D-/L-Lactic Acid (D-/L-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.

Enterococcus faecalis (E. faecalis) has been associated with the specific production of L-(+)-lactic acid. However, in this study, D-(−)-lactic acid production by E. faecalis was observed under specific pH conditions. E. faecalis PR31 exhibited a significant amount of D-(−)-lactic acid under a stirring culture in MRS broth at pH 4.5, 5.8, and 6.0, and the contents of D-(−)-lactic acid were 45.1, 35.9, and 36.2%, respectively. When the cell suspension prepared at a pH of 6.0 was reacted with L-(+)- or D-(−)-lactic acid, D-(−)- or L-(+)-lactic acid was produced, respectively, in a time- and dose-dependent manner. Therefore, this phenomenon of D-(−)-lactic acid production in PR31 was suggested to be due to the activation of the larA gene encoding lactate racemase that is present in PR31. However, even in the E. faecalis-type strain NBRC 100480, which contains neither larA nor vanH, encoding D-(−)-lactate dehydrogenase VanH, D-(−)-lactic acid was also produced at specific pH values. Therefore, the production of D-(−)-lactic acid in NBRC 100480 was thought to occur not via the activation of larA. The biological significance of D-(−)-lactic acid production in E. faecalis depending on the pH and the detailed underlying mechanism, including whether it is the same in PR31 and NBRC 100480, remain to be elucidated in future studies.

2-hydroxy acids are organic carboxylic acids ubiquitous in the living world and are important building blocks in organic synthesis. Recently, the lactate racemase (LarA) superfamily, a diverse superfamily of 2-hydroxy acid racemases and epimerases using the nickel-pincer nucleotide (NPN) cofactor, has been uncovered. In this study, we performed a taxonomic analysis of the LarA superfamily, showing the distribution of lactate racemase homologs (LarAHs) sequences across the three domains of life. Subsequently, we overexpressed and purified nine LarAHs and investigated their biochemical properties and substrate specificities. We show that LarAHs from the lactate racemases group are more promiscuous than previously thought, with some members showing high specificity towards glycerate or 2-hydroxybutyrate. In other phylogenetic groups, we identified a new malate racemase and 2-hydroxyglutarate racemase, as well as a new 2-gluconate epimerase from an eukaryotic organism. We show that some LarAHs are able to isomerize up to 16 different substrates, mostly 2-hydroxy acids with hydrophobic side chains, thereby identifying 14 novel 2-hydroxy acid racemization and epimerization reactions catalyzed by LarAHs. These include the racemization of glycerate, 2-hydroxybutyrate, 2,4-dihydroxybutyrate, 2-hydroxyvalerate, 2-hydroxycaproate, 2,3-dihydroxyisovalérate, 2-hydroxy-3,3-dimethylbutyrate, 3-(4-hydroxyphenyl)lactate, 2-hydroxy-4-phenylbutyrate, and 2-hydroxy-4-oxo-4-phenylbutyrate. Additionally, we observed the C2-epimerization of all 2,3-dihydroxybutyrate stereoisomers (4-deoxy-DL-threonate and 4-deoxy-DL-erythronate) and the C2-epimerization of D-arabinarate epimers. Finally, through comparative analysis of Alphafold structural predictions, we identified key residues likely involved in substrate specificity and predicted the function of half of the LarAHs from the LarA superfamily. In conclusion, this study widely expands the scope of substrates isomerized by NPN-dependent enzymes.

In this study a plant-based spread was developed using dextran-enriched ingredients derived from pea flours, supplemented with defatted durum wheat germ and almond flour. Optimization of fermentation with Leuconostoc pseudomesenteroides DSM 20193, both with and without enzymatic hydrolysis, aimed to enhance exopolysaccharide production and the nutritional value of pea flours. Best results were achieved through enzymatic hydrolysis with Veron PS protease followed by fermentation at 25°C, resulting in elevated dextran levels and increased peptides and total free amino acid concentration in green and yellow pea-based ingredients. The yellow pea-based ingredient was selected for the final plant-based spread formulation, blended at 35% w/w, with 45% w/w defatted durum wheat germ, and 20% w/w almond flour. The resultant spread exhibited elastic and solid-like characteristics like milk-based spreadable cheese and yogurt, boasting 'high protein' (12.49 g/100g) and 'high fiber' (11.01 g/100g) designations. It maintained chemical, biochemical, and microbiological stability over a 10-day shelf-life under refrigerated conditions. Sensory evaluation confirmed the acceptability of the plant-based spread (PBS), highlighting a well-balanced aroma and a grainy, adhesive texture. This research underscores the potential of an integrated approach utilizing food-grade enzymes and fermentation for the in-situ production of dextran to create innovative, clean label, and plant-based foods.

