Bacterial diversity using metagenomics of 16s rDNA in water kefir, an innovative source of probiotics for bee nutrition.

Water kefir is a sparkling, slightly acidic fermented beverage made from sugar, water, and water kefir grains, which are a mixture of yeast and bacteria. These grains produce a variety of fermentation compounds such as lactic acid, acetaldehyde, acetoin, ethanol and carbon dioxide. In this study, a high-throughput sequencing technique was used to characterize the bacterial composition of the original water kefir from which potential probiotics were obtained. We studied the bacterial diversity of both water kefir grains and beverages. DNA was extracted from three replicate samples of both grains and beverages using the Powerlyzer Microbial Kit. The hypervariable V1-V2 region of the bacterial 16S ribosomal RNA gene was amplified to prepare six DNA libraries. Between 1.4M and 2.4M base-pairs were sequenced for the library. In total, 28721971 raw reads were obtained from all the samples. Estimated species richness was higher in kefir beverage samples compared to grain samples. Moreover, a higher level of microbial alpha diversity was observed in the beverage samples. Particularly, the predominant bacteria in beverages were Anaerocolumna and Ralstonia, while in grains Liquorilactobacillus dominated, with lower levels of Leuconostoc and Oenococcus. Although the bacterial diversity in kefir grains was low because only three genera were the most represented, all of them are LAB bacteria with the potential to serve as probiotics in the artificial feeding of bees.

Innovative beverage creation through symbiotic microbial communities inspired by traditional fermented beverages: current status, challenges and future directions.

Fermented beverages (FBs) are facing challenges in functional performance and flavor complexity, necessitating the development of new multi-functional options. Traditional fermented beverages (TFBs), both alcoholic and nonalcoholic, have gained increased attention for their health-promoting effects during the COVID-19 pandemic. This review summarized the primary commercially available probiotics of FBs, along with the limitations of single and mixed probiotic FBs. It also examined the recent research progress on TFBs, emphasizing the typical microbial communities (MC) of TFBs, and TFBs made from crops (grains, vegetables, fruits, etc.) worldwide and their associated functions and health benefits. Furthermore, the construction, technical bottlenecks of the synthetic MC involved in developing innovative FBs were presented, and the promising perspective of FBs was described. Drawing inspiration from the MC of TFBs, developing of stable and multifunctional FBs using synthetic MC holds great promise for beverage industry. However, synthetic MC suffers from structural instability and poorly acknowledged interaction mechanisms, resulting in disappointing results in FBs. Future researches should prioritize creating synthetic MC fermentation that closely resemble natural fermentation, tailored to meet the needs of different consumers. Creating personalized FBs with high-tech intelligence is vital in attracting potential consumers and developing novel beverages for the future.

Comparative metatranscriptome analysis of Brazilian milk and water kefir beverages.

The present study compared bacterial and fungal diversity of kefir beverages produced using milk (MK) or sugared water (WK) as propagation matrices and grains from the cities of Curitiba (CU) or Salvador (SA), Brazil, by sequencing the complete set of RNA transcripts produced in four products. In Brazil, milk and sugared water are used as matrices to propagate kefir grains. In all beverages, the bacterial community was composed of Lactobacillaceae and Acetobacteraceae. Saccharomycetaceae was the yeast family more abundant in WK, and Dipodascaceae and Pichiaceae in MK. Regarding KEGG mapping of functional orthologs, the four kefir samples shared 70% of KO entries of yeast genes but only 36% of bacterial genes. Concerning main metabolic processes, the relative abundance of transcripts associated with metabolism (energy metabolism) and environmental information processing (membrane transport) had the highest water/milk kefir ratio observed in Firmicutes. In contrast, transcripts associated with genetic information processing (protein translation, folding, sorting, and degradation) oppositely had the lowest water/milk ratios. Concluding, milk and water kefir have quite different communities of microorganisms. Still, the main mapped functional processes are similar, with only quantitative variation in membrane transport and energy acquisition in the water kefir and protein synthesis and turnover in the milk kefir.

Probiotics and in-hive fermentation as a source of beneficial microbes to support the gut microbial health of honey bees.

