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.

The use of milling by-products as ingredients in food formulations has increased gradually over the past years, due to their well-recognized health properties. Fermentation performed with selected microbial strains or microbial consortia is the most promising way to reduce antinutritional factors of cereals and bran, while increasing their nutritional and functional properties. This work, developed within the BBI project INGREEN, was aimed to study the functional, nutritional and technological features of a pre-fermented ingredient obtained from the fermentation of a mixture of rye bran and wheat germ by a selected microbial consortium composed of yeasts (Kazachstania unispora and Kazachstania servazii) and lactic acid bacteria (Latilactobacillus curvatus) using as reference the unfermented mixture and the same mixture fermented by a baker’s yeast. The selected microbial consortium improved the complexity of the volatile molecules such as acids, alcohols and esters. A better retention of color parameters was maintained compared to the product fermented by a baker’s yeast. In addition, the fermentation by the selected consortium showed a significant increase in short chain fatty acids (more than 5-fold), antioxidant activity (22–24%), total phenol content (53-71%), bioactive peptides (39-52%), a reduction of 20-28% in phytic acid content and an increase in prebiotic activity not only compared to the unfermented product but also compared to the preferment obtained with a baker’s yeast. Overall, the fermentation by the selected microbial consortium can be considered a valuable way to valorize milling by-products and promote their exploitation as food ingredients.

OPA1, a dynamin-related GTPase mutated in autosomal dominant optic atrophy, is essential for the fusion of the inner mitochondrial membrane. Although OPA1 deficiency leads to impaired mitochondrial morphology, the role of OPA1 in central carbon metabolism remains unclear. Here, we aim to explore the functional role and metabolic mechanism of OPA1 in cell fitness beyond the control of mitochondrial fusion. We applied [U-¹³C]glucose and [U-¹³C]glutamine isotope tracing techniques to OPA1-knockout (OPA1-KO) mouse embryonic fibroblasts (MEFs) compared to OPA1 wild-type (OPA1-WT) controls. Furthermore, the resulting tracing data were integrated by metabolic flux analysis to understand the underlying metabolic mechanism through which OPA1 deficiency reprograms cellular metabolism. OPA1-deficient MEFs were depleted of intracellular citrate, which was consistent with the decreased oxygen consumption rate in these cells with mitochondrial fission that is not balanced by mitochondrial fusion. Whereas oxidative glucose metabolism was impaired, OPA1-deficient cells activated glutamine-dependent reductive carboxylation and subsequently relied on this reductive metabolism to produce cytosolic citrate as a predominant acetyl-CoA source for de novo fatty acid synthesis. Prevention of cytosolic glutamine reductive carboxylation by GSK321, an inhibitor of isocitrate dehydrogenase 1 (IDH1), largely repressed lipid synthesis and blocked cell proliferation in OPA1-deficient MEFs. Our data support that, when glucose oxidation failed to support lipogenesis and proliferation in cells with unbalanced mitochondrial fission, OPA1 deficiency stimulated metabolic anaplerosis into glutamine-dependent reductive carboxylation in an IDH1-mediated manner.

The potential use as malolactic starters of four indigenous strains of Oenococcus oeni was evaluated under different inoculation regimes. Among others, the fermentative capacity of strains, their degree of implantation, the main oenological parameters as well as their ability to modulate the aromatic profile of wines, were analysed. Main results elucidated that co-inoculation led to the prompt consecution of malolactic fermentation (MLF), highlighting the performance of indigenous Oenococcus oeni P2A strain and commercial O. oeni Viniflora OENOS strain which finished the process 20-30 days earlier compared to batches that undergo sequential inoculation. Moreover, inoculation strategy did also have an important influence on the volatile profile of wines. Co-inoculated wines significantly showed less concentration of volatile compounds. Main reduction was detected in higher alcohols and acids. Lower concentration of acids and higher alcohols may prevent the masking of desired aroma attributes. Indeed, in co-inoculated wines, the perception of ripe fruit aroma was highlighted over the others, and was extensively perceived by panellists in comparison with their respective sequentially inoculated wines. Above all, it was elucidated the suitability of the strain P2A, resulting an advantageous alternative to significantly reduce the overall winemaking time as well as to better control the fermentative process.

