Vitamin B12 Production Research Articles

Metabolic engineering and optimization of the fermentation medium for vitamin B12 production in Escherichia coli.

Vitamin B12 is a crucial fine chemical that is widely used in the pharmaceutical, food and chemical industries, and its production solely dependents on microbial fermentation. We previously constructed an artificial vitamin B12 biosynthesis pathway in Escherichia coli, but the yield of the engineered strains was low. Here, we removed metabolic bottlenecks of the vitamin B12 biosynthesis pathway in engineered E. coli strains. After screening cobB genes from different sources, optimizing the expression of cobN and customizing the ribosome binding sites of cobS and cobT, the vitamin B12 yield increased to 152.29μg/g dry cell weight (DCW). Optimization of the downstream module, which converts co(II)byrinic acid a,c-diamide into adenosylcobinamide phosphate, elevated the vitamin B12 yield to 249.04μg/g DCW. A comparison of a variety of equivalent components indicated that glucose and corn steep liquor are optimal carbon and nitrogen sources, respectively. Finally, an orthogonal array design was applied to determine the optimal concentrations of glucose and nitrogen sources including corn steep liquor and yeast extract, through which a vitamin B12 yield of 530.29μg/g DCW was obtained. The metabolic modifications and optimization of fermentation conditions achieved in this study offer a basis for further improving vitamin B12 production in E. coli and will hopefully accelerate its industrial application.

Bacterial vitamin B12 production enhances nematode predatory behavior

Although the microbiota is known to affect host development, metabolism, and immunity, its impact on host behavior is only beginning to be understood. In order to better characterize behavior modulation by host-associated microorganisms, we investigated how bacteria modulate complex behaviors in the nematode model organism Pristionchus pacificus. This nematode is a predator that feeds on the larvae of other nematodes, including Caenorhabditis elegans. By growing P. pacificus on different bacteria and testing their ability to kill C. elegans, we reveal large differences in killing efficiencies, with a Novosphingobium species showing the strongest enhancement. This enhanced killing was not accompanied by an increase in feeding, which is a phenomenon known as surplus killing, whereby predators kill more prey than necessary for sustenance. Our RNA-seq data demonstrate widespread metabolic rewiring upon exposure to Novosphingobium, which facilitated screening of bacterial mutants with altered transcriptional responses. We identified bacterial production of vitamin B12 as an important cause of such enhanced predatory behavior. Although vitamin B12 is an essential cofactor for detoxification and metabolite biosynthesis, shown previously to accelerate development in C. elegans, supplementation with this enzyme cofactor amplified surplus killing in P. pacificus, whereas mutants in vitamin B12-dependent pathways reduced surplus killing. By demonstrating that production of vitamin B12 by host-associated microbiota can affect complex host behaviors, we reveal new connections between animal diet, microbiota, and nervous system.

Simultaneous production of propionic acid and vitamin B12 from corn stalk hydrolysates by Propionibacterium freudenreichii in an expanded bed adsorption bioreactor

Vitamin B12 and propionic acid that were simultaneous produced by Propionibacterium freudenreichii are both favorable chemicals widely used in food preservatives, medicine, and nutrition. While the carbon source and propionic acid accumulation reflected fermentation efficiency. In this study, using corn stalk as a carbon source and fed-batch fermentation process in an expanded bed adsorption bioreactor was studied for efficient and economic biosynthesis of acid vitamin B12 and propionic. With liquid hot water pretreated corn stalk hydrolysates as carbon source, 28.65 mg L−1 of vitamin B12 and 17.05 g L−1 of propionic acid were attained at 168 h in batch fermentation. In order to optimize the fermentation outcomes, fed-batch fermentation was performed with hydrolyzed corn stalk in expanded bed adsorption bioreactor (EBAB), giving 47.6 mg L−1 vitamin B12 and 91.4 g L−1 of propionic acid at 258 h, which correspond to product yields of 0.37 mg g−1 and 0.75 g g−1, respectively. The present study provided a promising strategy for economically sustainable production of vitamin B12 and propionic acid by P. freudenreichii fermentation using biomass cornstalk as carbon source and expanded bed adsorption bioreactor.