Managed populations of honey bees (Apis mellifera Linnaeus; Hymenoptera: Apidae) are regularly exposed to infectious diseases. Good hive management including the occasional application of antibiotics can help mitigate infectious outbreaks, but new beekeeping tools and techniques that bolster immunity and help control disease transmission are welcome. In this review, we focus on the applications of beneficial microbes for disease management as well as to support hive health and sustainability within the apicultural industry. We draw attention to the latest advances in probiotic approaches as well as the integration of fermented foods (such as water kefir) with disease-fighting properties that might ultimately be delivered to hives as an alternative or partial antidote to antibiotics. There is substantial evidence from in vitro laboratory studies that suggest beneficial microbes could be an effective method for improving disease resistance in honey bees. However, colony level evidence is lacking and there is urgent need for further validation via controlled field trials experimentally designed to test defined microbial compositions against specific diseases of interest.

Microbiological and chemical characterization of water kefir: An innovative source of potential probiotics for bee nutrition.

We evaluated the microbial composition of water kefir grains and beverage over the course of one year to determine whether the number and type of microorganisms changed over the time. Bacteria and yeast colonies with different morphologies were isolated from water kefir and their antimicrobial activity was evaluated against Paenibacillus larvae and Ascosphaera apis. A chemical characterization of kefir was also carried out. Our results confirmed that bacteria and yeasts were more numerous in kefir grains compared with those in the beverage. The counts of microorganisms declined, although an important microbial community was still present in kefir after the long storage period. Eleven strains which inhibited bee pathogens were isolated from kefir. Genotypic results demonstrated that these isolates included Lentilactobacillus hilgardii, Lentilactobacillus buchneri and Saccharomyces cerevisiae. Thus, water kefir may be an innovative source of potential probiotic strains for bee nutrition in order to control honeybee diseases.

A review of nondairy kefir products: their characteristics and potential human health benefits.

Functional foods are foods that, in addition to having nutrients, contain in their composition ingredients that act specifically on body functions associated with the control and reduction of the risk of developing some diseases. In this sense, kefir, a group of microorganisms in symbiosis, mainly yeasts and lactic acid bacteria, stands out. The trend of ingesting kefir has been focused on the development of products that serve specific consumers, such as those who are lactose-intolerant, vegans and vegetarians, and consumers in general who seek to combine the consumption of functional products with the improvement of their health and lifestyle. This overview provides an insight into kefir, presenting the technological process to produce a nondairy beverage and evidence of the benefits of its use to reduce the risk of disease. We also discuss regulatory aspects of products fermented using kefir. Until now, the use of kefir (isolated microorganism, kefiran, or fermented product) has demonstrated the potential to promote an increase in the number of bifidobacteria in the colon and an increase in the glycemic control while reducing the blood cholesterol and balancing the intestinal microbiota, which helps in reducing constipation and diarrhea, improving intestinal permeability, and stimulating and balancing the immune system. However, the literature still has gaps that need to be clarified, such as the consumption dose of kefir or its products to cause some health benefit.

Water kefir: Factors affecting grain growth and health-promoting properties of the fermented beverage.

Nowadays, the interest in the consumption of healthy foods has increased as well as the homemade preparation of artisanal fermented product. Water kefir is an ancient drink of uncertain origin, which has been passed down from generation to generation and is currently consumed practically all over the world. Considering the recent and extensive updates published on sugary kefir, this work aims to shed light on the scientific works that have been published so far in relation to this complex ecosystem. We focused our review evaluating the factors that affect the beverage microbial and chemical composition that are responsible for the health attribute of water kefir as well as the grain growth. The microbial ecosystem that constitutes the grains and the fermented consumed beverage can vary according to the fermentation conditions (time and temperature) and especially with the use of different substrates (source of sugars, additives as fruits and molasses). In this sense, the populations of microorganisms in the beverage as well as the metabolites that they produce varies and in consequence their health properties. Otherwise, the knowledge of the variables affecting grain growth are also discussed for its relevance in maintenance of the starter biomass as well as the use of dextran for technological application.

Water kefir grains vs. milk kefir grains: Physical, microbial and chemical comparison.