As the human population grows on the planet so does the generation of waste and particularly that of food waste. In order to tackle the world sustainability crisis, efforts to recover products from waste are critical. Here, we anaerobically recovered volatile fatty acids (VFAs) from food waste and analysed the microbial populations underpinning the process. An increased contribution of fungi relative to bacteria was observed throughout the reactor operation, with both kingdoms implicated into the main three steps of anaerobic digestion occurring within our systems: hydrolysis, acidogenesis and acetogenesis. Overall, Ascomycota, Proteobacteria and Firmicutes were found to drive the anaerobic digestion of food waste, with butyrate as the most abundant VFA likely produced by Clostridium using lactate as a precursor. Taken together we demonstrate that the generation of products of added-value from food waste results from cross-kingdoms microbial activities implicating fungi and bacteria.

L-lactate is an essential organic chemical in food processing, clinical, chemical, and fermentation industries. AuNPs were synthesized by chemical reduction reaction method and functionalized with 3-aminophenylboronic acid (3-APBA). The structural, morphological, FTIR, DLS, zeta potential characterization studies were performed to optimize the sensing mechanism. First time reporting facile enzyme-free sensor for the detection of L-lactate content in milk. Initially, the sensor has been optimized for nano reagent concentration, pH and time. 3-APBA@AuNPs showed a stable SPR intensity and forms a reversible tetrahedral ester complex with the analyte L-lactate (100 mM) resulting the NPs aggregation and revealing the blue shift of UV-VIS absorption wavelength (λ = 300 nm to 288 nm). Nestle- Lactogen, an India commercial milk powder has been used for the standardization study of lactate sensing. The milk samples spiked with L-Lactate (1-20mM) showed an increase in UV-VIS absorption peak intensity with concentration, and estimated the lower detection limit (LOD) = 1.1 mM using the calibration graphs. The optical sensing mechanism is explained may be due to π-π* transition of AuNPs stabilized by tetragonal complex of L-lactate bonded to 3-boronate via nucleophilic attack on boron atom. The nanosensor data was validated with commercial Megazyme kit and conventional HPLC technique. In commercial product Amul-India milk, the L-lactate value estimated as 0.678Mm with 95% correlation. Hence, the proposed sensor technique could be an alternate to the high cost conventional methods and at next level, it can be interfaced to smart phone to develop an onsite food, pharma and clinical device applications.

Small-molecule inhibitors of enzyme function are critical tools for the study of cell biological processes and for treatment of human disease. Identifying inhibitors with suitable specificity and selectivity for single enzymes, however, remains a challenge. In this study we describe our serendipitous discovery that NMS-873, a compound that was previously identified as a highly selective allosteric inhibitor of the ATPase valosin-containing protein (VCP/p97), rapidly induces aerobic fermentation in cultured human and mouse cells. Our further investigation uncovered an unexpected off-target effect of NMS-873 on mitochondrial oxidative phosphorylation, specifically as a dual inhibitor of Complex I and ATP synthase. This work points to the need for caution regarding the interpretation of cell survival data associated with NMS-873 treatment and indicates that cellular toxicity associated with its use may be caused by both VCP/p97-dependent and VCP/p97-independent mechanisms.

The bioavailability of minerals, such as zinc and magnesium, has a significant impact on the fermentation process. These metal ions are known to influence the growth and metabolic activity of yeast, but there are few reports on their effects on lactic acid bacteria (LAB) metabolism during sour brewing. This study aimed to evaluate the influence of magnesium and zinc ions on the metabolism of Lactobacillus brevis WLP672 during the fermentation of brewers’ wort. We carried out lactic acid fermentations using wort with different mineral compositions: without supplementation; supplemented with magnesium at 60 mg/L and 120 mg/L; and supplemented with zinc at 0.4 mg/L and 2 mg/L. The concentration of organic acids, pH of the wort and carbohydrate use was determined during fermentation, while aroma compounds, real extract and ethanol were measured after the mixed fermentation. The addition of magnesium ions resulted in the pH of the fermenting wort decreasing more quickly, an increase in the level of L-lactic acid (after 48 h of fermentation) and increased concentrations of some volatile compounds. While zinc supplementation had a negative impact on the L. brevis strain, resulting in a decrease in the L-lactic acid content and a higher pH in the beer. We conclude that zinc supplementation is not recommended in sour beer production using L. brevis WLP672.

