analyzed had a rich diversity of bacteria and fungi the bacteriome was dominated by bacteria belonging to the Lactococcus and Streptococcus genera and the main bacterial species were Lactococcus cremoris, Streptococcus thermophiles, Streptococcus suis, Lactobacillus delbrueckii, and Streptococcus sp. HSISS1. The fungal microbiome was dominated by Zygotorulaspora and the most abundant fungal species included Zygotorulaspora florentina, Brettanomyces anomalus, and Kazachstania unispora. To the best of our knowledge, this is the first study in Ireland to use full-length nanopore sequencing to characterize both bacterial and fungal communities in retail kefirs.

Deregulated energy metabolism is a hallmark of cancer, characterized by increased glycolytic flux. Cancer-specific modification of 6-phosphofructo-1-kinase (PFK) impairs its ability to regulate the enzyme's activity which increases glycolytic flux. Consequently, excessive cytosolic NADH formation triggers a harmful redox imbalance in cancer cells, which is rapidly neutralized by the formation of lactic acid and superoxide (SOX). To learn more about deregulated glycolysis in cancer cells, a supercomputer used the atomic model of the crystal structure of human PFK1 for virtual screening a database of 4.5 million compounds by docking with the catalytic binding sites of the enzyme. The screening revealed two compounds capable of reducing modified, cancer-specific PFK1 activity and simultaneously suppressing lactate and SOX formation. A dose-dependent inhibition was observed in the cells treated by compounds in the following tumorigenic cells: Jurkat (Acute T cells leukemia); Caco-2 (colorectal adenocarcinoma); COLO 829 (melanoma); and MDA-MB-231 (breast gland adenocarcinoma). In addition, two selected compounds assessed for cytostatic and cytotoxic activity showed no negative effects on tumorigenic cells. However, during incubation, the strengths of inhibitions continuously decreased, both during lactate and SOX formation. No such effects were observed if compounds were sequentially submitted to the cells at low concentrations every 24 hours. Additional experiments performed by Jurkat cells revealed reduced respiration and glycolysis rates in the cells treated with compounds concerning the untreated cells. Inhibition of modified cancer-specific PFK1 activity reduces deregulated glycolytic flux, prevents abundant cytosolic NADH formation, and restores redox balance thus simultaneously preventing the formation of deleterious effects of lactate and SOX, two crucial players in cancer initiation and development.

Background: Cryptorchidism is the absence of one or both testicles in the scrotum at birth, being a risk factor for testis cancer and infertility. The most effective method to treat cryptorchidism is orchiopexy, followed by human chorionic gonadotropin (hCG) therapy; however, a portion of treated patients do not show a significant improvement in testis volume and vascularization after adjuvant therapy. Methods: In this study, we generated an in vitro model to predict the patient response to hCG by cultivating and treating primary cells derived from five cryptorchid patients’ biopsies of gubernaculum testis, the ligament that connects the testicle to the scrotum. On these in vitro cultured cells, we analyzed the effect of hCG on cell proliferation, tubular structure formation, cellular respiration, reactive oxygen species content, and proteome. Results: We demonstrate that in vitro hCG stimulates gubernacular cells to proliferate and form vessel-like structures to a different extent among the five cryptorchid patients’ cells, with a decrease in oxygen consumption and reactive oxygen species generation. Furthermore, from the proteomic analysis, we show that hCG regulates the intra- and extra-cellular organization of gubernacular cells together with a massive regulation of the antioxidant response. Conclusions: Hereby, we characterized the cellular and molecular effects of hCG, demonstrating that the diverse patient response to hCG may be ascribable to their age since young patients better respond in vitro to the hormone, supporting a prompt surgical procedure and subsequent therapy.