Lactic acid (LA) serves as a freshness marker in certain foods. In the present work, electrified interfaces of different nature (i.e., liquid-liquid and liquid-organogel) have been developed for the quantification of LA. Electrochemical sensing of LA at the liquid-organogel interface revealed that adsorptive stripping voltammetry, with a preconcentration time of 500 s offered better sensitivity. Electroanalytical ability of LA under optimized conditions displayed a detection limit of 0.97 μM and 0.71 μM with sensitivity of 2.84 nA μM⁻¹ and 3.59 nA μM⁻¹ for liquid-liquid and liquid-organogel interfaces respectively. Quantification of LA using the developed methodology has been demonstrated in buttermilk as the real matrix. Analysis demonstrate that electrified liquid-liquid and liquid-organogel interfaces are promising approach for sensing LA in buttermilk extract.

Breast milk is the best source of nutrition for infants. Human milk oligosaccharides (HMOs) and the corresponding HMOs-consuming Bifidobacterium positively influence infant health. This study aims to isolate and characterize Bifidobacterium from breast milk of healthy Chinese mothers, identifying the most efficacious strains for inclusion in simulated maternal milk formulas. Nine Bifidobacterium strains (two of B. breve and seven of B. infantis) were isolated, exhibiting a broad spectrum of probiotic potential. This included tolerance to simulated infant gastrointestinal conditions, notable adhesion, antibacterial, antioxidant activities, and HMOs utilization ability. Lacto-N-Tetraose (LNT) is preferred in early growth among Bifidobacterium isolates. B. breve showed a preference for LNT, whereas B. infantis showed a preference for fucosylated HMOs, and displayed reduced utilization of sialylated HMOs. They also exhibited robust safety profiles, including no hemolytic activity, an appropriate D/L lactate-producing ratio, and non-toxicity in an acute oral toxicity assay on mice. It is noteworthy that B. breve N-90, O-147, B. infantis O-161 and R-1 exhibited anti-inflammatory effects in LPS-induced RAW 264.7 cells. Specifically, a notable reduction in TNF-α levels was observed in pre-treatment, while a decrease in IL-1β and IL-6 levels in co-treatment. B. breve N-90 and B. infantis R-1 were identified finally as promising probiotic candidates. Their whole-genome sequencing analysis confirmed presence of functional genes associated with gastrointestinal colonization, antioxidation, and glycoside hydrolase activity on HMOs. The annotation for antibiotic resistance and virulence genes concurred with phenotypes, further validating the safety. Breast milk is a good source for Bifidobacteria isolation, while Bifidobacteria utilize HMOs in a strain-dependent manner. The two selected strains, B. breve N-90 and B. infantis R-1, are potential candidates for inclusion in simulated maternal milk formulas and deserved further in vivo investigation for their health-promoting effects.

The probiotic Bacillus subtilis 29784 (Bs29784) sustains chicken's intestinal health, enhancing animal resilience and performance through the production of the bioactive metabolites hypoxanthine (HPX), niacin (NIA), and pantothenate (PTH). Here, using enterocyte in vitro models, we determine the functional link between these metabolites and the three pillars of intestinal resilience: immune response, intestinal barrier, and microbiota. We evaluated in vitro the capacity of Bs29784 vegetative cells, spores, and metabolites to modulate global immune regulators (using HT-29-NF-κB and HT-29-AP-1 reporter cells), intestinal integrity (HT-29-MUC2 reporter cells and Caco-2 cells), and cytokine production (Caco-2 cells). Finally, we simulated intestinal fermentations using chicken's intestinal contents as inocula to determine the effect of Bs29784 metabolites on the microbiota and their fermentation profile. Bs29784 vegetative cells reduced the inflammatory response more effectively than spores, indicating that their benefit is linked to metabolic activity. To assess this hypothesis, we studied Bs29784 metabolites individually. The results showed that each metabolite had different beneficial effects. PTH and NIA reduced the activation of the pro-inflammatory pathways AP-1 and NF-κB. HPX upregulated mucin production by enhancing MUC2 expression. HPX, NIA, and PTH increased cell proliferation. PTH and HPX increased epithelial resilience to an inflammatory challenge by limiting permeability increase. In cecal fermentations, NIA increased acetate, HPX increased butyrate, whereas PTH increased acetate, butyrate, and propionate. In ileal fermentations, PTH increased butyrate. All molecules modulated microbiota, explaining the different fermentation patterns. Altogether, we show that Bs29784 influences intestinal health by acting on the three lines of resilience via its secreted metabolites.