There is a profound need in bioprocess manufacturing for low-cost single-use sensors that allow timely monitoring of critical product and production attributes. One such opportunity is screen-printed enzyme-based electrochemical sensors, which have the potential to enable low-cost online and/or off-line monitoring of specific parameters in bioprocesses. In this study, such a single-use electrochemical biosensor for lactate monitoring is designed and evaluated. Several aspects of its fabrication and use are addressed, including enzyme immobilization, stability, shelf-life and reproducibility. Applicability of the biosensor to off-line monitoring of bioprocesses was shown by testing in two common industrial bioprocesses in which lactate is a critical quality attribute (Corynebacterium fermentation and mammalian Chinese hamster ovary (CHO) cell cultivation). The specific response to lactate of the screen-printed biosensor was characterized by amperometric measurements. The usability of the sensor at typical industrial culture conditions was favorably evaluated and benchmarked with commonly used standard methods (HPLC and enzymatic kits). The single-use biosensor allowed fast and accurate detection of lactate in prediluted culture media used in industrial practice. The design and fabrication of the biosensor could most likely be adapted to several other critical bioprocess analytes using other specific enzymes. This makes this single-use screen-printed biosensor concept a potentially interesting and versatile tool for further applications in bioprocess monitoring.

Fermented dairy products have become attractive functional foods for the delivery of probiotics and their biologically active metabolites. The aim of this study was to examine the immunomodulatory activity of milk fermented with the probiotic lactic acid bacterium Lactobacillus rhamnosus R0011 (LrF) on macrophages challenged with lipopolysaccharide (LPS), a potent pro-inflammatory stimulus. To this end, human THP-1 or U937 monocytes were differentiated into resting macrophages then stimulated with LPS and co-incubated with the LrF or with milk controls. Levels of pro-inflammatory and immunoregulatory cytokines were determined by enzyme-linked immunosorbent assays. Culturing of LPS-stimulated U937 macrophages with either the whole or filtered LrF resulted in an increase in Interleukin (IL)-1Ra production relative to the negative control. THP-1 macrophages cultured with the LrF demonstrated an increase in LPS-induced IL-10 and IL-1β production, while production of LPS-induced IL-6, sCD54, IL-8, IL-1β, TNF-α, IL-12p70 and Transforming Growth Factor-β (TGF-β) was unaffected. Further, the LrF induced the expression of DC-SIGN and CD206, markers of immunoregulatory M2 macrophage polarization, in LPS-challenged THP-1 macrophages. Taken together, milk fermented with L. rhamnosus R0011 increased regulatory cytokine production from LPS-challenged U937 and THP-1 macrophages, while simultaneously up-regulating the production of IL-1β and expression of DC-SIGN and CD206, a profile characteristic of polarization into the immunoregulatory M2 macrophage phenotype.

A rapid fluorometric method is described for the determination of lactate and cholesterol by using ZnO nanowires (ZnO NWs). The assay is based on the detection of the hydrogen peroxide generated during the enzymatic reactions of the oxidation of lactate or cholesterol. Taking advantage of the electrostatic interactions between the enzymes and the ZnO NWs, two bioconjugates were prepared by mixing the nanomaterial and the enzymes, viz. lactate oxidase (LOx) or cholesterol oxidase (ChOx). The enzymatically generated hydrogen peroxide quenches the fluorescence of the ZnO NWs, which have emission peaks at 384 nm and at 520 nm under 330 nm photoexcitation. H₂O₂ quenches the 520 nm band more strongly. Response is linear up to 1.9 μM lactate concentration, and up to 1.1 μM cholesterol concentration. Relative standard deviation was found to be 5%. The detection limits for lactate and cholesterol are 0.54 and 0.24 μM, respectively.

