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Short-chain fatty acid and vitamin production potentials of Lactobacillus isolated from fermented foods of Khasi Tribes, Meghalaya, India

Short-chain fatty acid and vitamin production potentials of Lactobacillus isolated from fermented... Purpose Vitamins and SCFA (short-chain fatty acids) production from Lactobacillus isolates are studied due to its health benefits to the human hosts. Lactobacillus strains are widely used in fermented foods, and few of them are reported with vitamin and SCFA production potential. Therefore, in the present study, vitamins and SCFA production capability of isolates were studied to find the potent Lactobacillus cultures for value-added functional food product development. Methods Five Lactobacillus strains, i.e., KGL2, KGL3A, KGL4, RNS4, and WTS4, were isolated from rice-based traditional fermented foods of Garo Hills, Meghalaya, India. All the well grown isolates were morphologically, physiologically, and genetically characterized. Then, vitamins and SCFA were estimated using HPLC based methods. Vitamins produced in vitamins free assay medium and SCFA in milk medium are produced by Lactobacillus. Results Lactic acid bacteria produce essential vitamins like riboflavin, folate, cobalamin, and SCFA which have health impacts (anti-obesity, anti-diabetics, anti-microbial, and other chronic diseases prevention) to the host. These vitamins are essential for cellular and metabolic growth of living system. In the study, five potent Lactobacillus isolates viz., KGL2 (Lactobacillus fermentum), KGL3A (Lactobacillus plantarum), KGL4 (Lactobacillus fermentum), RNS4 (Lactobacillus rhamnosus), and WTS4 (Lactobacillus fermentum) were considered for vitamins (B ,B ,and B ) and SCFA productions (lactate, butyrate, 2 12 9 and acetate). However, KGL3A had shown highest B production (0.7 μg/ml) while KGL2 exhibited maximum B production 2 12 (0.05 μg/ml) after 36 h. Moreover, WTS4 attributed highest folate production (0.09 μg/ml) after 24 h. In addition, RNS4 reported the maximum short-chain fatty acid production (0.77 g/l acetic acid, 0.26 g/l lactic acid, and 0.008 g/l butyric acid respectively). Conclusions Potent Lactobacillus isolates from traditional fermented foods of Garo Hills, Meghalaya, India (North East Part of India) showed maximum production of B ,B ,and B as well as short-chain fatty acids and could be used for their application as 2 9 12 health beneficial functional fermented dairy products. . . . Keywords Lactobacillus Probiotics Short-chain fatty acids Vitamins Introduction Microorganisms are natural resource of diverse metabolites Subrota Hati and Maulik Patel contributed equally to this work. (vitamins, essential amino acids, organic acids, peptides), Electronic supplementary material The online version of this article which might be strain specific or species specific. Use of these (https://doi.org/10.1007/s13213-019-01500-8) contains supplementary value-added characteristics microbes as feed additives has in- material, which is available to authorized users. creased its demands due to ease of production and health benefits. Vitamins are vital micronutrients for every living cell * Subrota Hati acting as precursors or participating in many important enzy- subrota_dt@yahoo.com matic reactions or even in electron transport chain. 1 Microorganisms can generally biosynthesize B group vita- Dairy Microbiology Department, SMC College of Dairy Science, mins as per their requirements while humans cannot produce AAU, Anand, Gujarat, India 2 and hence they have to depend on external sources to fulfill Department of RDAP, North Eastern Hill University, Tura Campus, their daily requirement (Leblanc et al. 2011). Vitamin- Tura, Meghalaya, India 1192 Ann Microbiol (2019) 69:1191–1199 producing gut microbiome can synthesize and supply vita- most vitamin synthesis, humans take them as supplementary mins to the human hosts (Hill 1997). Therefore, scientists to the food in terms of plant origin (source of vitamins A, C, are working on finding the novel lactic acid bacteria which D, E, K) foods or microbial origin (source of vitamin B com- are having generally recognized as safe (GRAS) status and plex) foods. Supplementation of vitamins of microbial origin can also synthesize vital vitamins and other biomolecules. In has recently gained much attention, and much research is go- the study, we have attempted to isolate lactic acid bacteria ing on to identify the biosynthetic pathways of these vitamins (LAB) from rice-based traditional fermented foods of north- (Patel et al. 2013). B group vitamins are majorly involved in east region of India. This area belongs to Garo tribes, and food related energy metabolism; therefore, vitamin B– therefore, many traditional healthy fermented foods specific producing food-grade microbe identification and development to Garo tribes are available (Kumar et al. 2012; Goswami et al. of that microbe containing food are in demand. Vitamin B -, 2017). Chyne et al. (2017) reported that malnutrition problems folate-, and riboflavin-producing LAB incorporation into of children along with deficiencies in micronutrients (majorly fermented milk, soy milk, or yogurt could increase the vitamin in B-group vitamins) are higher in numbers among the Khasi concentration. The supplement of deficient vitamins to the tribes residing in West Khasi Hills District of Meghalaya due body through foods is the extra advantage to the host to deficiency of essential micronutrients. Although there is (Molina et al. 2012;Guet al. 2015). Riboflavin is a precursor availability of both cultivable and broader wild food resources for flavin mono-nucleotide (FMN) and flavin adenine dinu- in Khasi Hills, but nutritional anemia is majorly affecting cleotide (FAD) which plays major role in cellular metabolism, these areas because of deficiency in iron, folate, and vitamins and it is reported to produce by the gut microbiome (Hill A, B ,B ,and B . Not only in northeastern states, there are a 1997). Folic acid is the one of the key materials for DNA 2 6 12 huge number of vitamin B deficiencies among Indian pop- and RNA biosynthesis and routine metabolic activities of cell; ulation. The prevalence of folate deficiency is minimal as hence, supplementation of folate in food has many advantages compared to B .However,B deficiency is more prevalent such as reduce birth defects and also blood pressure in adults. 12 12 among the children and adolescents (Toteja and Gonmei Dairy product with the value-added folate has been reported; 2018). Therefore, we are looking to isolate the indigenous however, it has been shown that some industrially important potential microorganisms (GRAS status) with vitamins pro- strains of LAB (L. lactis, Lactobacillus acidophilus,and ducing ability in fermented foods. Eastern states of India in- Lactobacillus plantarum) are responsible behind it (LeBlanc cluding Meghalaya countless diverse fermented rice-based et al. 2011). Cobalamin, which is another essential vitamin of food items locally known as ki kpu, putharo, pusyep, pumaloi, B-complex, works as cofactor in DNA synthesis, hemoglobin pusaw, kanjika, kimoto, and various rice-based beverages are synthesis, and various fatty acids and amino acid metabo- being used since ancient time, which might be good source of lisms. Very few archaea and bacteria able to produce it and edible microbes such as LAB (Lactobacillus johnsonii, only few LAB can produce it (Bhushan et al. 2016). Santos Lactobacillus fermentum) with unique characteristics like et al. (2008)reported vitamin B – and B –producing strain 12 9 anti-bacterial potentials (Takahashi 2014;Murugan 2018). L. reuteri JCM11112 in fermented milk. The genus Lactobacillus is a group of lactic acid bacteria Apart from production of vitamins, recently, LAB are get- which plays an important role in human gut microbiota and ting more attention because of their organic acid production also found as active component in associated foods. Some which also has different therapeutic applications such as anti- Lactobacillus strains have been exploited for the development obesity and anti-diabetic (Salazar et al. 2011). Anti-microbial of novel functional foods by producing and or releasing the properties of lactic acid produced by LAB are well reported important metabolites. Genome analysis of various LAB spe- (Miquel et al. 2013;Mieszkinetal. 