Microbial fermentation of agro-industrial residues is gaining significant traction as a sustainable and economically viable approach in bioprocessing. This study explored lactic acid production from selected agro-industrial residues: pre-treated sugarcane waste, potato peel waste, or milk processing waste with alfalfa pellets using Lactobacilli strains of organic origin. Five homo-fermentative strains (VITJ1, VITJ2, VITJ3, VITJ4, and VITJ5) were assessed for compatibility and formed into 15 consortia. VITJ2 showed the highest individual production (147.1 ± 0.26 g/L at 48 h) in MRS media. The combination of sugarcane waste and alfalfa pellets yielded the highest crude lactic acid production (9.1%) after 48 h, suggesting its potential as a cost-competitive fermentation medium for industrial-scale lactic acid production. This study contributes to the growing body of evidence supporting the valorization of agro-industrial residues as feedstock for bioprocesses. Furthermore, it presents the novel concept of utilizing Lactobacilli consortia for lactic acid production.

Lactic acid (LA) is an important biobased platform chemical, with potential applications in synthetising a wide range of chemical products or serving as feedstock for various bioprocesses. Industrial LA production via pure culture fermentation is characterized by high operational costs and utilizes food-grade sugars, thereby reducing the feasibility of LA applications. In this context, our research focussed on valorising the largest dairy side stream, cheese whey permeate, through the use of mixed microbial communities. We evaluated the effect of different operational parameters (temperature, pH and hydraulic retention time) in non-axenic fermentations on productivity, yield, concentration, optical purity, and community. Our findings revealed that operating at mildly thermophilic conditions (45 °C) resulted in highly selective LA production, and significantly augmented the LA yield, and productivity, compared to higher temperatures (50–55 °C). In addition, operating at circumneutral pH conditions (6.0–6.5) led to significantly increased the LA fermentation performance compared to the conventional acid pH conditions (≤5.5). This led to an unprecedented LA productivity of 27.4 g/L/h with a LA yield of 70.0% which is 2.5 times higher compared to previous reported maximum. Additionally, varying pH levels influenced the optical purity of LA: we achieved an optical L-LA purity of 98.3% at pH 6.0–6.5, and an optical D-LA purity of 91.3% at a pH of 5.5. A short hydraulic retention time of less than 12 h was crucial for selective LA production. This process also yielded a microbial biomass composed of 90.3–98.6% Lactobacillus delbrueckii, which could be potentially valorised as probiotic or protein ingredient in food or feed products. Our work shows that by careful selection of operational conditions, the overall performance can be significantly increased compared to the state-of-the-art. These results highlight the potential of non-sterile LA fermentation and show that careful selection of simple reactor operation parameters can maximize process performance. A preliminary assessment suggests that valorising EU cheese whey permeate could increase LA and poly-LA production by 40 and 125 times, respectively. This could also lead to the production of 4,000 kton protein-rich biomass, potentially reducing CO₂ emissions linked to EU food and feed production by 4.87% or 2.77% respectively.

Various parasites alter their intermediate hostʼs phenotype in ways that increase parasite transmission to definitive hosts. To what extent infected intermediate hosts can recover from such “manipulation” is poorly documented, thus limiting our understanding of its proximate and ultimate causes. Here, we address the reversibility of several phenotypic alterations induced by the acanthocephalan Polymorphus minutus, a trophically-transmitted bird parasite, in its amphipod intermediate host. Using a recently developed laser-based technology, we selectively killed parasite larvae inside the body cavity of Gammarus fossarum, while preserving host viability. Following behavioral tests, parasite death was confirmed using DNA integrity assays. Alterations of geotaxis, locomotor activity and resting metabolic rate in infected gammarids remained unchanged one month after parasiteʼs death. In contrast, elevated brain lactate concentration and hemolymph total phenoloxidase activity of treated gammarids hosting a dead cystacanth returned to control (uninfected) levels. Interestingly, melanotic encapsulation response to dead cystacanths was rare up to two months after treatment, with only 5.6% of cystacanths being fully or partially melanized, thus suggesting long-lasting protection from the acellular outer envelope. Irreversible behavioral but reversible physiological alterations appear to be a cost-effective strategy of host manipulation, and point to a putative role of epigenetic alterations in parasite manipulation.