Assessment of Process Variables on Vitamin B12 Production in Fermented Dairy Product Including Propionic Acid

The fermented dairy products produced by various microorganism’s activity provide valuable nutrients for human. Fermentation affects the physicochemical and organoleptic characteristics of foods as well as human health. In the present review, we describe the production of vitamin B12 in a fermented dairy product by Propionibacterium species. The effect of the process variables on vitamin B12 production in fermented dairy products e.g. pH, temperature, different carbon and nitrogen sources as well as the type and size of inoculum, the fermentation time and fermentation strategy, etc. are discussed. Finally, fermentation strategy, inoculum preparation of Propionibacterium freudenreichii subsp. Shermanii, methods of determining biomass and Vitamin B12 concentration are reviewed and their effects on vitamin B12 production are mentioned. Propionibacteria are so popular due to their unique characteristics such as being safe, convenient and economical. Among all the highest efficiency was observed by P. freudenreichii. To achieve this purpose, some appropriate situations need to be considered. The best carbon source for this bacterium to produce biomass is lactate, the suitable pH for growth of the Propionibacterium species is in the range of 6 to 7 and the fed-batch is most preferable for vitamin B12 production.

Comparative Genomics Guides Elucidation of Vitamin B12 Biosynthesis in Novel Human-Associated Akkermansia Strains.

Akkermansia muciniphila is a mucin-degrading bacterium found in the gut of most humans and is considered a "next-generation probiotic." However, knowledge of the genomic and physiological diversity of human-associated Akkermansia sp. strains is limited. Here, we reconstructed 35 metagenome-assembled genomes and combined them with 40 publicly available genomes for comparative genomic analysis. We identified at least four species-level phylogroups (AmI to AmIV), with distinct functional potentials. Most notably, we identified genes for cobalamin (vitamin B12) biosynthesis within the AmII and AmIII phylogroups. To verify these predictions, 10 Akkermansia strains were isolated from adults and screened for vitamin B12 biosynthesis genes via PCR. Two AmII strains were positive for the presence of cobalamin biosynthesis genes, while all 9 AmI strains tested were negative. To demonstrate vitamin B12 biosynthesis, we measured the production of acetate, succinate, and propionate in the presence and absence of vitamin supplementation in representative strains of the AmI and AmII phylogroups, since cobalamin is an essential cofactor in propionate metabolism. Results showed that the AmII strain produced acetate and propionate in the absence of supplementation, which is indicative of vitamin B12 biosynthesis. In contrast, acetate and succinate were the main fermentation products for the AmI strains when vitamin B12 was not supplied in the culture medium. Lastly, two bioassays were used to confirm vitamin B12 production by the AmII phylogroup. This novel physiological trait of human-associated Akkermansia strains may affect how these bacteria interact with the human host and other members of the human gut microbiome.IMPORTANCE There is significant interest in the therapeutic and probiotic potential of the common gut bacterium Akkermansia muciniphila However, knowledge of both the genomic and physiological diversity of this bacterial lineage is limited. Using a combination of genomic, molecular biological, and traditional microbiological approaches, we identified at least four species-level phylogroups with differing functional potentials that affect how these bacteria interact with both their human host and other members of the human gut microbiome. Specifically, we identified and isolated Akkermansia strains that were able to synthesize vitamin B12 The ability to synthesize this important cofactor broadens the physiological capabilities of human-associated Akkermansia strains, fundamentally altering our understanding of how this important bacterial lineage may affect human health.