2017). Organic acids come cies has shown that habitat-specific gene addition and deletion under SCFA; mainly, acetate, propionate, and butyrate are takes place to adapt the surrounding territory (Goh 2009). vital for maintaining the health of the host when they are However, such novel trait specific to strains has to be identi- available in adequate amounts. SCFA exerts major role in fied from diverse natural niches and then to be selected for glucose, cholesterol, and lipids metabolism and related meta- their novel functional and technological attributes. Several bolic disorders (Besten et al. 2013). As a consequence, it can LAB species such as Lactococcus lactis, Lactobacillus affect the insulin level, sugar level, and weight of individual gasseri, and Lactobacillus reuteri and Bifidobacterium are (Kimura et al. 2014). Moreover, role of SCFA in types 2 reported for vitamins and SCFA production (Thirabunyanon diabetes and obesity control was reported (Cani 2013). et al. 2008; Ventura et al. 2009; Papagianni 2012). Moreover, SCFA roles in anti-inflammation and anti-cancer Nonetheless, these unique characteristics might help LAB to is reported by the study of Faecalibacterium prausnitzii (a niche specific survival (gut, diary food) and stimulate interac- butyrate producer), the leading anti-inflammatory commensal tions with surrounding environments (Besten et al. 2013). bacterium recognized on the basis of human clinical data of Vitamins are vital nutrient of human body for growth and the human intestinal microbiota (Miquel et al. 2013;Chen and various metabolic activities. However, due to inefficiency of Vitetta 2018), where role of F. prausnitzii strain A2-165 and Ann Microbiol (2019) 69:1191–1199 1193 its culture supernatant was reported to protect against 2,4,6- isolation kit procured from Genetix India Limited following trinitrobenzenesulfonic acid (TNBS)-induced colitis in mice. manufacturer’s instruction. The DNA was purified and ampli- In the present study, we evaluated the vitamins and organic fied using 16S ribosomal DNA primer 27F (AGAGTTTG acids producing ability of LAB (KGL2, KGL3A, KGL4, ATCMTGGCTCAG) and 519R (GWATTACCGC RNS4, and WTS4) isolated from fermented foods of Garo GGCKGCTG). For pcr amplification, methodology opted Hills of Meghalaya, India. The strains were examined for their from Pateland Patel(2015). Amplified product was se- B ,B ,and B vitamin production as well as SCFA produc- quenced, and nucleotide sequences were retrieved. Then, nu- 2 9 12 tion (acetic acid, lactic acid, and butyric acid) ability precisely, cleotide sequences were matched with NCBI BLAST data- using high-performance liquid chromatography (HPLC). base for the genus and species identification. All the sequence These Lactobacillus isolates could be used for the production of selected isolates was deposited in NCBI-GenBank along of SCFA in fermented foods for the value addition to the with accession numbers. traditional fermented foods with various health benefits asso- ciated with SCFA to the consumers. Certain LAB can Determination of vitamin B and B productions 2 12 biosynthesize B group vitamins for their growth and metabol- ic activities (Hill 1997; Leblanc et al. 2011;Thakur and Tomar Vitamin B and B productions were determined using a 2 12 2015). These strains can be used for the development of microbiological assay as described by Taranto et al. (2003) fermented foods to improve vitamin availability along with with some modifications. LAB isolates were inoculated in the beneficial bacteria. vitamin B – and B –free assay medium (HiMedia, India) 2 12 and subcultured three times in the same medium. Cells were incubated in 15 ml of the defined medium at 37 °C for 12, 24, Materials and methods 36, and 48 h and were harvested and washed thrice with 0.1 M phosphate buffer, pH 7.0. Then, washed cells were suspended Chemicals and reagents in 1 ml of extraction buffer (0.1 M Na HPO [pH 4.5 using 2 4 citric acid], 0.005% KCN) and disrupted the cells at 95 °C for All the selective media as well as vitamin production media 30 min, followed by vigorous vortexing for 1 min and centri- were purchased from Himedia, India. The HPLC standards for fuged at 10,000×g for 10 min. After centrifugation, the super- vitamins (B ,B ,and B ) and SCFA (acetic, lactic, and bu- natant was collected and passed through 0.45-μ syringe filter. 2 9 12 tyric acid) were purchased from Sigma-Aldrich, India. Then, the supernatant was analyzed further through HPLC for B and B productions. In HPLC analysis, 20-μlfiltrate was 2 12 LAB isolates injected in HPLC system (LC-10, Shimadzu, Japan) using microinjector for evaluation of B and B . An isocratic 2 12 Garo Hills is tribal area of north-eastern part of India. The HPLC system was fitted with RP18 endcapped column, tribes residing in these regions are consuming various rice- LiChroCART column (250 × 4.6 mm) (Chromolith-Merck), based fermented beverages as traditional medicines and ener- and a Guard column (40 × 4 mm). Sample was loaded using gy drink in their daily diets. Therefore, we had isolated several Hilton injector with 20 μl loop. The column was washed twice LAB from the fermented rice beverages for their functional with distilled water to remove salts and other contaminants for studies. The food samples from various fermented rice-based 2 h. After the cartridge was washed with water, the cobalamin beverages were collected, serially diluted, and plated on was eluted with volume of 50% acetonitrile with flow rate of Lactobacillus selective agar medium, i.e., MRS medium 0.3 ml/min and the oven temperature was kept at 40 °C. (HiMedia, India). The grown LAB were purified and exam- Absorbance of elute was monitored at 284 nm using UV ined for morphological and biochemical parameters (API Kit, detector. Biomerieux, Germany). Among them, five isolated LAB were used in present study for their potential SCFA and vitamin Determination of folate (B )production production potential determination. Five bacteria, i.e., KGL2, KGL3A, KGL4, RNS4, and WTS4, were isolated Folate production of the selected LAB strains was determined from rice-based traditional fermented foods of Garo Hills, by a microbiological assay as described by Panda et al. (2018) Meghalaya, India. The isolates were morphologically and with some modifications. LAB isolates were inoculated in physiologically examined. In addition, the isolates were bio- folate free assay medium (HiMedia, India) and subcultured chemically examined for catalase activity by inoculating in three times in the same medium. Cells incubated in 15 ml of hydrogen peroxide solution. All the purified cultures