Lactobacillus Kefiri Research Articles

Anthocyanin Addition to Kefir: Metagenomic Analysis of Microbial Community Structure.

The addition of anthocyanin to kefir for the production of more functional and bio-diversified kefir beverages has the potential to increase kefir's healthful activities. In the present study, anthocyanin extracts, obtained from black carrots, were added into kefir mixture during the fermentation process in different concentrations (1% and 5%, w/v). These kefir samples were then analyzed in terms of their microbiological qualities by metagenomic analysis. The results of the analyses show that the addition of anthocyanin has significant impacts on the community structure of kefir microbiome which in turn directly affects the expected health impacts of the beverage. Kefir with no anthocyanin included predominantly probiotic bacteria such as Lactococcus lactis (34%) and Lactobacillus kefiri (34%). On the other hand, kefir with 1% anthocyanin demonstrated a more balanced distribution of probiotic species like Lb. kefiri (17%), Leuconostoc mesenteroides (9%), and Lc. lactis (5%) at similar abundance rates. 5% anthocyanin kefir demonstrated the highest polarity in the community with a strong dominance of probiotic Lb. kefiri (72%), and distinctly less abundant bacteria such as Streptococcus salivarius subsp. thermophilus (3%). These findings provide that fortification with anthocyanins can be utilized to enhance the quality, composition, and beneficial functions of kefir.

Effects of Lactobacillus kefiri fermentation supernatant on skin aging caused by oxidative stress

• • Lactobacillus kefiri fermentation components(LAF) shows excellent antioxidant effect in vitro. • •Achievements of Lactobacillus kefiri fermentation components providing more possibilities for their application as functional raw materials in cosmetics. • •LAF can regulate the expression of oxidative stress, autophagy, and aging-related genes in damaged cells treated with H 2 O 2 , thereby delaying cell senescence. Lactobacillus kefiri is a beneficial bacteria that is often used to make yogurt only, and is beneficial to human health when it is used in food fermentation. In this article, a fermentation supernatant (LAF) is prepared by culturing Lactobacillus kefiri , and the anti-aging effects of LAF are evaluated at the biochemical, cellular, and molecular biology levels. The antioxidant activity of LAF is comprehensively evaluated by three free radical scavenging tests and the total antioxidant capacity (ABTS method) test. The toxicity of LAF on human skin fibroblasts (HSF) is detected using the MTT method, and the effects of LAF on H 2 O 2 -induced oxidative stress in terms of intracellular reactive oxygen species (ROS), Malondialdehyde (MDA) content, antioxidant enzyme activity are evaluated. The gene expression levels of LC3 and Beclin-1 are detected using the real-time reverse transcription-polymerase chain reaction (qRT-PCR) method, and the protein expression levels of LC3 are detected using the Western blot method to evaluate the effects of LAF on HSF autophagy. The expression of type I collagen (COL-1), Matrix metalloproteinase-1(MMP-1), AKT pathway, MAPK pathway, and other genes are detected by qRT-PCR to study the anti-aging effects of LAF. From the experimental results, it is found that LAF can delay the aging of HSF at the biochemical, cellular, and molecular biology levels, and can be utilized as a functional food ingredient.

Eco-Friendly Edible Packaging Systems Based on Live-Lactobacillus kefiri MM5 for the Control of Listeria monocytogenes in Fresh Vegetables.

To meet consumer requirements for high quality food free of chemical additives, according to the principles of sustainability and respect for the environment, new “green” packaging solutions have been explored. The antibacterial activity of edible bioactive films and coatings, based on biomolecules from processing by-products and biomasses, added with the bacteriocin producer Lactobacillus kefiri MM5, has been determined in vegetables against L. monocytogenes NCTC 10888 (i) “in vitro” by a modified agar diffusion assay and (ii) “on food” during storage of artificially contaminated raw vegetable samples, after application of active films and coatings. Both polysaccharides-based and proteins-based films and coatings showed excellent antilisterial activity, especially at 10 and 20 days. Protein-based films displayed a strong activity against L. monocytogenes in carrots and zucchini samples (p < 0.0001). After 10 days, both polysaccharide-based and protein-based films demonstrated more enhanced activity than coatings towards the pathogen. These edible active packagings containing live probiotics can be used both to preserve the safety of fresh vegetables and to deliver a beneficial probiotic bacterial strain. The edible ingredients used for the formulation of both films and coatings are easily available, at low cost and environmental impact.