AIMS: Although kefir has been known for centuries, there is confusion between the two types of kefir grains, for example, milk kefir (MK) grain and water kefir (WK) grain. This study aimed to unravel the differences and similarities between WK grain and MK grain. METHODS AND RESULTS: Microbiological analyses, identification of grains microbiota and enumeration of microbiological content of the grains as well as scanning electron microscope (SEM) imaging, dry matter, protein, ash, and mineral content, and colour analyses were carried out for the two types of grains. As a result, significant differences were found in microbiological content, chemical properties and colours (p < 0.05). Additionally, SEM images revealed the different intrinsic structures for the microbiota and the structure of the two types of grains. CONCLUSIONS: MK grain has more nutritional content compared to WK grain. Despite not as widely known and used as MK grain, WK grain is a good source for minerals and health-friendly micro-organisms such as lactic acid bacteria and yeasts. WK grain is possibly suitable for vegans and allergic individuals to fulfil nutritional requirements. Moreover, in this study, the variety of WK grain microbial consortia was wider than that of MK grains, and this significantly affected the resultant WK products. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that comprehensively compares two different kefir grains in microbial, chemical and physical properties.

Encapsulation of Russian Olive Water Kefir as an Innovative Functional Drink with High Antioxidant Activity.

Processing of Russian olive water kefir (RWK), as a fermented functional drink made with Russian olive juice and water kefir grains with high antioxidant activity, into powder is crucial for improving its stability for the commercialization of this product. For the first time, this study aimed to encapsulate water kefir microorganisms and bioactive compounds in RWK using carrier materials to develop a synbiotic functional powder using spray drying as an encapsulation method. The goal was maximizing antioxidant activity, product yield, and survival rate of water kefir microorganisms in the produced Russian olive water kefir powder. The optimal spray drying conditions were observed to be at an inlet air temperature of 120ºC, 35 % feed flow rate, and 7 % concentration of drying aid. The effects of spray drying conditions on the quality of microcapsules were assessed and modeled, and the validity of the model was verified. Also, the spray-dried powder's physicochemical properties were assessed and showed promising microbial and physicochemical characteristics compared with the freeze-dried powder.

Genomic and physiological insights into the lifestyle of Bifidobacterium species from water kefir.

Water kefir is a fermented beverage employing a natural microbial consortium, which harbours bifidobacteria, namely Bifidobacterium aquikefiri and Bifidobacterium tibiigranuli. However, little information is available on their metabolic properties or role in the consortium. In this study, we combined genomic and physiologic investigations to predict and characterize the properties of these organisms and their possible role in the consortium. When comparing the genomes of these psychrotrophic organisms with that of the three selected mesophilic probiotic Bifidobacterium strains, we could find 143 genes shared by the 3 known isolates of bifidobacteria from water kefir that do not occur in the probiotic strains. These include genes involved in acid and oxygen tolerance. In addition, their genomically predicted carbohydrate usage and transport suggest adaptation to sucrose and other plant-related sugars. Furthermore, they proved prototrophic for all amino acids in vitro, which enables them to cope with the strong amino acid limitation in water kefir.

Bifidobacterium tibiigranuli sp. nov. isolated from homemade water kefir.

Two Bifidobacterium strains, TMW 2.2057T and TMW 2.1764 were isolated from two different homemade water kefirs from Germany. Both strains were oxidase- and catalase-negative and Gram-staining-positive. Cells were non-motile, irregular rods that were aerotolerant anaerobes. On basis of fructose 6-phosphate phosphoketolase activity, they were assigned to the family Bifidobacteriaceae. Comparative analysis of 16S rRNA and concatenated housekeeping genes (clpC, dnaB, dnaG, dnaJ, hsp60 and rpoB) demonstrated that both strains represented a member of the genus Bifidobacterium, with Bifidobacterium subtile DSM 20096T as the closest phylogenetic relative (98.35 % identity). Both strains can be distinguished using randomly amplified polymorphic DNA fingerprinting. Analysis of concatenated marker gene sequences as well as average nucleotide identity by blast (ANIb) and in silico DNA-DNA hybridization (isDDH) calculations of their genome sequences confirmed Bifidobacterium subtile DSM 20096T as the closest relative (87.91 and 35.80 % respectively). All phylogenetic analyses allow differentiation of strains TMW 2.2057T and TMW 2.1764 from all hitherto described species of the genus Bifidobacterium with validly published names. We therefore propose a novel species with the name Bifidobacterium tibiigranuli, for which TMW 2.2057T (=DSM 108414T=LMG 31086T) is the type strain.