High-pressure processing (HPP) is a non-thermal preservation technology that can be applied as a control measure to inactivate pathogens and spoilage microorganisms once RTE meat products are packaged in a convenient format. HPP efficacy highly depends on product characteristics, but the impact of the sodium-reduced formulations and the effect of packaging atmosphere are scarcely known. The aim of the present work was to assess the effect of standard and sodium-reduced formulations from two different brands (A, B) under different packaging (vacuum and modified atmosphere packaging (MAP)) on the HPP inactivation kinetics of Listeria monocytogenes and spoilage lactic acid bacteria in cooked ham. Slices of cooked ham with standard and sodium-reduced formulations were inoculated with L. monocytogenes CTC1034 and Latilactobacillus sakei CTC746 (slime producer), packaged in vacuum and MAP (CO₂:N₂, 20:80), and pressurized (400 MPa/0–15 min) after 1 h (vacuum, MAP) or 24 h (MAP-exposed). Parameters of HPP inactivation kinetics were estimated by fitting the Weibull model to log reduction data. Results showed that the efficacy of HPP in sodium-reduced cooked hams tended to decrease compared to standard formulations, being the difference statistically significant for L. sakei. For L. monocytogenes, a significant enhancing effect of MAP was observed when HPP was applied just after packaging (1 h, MAP) of cooked ham of brand A. In the case of L. sakei, the inactivation by HPP was only enhanced in MAP-exposed samples. Therefore, the use of HPP as a control measure must be applied through a product-oriented approach considering the type of packaging and the time period between packaging and HPP.

The transition towards a sustainable model, particularly the circular economy, emphasizes the importance of redefining waste as a valuable resource, paving the way for innovative upcycling strategies. The olive oil industry, with its significant output of agricultural waste, offers a promising avenue for high-value biomass conversion into useful products through microbial processes. This study focuses on exploring new, high-value applications for olive leaves waste, utilizing a biotechnological approach with Lactobacillus casei for the production of second-generation lactic acid. Contrary to initial expectations, the inherent high polyphenol content and low fermentable glucose levels in olive leaves posed challenges for fermentation. Addressing this, an enzymatic hydrolysis step, following a preliminary extraction process, was implemented to increase glucose availability. Subsequent small-scale fermentation tests were conducted with and without nutrient supplements, identifying the medium that yielded the highest lactic acid production for scale-up. The scaled-up batch fermentation process achieved an enhanced conversion rate (83.58%) and specific productivity (0.26 g/L·h). This research confirms the feasibility of repurposing olive waste leaves for the production of lactic acid, contributing to the advancement of a greener economy through the valorization of agricultural waste.

The aim of the present study was to provide a first characterization of lacto-fermented garlic manufactured by local small-scale artisanal producers in the Lower Silesia Region (Poland). The lacto-fermented garlic samples showed high nutritional features in terms of antioxidant activity. A total of 86 compounds, belonging to various chemical classes, were identified by headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC/MS). Most of these compounds belonged to six main classes, being sulfur compounds, esters and acetates, oxygenated monoterpenes, monoterpene hydrocarbons, and alcohols. Aldehydes, acids, ketones, furans, and phenols were also identified. In the analyzed samples, counts up to 8 log cfu g⁻¹ were observed for lactic acid bacteria. Metataxonomic analysis revealed the presence of Levilactobacillus, Lactiplantibacillus, Latilactobacillus, Secundilactobacillus, Weissella, Leuconostoc, Lactococcus, Pediococcus, and Lacticaseibacillus among the major taxa. These results were confirmed by the isolation and characterization of viable lactic acid bacteria.

The principal objective of this study was to isolate and identify the microorganisms present in commercial kefir grains, a novel kefir-fermented coconut water (CWK) and a novel coconut water kefir-fermented sourdough using phenotypic identification and Sanger sequencing and examine the microbial diversity of CWK and CWK-fermented sourdough throughout the fermentation process using the MiSeq Illumina sequencing method. The phenotypic characterisation based on morphology identified ten isolates of LAB, five AAB and seven yeasts from kefir (K), CWK and CWK-fermented sourdough (CWKS). The results confirm the presence of the LAB species Limosilactobacillus fermentum, Lactobacillus. plantarum, L. fusant, L. reuteri and L. kunkeei; the AAB species Acetobacter aceti, A. lovaniensis and A. pasteurianus; and the yeast species Candida kefyr, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, C. guilliermondii and C. colliculosa. To the best of our knowledge, the identification of Rhodotorula from kefir is being reported for the first time. This study provides important insights into the relative abundances of the microorganisms in CWKS. A decrease in pH and an increase in the titratable acidity for CWK- and CWK-fermented sourdough corresponded to the increase in D- and L-lactic acid production after 96 h of fermentation. Significant reductions in the pHs of CWK and CWKS were observed between 48 and 96 h of fermentation, indicating that the kefir microorganisms were able to sustain highly acidic environments. There was also increased production of L-lactic acid with fermentation, which was almost twice that of D-lactic acid in CWK.