Crystallization of Rationally Engineered Lactobacillus kefir Alcoholdehydrogenases Monitored by Machine‐Learning Based Protein Crystal Detection

Crystallization of Rationally Engineered <i>Lactobacillus kefir</i> Alcoholdehydrogenases Monitored by Machine‐Learning Based Protein Crystal Detection

Growth and Metabolism of Lacticaseibacillus casei and Lactobacillus kefiri Isolated from Qymyz, a Traditional Fermented Central Asian Beverage

The growth characteristics of two strains of lactic acid bacteria (LAB), Lacticaseibacillus casei and Lactobacillus kefiri, isolated from qymyz, a traditional fermented mare milk beverage, were studied and modeled, including the effect of different carbohydrates, pH, and temperature. Along with population, substrates, and metabolites, lactic acid and ethanol were monitored by HPLC. Growth parameters were obtained from mono- and biphasic logistic growth models that fit the population evolution of L. casei and L. kefiri, respectively. The effect of temperature and pH on the growth rate was represented with the gamma concept model, while the effect of the limiting substrate was evaluated according to the Monod equation. Lastly, a simplified Luedeking and Piret equation was used to represent metabolite production. The optimum values of pH and temperature were 6.69 ± 0.20, 38.63 ± 0.32 °C, 5.93 ± 0.08, and 33.15 ± 0.53 °C, with growth rate values of 0.66 ± 0.01 h−1 and 0.29 ± 0.01 h−1 for L. casei and L. kefiri, respectively. L. casei had a homofermentative pathway, while L. kefiri was heterofermentative, with an ethanol production rate of 2.90 × 10−9 mg·CFU−1. The Monod model showed that L. casei had the lowest Ks value for lactose, while for L. kefiri, it was the highest among milk carbohydrates. These results show that the population of the two LAB strains and therefore the concentrations of acid and ethanol can be controlled by the fermentation conditions and that our model can help to significantly improve the production of qymyz.

Biocatalytic hydrogen-transfer to access enantiomerically pure proxyphylline, xanthinol, and diprophylline

Alcohol dehydrogenases (ADHs; EC 1.1.1.1) have been widely used for the reversible redox reactions of carbonyl compounds (i.e., aldehydes and ketones) and primary or secondary alcohols, often resulting in optically pure hydroxyl products with high added value. In this work, we report a concise chemoenzymatic route toward xanthine-based enantiomerically pure active pharmaceutical ingredients (API) – proxyphylline, xanthinol, and diprophylline employing various recombinant short-chain ADHs with (R)- or (S)-selectivity as key biocatalysts. By choosing the appropriate ADH, the (R)- as well as the (S)-enantiomer of proxyphylline was prepared in excellent enantiomeric excess (99–99.9% ee), >99% conversion, and the isolated yield ranging from 65% to 74%, depending on the used biocatalyst (ADH-A from Rhodococcus ruber or a variant derived from Lactobacillus kefir, Lk-ADH-Lica). In turn, E. coli/ADH-catalyzed bioreduction of the carbonylic precursor of xanthinol and diprophylline furnished the corresponding (S)-chlorohydrin in >99% ee, >99% conversion, and 80% yield (in the case of Lk-ADH-Lica); while the (R)-counterpart was afforded in 94% ee, 64% conversion, and 41% yield (in the case of SyADH from Sphingobium yanoikuyae). After further chemical functionalization of the key (S)-chlorohydrin intermediate, the desired homochiral (R)-xanthinol (>99% ee) was obtained in 97% yield and (S)-diprophylline (>99% ee) in 90% yield. The devised biocatalytic method is straightforward and thus might be considered practical in the manufacturing of title pharmaceuticals.

A Biocatalytic Platform for the Synthesis of Enantiopure Propargylic Alcohols and Amines.

Propargylic alcohols and amines are versatile building blocks in organic synthesis. We demonstrate a straightforward enzymatic cascade to synthesize enantiomerically pure propargylic alcohols and amines from readily available racemic starting materials. In the first step, the peroxygenase from Agrocybe aegerita converted the racemic propargylic alcohols into the corresponding ketones, which then were converted into the enantiomerically pure alcohols using the (R)-selective alcohol dehydrogenase from Lactobacillus kefir or the (S)-selective alcohol dehydrogenase from Thermoanaerobacter brokii. Moreover, an enzymatic Mitsunobu-type conversion of the racemic alcohols into enantiomerically enriched propargylic amines using (R)-selective amine transaminase from Aspergillus terreus or (S)-selective amine transaminase from Chromobacterium violaceum was established. The one-pot two-step cascade reaction yielded a broad range of enantioenriched alcohol and amine products in 70–99% yield.

