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

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00:08  Introduction
01:13    Principle
02:43   Reagent Preparation
03:23   Procedure
07:54   Calculations

D- L-Lactic Acid D- L-Lactate Rapid Assay Kit K-DLATE Scheme
Product code: K-DLATE

100 assays (50 of each) per kit

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Content: 100 assays (50 of each) per kit
Shipping Temperature: Ambient
Storage Temperature: Short term stability: 2-8oC,
Long term stability: See individual component labels
Stability: > 1 year under recommended storage conditions
Analyte: D-Lactic Acid, L-Lactic Acid
Assay Format: Spectrophotometer
Detection Method: Absorbance
Wavelength (nm): 340
Signal Response: Increase
Linear Range: 0.5 to 30 µg of D- or L-lactic acid per assay
Limit of Detection: 0.21 mg/L
Reaction Time (min): ~ 10 min (L-lactic acid),
~ 5 min (D-lactic acid)
Application examples: Wine, soft drinks, milk, dairy products, foods containing milk (e.g. dietetic foods, bakery products, baby food, chocolate, sweets and ice-cream), vinegar, fruit and vegetables, processed fruit and vegetables, meat products, food additives, paper (and cardboard), cosmetics, pharmaceuticals and other materials (e.g. biological cultures, samples, etc.).
Method recognition: Methods based on this principle have been accepted by DIN, GOST, IDF, EEC, EN, ISO, OIV, IFU, AIJN and MEBAK

The D-/L-Lactic Acid (D-/L-Lactate) (Rapid) test kit is used for the rapid and specific concurrent measurement and analysis of L-lactic acid (L-lactate) and D-lactic acid (D-lactate) in beverages, meat, dairy and food products.

Note for Content: The number of manual tests per kit can be doubled if all volumes are halved.  This can be readily accommodated using the MegaQuantTM  Wave Spectrophotometer (D-MQWAVE).

Explore more organic acid assay kit products.

Scheme-K-DLATE DLATE Megazyme

  • Extended cofactors stability. Dissolved cofactors stable for > 1 year at 4oC.
  • Rapid total analysis time (concurrent / flexible D and L-lactic acid reaction format) 
  • D-lactate dehydrogenase reaction very rapid with most samples (~ 5 min) 
  • Very competitive price (cost per test) 
  • All reagents stable for > 2 years after preparation 
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing 
  • Standard included 
Certificate of Analysis
Safety Data Sheet
FAQs Assay Protocol Data Calculator Product Performance Validation Report
Megazyme publication

Megazyme “advanced” wine test kits general characteristics and validation.

Charnock, S. J., McCleary, B. V., Daverede, C. & Gallant, P. (2006). Reveue des Oenologues, 120, 1-5.

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.

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Megazyme publication

Grape and wine analysis: Oenologists to exploit advanced test kits.

Charnock, S. C. & McCleary, B. V. (2005). Revue des Enology, 117, 1-5.

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.

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Effect of Yogurt Acid Whey on the Quality of Maize Silage.

Palamidi, I., Paraskeuas, V. V., Kotsampasi, B., Hadjigeorgiou, I., Politis, I. & Mountzouris, K. C. (2023). Fermentation, 9(12), 994.

The increasing popularity of Greek yogurt generates large amounts of acid whey worldwide. The use of yogurt acid whey in animal nutrition is limited. The aim of this study was to determine the effect of a yogurt acid whey powder (YAWP) addition to maize forage prior to ensiling on the nutritional, microbial and fermentation quality of maize silage. Depending on the addition level of the YAWP to maize forage, there were the following four experimental treatments: YAWP 0, 2.5, 5 and 10% w/w. An increasing YAWP inclusion level linearly increased the maize silage dry matter, crude protein and ash concentrations, whereas it reduced the crude fiber, neutral-detergent fiber and acid-detergent fiber concentrations. The silage pH decreased quadratically with the increasing YAWP level, with the lower plateau noted for the YAWP 5% addition. Concentrations of total bacteria in the silage and Lactobacillus spp. decreased linearly with the YAWP increase. The silage acetic acid content decreased linearly, whereas propionic acid, lactic acid and the ratio of lactic to acetic acid increased linearly with the increasing YAWP level. The ammonia-N content decreased linearly with the increasing YAWP level. In conclusion, the incorporation of the 5 and 10% YAWP addition in silage preparation improved the nutritional and fermentative quality of the produced silage.