Macrophage responses to activation are fluid and dynamic in their ability to respond appropriately to challenges, a role integral to host defence. While bacteria can influence macrophage differentiation and polarization into pro-inflammatory and alternatively activated phenotypes through direct interactions, many questions surround indirect communication mechanisms mediated through secretomes derived from gut bacteria, such as lactobacilli. We examined effects of secretome-mediated conditioning on THP-1 human monocytes, focusing on the ability of the Lacticaseibacillus rhamnosus R0011 secretome (LrS) to drive macrophage differentiation and polarization and prime immune responses to subsequent challenge with lipopolysaccharide (LPS). Genome-wide transcriptional profiling revealed increased M2-associated gene transcription in response to LrS conditioning in THP-1 cells. Cytokine and chemokine profiling confirmed these results, indicating increased M2-associated chemokine and cytokine production (IL-1Ra, IL-10). These cells had increased cell-surface marker expression of CD11b, CD86, and CX₃CR1, coupled with reduced expression of the M1 macrophage-associated marker CD64. Mitochondrial substrate utilization assays indicated diminished reliance on glycolytic substrates, coupled with increased utilization of citric acid cycle intermediates, characteristics of functional M2 activity. LPS challenge of LrS-conditioned THP-1s revealed heightened responsiveness, indicative of innate immune priming. Resting stage THP-1 macrophages co-conditioned with LrS and retinoic acid also displayed an immunoregulatory phenotype with expression of CD83, CD11c and CD103 and production of regulatory cytokines. Secretome-mediated conditioning of macrophages into an immunoregulatory phenotype is an uncharacterized and potentially important route through which lactic acid bacteria and the gut microbiota may train and shape innate immunity at the gut-mucosal interface.

Viili, a Finnish ropy fermented milk, is traditionally manufactured through spontaneous fermentation, by mesophilic lactic acid bacteria and yeast-like fungi, or back-slopping. This study evaluated four natural viili starters as sources of lactic acid bacteria for dairy production. Back-slopping activation of the studied viili samples was monitored through pH and titratable acidity measurements and enumeration of mesophilic lactic acid bacteria. Sixty lactic acid bacteria isolates were collected, molecularly identified, and assayed for acidification performance, enzymatic activities, production of exopolysaccharides (EPSs), presence of the histidine decarboxylase (hdcA) gene of Gram-positive bacteria, and production of bacteriocins. A neat predominance of Lactococcus lactis emerged among the isolates, followed by Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Enterococcus lactis, and Lactococcus cremoris. Most isolates exhibited proteolytic activity, whereas only a few enterococci showed lipase activity. Five isolates identified as L. cremoris, L. lactis, and E. faecalis showed a good acidification performance. Most of the isolates tested positive for leucine arylamidase, whereas only one E. durans and two L. lactis isolates were positive for valine arylamidase. A few isolates also showed a positive reaction for beta-galactosidase and alpha- and beta-glucosidase. None of the isolates produced EPSs or bacteriocins. The hdcA gene was detected in five isolates identified as L. lactis and E. faecium. A few L. cremoris and L. lactis isolates for potential use as starter or adjunct cultures for dairy processing were finally identified.

This study aimed to explore the safety and properties of Lacticaseibacillus paracasei IDCC 3401 as a novel probiotic strain via genomic and phenotypic analyses. In whole-genome sequencing, the genes associated with antibiotic resistance and virulence were not detected in this strain. The minimum inhibitory concentration test revealed that L. paracasei IDCC 3401 was susceptible to all the antibiotics tested, except for kanamycin. Furthermore, the strain did not produce toxigenic compounds, such as biogenic amines and D-lactate, nor did it exhibit significant toxicity in a single-dose acute oral toxicity test in rats. Phenotypic characterization of carbohydrate utilization and enzymatic activities indicated that L. paracasei IDCC 3401 can utilize various nutrients, allowing it to grow in deficient conditions and produce health-promoting metabolites. The presence of L. paracasei IDCC 3401 supernatants significantly inhibited the growth of enteric pathogens (p < 0.05). In addition, the adhesion ability of L. paracasei IDCC 3401 to intestinal epithelial cells was found to be as superior as that of Lacticaseibacillus rhamnosus GG. These results suggest that L. paracasei IDCC 3401 is safe for consumption and provides health benefits to the host.