Vitamin B<sub>12</sub> production in soybean fermentation for tempeh

Most studies have found that vitamin B12 in tempeh is produced by contaminating bacteria specifically, Klebsiella sp. and Citrobacter freundii, during fungal fermentation. This study is to evaluate the effect of starter culture on the vitamin B12 and isoflavone aglicone content of soybean fermentation for tempeh production. In this study, soybeans were washed, soaked in water overnight, dehulled and sterilized by boiling at 100 ℃ for 30 min. Three starter cultures (103 CFU g-1) namely Rhizopus oligosporus, Klebsiella sp. and Saccharomyces cerevisiae were then inoculated as follows, soybeans + R. oligosporus + Klebsiella sp. (SRK), soybeans + R. oligosporus + S. cerevisiae (SRSc), soybeans + R. oligosporus + S. cerevisiae + Klebsiella sp. (SRScK), and Soy + R. oligosporus (SR) and soybeans + Klebsiella sp (SK). Inoculated soybeans were then incubated at 30 ±2 ℃ for 40 hours (tempeh-style). The growth of Klebsiella sp., S. cerevisiae, R. oligosporus and the production of vitamin B12 as well as isoflavone aglicones were observed. The results showed the highest vitamin B12 3.15 mg 100 g-1 was found in tempeh SRSc, followed by 2.88 mg 100 g-1 and 1.64 mg 100 g-1 in tempeh SR and SRScK, respectively. In addition, vitamin B12 in tempeh SRK was the lowest (0.81 mg 100 g-1). All the starter cultures were able to hydrolyze daidzin and genistin, but the amount of daidzein and genistein was tripled and doubled, respectively when Klebsiella sp was inoculated to the soybean fermentation. The study suggested that S. cerevisiae contributes to the production of vitamin B12, while Klebsiella sp contributes to production of daidzein and genistein in soybean fermentation for tempeh production.

Betaine biosynthesis in a heterologous expression system based on the B12 producer Pseudomonas denitrificans

Glycine betaine is an important donor of methyl groups in various metabolic processes of the cell and acts as an osmoprotector when exposed to various abiotic stresses in pro- and eukaryotic organisms. Moreover, exogenous application of betaine activates the production of vitamin B12 in industrial strains-producers. In this work, we have developed a new technology for microbiological betaine synthesis that can be used in biotechnology to activate B12 biosynthesis during large-scale fermentation of Pseudomonas denitrificans.

Cobamides.

Vitamin B12 is the only known essential human micronutrient made exclusively by prokaryotes. Kennedy and Taga introduce us to the world of cobamides-those cobalt-containing compounds, like B12, that appear to be the proprietary domain of our microbial partners.

Optimization of semi-anaerobic vitamin B12 (cyanocobalamin) production from rice bran oil using Propionibacterium freudenreichii PTCC1674

Abstract Vitamin B12 contributes many substantial metabolic cycles in the living organisms. Since human beings cannot produce such co-factor by their metabolism, they have to receive this vitamin from foods and supplements. Dimethylbenzimidazole (DMBI) distinguishes the active form of vitamin B12 from pseudo-vitamin B12. De Novo total biosynthesis of vitamin B12 in the bacteria should include DMBI biosynthesis through riboflavin pathway. Propionibacterium freudenreichii can produce vitamin B12 through anaerobic biosynthesis pathway. As vitamin B12 production by P. freudenreichii is the growth-associated phenomena, the effect of different carbon sources (rice bran oil, argan oil), nutrients (DMBI) and amino acids (L-Serin, L-Tryptophan, l -cysteine, l -Methionine) on the growth of Propionibacterium freudenreichii PTCC1674 (pfre) were investigated. Through the statistical analysis of vitamin B12 production, rice bran oil (RBO) was selected as the sole carbon source. By applying Plackett-Burman method, significant parameters of vitamin B12 production were extracted and optimized based on Box-Behnken design of experiments. RBO, DMBI and CaCl2.2H2O concentrations and temperature were the four main effective parameters of vitamin B12 production. Via implemented response to surface methodology (RSM), the response was optimized to 2.94 mg/L, while 14% increase of vitamin B12 (cyanocobalamin) production was obtained at RBO concentration of 8.648% V/V, temperature of 38.3 (°C), DMBI concentration of 55.758 (mg/L) and elemental solution concentration of 2 (mg/L). It was concluded that pfre can grow on rice bran oil as a new carbon source while the changing of culture media composition alters the growth profile. Box-Behnken design effectively optimized parameters achieved from Plackett-Burman screening method.