were the defined medium at 37 °C for 12, 24, 36, and 48 h were further identified based on 16S rDNA–based molecular iden- harvested and washed thrice with 0.1 M phosphate buffer, tification technique. The isolates were grown in 5 ml MRS pH 7.0. Washed cells were resuspended in 1 ml of extraction broth at 37 °C overnight. The DNA was extracted with DNA buffer (20 mM sodium phosphate buffer, with pH 6.2) and 1194 Ann Microbiol (2019) 69:1191–1199 disrupted at 95 °C for 30 min, followed by vigorous vortexing phylogenetic tree divides all 5 isolates in two major clusters; for 1 min, and centrifuged at 10,000×g for 10 min. After one cluster is of L. fermentum isolates and their closely related centrifugation, the supernatant was collected and passed L. fermentum strains from NCBI database, while other cluster through 0.45-μ syringe filter. The collected supernatant was is subdivided into two subclusters describing one cluster has analyzed through HPLC for folate estimation. Five percent of L. plantarum and associated strains and other cluster has acetonitrile in 20 mM sodium phosphate buffer, with pH 6.2 Lactobacillus rhamnosus and associated strains. The phyloge- used as mobile phase and flow rate was 1 ml/min. A UV– netic tree suggests each isolate is genetically and evolutionary Visible detector was used at 280 nm (Panda et al. 2018. distinct from other isolates. Determination of SCFA production Vitamin B production of the Lactobacillus strains SCFA produced by LAB isolates were measured using HPLC Riboflavin is important for cell metabolic activities particular- system suggested by LeBlanc et al. (2017)with some modi- ly in oxidation-reduction reactions and cell growth. The mi- fication. The active bacterial strain was inoculated in 10 ml crobiological assay with chemically defined B -free medium sterilized skimmed milk and incubated for 24 h at 37 °C. After has been used for the riboflavin production for the five iso- 24 h, 5-ml sample was mixed with 45 ml water and vortexed lates. KGL3A exhibited highest B production (0.72 μg/ml) for 5 min. After vortexing, samples were allowed to settle for after 36 h. While RNS4 reported maximum B production 10 min and filtered by Whatman paper no. 42. The filtered (0.55 μg/ml) after 24 h followed by decline in vitamin in samples were further passed through 0.45-μ syringe filter and 36 h and 48 h respectively. But WTS4, KGL2, and KGL4 also injected into HPLC system. For HPLC analysis, the column produced 0.3 to 0.5 μg/ml in 24 to 36 h (Fig. 2). In initial, 12-h was washed twice with 0.01% phosphoric acid to remove salts cultures showed no or very minute riboflavin production and other contaminants. After the cartridge was washed, the followed by gradual increase in riboflavin production. organic acid was eluted with 0.01% phosphoric acid with flow However, RNS4 showed production up to 24 h followed by rate of 0.5 ml/min and the oven temperature was kept at 40 °C. decrease in riboflavin concentration, while KGL4, WTS4, Absorbance of elute was monitored at 210 nm. KGL2, and KGL3A produced riboflavin up to 36 h, followed by decreasing the content after 48 h. The increase in concen- Statistical analysis tration in log phase and early stationary phase is associated with major role in cell metabolic activities where riboflavin All data presented here are as means (± SEM) of three repli- could be utilized by microbes for their multiplication and cates (n = 3). The vitamins and organic acid concentrations growth (Kaprasob et al. 2018). Guru and Viswanathan obtained by HPLC were analyzed using one-way ANOVA (2013) had reported similar results in their experiments with with Bonferroni posttest. P values less than 0.05 were consid- L. acidophilus and L. lactis fermented whey and skim milk. ered significant. For graphical presentation and data analysis, They reported higher B (2930 μg/l and 2610 μg/l respective- Origin (version 8.0) was used. ly) production in whey compared to skimmed milk. In addi- tion, riboflavin production in MRS medium was reported by Thakur and Tomar (2015) with 2.13 mg/l and 2.36 mg/l of Results and discussion riboflavin by strain KTLF1 (L. fermentum) and KTLP13 (L. plantarum) respectively. The major objective of this work Isolation and identification of Lactobacillus strains was to study the riboflavin production potential of isolates and their use in food to complement the daily needs of riboflavin. Five isolates viz., KGL4, RNS4, WTS4, KGL2, and KGL3A Likewise, approach was advocated by Russo et al. (2014)for were considered for vitamins and SCFA productions. The administration of higher riboflavin producing L. fermentum bacterial isolates were physiologically, morphologically, and PBCC11.5 and its parental strain to fortify bread and achieved biochemically examined for purity and other parameters from 3.3 to 7.0 μg/g riboflavin in bread. (Table 1). All the bacterial isolates were preliminary examined for toxicity and pathogenicity which confers nonharmful char- Vitamin B productions of the Lactobacillus strains acteristics of all isolates. Identification of LAB species was also carried out by 16SrRNA gene amplification using PCR Preliminary, folate producing potential was analyzed by method followed by sequencing of PCR product. Based on chemically defined folate free medium where all the five iso- sequencing analysis through NCBI blast, the identified LAB lates shown folate production potential. In the study, WTS4 species are presented in Table 1, with respective accession produced 0.092 μg/ml folate after 24 h and decreasing the numbers. Moreover, the derived phylogenetic tree of isolates contents after 36 h. KGL2, KGL3A, KGL4, and RNS4 along with their closely related strains was given in Fig. 1.The showed folate production in the range from 0.05 to Ann Microbiol (2019) 69:1191–1199 1195 Table 1 List of bacterial cultures used in the study with biochemical characteristics Culture Species Accession Morphology (Gram Growth against Catalase Hemolytic Dnase Susceptibility to code no. reaction and shape) 6.5% NaCl test test test antibiotics (Amp, Tet, ERY, Kan, Met) KGL4 L. fermentum MF951099 Gram +, short road Negative Negative Negative Negative Susceptible RNS4 L. rhamnosus MG027692 Gram +, short road Negative Negative Negative Negative Susceptible WTS4 L. fermentum MG027694 Gram +, short road Negative Negative Negative Negative Susceptible KGL2 L. fermentum MG561927 Gram +, short road Negative Negative Negative Negative Susceptible KGL3A L. plantarum MG722814 Gram +, short road Negative Negative Negative Negative Susceptible Amp ampicillin (10 mcg), Tet tetracycline (30 mcg), ERY erythromycin (15 mcg), Kan kanamycin (30 mcg), Met methicillin (5 mcg) 0.082 μg/ml after 12 h, followed by decreasing in between 24 folate (Rossi et al. 2011). The four strains used in our studies and 36 h (Fig. 3). Microbes synthesize mainly two types of belong to L. fermentum (KGL2, KGL4, and WTS4) and folate, namely 5-methyl tetrahydrofolate (5-MTHF) and tetra- L. plantarum (KGL3A) which advocate authentication of hydrofolate (THF). However, all the isolates were producing our results with scientifically reported data elsewhere 5-MTHF during HPLC analysis. Panda et al. (2018)and (Leblanc et al. 2011). Two good folate producers reveled in Gangadharan et al. (2010) reported the 5-MTHF producing the study WTS4 and KGL3A could be used in the future for LAB. Precisely, L. rhamnosus (IFM-4), L. cremoris (CM22), fortification of folate in fermented dairy products. and L. lactis (CM28) studied with 35 