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Combined use of Trametes versicolor extract and sourdough fermentation to extend the microbiological shelf-life of baked goods.

Torreggiani, A., Beccaccioli, M., Verni, M., Cecchetti, V., Minisci, A., Reverberi, M., Pontonio, E. & Rizzello, C. G. (2023). LWT, 189, 115467.

Fungal spoilage is the main responsible for the short shelf-life of baked goods. Indeed, many chemical preservatives (e.g., calcium propionate and ethanol) are often included in their formulation leading to consumer dissatisfaction. Here, an in-vitro and in-situ integrated approach was used to investigate the potential antifungal activity of the extract obtained from Trametes versicolor as biological preservative. An intense inhibition towards most of the Aspergillus and Penicillium species and a broad spectrum of activity on typical spoilage fungi of the bakery products, as well as high thermal stability and intense activity at pH 4.00 characterized the Trametes extract. The antifungal potential of the extract has been exploited in sour bread made with organic acids (chemical) or type II-sourdough (biological). Compared to baker's yeast bread produced as control (pH 5.6), the acidified samples were characterized by a longer mold-free shelf-life, with indicator mycelia that became visible after 6–9 days of storage at room temperature. Sourdough was effective to counteract the negative effect of the extract supplementation on the leavening performances, textural and sensory features of the bread samples.

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Application of whole-cell biosensors for analysis and improvement of L-and D-lactic acid fermentation by Lactobacillus spp. from the waste of glucose syrup production.

Augustiniene, E., Jonuskiene, I., Kailiuviene, J., Mazoniene, E., Baltakys, K. & Malys, N. (2023). Microbial cell factories, 22(1), 223.

Background: Lactic acid is one of the most important organic acids, with various applications in the food, beverage, pharmaceutical, cosmetic, and chemical industries. Optically pure forms of L- and D-lactic acid produced via microbial fermentation play an important role in the synthesis of biodegradable polylactic acid. Alternative substrates, including by-products and residues from the agro-food industry, provide a cost-effective solution for lactic acid production and are a promising avenue for the circular economy. Results: In this study, the transcription factor (TF)-based whole-cell biosensor strategy was developed for the L- and D-lactic acid determination. It was cross validated with commonly used high-performance liquid chromatography and enzymatic methods. The utility of biosensors as an efficient analytical tool was demonstrated by their application for the lactic acid determination and fermentation improvement. We explored the ability of Lacticaseibacillus paracasei subsp. paracasei, Lactobacillus delbrueckii subsp. lactis, and Lactobacillus amylovorus to biosynthesize optically pure L-lactic acid, D-lactic acid or mixture of both from organic-rich residual fraction (ORRF), a waste of glucose syrup production from wheat starch. The fermentation of this complex industrial waste allowed the production of lactic acid without additional pretreatment obtaining yields from 0.5 to 0.9 Cmol/Cmol glucose. Conclusions: This study highlights the utility of whole cell biosensors for the determination of L- and D-forms of lactic acid. The fermentation of L-lactic acid, D-lactic acid and mixture of both by L. paracasei, L. lactis, and L. amylovorus, respectively, was demonstrated using waste of glucose syrup production, the ORRF.

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Selenium-Fortified Kombucha-Pollen Beverage by In Situ Biosynthesized Selenium Nanoparticles with High Biocompatibility and Antioxidant Activity.

Tritean, N., Dima, Ș. O., Trică, B., Stoica, R., Ghiurea, M., Moraru, I., Cimpean, A., Oancea, F. & Constantinescu-Aruxandei, D. (2023). Antioxidants, 12(9), 1711.