Metabolic shift in the production of corrinoid compounds by Lactobacillus coryniformis in the absence of purines

Lactobacillus coryniformis CRL 1001 and L. reuteri CRL 1098 have the complete genes necessary to synthesize pseudo-cobalamin as final product in a vitamin B12 free commercial medium. Unlike vitaminB12 (the most biologically active form), the pseudo-cobalamin contains adenine instead of 5,6-dimethlbenzimidazole (DMB) in the Coα-ligand. Considering the vitamin B12-gene clusters of these bacteria, the aim of this work was to analyze the production of corrinoids with DMB (vitamin B12) instead of adenine (pseudo-B12) as lower ligand base in a vitamin B12 free chemically defined medium (CDM) without purines. Genome-wide screening of genes related to purine metabolism showed that both strains possess all pur genes necessary for the synthesis of inositol monophosphate, the main precursor for purine biosynthesis. Accordingly, both strains were able to grow in B12 free CDM without purines, with the supplementation of different synthetic intermediaries. Isolated compounds with positive vitamin B12 activity were quantified and characterized by LC/MS-MS. Total corrinoids values were higher for both strains in comparison to those obtained in vitaminB12 free commercial medium. Interestingly, CRL 1001 strain synthesized cobalamin, suggesting that this strain is able to activate DMB as nitrogenous base instead adenine when it is in excess in a purine-free medium. The present paper represents the first demonstration of a partial metabolic shift to produce vitamin B12 in a Lactobacillus strain.

Identification and Antibiotic Resistance Assessment of Ensifer adhaerens YX1, a Vitamin B12 -Producing Strain Used as a Food and Feed Additive.

This study provides phenotypic and molecular analyses of the antibiotic resistance of Ensifer adhaerens strain YX1 (CICC 11008s), a strain that was identified using a polyphasic taxonomy approach. The antibiotic resistance profile of E. adhaerens YX1 was assessed using the Clinical & Laboratory Standards Inst. (CLSI) method. The strain was susceptible to ciprofloxacin, levofloxacin, norfloxacin, ofloxacin, gentamicin, tobramycin, chloramphenicol, tetracycline, imipenem, and ceftazidime, and resistant to kanamycin, streptomycin, fosfomycin, and nitrofurantoin. The antibiotic resistance genes nsfA, nsfB, fosA, aph, and aadA1 were not detected in E. adhaerens YX1 via PCR using gene-specific primers. Subsequently, the genome sequence of E. adhaerens was screened for antibiotic genes. Although no antibiotic resistance genes were identified using the ResFinder database, five genes copies of one resistance gene, adeF, were detected using the Comprehensive Antibiotic Resistance Database (CARD). The results of this study will be useful for understanding the phenotypic and genotypic aspects of E. adhaerens antibiotic resistance. No safety issues were identified for E. adhaerens YX1 in terms of antibiotic resistance. Performing similar studies will be conducive to the safety assessment and control of the use of E. adhaerens in the food and feed industry. PRACTICAL APPLICATION: Few relevant reports are currently available regarding antibiotic resistance assessments or other safety evaluations for Ensifer adhaerens. Because of a lack of relevant information on the safety of this bacterium, including the genetic basis of antibiotic resistance in the production strain, it has not been recommended for use in the "qualified presumption of safety" (QPS) list and subsequent updated lists. The current study shows no safety issue of E. adhaerens YX1 in terms of its antibiotic resistance. These results are important as they provide an initial basis for an understanding of the antibiotic resistance/susceptibility of E. adhaerens YX1 (CICC 11008s), which produces vitamin B12 and is widely used in the food and feed industry.