ng/ml, 12.5 ng/ml, and 14.2 ng/ml folate production respectively. The major role of Vitamin B production of the Lactobacillus strains folate is in nucleic acid biosynthesis; hence, it is require in high amount in early log phase. Microbes used in the studies All the LAB isolates were grown in vitamin B –free assay are capable to synthesize their own requirement of folate. In medium, and therefore, HPLC quantification was carried out. high amount of folate production in early growth phase (12 h), HPLC analysis shown KGL2 has highest 0.058 μg/ml B results can be correlated for nucleic acid synthesis and cell production, followed by KGL3A (0.046 μg/ml) after 36 h of growth while decline with time is associated with less require- incubation (Fig. 4). Both the reported strains achieved maxi- ment in matured cells. These results clearly exhibited the role mum B production in 36 h followed by depletion after 48 h. of folate in early development of cells and its importance. Possible reason is due to the deficient in nutrient concentration Sequencing information from the available data revealed that leading to reduction in metabolic activity. Similar results of L. johnsonii, L. acidophilus, Lactobacillus salivarius, B producing LAB namely L. plantarum, L. casei, L. reuteri, Lactobacillus brevis, Lactobacillus casei, L. gasseri, and Lactobacillus coryniformis were reported (Masuda et al. L. rhamnosus,and Lactobacillus crispatus lack the genes for 2012;Guetal. 2015). While WTS4 had exhibited folate biosynthesis production while L. lactis, L. plantarum, 0.024 μg/ml B production after 24 h, followed by decline Lactobacillus sakei, Lactobacillus delbrueckii, L. reuteri, in 36 h and achieved its maximum B production Lactobacillus helveticus,and L. fermentum have folate bio- (0.028 μg/ml) after 48 h. Moreover, KGL4 and RNS4 have synthesis gene clusters and assume that they could produce shown sequential increase in vitamin production (0.023 μg/ml NR_043408.1 Lactobacillus rhamnosus strain JCM 1... Fig. 1 Phylogenetic tree of LAB isolates with their closest bacteria NR_113332.1 Lactobacillus rhamnosus strain NBRC ... from NCBI Database MG027692.1 Lactobacillus rhamnosus strain RNS4 NR_117813.1 Lactobacillus plantarum strain JCM 1149 NR_104573.1 Lactobacillus plantarum strain CIP 103... MG722814.1 Lactobacillus plantarum strain KGL3A NR_104927.1 Lactobacillus fermentum strain CIP NR_113335.1 Lactobacillus fermentum strain NBRC ... MG027694.1 Lactobacillus fermentum strain WTS4 MF951099.1 Lactobacillus fermentum strain KGL4 MG561927.1 Lactobacillus fermentum strain KGL2 1196 Ann Microbiol (2019) 69:1191–1199 Fig. 2 Vitamin B production by 12 H 24 H 36 H 48H Lactobacillus isolates. Means with different letters (a, b, c) 0.8 a indicating significant difference (P <0.05), n = 3, means ± S.D. 0.7 a,b 0.6 0.5 0.4 0.3 0.2 0.1 KGL4 RNS4 WTS4 KGL2 KGL3A Culture and 0.035 μg/ml respectively) up to 24 h, followed by de- deficiencies and consequences to the human health, people are creasing after 36 h and 48 h. However, few vitamins B looking for vitamin B enriched foods. Therefore, the use of 12 12 producing LAB are reported compared to folate or riboflavin vitamin B –producing LAB in fermented or dairy foods production. The first evidence of B production in LAB was could be a good alternative to mitigate B deficiencies in 12 12 reported in L. reuteri CRL1908 (Taranto et al. 2003). The community. chromatographic data of CRL1908 cell extract confirmed the presence of cobalamin like compound and further identi- fied as cobalamin. Recently, during the genome analysis of Determination of SCFA production Lactobacillus rossiae, a complete de novo biosynthesis path- way of vitamin B was constructed and identified in few Increasing health awareness have recently exploded the role of microbes (Angelis et al. 2014). Moreover, latest identification SCFA in gastrointestinal tract and associated health benefits of important B structural genes (cobT, cbiB and cbiA) and (LeBlanc et al. 2017). Therefore, people are looking for prod- previously known cbiK would further help to the researchers ucts having good sources of health supporting SCFA. Dairy to identify the B production mechanism (Bhushan et al. products associated with milk fermentation by LAB are good 2016) and also to design strategies to execute overproduction sources of these organic acids. LAB-fermented products are of these vitamins for future application using cloning rich in lactic acid, followed by acetic acid, and also contain methods. With increasing the awareness of vitamin B related minute quantity of propionic acid and butyric acid. Therefore, 12 H 24 H 36 H 48H 0.07 0.12 12h 24h 36h 48h 0.06 0.1 0.05 0.08 0.04 0.06 0.03 0.04 0.02 0.02 a 0.01 KGL4 RNS4 WTS4 KGL2 KGL3A KGL4 RNS4 WTS4 KGL2 KGL3A Culture Culture Fig. 3 Vitamin B production by Lactobacillus isolates. Means with Fig. 4 Vitamin B production by Lactobacillus isolates. Means with 9 12 different letters (a, b) indicating significant difference (P <0.05), n =3, different letters (a, b) indicating significant difference (P <0.05), n =3, means ± S.D. means ± S.D. Folate Concerntration (μg/ml) B Concerntration (µg/ml) B Concerntraon (μg/ml) 12 Ann Microbiol (2019) 69:1191–1199 1197 (a) Acetic acid KGL4 RNS4 WTS4 KGL2 KGL3A Culture (b) Lactic acid KGL4 RNS4 WTS4 KGL2 KGL3A Culture (c) Butyric acid 0.25 0.20 0.15 0.10 0.05 0.00 KGL4 RNS4 WTS4 KGL2 KGL3A Culture Fig. 5 SCFA production by Lactobacillus isolates after 24 h. a Acetic acid. b Lactic acid. c Butyric acid (P <0.05), n = 3, means ± S.D. Butyric acid (ug/ml) Acetic acid (ug/ml) Lactic acid (ug/ml) 1198 Ann Microbiol (2019) 69:1191–1199 the SCFA-producing ability of five LAB isolates was studied as well as short-chain fatty acids. These five Lactobacillus in this study. RNS4 (L. rhamnosus) showed highest overall cultures could be used for the development of functional SCFA production among all the isolates with 15.41 μg/ml fermented dairy foods. acetic acid (Fig. 5a), 5.18 μg/ml lactic acid (Fig. 5b), and Funding This research was financed by the Department of 0.16 μg/ml butyric acid (Fig. 5c), followed by KGL2 with Biotechnology, New Delhi, Government of India (Agri/2015/02). 12.9 μg/ml, 4.39 μg/ml, and 0.23 μg/ml acetic acid, lactic acid, and butyric acid respectively. Similar result of SCFA Compliance with ethical standards This article does not production reported in L. rhamnosus strain GG along with contain any studies with human participants or animals performed by propionic acid (89 μM) production in MRS medium any of the authors. (LeBlanc et al. 2017), while KGL4 (16 μg/ml), RNS2 (5.1916 μg/ml), and KGL2 (0.2516 μg/ml) showed maximum Conflict of interest The authors declare that they have no conflict of interest. acetic acid, lactic acid, and butyric acid production after 24 h. The variation in acid production is particularly strain specific due to inherent potential of each strain at individual level rather than species level (Macfarlane and Macfarlane 2003). References Carbohydrate fermentation by LAB can leads to various short- Angelis MD, Bottacini F, Fosso B, Kelleher P, Calasso M, Cagno RD, chain fatty acid productions (Pessione 2012). Even in carbon Ventura M, Picardi E, Sinderen DV, Gobbetti M (2014) depletion and anaerobic condition, lactic acid can be further Lactobacillus rossiae, a vitamin B12 producer, represents a meta- converted into acetic acid by several LAB (Oude Elferink bolically versatile species within the genus Lactobacillus.PLoS One et al. 2001). 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PLoS Genet 5. https://doi.org/10.1371/journal.pgen.1000785 Panda SH, Das S, Bal P, Panda SK, Mohanty N (2018) Characterization Publisher’snote Springer Nature remains neutral with regard to of novel folate producing Lactobacillus rhamnosus and its appliance jurisdictional claims in published maps and institutional affiliations. in fortification of ragi (Eleusine coracana)gruel. Food Biosci 21: 100–106 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Short-chain fatty acid and vitamin production potentials of Lactobacillus isolated from fermented foods of Khasi Tribes, Meghalaya, India