Biogenic selenium nanoparticles (SeNPs) have been shown to exhibit increased bioavailability. Fermentation of pollen by a symbiotic culture of bacteria and yeasts (SCOBY/Kombucha) leads to the release of pollen content and enhances the prebiotic and probiotic effects of Kombucha. The aim of this study was to fortify Kombucha beverage with SeNPs formed in situ by Kombucha fermentation with pollen. Response Surface Methodology (RSM) was used to optimize the biosynthesis of SeNPs and the pollen-fermented Kombucha beverage. SeNPs were characterized by Transmission electron microscopy energy-dispersive X-ray spectroscopy (TEM-EDX), Fourier-transform infrared spectroscopy (FTIR), Dynamic light scattering (DLS), and Zeta potential. The pollen-fermented Kombucha beverage enriched with SeNPs was characterized by measuring the total phenolic content, antioxidant activity, soluble silicon, saccharides, lactic acid, and the total content of Se0. The polyphenols were identified by liquid chromatography–mass spectrometry (LC-MS). The pollen and the bacterial (nano)cellulose were characterized by scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDX), FTIR, and X-Ray diffraction (XRD). We also assessed the in vitro biocompatibility in terms of gingival fibroblast viability and proliferation, as well as the antioxidant activity of SeNPs and the pollen-fermented Kombucha beverage enriched with SeNPs. The results highlight their increased biological performance in this regard.

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Physicochemical changes during controlled laboratory fermentation of cocoa (CCN-51) with the inclusion of fruits and on-farm inoculation.

Peña González, M. A., Ortiz Urgiles, J. P., Santander Pérez, F. A., Lazo Vélez, M. A. & Caroca Cáceres, R. S. (2023). Brazilian Journal of Food Technology, 26, e2023013.

Fermentation is key to developing the organoleptic characteristics of cocoa beans, as dynamic changes in metabolites have a significant impact on flavors and aromas, hence modifications of this process have been investigated. In this research, the mucilage of CCN-51 cocoa beans was replaced by a mixture of passion fruit (Passiflora edulis) and plantain (Musa paradisiaca L.) pulp, and a controlled fermentation of this mixture was carried out after its spontaneous on-farm inoculation. The physicochemical changes and correlations during the five days of fermentation were evaluated. At the end of the process, the temperature reached 47 ºC in the fermentation mass and pH 5.64 was recorded in the cotyledon. In the first 48 hours, citric acid and fructose were high but at the end of fermentation were 71% and 41.17% lower than at the start of fermentation, respectively. As glucose and fructose were consumed during fermentation, acetic acid and lactic acid levels increased from day two onward, reaching values at the end of the process of 22.48 mg/g and 16.01 mg/g, respectively. In contrast, the bromatological parameters did not show greater variability when comparing each day of fermentation. The data generated and results presented in this study will contribute to the knowledge of possible sensory improvements achieved with the inclusion of pulp fruits in the fermentation stage.

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A study of electron source preference and its impact on hydrogen production in microbial electrolysis cells fed with synthetic fermentation effluent.

Choi, Y., Kim, D., Choi, H., Cha, J., Baek, G. & Lee, C. (2023). Bioengineered, 14(1), 2244759.

Fermentation effluents from organic wastes contain simple organic acids and ethanol, which are good electron sources for exoelectrogenic bacteria, and hence are considered a promising substrate for hydrogen production in microbial electrolysis cells (MECs). These fermentation products have different mechanisms and thermodynamics for their anaerobic oxidation, and therefore the composition of fermentation effluent significantly influences MEC performance. This study examined the microbial electrolysis of a synthetic fermentation effluent (containing acetate, propionate, butyrate, lactate, and ethanol) in two-chamber MECs fitted with either a proton exchange membrane (PEM) or an anion exchange membrane (AEM), with a focus on the utilization preference between the electron sources present in the effluent. Throughout the eight cycles of repeated batch operation with an applied voltage of 0.8 V, the AEM-MECs consistently outperformed the PEM-MECs in terms of organic removal, current generation, and hydrogen production. The highest hydrogen yield achieved for AEM-MECs was 1.26 L/g chemical oxygen demand (COD) fed (approximately 90% of the theoretical maximum), which was nearly double the yield for PEM-MECs (0.68 L/g COD fed). The superior performance of AEM-MECs was attributed to the greater pH imbalance and more acidic anodic pH in PEM-MECs (5.5-6.0), disrupting anodic respiration. Although butyrate is more thermodynamically favorable than propionate for anaerobic oxidation, butyrate was the least favored electron source, followed by propionate, in both AEM- and PEM-MECs, while ethanol and lactate were completely consumed. Further research is needed to better comprehend the preferences for different electron sources in fermentation effluents and enhance their microbial electrolysis.

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Dual role of probiotic lactic acid bacteria cultures for fermentation and control pathogenic bacteria in fruit-enriched fermented milk.