Co-fermentation of Propionibacterium freudenreichii and Lactobacillus brevis in Wheat Bran for in situ Production of Vitamin B12.

The present study investigated the effect of co-fermentation on vitamin B12 content and microbiological composition of wheat bran. Propionibacterium freudenreichii DSM 20271 was used as the producer of vitamin while Lactobacillus brevis ATCC 14869 was selected to ensure the microbial safety of the bran dough. Fermentation trials were conducted in bioreactors to monitor and adjust the pH of the ferments. Vitamin B12 level reached 357 ± 8 ng/g dry weight (dw) after 1 day of pH-controlled fermentation with P. freudenreichii monoculture and remained stable thereafter. In co-fermentation with L. brevis, slightly less vitamin B12 (255 ± 31 ng/g dw) was produced in 1 day and an effective inhibition of the growth of total Enterobacteriaceae and Bacillus cereus was obtained. On day 3, vitamin B12 content in pH-controlled co-fermentation increased to 332 ± 44 ng/g dw. On the other hand, without a pH control, co-fermentation resulted in a stronger inhibition of Enterobacteriaceae and B. cereus but a lower level of vitamin B12 (183 ± 5 ng/g dw on day 3). These results demonstrated that wheat bran fermented by P. freudenreichii and L. brevis can be a promising way to produce vitamin B12 fortified plant-origin food ingredients, which could reduce cereal waste streams and contribute to a more resilient food chain.

Vitamin B12: From Deficiency to Biotechnological Solution

Vitamin B12 production by using propionibacteria and enriching food to produce functional foods is an important subject for researches. Some microorganisms have the potential to produce a wide range of components that are health promoting for human. Among them Propionibacteria has been identified as an effective producer of vitamin B12 and anti-microbial compounds such as propionic acid for decades. In this study at first, the structure, health beneficial effects and properties of vitamin B12 as well as scaled up production of vitamin are mentioned. Then biotechnological strategy is described as a solution to overcome vitamin deficiency and production of functional food. Finally, the specification of propionibacteria and its growth condition as well as bacterium ability to produce some other interesting metabolite in human food as byproduct are discussed.

Microalgal assimilation of vitamin B12 toward the production of a superfood.

A network of components from different metabolic pathways is the building scaffold of an indispensable compound in the human organism-vitamin B12 . The biosynthesis of this compound is restricted to a limited number of representatives of bacteria and archaea, while vitamin B12 -dependent enzymes are spread through several domains of life. Different attempts have been performed to increase vitamin B12 levels in dietary products, particularly in vegetarian and vegan dietary regimes. The integration of vitamin B12 in microalgae through symbiosis with microorganisms generally recognized as safe, for example the probiotic Lactobacillus reuteri, can even increase the nutritional value of the microalgal biomass. This study reviews the microbial production of vitamin B12 based on genetic analyses and chemical studies. Recent genetic approaches are focused, particularly potential metabolic engineering targets to increase vitamin B12 production. The bioincorporation of vitamin B12 in microalgae as an attempt to provide a superfood is also reviewed. PRACTICAL APPLICATIONS: Novel food habits (i.e., vegan lifestyle) may lack relevant nutrients, including vitamin B12 . Therefore, there is an increased demand for dietary products rich in vitamin B12 . Of potential interest is the provision of microbial-based superfood rich in numerous nutrients, including this vitamin. This manuscript provides an in-depth and timely overview on vitamin B12 biosynthesis and the major advances on metabolic engineering for improved vitamin B12 production by probiotic bacteria and other microorganisms generally recognized as safe. A relevant advance would result from the bioincorporation of vitamin B12 in alternative microorganisms (non-vitamin B12 producers) increasingly recognized as superfood, that is microalgae.

One-vector CRISPR/Cas9 genome engineering of the industrial fungus Ashbya gossypii.