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Springer Journals
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Copyright © 2019 by Università degli studi di Milano
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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1590-4261
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1869-2044
DOI
10.1007/s13213-019-01500-8
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Abstract

Purpose Vitamins and SCFA (short-chain fatty acids) production from Lactobacillus isolates are studied due to its health benefits to the human hosts. Lactobacillus strains are widely used in fermented foods, and few of them are reported with vitamin and SCFA production potential. Therefore, in the present study, vitamins and SCFA production capability of isolates were studied to find the potent Lactobacillus cultures for value-added functional food product development. Methods Five Lactobacillus strains, i.e., KGL2, KGL3A, KGL4, RNS4, and WTS4, were isolated from rice-based traditional fermented foods of Garo Hills, Meghalaya, India. All the well grown isolates were morphologically, physiologically, and genetically characterized. Then, vitamins and SCFA were estimated using HPLC based methods. Vitamins produced in vitamins free assay medium and SCFA in milk medium are produced by Lactobacillus. Results Lactic acid bacteria produce essential vitamins like riboflavin, folate, cobalamin, and SCFA which have health impacts (anti-obesity, anti-diabetics, anti-microbial, and other chronic diseases prevention) to the host. These vitamins are essential for cellular and metabolic growth of living system. In the study, five potent Lactobacillus isolates viz., KGL2 (Lactobacillus fermentum), KGL3A (Lactobacillus plantarum), KGL4 (Lactobacillus fermentum), RNS4 (Lactobacillus rhamnosus), and WTS4 (Lactobacillus fermentum) were considered for vitamins (B ,B ,and B ) and SCFA productions (lactate, butyrate, 2 12 9 and acetate). However, KGL3A had shown highest B production (0.7 μg/ml) while KGL2 exhibited maximum B production 2 12 (0.05 μg/ml) after 36 h. Moreover, WTS4 attributed highest folate production (0.09 μg/ml) after 24 h. In addition, RNS4 reported the maximum short-chain fatty acid production (0.77 g/l acetic acid, 0.26 g/l lactic acid, and 0.008 g/l butyric acid respectively). Conclusions Potent Lactobacillus isolates from traditional fermented foods of Garo Hills, Meghalaya, India (North East Part of India) showed maximum production of B ,B ,and B as well as short-chain fatty acids and could be used for their application as 2 9 12 health beneficial functional fermented dairy products. . . . Keywords Lactobacillus Probiotics Short-chain fatty acids Vitamins Introduction Microorganisms are natural resource of diverse metabolites Subrota Hati and Maulik Patel contributed equally to this work. (vitamins, essential amino acids, organic acids, peptides), Electronic supplementary material The online version of this article which might be strain specific or species specific. Use of these (https://doi.org/10.1007/s13213-019-01500-8) contains supplementary value-added characteristics microbes as feed additives has in- material, which is available to authorized users. creased its demands due to ease of production and health benefits. Vitamins are vital micronutrients for every living cell * Subrota Hati acting as precursors or participating in many important enzy- subrota_dt@yahoo.com matic reactions or even in electron transport chain. 1 Microorganisms can generally biosynthesize B group vita- Dairy Microbiology Department, SMC College of Dairy Science, mins as per their requirements while humans cannot produce AAU, Anand, Gujarat, India 2 and hence they have to depend on external sources to fulfill Department of RDAP, North Eastern Hill University, Tura Campus, their daily requirement (Leblanc et al. 2011). Vitamin- Tura, Meghalaya, India 1192 Ann Microbiol (2019) 69:1191–1199 producing gut microbiome can synthesize and supply vita- most vitamin synthesis, humans take them as supplementary mins to the human hosts (Hill 1997). Therefore, scientists to the food in terms of plant origin (source of vitamins A, C, are working on finding the novel lactic acid bacteria which D, E, K) foods or microbial origin (source of vitamin B com- are having generally recognized as safe (GRAS) status and plex) foods. Supplementation of vitamins of microbial origin can also synthesize vital vitamins and other biomolecules. In has recently gained much attention, and much research is go- the study, we have attempted to isolate lactic acid bacteria ing on to identify the biosynthetic pathways of these vitamins (LAB) from rice-based traditional fermented foods of north- (Patel et al. 2013). B group vitamins are majorly involved in east region of India. This area belongs to Garo tribes, and food related energy metabolism; therefore, vitamin B– therefore, many traditional healthy fermented foods specific producing food-grade microbe identification and development to Garo tribes are available (Kumar et al. 2012; Goswami et al. of that microbe containing food are in demand. Vitamin B -, 2017). Chyne et al. (2017) reported that malnutrition problems folate-, and riboflavin-producing LAB incorporation into of children along with deficiencies in micronutrients (majorly fermented milk, soy milk, or yogurt could increase the vitamin in B-group vitamins) are higher in numbers among the Khasi concentration. The supplement of deficient vitamins to the tribes residing in West Khasi Hills District of Meghalaya due body through foods is the extra advantage to the host to deficiency of essential micronutrients. Although there is (Molina et al. 2012;Guet al. 2015). Riboflavin is a precursor availability of both cultivable and broader wild food resources for flavin mono-nucleotide (FMN) and flavin adenine dinu- in Khasi Hills, but nutritional anemia is majorly affecting cleotide (FAD) which plays major role in cellular metabolism, these areas because of deficiency in iron, folate, and vitamins and it is reported to produce by the gut microbiome (Hill A, B ,B ,and B . Not only in northeastern states, there are a 1997). Folic acid is the one of the key materials for DNA 2 6 12 huge number of vitamin B deficiencies among Indian pop- and RNA biosynthesis and routine metabolic activities of cell; ulation. The prevalence of folate deficiency is minimal as hence, supplementation of folate in food has many advantages compared to B .However,B deficiency is more prevalent such as reduce birth defects and also blood pressure in adults. 