An experiment was conducted to evaluate Pediococcus spp.-fermented chicken meat as a snack for dogs. The fermented or non-fermented snacks used in this study were prepared through the following process; meat mixtures containing 52.8% MDCM, 35.2% chicken breast meat (CBM) and 9.7% corn starch were inoculated with or without Pediococcus spp., incubated at 37°C for 24 h and then sterilized at 121°C for 20 min. During the 24-h fermentation, the pH of fermented chicken snack dropped rapidly with concomitant increase in number of lactic acid bacteria. The nutritional composition was not altered by fermentation. In vitro pepsin nitrogen digestibility was higher (p < 0.05) in the fermented snack compared with the non-fermented snack. Upon storage at room temperature for 14 days, bacteria grew slowly in fermented vs. non-fermented snack samples. In a palatability trial, dogs preferred non-fermented over fermented snack food. In 12-d-long feeding trial, fecal ammonia content was lowered, but fecal lactic acid content was increased in dogs fed the fermented vs. non-fermented snack food. Our study shows that the fermented MDCM-based snack exhibited good preservability upon storage, and improved in vitro nitrogen digestibility and fecal characteristics in dogs.

Immobilization of Lactobacillus rhamnosus ATCC7469 in poly(vinyl alcohol)/calcium alginate (PVA/Ca-alginate) matrix using “freezing–thawing” technique for application in lactic acid (LA) fermentation was studied in this paper. PVA/Ca-alginate beads were made from sterile and non-sterile PVA and sodium alginate solutions. According to mechanical properties, the PVA/Ca-alginate beads expressed a strong elastic character. Obtained PVA/Ca-alginate beads were further applied in batch and repeated batch LA fermentations. Regarding cell viability, L. rhamnosus cells survived well rather sharp immobilization procedure and significant cell proliferation was observed in further fermentation studies achieving high cell viability (up to 10.7 log CFU g⁻¹) in sterile beads. In batch LA fermentation, the immobilized biocatalyst was superior to free cell fermentation system (by 37.1%), while the highest LA yield and volumetric productivity of 97.6% and 0.8 g L⁻¹ h⁻¹, respectively, were attained in repeated batch fermentation. During seven consecutive batch fermentations, the biocatalyst showed high mechanical and operational stability reaching an overall productivity of 0.78 g L⁻¹ h⁻¹. This study suggested that the “freezing–thawing” technique can be successfully used for immobilization of L. rhamnosus in PVA/Ca-alginate matrix without loss of either viability or LA fermentation capability.

Cyanobacterial cell factories are widely researched for the sustainable production of compounds directly from CO₂. Their application, however, has been limited for two reasons. First, traditional approaches have been shown to lead to unstable cell factories that lose their production capability when scaled to industrial levels. Second, the alternative approaches developed so far are mostly limited to growing conditions, which are not always the case in industry, where nongrowth periods tend to occur (e.g., darkness). We tackled both by generalizing the concept of growth-coupled production to fitness coupling. The feasibility of this new approach is demonstrated for the production of fumarate by constructing the first stable dual-strategy cell factory. We exploited circadian metabolism using both systems and synthetic biology tools, resulting in the obligatorily coupling of fumarate to either biomass or energy production. Resorting to laboratory evolution experiments, we show that this engineering approach is more stable than conventional methods.