Borgonovi, T. F., Fugaban, J. I. I., Bucheli, J. E. V., Casarotti, S. N., Holzapfel, W. H., Todorov, S. D. & Penna, A. L. B. (2023). Probiotics and Antimicrobial Proteins, 1-16.

The food industry has been developing new products with health benefits, extended shelf life, and without chemical preservation. Bacteriocin-producing lactic acid bacteria (LAB) strains have been evaluated for food fermentation to prevent contamination and increase shelf life. In this study, potentially probiotic LAB strains, Lactiplantibacillus (Lb.) plantarum ST8Sh, Lacticaseibacillus (Lb.) casei SJRP38, and commercial starter Streptococcus (St.) thermophilus ST080, were evaluated for their production of antimicrobial compounds, lactic acid and enzyme production, carbohydrate assimilation, and susceptibility to antibiotics. The characterization of antimicrobial compounds, the proteolytic activity, and its inhibitory property against Listeria (List.) monocytogenes and Staphylococcus (Staph.) spp. was evaluated in buriti and passion fruit-supplemented fermented milk formulations (FMF) produced with LAB strains. Lb. plantarum ST8Sh was found to inhibit List. monocytogenes through bacteriocin production and produced both L(+) and D(−) lactic acid isomers, while Lb. casei SJRP38 mainly produced L(+) lactic acid. The carbohydrate assimilation profiles were compatible with those usually found in LAB. The potentially probiotic strains were susceptible to streptomycin and tobramycin, while Lb. plantarum ST8Sh was also susceptible to ciprofloxacin. All FMF produced high amounts of L(+) lactic acid and the viability of total lactobacilli remained higher than 8.5 log CFU/mL during monitored storage period. Staph. aureus ATCC 43300 in fermented milk with passion fruit pulp (FMFP) and fermented milk with buriti pulp (FMB), and Staph. epidermidis KACC 13234 in all formulations were completely inhibited after 14 days of storage. The combination of Lb. plantarum ST8Sh and Lb. casei SJRP38 and fruit pulps can provide increased safety and shelf-life for fermented products, and natural food preservation meets the trends of the food market.

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Up-cycling grape pomace through sourdough fermentation: Characterization of phenolic compounds, antioxidant activity, and anti-inflammatory potential.

Torreggiani, A., Demarinis, C., Pinto, D., Papale, A., Difonzo, G., Caponio, F., Pontonio, E., Verni, M. & Rizzello, C. G. (2023).. Antioxidants12(8), 1521.

Despite its appealing composition, because it is rich in fibers and polyphenols, grape pomace, the major by-product of the wine industry, is still discarded or used for feed. This study aimed at exploiting grape pomace functional potential through fermentation with lactic acid bacteria (LAB). A systematic approach, including the progressively optimization of the grape pomace substrate, was used, evaluating pomace percentage, pH, and supplementation of nitrogen and carbon sources. When grape pomace was used at 10%, especially without pH correction, LAB cell viability decreased up to 2 log cycles. Hence, the percentage was lowered to 5 or 2.5% and supplementations with carbon and nitrogen sources, which are crucial for LAB metabolism, were considered aiming at obtaining a proper fermentation of the substrate. The optimization of the substrate enabled the comparison of strains performances and allowed the selection of the best performing strain (Lactiplantibacillus plantarum T0A10). A sourdough, containing 5% of grape pomace and fermented with the selected strain, showed high antioxidant activity on DPPH and ABTS radicals and anti-inflammatory potential on Caco2 cells. The anthocyanins profile of the grape pomace sourdough was also characterized, showing qualitative and quantitative differences before and after fermentation. Overall, the grape pomace sourdough showed promising applications as a functional ingredient in bread making.

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Gluten-Free Bread Enriched with Artichoke Leaf Extract In Vitro Exerted Antioxidant and Anti-Inflammatory Properties.

Vacca, M., Pinto, D., Annunziato, A., Ressa, A., Calasso, M., Pontonio, E., Celano, G. & De Angelis, M. (2023). Antioxidants12(4), 845.