12 12 among the children and adolescents (Toteja and Gonmei Dairy product with the value-added folate has been reported; 2018). Therefore, we are looking to isolate the indigenous however, it has been shown that some industrially important potential microorganisms (GRAS status) with vitamins pro- strains of LAB (L. lactis, Lactobacillus acidophilus,and ducing ability in fermented foods. Eastern states of India in- Lactobacillus plantarum) are responsible behind it (LeBlanc cluding Meghalaya countless diverse fermented rice-based et al. 2011). Cobalamin, which is another essential vitamin of food items locally known as ki kpu, putharo, pusyep, pumaloi, B-complex, works as cofactor in DNA synthesis, hemoglobin pusaw, kanjika, kimoto, and various rice-based beverages are synthesis, and various fatty acids and amino acid metabo- being used since ancient time, which might be good source of lisms. Very few archaea and bacteria able to produce it and edible microbes such as LAB (Lactobacillus johnsonii, only few LAB can produce it (Bhushan et al. 2016). Santos Lactobacillus fermentum) with unique characteristics like et al. (2008)reported vitamin B – and B –producing strain 12 9 anti-bacterial potentials (Takahashi 2014;Murugan 2018). L. reuteri JCM11112 in fermented milk. The genus Lactobacillus is a group of lactic acid bacteria Apart from production of vitamins, recently, LAB are get- which plays an important role in human gut microbiota and ting more attention because of their organic acid production also found as active component in associated foods. Some which also has different therapeutic applications such as anti- Lactobacillus strains have been exploited for the development obesity and anti-diabetic (Salazar et al. 2011). Anti-microbial of novel functional foods by producing and or releasing the properties of lactic acid produced by LAB are well reported important metabolites. Genome analysis of various LAB spe- (Miquel et al. 2013;Mieszkinetal. 2017). Organic acids come cies has shown that habitat-specific gene addition and deletion under SCFA; mainly, acetate, propionate, and butyrate are takes place to adapt the surrounding territory (Goh 2009). vital for maintaining the health of the host when they are However, such novel trait specific to strains has to be identi- available in adequate amounts. SCFA exerts major role in fied from diverse natural niches and then to be selected for glucose, cholesterol, and lipids metabolism and related meta- their novel functional and technological attributes. Several bolic disorders (Besten et al. 2013). As a consequence, it can LAB species such as Lactococcus lactis, Lactobacillus affect the insulin level, sugar level, and weight of individual gasseri, and Lactobacillus reuteri and Bifidobacterium are (Kimura et al. 2014). Moreover, role of SCFA in types 2 reported for vitamins and SCFA production (Thirabunyanon diabetes and obesity control was reported (Cani 2013). et al. 2008; Ventura et al. 2009; Papagianni 2012). Moreover, SCFA roles in anti-inflammation and anti-cancer Nonetheless, these unique characteristics might help LAB to is reported by the study of Faecalibacterium prausnitzii (a niche specific survival (gut, diary food) and stimulate interac- butyrate producer), the leading anti-inflammatory commensal tions with surrounding environments (Besten et al. 2013). bacterium recognized on the basis of human clinical data of Vitamins are vital nutrient of human body for growth and the human intestinal microbiota (Miquel et al. 2013;Chen and various metabolic activities. However, due to inefficiency of Vitetta 2018), where role of F. prausnitzii strain A2-165 and Ann Microbiol (2019) 69:1191–1199 1193 its culture supernatant was reported to protect against 2,4,6- isolation kit procured from Genetix India Limited following trinitrobenzenesulfonic acid (TNBS)-induced colitis in mice. manufacturer’s instruction. The DNA was purified and ampli- In the present study, we evaluated the vitamins and organic fied using 16S ribosomal DNA primer 27F (AGAGTTTG acids producing ability of LAB (KGL2, KGL3A, KGL4, ATCMTGGCTCAG) and 519R (GWATTACCGC RNS4, and WTS4) isolated from fermented foods of Garo GGCKGCTG). For pcr amplification, methodology opted Hills of Meghalaya, India. The strains were examined for their from Pateland Patel(2015). Amplified product was se- B ,B ,and B vitamin production as well as SCFA produc- quenced, and nucleotide sequences were retrieved. Then, nu- 2 9 12 tion (acetic acid, lactic acid, and butyric acid) ability precisely, cleotide sequences were matched with NCBI BLAST data- using high-performance liquid chromatography (HPLC). base for the genus and species identification. All the sequence These Lactobacillus isolates could be used for the production of selected isolates was deposited in NCBI-GenBank along of SCFA in fermented foods for the value addition to the with accession numbers. traditional fermented foods with various health benefits asso- ciated with SCFA to the consumers. Certain LAB can Determination of vitamin B and B productions 2 12 biosynthesize B group vitamins for their growth and metabol- ic activities (Hill 1997; Leblanc et al. 2011;Thakur and Tomar Vitamin B and B productions were determined using a 2 12 2015). These strains can be used for the development of microbiological assay as described by Taranto et al. (2003) fermented foods to improve vitamin availability along with with some modifications. LAB isolates were inoculated in the beneficial bacteria. vitamin B – and B –free assay medium (HiMedia, India) 2 12 and subcultured three times in the same medium. Cells were incubated in 15 ml of the defined medium at 37 °C for 12, 24, Materials and methods 36, and 48 h and were harvested and washed thrice with 0.1 M phosphate buffer, pH 7.0. Then, washed cells were suspended Chemicals and reagents in 1 ml of extraction buffer (0.1 M Na HPO [pH 4.5 using 2 4 citric acid], 0.005% KCN) and disrupted the cells at 95 °C for All the selective media as well as vitamin production media 30 min, followed by vigorous vortexing for 1 min and centri- were purchased from Himedia, India. The HPLC standards for fuged at 10,000×g for 10 min. After centrifugation, the super- vitamins (B ,B ,and B ) and SCFA (acetic, lactic, and bu- natant was collected and passed through 0.45-μ syringe filter. 2 9 12 tyric acid) were purchased from Sigma-Aldrich, India. Then, the supernatant was analyzed further through HPLC for B and B productions. In HPLC analysis, 20-μlfiltrate was 2 12 LAB isolates injected in HPLC system (LC-10, Shimadzu, Japan) using microinjector for evaluation of B and B . An isocratic 2 12 Garo Hills is tribal area of north-eastern part of India. The HPLC system was fitted with RP18 endcapped column, tribes residing in these regions are consuming various rice- LiChroCART column (250 × 4.6 mm) (Chromolith-Merck), based fermented beverages as traditional medicines and ener- and a Guard column (40 × 4 mm). Sample was loaded using gy drink in their daily diets. Therefore, we had isolated several Hilton injector with 20 μl loop. The column was washed twice LAB from the fermented rice beverages for their functional with distilled water to remove salts and other contaminants for studies. The food samples from various fermented rice-based 2 h. After the cartridge was washed with water, the cobalamin beverages were collected, serially diluted, and plated on was eluted with volume of 50% acetonitrile with flow rate of Lactobacillus selective agar medium, i.e., MRS medium 0.3 ml/min and the oven temperature was kept at 40 °C. (HiMedia, India). The grown LAB were purified and exam- Absorbance of elute was monitored at 284 nm using UV ined for morphological and biochemical parameters (API Kit, detector. Biomerieux, Germany). Among them, five isolated LAB