The metagenome-assembled genome of Candidatus Oenococcus aquikefiri from water kefir represents the species Oenococcus sicerae.

Recently, a metagenomic study of a water kefir fermentation ecosystem enabled the reconstruction of a metagenome-assembled genome (MAG) of an Oenococcus species that was different from the three species of this genus known so far. Therefore, the name Candidatus Oenococcus aquikefiri was proposed for this novel Oenococcus species. In the meantime, however, a fourth member of the genus, Oenococcus sicerae, isolated from French cider, was reported. The comparison of its genome sequence with the Candidatus O. aquikefiri MAG showed an average nucleotide identity (ANI) value of 98.53%. In addition, the 16S rRNA and pheS genes of the two species were 99.4% and 99.9% identical, respectively. As the presence of O. sicerae in a water kefir metagenome was also revealed by metagenomic recruitment plotting, it can be stated that Candidatus O. aquikefiri and O. sicerae belong to the same species. Intraspecies variations include the presence or absence of a citrate lyase operon and components of various phosphotransferase (PTS) transport systems.

Identification and comparison of two closely related dextransucrases released by water kefir borne Lactobacillus hordei TMW 1.1822 and Lactobacillus nagelii TMW 1.1827.

Dextransucrases are extracellular enzymes, which are exclusively expressed by lactic acid bacteria (LAB) and produce α-1→6 linked glucose polymers from sucrose. In this study, two dextransucrases derived from water kefir borne Lactobacillus hordei TMW 1.1822 and Lactobacillus nagelii TMW 1.1827 were identified and comparatively investigated. Differences between both proteins mainly arise from an additional C-terminal glucan-binding domain and the presence of a signal motif in the L. nagelii TMW 1.1827 dextransucrase. L. hordei TMW 1.1822 released the enzyme only in the presence of its substrate sucrose in contrast to L. nagelii TMW 1.1827, while both strains functionally expressed the dextransucrases independently of sucrose. Both enzymes could be recovered as crude protein extracts in culture supernatants, as they are not covalently bound to the cell surface. This enabled the formation of dextrans at equal reaction conditions as well as their subsequent structural analysis in terms of molecular structure and molecular weight. The volumetric transglycosylation and hydrolysis activities were distinctly different for both enzymes, which produced O3-branched dextrans with a comparable degree of branching. Moreover, identical oligosaccharides were obtained for both dextrans upon endo-dextranase digestion, while some differences in the polysaccharide fine structures could be identified from the varying portions of certain oligosaccharides. Dextrans synthesized by the dextransucrase released by L. nagelii exhibited an averaged molecular weight (Mw) of 7.9×107 Da, while those produced by the dextransucrase released by L. hordei exhibited an Mw of 6.1×107 Da. Moreover, glycosylation of glucansucrases by LAB was identified for the first time for the released dextransucrase of L. nagelii TMW 1.1827. Our study therefore reveals new molecular insights into how dextransucrases released by water kefir borne L. hordei TMW 1.1822 and L. nagelii TMW 1.1827 contribute to the complex formation of the traditional beverage water kefir.

Oxygen and diverse nutrients influence the water kefir fermentation process.