Due to its high nutritional value and broad beneficial effects, the artichoke plant (Cynara cardunculus L.) is an excellent healthy food candidate. Additionally, the artichoke by-products are usually discarded even though they still contain a huge concentration of dietary fibers, phenolic acids, and other micronutrients. The present work aimed to characterize a laboratory-made gluten-free bread (B) using rice flour supplemented with a powdered extract from artichoke leaves (AEs). The AE, accounting for the 5% of titratable chlorogenic acid, was added to the experimental gluten-free bread. Accounting for different combinations, four different bread batches were prepared. To evaluate the differences, a gluten-free type-II sourdough (tII-SD) was added in two doughs (SB and SB-AE), while the related controls (YB and YB-AE) did not contain the tII-SD. Profiling the digested bread samples, SB showed the lowest glycemic index, while SB-AE showed the highest antioxidant properties. The digested samples were also fermented in fecal batches containing viable cells from fecal microbiota samples obtained from healthy donors. Based on plate counts, no clear tendencies emerged concerning the analyzed microbial patterns; by contrast, when profiling volatile organic compounds, significant differences were observed in SB-AE, exhibiting the highest scores of hydrocinnamic and cyclohexanecarboxylic acids. The fecal fermented supernatants were recovered and assayed for healthy properties on human keratinocyte cell lines against oxidative stress and for effectiveness in modulating the expression of proinflammatory cytokines in Caco-2 cells. While the first assay emphasized the contribution of AE to protect against stressor agents, the latter enlightened how the combination of SB with AE decreased the cellular TNF-α and IL1-β expression. In conclusion, this preliminary study suggests that the combination of AE with sourdough biotechnology could be a promising tool to increase the nutritional and healthy features of gluten-free bread.

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Transcriptional analysis of the molecular mechanism underlying the response of Lactiplantibacillus plantarum to lactic acid stress conditions.

Jang, H. Y., Kim, M. J., Bae, M., Hwang, I. M. & Lee, J. H. (2023). Heliyon, 9(6).

Lactic acid bacteria (LAB) present various benefits to humans; they play key roles in the fermentation of food and as probiotics. Acidic conditions are common to both LAB in the intestinal tract as well as fermented foods. Lactiplantibacillus plantarum is a facultative homofermentative bacterium, and lactic acid is the end metabolite of glycolysis. To characterize how L. plantarum responds to lactic acid, we investigated its transcriptome following treatment with hydrochloride (HCl) or dl-lactic acid at an early stage of growth. Bacterial growth was more attenuated in the presence of lactic acid than in the presence of HCl at the same pH range. Bacterial transcriptome analysis showed that the expression of 67 genes was significantly altered (log2FC > 2 or < 2). A total of 31 genes were up- or downregulated under both conditions: 19 genes in the presence of HCl and 17 genes in the presence of dl-lactic acid. The fatty acid synthesis-related genes were upregulated in both acidic conditions, whereas the lactate racemization-related gene (lar) was only upregulated following treatment with dl-lactic acid. In particular, lar expression increased following l-lactic acid treatment but did not increase following HCl or d-lactic acid treatment. Expression of lar and production of d-lactic acid were investigated with malic and acetic acid; the results revealed a higher expression of lar and production of d-lactic acid in the presence of malic acid than that in the presence of acetic acid.

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Simultaneous saccharification and fermentation with Weizmannia coagulans for recovery of synthetic fibers and production of lactic acid from blended textile waste.

Mihalyi, S., Tagliavento, M., Boschmeier, E., Archodoulaki, V. M., Bartl, A., Quartinello, F. & Guebitz, G. M. (2023). Resources, Conservation and Recycling, 196, 107060.

Textile waste is still mostly landfilled or incinerated while a lot of fabrics consist of blended fibers that represent a challenge for recycling. In this study, enzymes were used to specifically decompose cellulose from blends for recovery of synthetic fibers and microbial conversion of resulting glucose into lactic acid (LA) as a building block of the biobased polyester PLA. To overcome enzyme inhibition by glucose and to reduce process cost, simultaneous saccharification and fermentation (SSF) by Weizmannia coagulans in presence of cellulolytic enzymes were investigated. Indeed, viscose fibers in blends were completely hydrolyzed according to weight loss and HPLC quantification of formed glucose which was converted into pure l-LA (25.6 g/L). In parallel, pure synthetic fibers (polyester and polyamide) were recovered as confirmed by SEM, FTIR, and TGA analyses. This approach represents a circular economy concept to recycle and simultaneously valorize each component of a blended textile waste stream.

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Differential behavior of Lactobacillus helveticus B1929 and ATCC 15009 on the hydrolysis and angiotensin-I-converting enzyme inhibition activity of fermented ultra-high temperature milk and nonfat dried milk powder.