SummaryThe filamentous fungus Ashbya gossypii is currently used for the industrial production of vitamin B2. Furthermore, the ability of A. gossypii to grow using low‐cost substrates together with the inexpensive downstream processing makes this fungus an attractive biotechnological chassis. Indeed, the production in A. gossypii of other high‐added value compounds such as folic acid, nucleosides and biolipids has been described. Hence, the development of new methods to expand the molecular toolkit for A. gossypii genomic manipulation constitutes an important issue for the biotechnology of this fungus. In this work, we present a one‐vector CRISPR/Cas9 system for genomic engineering of A. gossypii. We demonstrate the efficiency of the system as a marker‐less approach for nucleotide deletions and substitutions both with visible and invisible phenotypes. Particularly, the system has been validated for three types of genomic editions: gene inactivation, the genomic erasure of loxP scars and the introduction of point mutations. We anticipate that the use of the CRISPR/Cas9 system for A. gossypii will largely contribute to facilitate the genomic manipulations of this industrial fungus in a marker‐less manner.

Fed-Batch Production of a Fermented Beverage Containing Vitamin B12

Production of fermented functional foods containing micronutrients is required for their health beneficial properties. The impact of 11 process variables on vitamin B12 production in a dairy beverage containing propionic acid was investigated. Propionibacterium freudenreichii ssp. shermanii was applied in a 3-l fermentor in the fed-batch fermentation system. The most suitable conditions for vitamin B12 production were achieved by 5% v/v inoculum size containing Propionibacterium freudenreichii (without Lactobacillus acidophilus) and continuous feeding of lactose with the rate of 0.04 l/h at 36°C in a medium containing 25 g/L molasses, 10 g/L corn steep liquor, at pH=6.5, after 96 h fermentation. Maximum vitamin concentration (30 mg/L) and productivity (7.5 mg/L.day) were obtained in trial 9. Organoleptic properties of the fermented beverage were also acceptable for panelists and no significant difference was observed between samples and control during 6 days refrigerated storage.

Enhanced Vitamin B12 Production using Chlorella vulgaris

Microalgae Chlorella Vulgaris is enriched in vitamin B12 and cobalt ion which is positioned in the center of the vitamin molecule. This study aimed to investigate how different concentrations of cobalt chloride salt affected the vitamin B12 production by utilizing CO2 gas, to assess C. vulgaris biomass. Therefore, Bold’s basal medium used as the medium and 0.5, 1.5, 2 and 2.5 μM cobalt chloride salt was added to C. vulgaris culture. Under four cobalt chloride salt treatments, the best growth rate was obtained at the 2 μM of cobalt chloride salt (0.186 + 0.07 g /L.d). CO2 gas was supplied by 5% CO2 gas cylinder and fermented milk as a novel biological CO2 gas generator (CO2,10%). Use of fermented milk is a practical approach for elimination of waste gas emission and converting CO2 into biomass. The results revealed that, in the presence of 5% CO2 gas, C. vulgaris vitamin B12 content at 2 and 2.5 μM cobalt chloride, were 166.23 ± 1.78 and 173.32 ± 4.23 μg /100 g of dry biomass (7 and 12% higher than control), respectively. However, under controlled condition (ambient air and 2 μM cobalt chloride) vitamin B12 content was 154.9 ± 1.14 μg / 100 g of dry biomass.

Phycocomes zhengii gen. nov., sp. nov., a marine bacterium of the family Rhodobacteraceae isolated from the phycosphere of Chlorella vulgaris.