Multi-marker tests hold promise for identifying symptomatic women at risk of imminent preterm delivery (PTD, <37 week’s gestation). This study sought to determine the relationship of inflammatory mediators and metabolites in cervicovaginal fluid (CVF) with spontaneous PTD (sPTD) and delivery within 14 days of presentation with symptoms of preterm labour (PTL). CVF samples from 94 (preterm = 19, term = 75) singleton women with symptoms of PTL studied between 19⁺⁰-36⁺⁶ weeks’ gestation were analysed for cytokines/chemokines by multiplexed bead-based immunoassay, while metabolites were quantified by enzyme-based spectrophotometry in a subset of 61 women (preterm = 16, term = 45). Prevalence of targeted vaginal bacterial species was determined for 70 women (preterm = 14, term = 66) by PCR. Overall, 10 women delivered within 14 days of sampling. Predictive capacities of individual biomarkers and cytokine-metabolite combinations for sPTD and delivery within 14 days of sampling were analysed by logistic regression models and area under the receiver operating characteristic curve. Fusobacterium sp., Mubiluncus mulieris and Mycoplasma hominis were detected in more preterm-delivered than term women (P<0.0001), while, M. curtisii was found in more term-delivered than preterm women (P<0.0001). RANTES (0.91, 0.65-1.0), IL-6 (0.79, 0.67-0.88), and Acetate/Glutamate ratio (0.74, 0.61-0.85) were associated with delivery within 14 days of sampling (AUC, 95% CI). There were significant correlations between cytokines and metabolites, and several cytokine-metabolite combinations were associated with sPTD or delivery within 14 days of sampling (e.g. L/D-lactate ratio+Acetate/Glutamate ratio+IL-6: 0.84, 0.67-0.94). Symptomatic women destined to deliver preterm and within 14 days of sampling express significantly higher pro-inflammatory mediators at mid to late gestation. In this cohort, IL-6, Acetate/Glutamate ratio and RANTES were associated with delivery within 14 days of sampling, consistent with their roles in modulating infection-inflammation-associated preterm labour in women presenting with symptoms of preterm birth. Replication of these observations in larger cohorts of women could show potential clinical utility.

Enzymatic browning (EB) has impeded the commercialization of fresh-cut (FC) potato. However, recently introduced Innate^®-engineered cultivars with silenced polyphenol oxidase (PPO) have overcome this obstacle. As a supplement to refrigeration, low O₂ atmosphere may extend the shelf-life of FC potato by reducing wound-induced respiration, associated dry matter loss, EB, and low-temperature sweetening (LTS). Determining the O₂ concentration that minimizes these deteriorative physiological processes without invoking anaerobic metabolism is prerequisite to designing effective modified atmosphere packaging (MAP). Accordingly, FC tubers of cultivars Russet Burbank (RB), Ranger Russet (RR), and their Innate^® counterparts, CultivATE^®, GenerATE^®, and GlaciATE^®, were stored (4°C) in ten O₂ atmospheres ranging from 0 to 21 kPa to determine the lower oxygen limit (LOL) for aerobic respiration and effects on LTS and EB. FC tissue stored at 21 kPa O₂ displayed a prominent cold-induced respiratory acclimation response (RAR), characterized by an initial decline in respiration rate followed by a steady increase through 48 h. The RAR decreased with O₂ and was extinguished at ≤1.5 kPa. Lowering O₂ from 21-7 kPa had little effect on tissue respiration; however, rates fell from 4.23 to 3.41 μg kg⁻¹ s⁻¹ as O₂ decreased from 7 to 3.5 kPa, followed by a further 79% reduction to 0.70 μg kg⁻¹ s⁻¹ at 0 kPa O₂. Tissue lactate profiles revealed the onset of anaerobic metabolism at ca 1.5 kPa O₂ for all cultivars. Importantly, lactate and ethanol accumulation were negligible through 16 d at ≥2 kPa O₂ (= LOL) but increased considerably at ≤1 kPa. Low O₂ attenuated the cold-induced synthesis of sucrose and reducing sugars in FC tissue from RB, and sucrose from GlaciATE^® tubers in which acid invertase is silenced. Enzymatic browning of FC RB and RR tubers was inhibited by anoxia, but extensive at ≥0.5 kPa O₂. FC Innate^® tubers exhibited only minor EB at all O₂ concentrations. Collectively, these data inform the further development of MAP for FC potato.