Response surface optimization of millet milk fermented by Lactobacillus kefir

Based on the amino acid score (AAS), chemical score (CS), and amino acid proportional coefficient score (SRCAA) as evaluation indicators, millet powder, whey protein isolate powder, and soybean protein isolate powder were mixed to make millet compound powder. After the millet compound powder and water were uniformly mixed at a material-to-liquid ratio of 1:20, the kefir fermented millet nutritional milk was prepared by beating, gelatinizing, liquefying, saccharifying, and fermenting. The protein content, polyphenol content, viable count and acidity of kefir millet fermented milk were significantly higher than those of the other two commercially available similar beverages. The scavenging rates of DPPH free radicals and hydroxyl radicals in the kefir millet fermented milk group at three stages (before undigested, after gastric digestion for 2.5 h, and after intestinal digestion for 2.5 h) were significantly higher than those in the unfermented millet pulp group. Novelty impact statement In this study, based on the amino acid pattern and amino acid nutrition evaluation methods (AAS, CS, SRCAA), millet flour and two protein powders with rich lysine content (whey protein isolate powder and soybean protein isolate powder) were rationally compounded as amino acid balanced millet compound powder. The millet compound powder is fermented by Lactobacillus kefir into millet nutritional fermented milk with unique flavor of probiotic fermentation. The antioxidant activity of millet nutritional fermented milk and millet pulp before fermentation was compared and analyzed by gastrointestinal digestion simulation in vitro, and it was determined that millet nutritional fermented milk had strong antioxidant activity.

Efficient Synthesis of cis-4-Propylcyclohexanol Using a Mutant Alcohol Dehydrogenase Coupled with Glucose Dehydrogenase

cis-4-Propylcyclohexanol is an important intermediate for synthesizing trans-2-(4-propylcyclohexyl)-1,3-propanediol, which is widely used in the manufacture of liquid crystal displays. In this study, cis-4-propylcyclohexanol was prepared using a mutant alcohol dehydrogenase from Lactobacillus kefir (LK-TADH, A94T/F147L/L199H/A202L) coupled with glucose dehydrogenase. Using the optimal catalytic conditions, 125 g/L (250 g) of 4-propylcyclohexanone was completely transformed after 5 h, and 225.8 g of cis-4-propylcyclohexanol (cis/trans ratio of 99.5:0.5) was obtained through extraction and rotary evaporation at a yield of 90.32%. This study reports a potential method for the green production of cis-4-propylcyclohexanol as the key intermediate of trans-2-(4-propylcyclohexyl)-1,3-propanediol at an industrial level.

A Peroxygenase-Alcohol Dehydrogenase Cascade Reaction to Transform Ethylbenzene Derivatives into Enantioenriched Phenylethanols.

In this study, we developed a new bienzymatic reaction to produce enantioenriched phenylethanols. In a first step, the recombinant, unspecific peroxygenase from Agrocybe aegerita (rAaeUPO) was used to oxidise ethylbenzene and its derivatives to the corresponding ketones (prochiral intermediates) followed by enantioselective reduction into the desired (R)‐ or (S)‐phenylethanols using the (R)‐selective alcohol dehydrogenase (ADH) from Lactobacillus kefir (LkADH) or the (S)‐selective ADH from Rhodococcus ruber (ADH‐A). In a one‐pot two‐step cascade, 11 ethylbenzene derivatives were converted into the corresponding chiral alcohols at acceptable yields and often excellent enantioselectivity.

New Perspective on chronic functional constipation in children with the identification of the microbiota

Chronic functional constipation is a common gastrointestinal disorder in children with no underlying organic cause. Although reasons such as bad eating habits and early transition to solid food are blamed for constipation, the etiology of the disease has not been fully elucidated. Our study aims to determine the differences in stool microbiota between children with chronic functional constipation and healthy children. Feces samples of 49 patients and 40 healthy children who meet Rome IV criteria were analyzed by 16s rRNA /PCR (Polymerase Chain Reaction) method. Within the sample microbial diversity, the Shannon diversity index was calculated based on the profiles obtained using the R 2.15.2 software package programe. In our study, no statistically significant difference was found between the study group and the control group in terms of the Shannon Diversity Index (p &lt;0.05). The species found in healthy children (n = 34) but not in constipated children were determined as Lactobacillus kefiri and Bifidobacterium infantis. In addition, Lactobacillus casei and Lactobacillus acidophilus were detected with a higher rate in constipated children (p&gt; 0.005). Although there is no significant difference between microbiota subtypes, the fact that Lactobacillus kefiri and Bifidobacterium infantis were detected only in healthy children may guide the supportive treatments to be given to constipated children. The results of our study also show that there is a need for more comprehensive studies in large populations, supporting other literature studies showing that the gastrointestinal microbiota is different in constipated and normal children.