Eight water kefir fermentation series differing in the presence of oxygen, the nutrient concentration, and the nutrient source were studied during eight consecutive backslopping steps. The presence of oxygen allowed the proliferation of acetic acid bacteria, resulting in high concentrations of acetic acid, and decreased the relative abundance of Bifidobacterium aquikefiri. Low nutrient concentrations resulted in slow water kefir fermentation and high pH values, which allowed the growth of Comamonas testosteroni/thiooxydans. Further, low nutrient concentrations favored the growth of Lactobacillus hilgardii and Dekkera bruxellensis, whereas high nutrient concentrations favored the growth of Lactobacillus nagelii and Saccharomyces cerevisiae. Dried figs, dried apricots, and raisins resulted in stable water kefir fermentation. Water kefir fermentation with dried apricots resulted in the highest pH and water kefir grain growth, whereas that with raisins resulted in the lowest pH and water kefir grain growth. Further, water kefir fermentation with raisins resembled fermentations with low nutrient concentrations, that with dried apricots resembled fermentations with normal nutrient concentrations, and that with fresh figs or a mixture of yeast extract and peptone resembled fermentations with high nutrient concentrations.

The water kefir grain inoculum determines the characteristics of the resulting water kefir fermentation process.

AIMS: To investigate the influence of the water kefir grain inoculum on the characteristics of the water kefir fermentation process. METHODS AND RESULTS: Three water kefir fermentation processes were started with different water kefir grain inocula and followed as a function of time regarding microbial species diversity, community dynamics, substrate consumption profile and metabolite production course. The inoculum determined the water kefir grain growth, the viable counts on the grains, the time until total carbohydrate exhaustion, the final metabolite concentrations and the microbial species diversity. There were always 2-10 lactic acid bacterial cells for every yeast cell and the majority of these micro-organisms was always present on the grains. Lactobacillus paracasei, Lactobacillus hilgardii, Lactobacillus nagelii and Saccharomyces cerevisiae were always present and may be the key micro-organisms during water kefir fermentation. Low water kefir grain growth was associated with small grains with high viable counts of micro-organisms, fast fermentation and low pH values, and was not caused by the absence of exopolysaccharide-producing lactic acid bacteria. CONCLUSIONS: The water kefir grain inoculum influences the microbial species diversity and characteristics of the fermentation process. A select group of key micro-organisms was always present during fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study allows a rational selection of a water kefir grain inoculum.

Investigation of the instability and low water kefir grain growth during an industrial water kefir fermentation process.

A poorly performing industrial water kefir production process consisting of a first fermentation process, a rest period at low temperature, and a second fermentation process was characterized to elucidate the causes of its low water kefir grain growth and instability. The frozen-stored water kefir grain inoculum was thawed and reactivated during three consecutive prefermentations before the water kefir production process was started. Freezing and thawing damaged the water kefir grains irreversibly, as their structure did not restore during the prefermentations nor the production process. The viable counts of the lactic acid bacteria and yeasts on the water kefir grains and in the liquors were as expected, whereas those of the acetic acid bacteria were high, due to the aerobic fermentation conditions. Nevertheless, the fermentations progressed slowly, which was caused by excessive substrate concentrations resulting in a high osmotic stress. Lactobacillus nagelii, Lactobacillus paracasei, Lactobacillus hilgardii, Leuconostoc mesenteroides, Bifidobacterium aquikefiri, Gluconobacter roseus/oxydans, Gluconobacter cerinus, Saccharomyces cerevisiae, and Zygotorulaspora florentina were the most prevalent microorganisms. Lb. hilgardii, the microorganism thought to be responsible for water kefir grain growth, was not found culture-dependently, which could explain the low water kefir grain growth of this industrial process.

Characterisation of the protein quality and composition of water kefir-fermented casein.

This study aimed to assess the technique of natural fermentation by applying water kefir to the casein protein. The diverse microorganisms and their enzymes found naturally in the water kefir can influence casein's characteristics. The fermented casein's protein quality (digestibility and secondary protein structure) and composition (total soluble solids and nutritive and non-nutritive substances) were investigated. Our findings revealed that the fermented casein's protein digestibility and total phenolic content increased from 82.46 to 88.60 % and 7.6 to 8.0 mg gallic acid equivalent/100 g, respectively. In addition, their surface charge and hydrophobicity changed from -30.06 to -34.93 mV and 286.9 to 213.7, respectively. Furthermore, the fermented casein's secondary protein components, α-helix (decreased from 13.66 to 8.21 %) and random coil (increased from 16.88 to 19.61 %), were also altered during the fermentation. Based on these findings, the water kefir fermentation approach could be an effective, practical, non-thermal approach for improving casein's protein quality and composition.