The application of by‐products from the brewing industry in lactic acid (LA) production was investigated in order to replace expensive nitrogen sources (such as yeast extract) with cheaper and renewable nitrogenous materials such as brewer's yeast (BY). In this study, brewer's spent grain (BSG) hydrolysate was used for L‐(+)‐LA fermentation by Lactobacillus rhamnosus ATCC 7469. The effect of pH control during the fermentation and the addition of various BY contents (5-50 g/L) in BSG hydrolysate on fermentation parameters was evaluated. BY addition significantly increased free amino nitrogen (FAN) concentration (by 25.2% at 5 g/L to 616% at 50 g/L). A strong positive correlation between FAN concentration in the hydrolysate and concentration of L‐(+)‐LA produced was observed (correlation coefficient of 0.913). A high cell viability of L. rhamnosus ATCC 7469 (1.95–3.32 × 10⁹ CFU/mL at the end of fermentation) was achieved in all fermentations with the addition of brewer's yeast. The addition of BY increased L‐(+)‐lactic acid yield and volumetric productivity up to 8.4% (5 g/L) and 48.3% (50 g/L). The highest L‐(+)‐LA yield (89%) and volumetric productivity (0.89 g/L h⁻¹) were achieved in fermentation of BSG hydrolysate with 50 g/L of BY.

Natural fluctuations in dissolved oxygen are an important physiological challenge faced by marine organisms. This is particularly true of intertidal species that may become trapped in bodies of water where oxygen becomes rapidly depleted, or which may be emersed and exposed to elevated oxygen, but without respiratory organs adapted for uptake of oxygen from air. We sought to determine whether oxygen handling approaches differ in two species of sea cucumber, Cucumaria miniate and Parastichopus californicus, which inhabit distinct niches that vary in dissolved oxygen profiles. C. miniate occupies the rocky intertidal zone and thus experiences daily oscillations in oxygen content, whereas P. californicus inhabits the more oxygen stable, subtidal zone. Using closed chamber respirometry, our data showed that C. miniate have a higher basal metabolic rate than P. californicus, attributed to continuous filter-feeding, a phenomenon that contrasts with the sporadic feeding habits of the sediment-feeding P. californicus. Exposure to 6-h of anoxia or 6-h of emersion had no effect on coelomic fluid glucose concentrations in either species, however P. californicus body wall glucose concentrations increased ~6-fold relative to immersed normoxic controls under both treatments. Lactate production, a marker of anaerobic metabolism, was unaltered in both species under both anoxia and emersion conditions. These data demonstrate that habitat influences anoxia tolerance in sea cucumbers, with the intertidal C. miniate displaying a higher tolerance to both low dissolved oxygen and emersion, than the subtidal species, P. californicus.

In recent years, there is an increasing interest from the winemaking industry for the use of mixed fermentations with Starmerella bacillaris (synonym Candida zemplinina) and Saccharomyces cerevisiae, due to their ability to modulate metabolites of oenological interest. The current study was carried out to elucidate the effect of this fermentation protocol on the growth and malolactic activity of lactic acid bacteria (LAB) used for malolactic fermentation (MLF) and on the chemical and volatile profile of Nebbiolo wines and their chromatic characteristics. To this end, two LAB species, namely Lactobacillus plantarum and Oenococcus oeni, were inoculated at the beginning and at the end of the alcoholic fermentation (AF) performed by pure and mixed yeast using the abovementioned yeasts. The different yeast inoculation protocols and the combination of species tested influenced greatly the interactions and behavior of the inoculated yeasts and LAB during AF and MLF. For both LAB species, inoculation timing was critical to how rapidly MLF started and finished. Fermentation inoculated with L. plantarum, at the beginning of the AF, completed MLF faster than those inoculated with O. oeni. The presence of Starm. Bacillaris in mixed fermentation promoted LAB growth and activity, in particular, O. oeni. Furthermore, LAB species choice had a greater impact on the volatile and chromatic profile of the wines than inoculation time. These findings reveal new knowledge about the importance of LAB species choice and inoculation time to ensure fast MLF completion and to improve wine characteristics in mixed fermentation with Starm. Bacillaris and S. cerevisiae.