were used in present study for their potential SCFA and vitamin Determination of folate (B )production production potential determination. Five bacteria, i.e., KGL2, KGL3A, KGL4, RNS4, and WTS4, were isolated Folate production of the selected LAB strains was determined from rice-based traditional fermented foods of Garo Hills, by a microbiological assay as described by Panda et al. (2018) Meghalaya, India. The isolates were morphologically and with some modifications. LAB isolates were inoculated in physiologically examined. In addition, the isolates were bio- folate free assay medium (HiMedia, India) and subcultured chemically examined for catalase activity by inoculating in three times in the same medium. Cells incubated in 15 ml of hydrogen peroxide solution. All the purified cultures were the defined medium at 37 °C for 12, 24, 36, and 48 h were further identified based on 16S rDNA–based molecular iden- harvested and washed thrice with 0.1 M phosphate buffer, tification technique. The isolates were grown in 5 ml MRS pH 7.0. Washed cells were resuspended in 1 ml of extraction broth at 37 °C overnight. The DNA was extracted with DNA buffer (20 mM sodium phosphate buffer, with pH 6.2) and 1194 Ann Microbiol (2019) 69:1191–1199 disrupted at 95 °C for 30 min, followed by vigorous vortexing phylogenetic tree divides all 5 isolates in two major clusters; for 1 min, and centrifuged at 10,000×g for 10 min. After one cluster is of L. fermentum isolates and their closely related centrifugation, the supernatant was collected and passed L. fermentum strains from NCBI database, while other cluster through 0.45-μ syringe filter. The collected supernatant was is subdivided into two subclusters describing one cluster has analyzed through HPLC for folate estimation. Five percent of L. plantarum and associated strains and other cluster has acetonitrile in 20 mM sodium phosphate buffer, with pH 6.2 Lactobacillus rhamnosus and associated strains. The phyloge- used as mobile phase and flow rate was 1 ml/min. A UV– netic tree suggests each isolate is genetically and evolutionary Visible detector was used at 280 nm (Panda et al. 2018. distinct from other isolates. Determination of SCFA production Vitamin B production of the Lactobacillus strains SCFA produced by LAB isolates were measured using HPLC Riboflavin is important for cell metabolic activities particular- system suggested by LeBlanc et al. (2017)with some modi- ly in oxidation-reduction reactions and cell growth. The mi- fication. The active bacterial strain was inoculated in 10 ml crobiological assay with chemically defined B -free medium sterilized skimmed milk and incubated for 24 h at 37 °C. After has been used for the riboflavin production for the five iso- 24 h, 5-ml sample was mixed with 45 ml water and vortexed lates. KGL3A exhibited highest B production (0.72 μg/ml) for 5 min. After vortexing, samples were allowed to settle for after 36 h. While RNS4 reported maximum B production 10 min and filtered by Whatman paper no. 42. The filtered (0.55 μg/ml) after 24 h followed by decline in vitamin in samples were further passed through 0.45-μ syringe filter and 36 h and 48 h respectively. But WTS4, KGL2, and KGL4 also injected into HPLC system. For HPLC analysis, the column produced 0.3 to 0.5 μg/ml in 24 to 36 h (Fig. 2). In initial, 12-h was washed twice with 0.01% phosphoric acid to remove salts cultures showed no or very minute riboflavin production and other contaminants. After the cartridge was washed, the followed by gradual increase in riboflavin production. organic acid was eluted with 0.01% phosphoric acid with flow However, RNS4 showed production up to 24 h followed by rate of 0.5 ml/min and the oven temperature was kept at 40 °C. decrease in riboflavin concentration, while KGL4, WTS4, Absorbance of elute was monitored at 210 nm. KGL2, and KGL3A produced riboflavin up to 36 h, followed by decreasing the content after 48 h. The increase in concen- Statistical analysis tration in log phase and early stationary phase is associated with major role in cell metabolic activities where riboflavin All data presented here are as means (± SEM) of three repli- could be utilized by microbes for their multiplication and cates (n = 3). The vitamins and organic acid concentrations growth (Kaprasob et al. 2018). Guru and Viswanathan obtained by HPLC were analyzed using one-way ANOVA (2013) had reported similar results in their experiments with with Bonferroni posttest. P values less than 0.05 were consid- L. acidophilus and L. lactis fermented whey and skim milk. ered significant. For graphical presentation and data analysis, They reported higher B (2930 μg/l and 2610 μg/l respective- Origin (version 8.0) was used. ly) production in whey compared to skimmed milk. In addi- tion, riboflavin production in MRS medium was reported by Thakur and Tomar (2015) with 2.13 mg/l and 2.36 mg/l of Results and discussion riboflavin by strain KTLF1 (L. fermentum) and KTLP13 (L. plantarum) respectively. The major objective of this work Isolation and identification of Lactobacillus strains was to study the riboflavin production potential of isolates and their use in food to complement the daily needs of riboflavin. Five isolates viz., KGL4, RNS4, WTS4, KGL2, and KGL3A Likewise, approach was advocated by Russo et al. (2014)for were considered for vitamins and SCFA productions. The administration of higher riboflavin producing L. fermentum bacterial isolates were physiologically, morphologically, and PBCC11.5 and its parental strain to fortify bread and achieved biochemically examined for purity and other parameters from 3.3 to 7.0 μg/g riboflavin in bread. (Table 1). All the bacterial isolates were preliminary examined for toxicity and pathogenicity which confers nonharmful char- Vitamin B productions of the Lactobacillus strains acteristics of all isolates. Identification of LAB species was also carried out by 16SrRNA gene amplification using PCR Preliminary, folate producing potential was analyzed by method followed by sequencing of PCR product. Based on chemically defined folate free medium where all the five iso- sequencing analysis through NCBI blast, the identified LAB lates shown folate production potential. In the study, WTS4 species are presented in Table 1, with respective accession produced 0.092 μg/ml folate after 24 h and decreasing the numbers. Moreover, the derived phylogenetic tree of isolates contents after 36 h. KGL2, KGL3A, KGL4, and RNS4 along with their closely related strains was given in Fig. 1.The showed folate production in the range from 0.05 to Ann Microbiol (2019) 69:1191–1199 1195 Table 1 List of bacterial cultures used in the study with biochemical characteristics Culture Species Accession Morphology (Gram Growth against Catalase Hemolytic Dnase Susceptibility to code no. reaction and shape) 6.5% NaCl test test test antibiotics (Amp, Tet, ERY, Kan, Met) KGL4 L. fermentum MF951099 Gram +, short road Negative Negative Negative Negative Susceptible RNS4 L. rhamnosus MG027692 Gram +, short road Negative Negative Negative Negative Susceptible WTS4 L. fermentum MG027694 Gram +, short road Negative Negative Negative Negative Susceptible KGL2 L. fermentum MG561927 Gram +, short road Negative Negative Negative Negative Susceptible KGL3A L. plantarum MG722814 Gram +, short road Negative Negative Negative Negative Susceptible Amp ampicillin (10 mcg), Tet tetracycline (30 mcg), ERY erythromycin (15 mcg), Kan kanamycin (30 mcg), Met methicillin (5 mcg) 0.082 μg/ml after 12 h, followed by decreasing