A Gram-stain-negative, motile with flagellum, ovoid- or rod-shaped, aerobic bacterium, was isolated from the phycosphere of the microalga Chlorellavulgaris and designated as strain LMIT002T. The bacterium formed white, circular and smooth colonies on marine agar 2216 after 48 h incubation at 25 °C. On the basis of 16S rRNA gene sequence analysis, strain LMIT002T was found to be affiliated with the family Rhodobacteraceae of the order Rhodobacterales, and formed a distinct group. The 16S rRNA gene sequence similarities between strain LMIT002T and type strains in the family including Poseidonocella pacifica DSM 29316T, Roseobacter litoralis and Rhodovulum sulfidophilum were 95.1, 95.0 and 95.0 %, respectively. Strain LMIT002Tgrew optimally at 25 °C, pH 6.0 and in the presence of 2.0 % (w/v) NaCl. The genomic DNA G+C content was 67.0 mol% (the thermal denaturation method) or 66.9 % (genome sequencing). The sole respiratory quinone was ubiquinone-8, while the major fatty acids were summed feature 8 (C18 : 1 ω7с/C18 : 1 ω6с). The draft genome of strain LMIT002T was sequenced and annotated, with the results showing that it had a total size of 4 607 780 bp and comprised 4557 genes. Functional genes encoding the production of vitamin B12, indole-3-acetic acid and transform dimethylsulphoniopropionate were detected. Given its distinct genomic, morphological and physiological differences from previously described type strains, strain LMIT002T is proposed as a representative of a novel genus of the family Rhodobacteraceae, with the name Phycocomes zhengii gen. nov., sp. nov. The type strain is LMIT002T (=KCTC 62390T=CICC 24357T).

Metabolic engineering of Escherichia coli for de novo biosynthesis of vitamin B12

The only known source of vitamin B12 (adenosylcobalamin) is from bacteria and archaea. Here, using genetic and metabolic engineering, we generate an Escherichia coli strain that produces vitamin B12 via an engineered de novo aerobic biosynthetic pathway. In vitro and/or in vivo analysis of genes involved in adenosylcobinamide phosphate biosynthesis from Rhodobacter capsulatus suggest that the biosynthetic steps from co(II)byrinic acid a,c-diamide to adocobalamin are the same in both the aerobic and anaerobic pathways. Finally, we increase the vitamin B12 yield of a recombinant E. coli strain by more than ∼250-fold to 307.00 µg g−1 DCW via metabolic engineering and optimization of fermentation conditions. Beyond our demonstration of E. coli as a microbial biosynthetic platform for vitamin B12 production, our study offers an encouraging example of how the several dozen proteins of a complex biosynthetic pathway can be transferred between organisms to facilitate industrial production.

Use of a Potential Probiotic, Lactobacillus casei L4, in the Preparation of Fermented Coconut Water Beverage

Coconut water (CW) is a clear, nutritive liquid found as the coconut endosperm of green coconuts such as Cocos nucifera L., and its widespread consumption owes to its unique composition of sugars, minerals, vitamins, enzymes, and hormones. Probiotic fermentation of CW may facilitate the development of an improved functional beverage with probiotic benefits; therefore, we aimed to produce a fermented CW beverage using the potential probiotic Lactobacillus casei L4. CW was fermented with L. casei L4 for 48 h at 35°C, and the pH, organic acid-production rate, antioxidant activity, antibacterial activity, sugar, mineral, vitamin B12 levels, and total viable bacteria counts were investigated at 24 and 48 h. We demonstrated that the fermentation of CW with probiotic lactobacilli increased the cell viability count. Vitamin B12 production was highest in the extracellular environment at 48 h (11.47 μg/mL), while the total phenolic content was significantly (p < 0.05) higher in the fermented CW at 48 h (72.1 μg/mL gallic acid equivalents) than observed with the other investigated groups or time points. The fermented materials exhibited the highest 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical-scavenging activities at 48 h (58.4 and 69.2%, respectively). The levels of most minerals remained unchanged in the fermented CW, except for calcium, manganese, phosphorus, and sodium. Furthermore, the culture supernatant from fermented CW inhibited the growth of foodborne pathogens such as Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, and Salmonella typhi, although the degree of inhibition varied between the species. Moreover, adding 15% honey and artificial coconut flavor to the fermented CW resulted in a better-tasting product, as demonstrated by a sensory-evaluation test. The obtained results indicated that the CW product fermented by L. casei L4 may be used as a novel functional beverage containing both electrolytes and probiotics, and can serve as a good vehicle for preparing a wider range of novel products.