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.

This study was performed to evaluate the effects of rye silage treated with sodium formate (Na-Fa) and lactic acid bacteria (LAB) inoculants on the ruminal fermentation characteristics, methane yield and energy balance in Hanwoo steers. Forage rye was harvested in May 2019 and ensiled without additives (control) or with either a LAB inoculant or Na-Fa. The LAB (Lactobacillus plantarum) were inoculated at 1.5 × 10¹⁰ CFU/g fresh matter, and the inoculant was sprayed onto the forage rye during wrapping at a rate of 4 L/ton of fresh rye forage. Sixteen percent of the Na-Fa solution was sprayed at a rate of approximately 6.6 L/ton. Hanwoo steers (body weight 275 ± 8.4 kg (n = 3, group 1); average body weight 360 ± 32.1 kg (n = 3, group 2)) were allocated into two pens equipped with individual feeding gates and used in duplicated 3 × 3 Latin square design. The experimental diet was fed twice daily (09:00 and 18:00) during the experimental period. Each period comprised 10 days for adaptation to the pen and 9 days for measurements in a direct respiratory chamber. The body weights of the steers were measured at the beginning and at the end of the experiment. Feces and urine were collected for 5 days after 1 day of adaptation to the chamber, methane production was measured for 2 days, and ruminal fluid was collected on the final day. In the LAB group, the ratio of acetic acid in the rumen fluid was significantly lower (p = 0.044) and the ratio of propionic acid in the rumen fluid was significantly higher (p = 0.017). Methane production per DDMI of the Na-FA treatment group was lower than that of the other groups (p = 0.052), and methane production per DNDFI of the LAB treatment group was higher than that of the other groups (p = 0.056). The use of an acid-based additive in silage production has a positive effect on net energy and has the potential to reduce enteric methane emissions in ruminants.

One of the objectives of the European Union (EU) 2030 strategy is the sustainable growth through a series of measures focused on the application of the “Circular Economy” (ec.europa.eu, 2020) with many benefits also for climate, environment, and society. Food waste and sewage sludge management is part of this actions plan. Both streams are ideal source for microbial valorization processes to produce biofuels in a sustainable way. Anaerobic digestion (AD) is the benchmark robust technology for biogas production; however, the dark fermentation (DF) is a higher-rate process which offers the possibility to accumulate important building blocks (volatile fatty acids; VFA) and hydrogen from renewable resources, at reduced volumetric impact compared to AD. Focusing on this goal, the effectiveness of the hydrogenase enzymes needs to be favoured; some strategies have been already adopted: maintaining pH between 5.0-6.0; limiting the activities of methanogenic bacteria, not responsible for VFA and H2 accumulation, by decreasing the hydraulic retention time (HRT).

Urinary tract infections (UTIs) are one of the most common bacterial infections in humans, with ~400 million cases across the globe each year. Uropathogenic Escherichia coli (UPEC) is the major cause of UTI and increasingly associated with antibiotic resistance. This scenario has been worsened by the emergence and spread of pandemic UPEC sequence type 131 (ST131), a multidrug-resistant clone associated with extraordinarily high rates of infection. Here, we employed transposon-directed insertion site sequencing in combination with metabolomic profiling to identify genes and biochemical pathways required for growth and survival of the UPEC ST131 reference strain EC958 in human urine (HU). We identified 24 genes required for growth in HU, which mapped to diverse pathways involving small peptide, amino acid and nucleotide metabolism, the stringent response pathway, and lipopolysaccharide biosynthesis. We also discovered a role for UPEC resistance to fluoride during growth in HU, most likely associated with fluoridation of drinking water. Complementary nuclear magnetic resonance (NMR)-based metabolomics identified changes in a range of HU metabolites following UPEC growth, the most pronounced being L-lactate, which was utilized as a carbon source via the L-lactate dehydrogenase LldD. Using a mouse UTI model with mixed competitive infection experiments, we demonstrated a role for nucleotide metabolism and the stringent response in UPEC colonization of the mouse bladder. Together, our application of two omics technologies combined with different infection-relevant settings has uncovered new factors required for UPEC growth in HU, thus enhancing our understanding of this pivotal step in the UPEC infection pathway.