Guron, G. K., Qi, P. X., McAnulty, M. J., Renye Jr, J. A., Miller, A. L., Oest, A. M., Wickham, E. D. & Harron, A. (2023). Journal of Dairy Science, In Press.

Consumers' growing interest in fermented dairy foods necessitates research on a wide array of lactic acid bacterial strains to be explored and used. This study aimed to investigate the differences in the proteolytic capacity of Lactobacillus helveticus strains B1929 and ATCC 15009 on the fermentation of commercial ultra-pasteurized (UHT) skim milk and reconstituted nonfat dried milk powder (at a comparable protein concentration, 4%). The antihypertensive properties of the fermented milk, measured by angiotensin-I-converting enzyme inhibitory (ACE-I) activity, were compared. The B1929 strain lowered the pH of the milk to 4.13 ± 0.09 at 37°C after 24 h, whereas ATCC 15009 needed 48 h to drop the pH to 4.70 ± 0.18 at 37°C. Two soluble protein fractions, one (CFS1) obtained after fermentation (acidic conditions) and the other (CFS2) after the neutralization (pH 6.70) of the pellet from CFS1 separation, were analyzed for d-/l-lactic acid production, protein concentration, the degree of protein hydrolysis, and ACE-I activity. The CFS1 fractions, dominated by whey proteins, demonstrated a greater degree of protein hydrolysis (7.9%) than CFS2. On the other hand, CFS2, mainly casein proteins, showed a higher level of ACE-I activity (33.8%) than CFS1. Significant differences were also found in the d- and l-lactic acid produced by the UHT milk between the 2 strains. These results attest that milk casein proteins possessed more detectable ACE-I activity than whey fractions, even without a measurable degree of hydrolysis. Findings from this study suggest that careful consideration must be given when selecting the bacterial strain and milk substrate for fermentation.

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In vitro safety assessment of electrohydrodynamically encapsulated Lactiplantibacillus plantarum CRD7 and Lacticaseibacillus rhamnosus CRD11 for probiotics use.

Varada, V. V., Panneerselvam, D., Pushpadass, H. A., Mallapa, R. H., Ram, C. & Kumar, S. (2023). Current Research in Food Science, 6, 100507.

The current study aimed to validate the safety of electrohydrodynamically encapsulated Lactiplantibacillus plantarum CRD7 and Lacticaseibacillus rhamnosus CRD11 in accordance with guidelines of FAO/WHO and ICMR/DBT. In vitro assays such as mucin degradation, hemolysis of blood cells, antimicrobial susceptibility pattern, possession of virulence factors, biogenic amine, and ammonia production were assessed. In results, the cross-streak and co-culture techniques revealed that CRD7 and CRD11 were compatible in vitro. Upon visual inspection through scanning electron and fluorescence microscopy, the integrity of bacterial cell membrane was confirmed even after the encapsulation process. CRD7 and CRD11 were non-hemolytic and showed negative responses to gelatinase, urease, and DNase activities. Non-mucinolytic activity of CRD7 and CRD11 was verified by measuring cell growth rate (p < 0.05) in different modified media followed by SDS-PAGE. High-performance liquid chromatography analysis revealed that both the strains did not produce biogenic amines (putrescine, cadaverine, histamine, and tyramine). Neither of the Lactobacillus strains produced ammonia after growing in BHI broth for 5 days at 37 °C. L-lactate production was highest (p < 0.05) in CRD11 (8.83 g/L), followed by CRD7 (8.16 g/L), whereas the lowest (p < 0.05) D-lactate was registered for encapsulated CRD11 (0.33 g/L) and CRD7 (0.49 g/L). The antibiogram profile determined through minimum inhibitory concentration showed that CRD7 and CRD11 were sensitive to key antibiotics suggested by EFSA except for gentamycin and kanamycin. PCR data on virulence genes demonstrated that both strains were safe for probiotic use. Moreover, CRD7 and CRD11 strains caused insignificant (p > 0.05) changes in the cell viability of Caco-2 cells as estimated by MTT (98.94-99.50%) and NR uptake (95.42–97.03%) assays and showed sensitivity to human serum. According to the results of these evaluated attributes, it is concluded that L. plantarum CRD7 and L. rhamnosus CRD11 are safe, non-toxic to human epithelial cells, and thus may be potentially suitable for various food/feed applications.

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