Microbial viability and nutritional content of water kefir grains under different storage conditions.

Water kefir grains are an important source of probiotics, mainly containing lactic acid bacteria and yeasts. The aim of this study is to investigate the changes in microbial and chemical properties of water kefir grains during 1-month storage at +4°C and -18°C. The initial content of lactobacilli, lactococci, and yeast in water kefir grains was 6.06, 6.33, and 5.93 log CFU/g, respectively. The number of lactobacilli, Lactobacillus acidophilus, and Bifidobacterium spp. in the water kefir grains were comparable, with slight changes at the end of refrigerated storage (p > .05). Lactococci and yeasts decreased significantly after both storage conditions compared to the initial content (p < .05). The dry matter and ash contents remained unchanged during storage (p > .05). Water kefir grains contained significant amounts of calcium, vitamin B2, vitamin B6, vitamin B7, and vitamin B12. Storage at both +4°C and -18°C did not affect the mineral and vitamin contents, except for Cu and Vitamin B2. The results indicate that the water kefir grains remained viable after storage at both temperatures. If water kefir grains need to be stored, it is recommended to store them at +4°C in sugared water as it ensures better survivability of the microbiota of the grains.

Microbial Composition of Water Kefir Grains and Their Application for the Detoxification of Aflatoxin B1.

Water kefir grains (WKGs), the starter used to develop a traditional beverage named water kefir, consist of a symbiotic mixture of probiotics with diverse bioactivities, but little is known about their abilities to remove mycotoxins that have serious adverse effects on humans and animals. This study investigated the ability of WKGs to remove aflatoxin B1 (AFB1), one of the most toxic mycotoxins, under different settings, and determined the mechanism of absorption mediated by WKGs and the effect of WKGs on the toxicity induced by AFB1 and the reduction in AFB1 in cow milk and tea soups. The results showed the WKGs used herein were dominated by Lactobacillus, Acetobacter, Phenylobacterium, Sediminibacterium, Saccharomyces, Issatchenkia, and Kodamaea. HPLC analysis demonstrated that the WKGs effectively removed AFB1 at concentrations ranging from 1 to 5 µg/mL, pH values ranging from 3 to 9, and temperatures ranging from 4 to 45 °C. Additionally, the removal of AFB1 mainly depended on absorption, which was consistent with the Freundlich and pseudo-second-order kinetic models. Moreover, only 49.63% of AFB1 was released from the AFB1-WKG complex after four washes when the release of AFB1 was non-detectable. Furthermore, WKG treatment caused a dramatic reduction in the mutagenicity induced by AFB1 according to an Ames test and reduced more than 54% of AFB1 in cow milk and three tea soups. These results suggested that WKGs can act as a potential bio-absorbent with a high binding ability to detoxify AFB1 in food and feed via a chemical action step and multi-binding sites of AFB1 absorption in a wide range of scenarios.

Challenges in water kefir production and limitations in human consumption: A comprehensive review of current knowledge.

Water kefir is a convenient dairy-free alternative to dairy-based fermented beverages. It is prepared by fermenting a sucrose solution with fresh and dried fruits using water kefir grains, and demineralized whey can be used in water kefir production. This review describes current knowledge on water kefir production and its health effects. The main aims of this paper are to focus on the microbial composition, potential health-promoting properties, limitations in human consumption, and challenges in the production of water kefir. Water kefir grains and substrates, including brown sugar, dried and fresh fruits, vegetables, and molasses, used in the production influence the fermentation characteristics and composition of water kefir. Lactic acid bacteria, acetic acid bacteria, and yeasts are the microorganisms involved in the fermentation process. Lactobacillus species are the most common microorganisms found in water kefir. Water kefir contains various bioactive compounds that have potential health benefits. Water kefir may inhibit the growth of certain pathogenic microorganisms and food spoilage bacteria, resulting in various health-promoting properties, including immunomodulatory, antihypertensive, anti-inflammatory, anti-ulcerogenic, antiobesity, hypolipidemic, and hepatoprotective activities.