in between 24 folate (Rossi et al. 2011). The four strains used in our studies and 36 h (Fig. 3). Microbes synthesize mainly two types of belong to L. fermentum (KGL2, KGL4, and WTS4) and folate, namely 5-methyl tetrahydrofolate (5-MTHF) and tetra- L. plantarum (KGL3A) which advocate authentication of hydrofolate (THF). However, all the isolates were producing our results with scientifically reported data elsewhere 5-MTHF during HPLC analysis. Panda et al. (2018)and (Leblanc et al. 2011). Two good folate producers reveled in Gangadharan et al. (2010) reported the 5-MTHF producing the study WTS4 and KGL3A could be used in the future for LAB. Precisely, L. rhamnosus (IFM-4), L. cremoris (CM22), fortification of folate in fermented dairy products. and L. lactis (CM28) studied with 35 ng/ml, 12.5 ng/ml, and 14.2 ng/ml folate production respectively. The major role of Vitamin B production of the Lactobacillus strains folate is in nucleic acid biosynthesis; hence, it is require in high amount in early log phase. Microbes used in the studies All the LAB isolates were grown in vitamin B –free assay are capable to synthesize their own requirement of folate. In medium, and therefore, HPLC quantification was carried out. high amount of folate production in early growth phase (12 h), HPLC analysis shown KGL2 has highest 0.058 μg/ml B results can be correlated for nucleic acid synthesis and cell production, followed by KGL3A (0.046 μg/ml) after 36 h of growth while decline with time is associated with less require- incubation (Fig. 4). Both the reported strains achieved maxi- ment in matured cells. These results clearly exhibited the role mum B production in 36 h followed by depletion after 48 h. of folate in early development of cells and its importance. Possible reason is due to the deficient in nutrient concentration Sequencing information from the available data revealed that leading to reduction in metabolic activity. Similar results of L. johnsonii, L. acidophilus, Lactobacillus salivarius, B producing LAB namely L. plantarum, L. casei, L. reuteri, Lactobacillus brevis, Lactobacillus casei, L. gasseri, and Lactobacillus coryniformis were reported (Masuda et al. L. rhamnosus,and Lactobacillus crispatus lack the genes for 2012;Guetal. 2015). While WTS4 had exhibited folate biosynthesis production while L. lactis, L. plantarum, 0.024 μg/ml B production after 24 h, followed by decline Lactobacillus sakei, Lactobacillus delbrueckii, L. reuteri, in 36 h and achieved its maximum B production Lactobacillus helveticus,and L. fermentum have folate bio- (0.028 μg/ml) after 48 h. Moreover, KGL4 and RNS4 have synthesis gene clusters and assume that they could produce shown sequential increase in vitamin production (0.023 μg/ml NR_043408.1 Lactobacillus rhamnosus strain JCM 1... Fig. 1 Phylogenetic tree of LAB isolates with their closest bacteria NR_113332.1 Lactobacillus rhamnosus strain NBRC ... from NCBI Database MG027692.1 Lactobacillus rhamnosus strain RNS4 NR_117813.1 Lactobacillus plantarum strain JCM 1149 NR_104573.1 Lactobacillus plantarum strain CIP 103... MG722814.1 Lactobacillus plantarum strain KGL3A NR_104927.1 Lactobacillus fermentum strain CIP NR_113335.1 Lactobacillus fermentum strain NBRC ... MG027694.1 Lactobacillus fermentum strain WTS4 MF951099.1 Lactobacillus fermentum strain KGL4 MG561927.1 Lactobacillus fermentum strain KGL2 1196 Ann Microbiol (2019) 69:1191–1199 Fig. 2 Vitamin B production by 12 H 24 H 36 H 48H Lactobacillus isolates. Means with different letters (a, b, c) 0.8 a indicating significant difference (P <0.05), n = 3, means ± S.D. 0.7 a,b 0.6 0.5 0.4 0.3 0.2 0.1 KGL4 RNS4 WTS4 KGL2 KGL3A Culture and 0.035 μg/ml respectively) up to 24 h, followed by de- deficiencies and consequences to the human health, people are creasing after 36 h and 48 h. However, few vitamins B looking for vitamin B enriched foods. Therefore, the use of 12 12 producing LAB are reported compared to folate or riboflavin vitamin B –producing LAB in fermented or dairy foods production. The first evidence of B production in LAB was could be a good alternative to mitigate B deficiencies in 12 12 reported in L. reuteri CRL1908 (Taranto et al. 2003). The community. chromatographic data of CRL1908 cell extract confirmed the presence of cobalamin like compound and further identi- fied as cobalamin. Recently, during the genome analysis of Determination of SCFA production Lactobacillus rossiae, a complete de novo biosynthesis path- way of vitamin B was constructed and identified in few Increasing health awareness have recently exploded the role of microbes (Angelis et al. 2014). Moreover, latest identification SCFA in gastrointestinal tract and associated health benefits of important B structural genes (cobT, cbiB and cbiA) and (LeBlanc et al. 2017). Therefore, people are looking for prod- previously known cbiK would further help to the researchers ucts having good sources of health supporting SCFA. Dairy to identify the B production mechanism (Bhushan et al. products associated with milk fermentation by LAB are good 2016) and also to design strategies to execute overproduction sources of these organic acids. LAB-fermented products are of these vitamins for future application using cloning rich in lactic acid, followed by acetic acid, and also contain methods. With increasing the awareness of vitamin B related minute quantity of propionic acid and butyric acid. Therefore, 12 H 24 H 36 H 48H 0.07 0.12 12h 24h 36h 48h 0.06 0.1 0.05 0.08 0.04 0.06 0.03 0.04 0.02 0.02 a 0.01 KGL4 RNS4 WTS4 KGL2 KGL3A KGL4 RNS4 WTS4 KGL2 KGL3A Culture Culture Fig. 3 Vitamin B production by Lactobacillus isolates. Means with Fig. 4 Vitamin B production by Lactobacillus isolates. Means with 9 12 different letters (a, b) indicating significant difference (P <0.05), n =3, different letters (a, b) indicating significant difference (P <0.05), n =3, means ± S.D. means ± S.D. Folate Concerntration (μg/ml) B Concerntration (µg/ml) B Concerntraon (μg/ml) 12 Ann Microbiol (2019) 69:1191–1199 1197 (a) Acetic acid KGL4 RNS4 WTS4 KGL2 KGL3A Culture (b) Lactic acid KGL4 RNS4 WTS4 KGL2 KGL3A Culture (c) Butyric acid 0.25 0.20 0.15 0.10 0.05 0.00 KGL4 RNS4 WTS4 KGL2 KGL3A Culture Fig. 5 SCFA production by Lactobacillus isolates after 24 h. a Acetic acid. b Lactic acid. c Butyric acid (P <0.05), n = 3, means ± S.D. Butyric acid (ug/ml) Acetic acid (ug/ml) Lactic acid (ug/ml) 1198 Ann Microbiol (2019) 69:1191–1199 the SCFA-producing ability of five LAB isolates was studied as well as short-chain fatty acids. These five Lactobacillus in this study. RNS4 (L. rhamnosus) showed highest overall cultures could be used for the development of functional SCFA production among all the isolates with 15.41 μg/ml fermented dairy foods. acetic acid (Fig. 5a), 5.18 μg/ml lactic acid (Fig. 5b), and Funding This research was financed by the Department of 0.16 μg/ml butyric acid (Fig. 5c), followed by KGL2 with Biotechnology, New Delhi, Government of India (Agri/2015/02). 12.9 μg/ml, 4.39 μg/ml, and 0.23 μg/ml acetic acid, lactic acid, and butyric acid respectively. Similar result of SCFA Compliance with ethical standards This article does not production reported in L. rhamnosus strain GG along with contain any studies with human participants or animals performed by propionic acid (89 μM) production in MRS medium any of the authors. 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Annals of Microbiology – Springer Journals

Published: Jul 22, 2019

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