Lingonberry is a common wild berry that is often sold as jams and beverages. It naturally contains high amounts of the weak acid preservative benzoic acid making it an interesting ingredient for shelf-life extension. Despite this, their use as a raw ingredient is limited by the inherently intense sour taste. This study aimed to improve the taste of lingonberry juice by subjecting it to malolactic fermentation in order to reduce the sourness, and to investigate the benzoic acid in lingonberries as a natural preservative in juice blends by determining the microbial stability. After initial screening of lactic acid bacteria, a Lactiplantibacillus plantarum strain was used as the starter for subsequent investigations. Upon raising the pH, all malic acid was completely converted to lactic acid after seven days. The fermented juice was mixed with blackcurrant juice in different proportions. Challenge tests of the blends showed Listeria monocytogenes could not grow in any juice samples, while Candida albicans only grew in the pure blackcurrant juice. Aspergillus brasiliensis growth was delayed in all samples containing benzoic acid in a concentration-dependent manner. The sourness and astringency were substantially reduced in the juice with added L. plantarum compared to the unfermented juice.

Paneth cells (PCs), a subset of intestinal epithelial cells (IECs) found at the base of small intestinal crypts, play an essential role in maintaining intestinal homeostasis. Altered PCs function is associated with diverse intestinal pathologies, including ileal Crohn’s disease (CD). CD patients with ileal involvement have been previously demonstrated to display impairment in PCs and decreased levels of anti-microbial peptides. Although the immunosuppressive drug Azathioprine (AZA) is widely used in CD therapy, the impact of AZA on IEC differentiation remains largely elusive. In the present study, we hypothesized that the orally administered drug AZA also exerts its effect through modulation of the intestinal epithelium and specifically via modulation of PC function. AZA-treated CD patients exhibited an ileal upregulation of AMPs on both mRNA and protein levels compared to non-AZA treated patients. Upon in vitro AZA stimulation, intestinal epithelial cell line MODE-K exhibited heightened expression levels of PC marker in concert with diminished cell proliferation but boosted mitochondrial OXPHOS activity. Moreover, differentiation of IECs, including PCs differentiation, was boosted in AZA-treated murine small intestinal organoids and was associated with decreased D-glucose consumption and decreased growth rates. Of note, AZA treatment strongly decreased Lgr5 mRNA expression as well as Ki67 positive cells. Further, AZA restored dysregulated PCs associated with mitochondrial dysfunction. AZA-dependent inhibition of IEC proliferation is accompanied by boosted mitochondria function and IEC differentiation into PC.

The rate of pH decline, early post-mortem, has been identified as a key factor that impacts the tenderness of meat, and manipulating this rate of pH decline is highly relevant to ensure consistent high quality meat. Ultrasound is a potential intervention in early post – mortem muscle that may have an impact on the rate of glycolysis through its ability to alter enzyme activity. Following a variety of different ultrasound treatments frequencies (25 and 45 kHz) and durations (15, 30 and 45 min), it was found, when analysed in muscle, that ultrasound treatment duration, specifically the 30 min treatment, and interaction between treatment duration and frequency, had a significant impact on the rate of pH decline, post – treatment. Frequency did not have a significant effect on the rate of pH decline, post – treatment, in muscle. Ultrasound did not have a significant permanent effect on the activity of glycolytic enzymes present in bovine Longissimus lumborum et thoracis muscle, where no significant differences were observed on the rate of pH decline and rate of change of reducing sugars, glycogen and lactic acid, when analysed in an in vitro glycolytic buffer. It seems that the impact observed in intact muscle is not as a consequence of a permanent change in enzymatic activity, instead indicating an impact on conditions in the muscle which enhanced enzyme activity.