ПЕРВИЧНАЯ ИДЕНТИФИКАЦИЯ МИКРОБИМА КЕФИРНЫХ ЗЕРЕН ИЗ РАЗНЫХ ТЕРРИТОРИАЛЬНЫХ ЗОН

From samples of kefir grains (KG) of home-made kefirs from different traditional territorial zones of their production and use: the Caucasus (North Ossetia), China (Tibet) and Russia (Moscow region), 2 species of lactobacilli were isolated and identified: Lactobacillus kefiri and L.casei, 3 yeast species Pichia fermentans (from Moscow), Yarrowia lipolytica and Pichia fermentans (from Tibet), Galactomyces candidus (from North Ossetia). The ability of the studied cultures to form biofilms was shown, which is an important indicator for the creation of sustainable probiotic microbial communities and functional food products needed during the coronavirus pandemic.

Growth and Metabolism of Lactobacillus Casei and Lactobacillus Kefiri Isolated from Qymyz, a Traditional Fermented Central Asian Beverage

Growth and Metabolism of Lactobacillus Casei and Lactobacillus Kefiri Isolated from Qymyz, a Traditional Fermented Central Asian Beverage

Growth and Metabolism of Lactobacillus Casei and Lactobacillus Kefiri Isolated from Qymyz, a Traditional Fermented Central Asian Beverage

Growth and Metabolism of Lactobacillus Casei and Lactobacillus Kefiri Isolated from Qymyz, a Traditional Fermented Central Asian Beverage

Antifungal activity and mode of action of lactic acid bacteria isolated from kefir against Penicillium expansum

Penicillium expansum is the most common spoilage-associated organism found in fruit, which causes serious economic loss and public health concerns. The aim of this study was to isolate lactic acid bacteria (LAB) from kefir in order to study its antifungal effect on mold growth and further analyze the possible antifungal mode of action of LAB, including antifungal component, lytic enzymes activity and nutrient competition. Lactobacillus kefiri M4 and Pediococcus acidilactici MRS-7 were isolated from kefir, and the overlay method was used as a qualitative approach to evaluate their antifungal activities against Penicillium expansum , the results showed that all tested strains were inhibited by the isolates, except for P. expansum LPH6. Different treatments were carried out to characterize the nature of the molecules that contributed to the antifungal activity of the cell-free supernatants produced by the isolates, and it was shown that all supernatants were pH-dependent, partly heat sensitive, and were not influenced by proteinaceous treatment. The cell-free supernatants of both species of LAB were analyzed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS), and it was found that the main antifungal compounds were organic acids and carboxylic acids. Both LAB species were able to produce lytic enzymes, such as cellulases, proteases, and glucanases, which formed a halo zone on the plate. L. kefiri M4 and P. expansum LPH10 exhibited nutrient competition when co-cultured together in a tissue culture plate insert. The results of this study showed that L. kefiri M4 and P. acidilactici MRS-7 isolated from kefir possess antifungal activity, which provides useful information of how P. expansum response to the LAB isolated from kefir. • Two LAB strains had antifungal effect on Penicillium expansum . • Organic acids and carboxylic acids were responsible for antifungal activity. • Both strains can produce cellulase, protease and glucanase. • L. kefiri M4 and P. expansum LPH10 have nutrients competition.

Chemoenzymatic Asymmetric Synthesis of Pyridine-Based α-Fluorinated Secondary Alcohols.

Fluoro‐substituted and heteroaromatic compounds are valuable intermediates for a variety of applications in pharma‐ and agrochemistry and synthetic chemistry. This study investigates the chemoenzymatic preparation of chiral alcohols bearing a heteroaromatic ring with an increasing degree of fluorination in α‐position. Starting from readily available picoline derivatives prochiral α‐halogenated acyl moieties were introduced with excellent selectivity and 64–95 % yield. The formed carbonyl group was subsequently reduced to the corresponding alcohols using the alcohol dehydrogenase from Lactobacillus kefir, yielding an enantiomeric excess of 95–>99 % and up to 98 % yield.

Biocatalytic preparation of a key intermediate of antifungal drugs using an alcohol dehydrogenase with high organic tolerance

In this study, an alcohol dehydrogenase derived from Lactobacillus kefir (LkADH) was engineered and a simple and practical bioreduction system was developed for the preparation of (R)-2-chloro-1-(2, 4-dichlorophenyl) ethanol ((R)-CDPO), a key intermediate for the synthesis of antifungal drugs. Through active pocket iterative saturation mutagenesis, mutant LkADH-D18 (Y190C/V196L/M206H/D150H) was obtained with high stereoselectivity (99% ee, R vs 87% ee, S) and increased activity (0.44 µmol·min−1·mg−1). LkADH-D18 demonstrated NAD(P)H regeneration capability using a high concentration of isopropanol (IPA) as a co-substrate. Using 40% IPA (v/v), 400 mM of (R)-CDPO (90.1 g·L-1) was obtained via complete substrate conversion using 40 mg·mL−1 LkADH-D18 wet cells. The biocatalytic process catalyzed at constant pH with the cheap co-solvent IPA contributed to improved isolated yield of (R)-CDPO (97%), lower reaction cost, and simpler downstream purification, indicating the potential utility of LkADH-D18 in future industrial applications.

Transfer of a Rational Crystal Contact Engineering Strategy between Diverse Alcohol Dehydrogenases

Protein crystallization can serve as a purification step in biotechnological processes but is often limited by the non-crystallizability of proteins. Enabling or improving crystallization is mostly achieved by high-throughput screening of crystallization conditions and, more recently, by rational crystal contact engineering. Two selected rational crystal contact mutations, Q126K and T102E, were transferred from the alcohol dehydrogenases of Lactobacillus brevis (LbADH) to Lactobacillus kefir (LkADH). Proteins were expressed in E. coli and batch protein crystallization was performed in stirred crystallizers. Highly similar crystal packing of LkADH wild type compared to LbADH, which is necessary for the transfer of crystal contact engineering strategies, was achieved by aligning purification tag and crystallization conditions, as shown by X-ray diffraction. After comparing the crystal sizes after crystallization of LkADH mutants with the wild type, the mean protein crystal size of LkADH mutants was reduced by 40–70% in length with a concomitant increase in the total amount of crystals (higher number of nucleation events). Applying this measure to the LkADH variants studied results in an order of crystallizability T102E &gt; Q126K &gt; LkADH wild type, which corresponds to the results with LbADH mutants and shows, for the first time, the successful transfer of crystal contact engineering strategies.

Continuous-Flow Asymmetric Synthesis of (3R)-3-Hydroxyl-5-hexenoates with Co-Immobilized Ketoreductase and Lactobacillus kefir Dehydrogenase Integrating Greener Inline Microfluidic Liquid–Liquid Extractors and Membrane Separators

Continuous-Flow Asymmetric Synthesis of (3<i>R</i>)-3-Hydroxyl-5-hexenoates with Co-Immobilized Ketoreductase and <i>Lactobacillus kefir</i> Dehydrogenase Integrating Greener Inline Microfluidic Liquid–Liquid Extractors and Membrane Separators

Efficient preparation of the chiral intermediate of luliconazole with Lactobacillus kefir alcohol dehydrogenase through rational rearrangement of the substrate binding pocket

Abstract Luliconazole has been approved by the FDA for fungal infection therapy because of its broad-spectrum antifungal activity. However, low efficiency and harsh reaction conditions in the current synthesis of the luliconazole chiral intermediate (S)-2-chloro-1-(2,4-dichlorophenyl) ethanol ((S)-TCPE) limits its application. In the present study, a novel biocatalytic process for producing (S)-TCPE was developed. Based on a structural analysis at the atomic level on the wild-type (WT) Lactobacillus kefir ketoreductase (L. kefir KRED), a rational design of L. kefir KRED was performed with 2,2’,4’-trichloroacetophenone (TCAP) as the substrate. An enantioselective triple mutant A94S/S96E/E145A displaying a 117-fold increase in its relative activity compared to the WT was evolved, and the catalytic efficiency was raised to 5.17 s−1mM−1 from non-detectable. With 106 kg of wet cells of the triple variant loading, 275 kg TCAP was asymmetrically reduced with the yield of (S)-TCPE achieving 91% within 12 h, thus showing great potential for industrial applications. Furthermore, the molecular mechanism supporting the promotion of activity was revealed to be related to the rearrangement of the substrate binding pocket of the L. kefir KRED. By engineering an enzyme that serves as a biocatalyst for the efficient preparation of enantiomerically pure (S)-TCPE, the current study presents a reproducible strategy for the promotion of catalytic activity based on rational design.