Screening of Riboflavin-Producing Lactobacilli by a Polymerase

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Screening of riboflavin producing lactobacilli by PCR based approach and microbiological assay KIRAN THAKUR, Sudhir K. Tomar, Biswajit Brahma, and Sachinandan De J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b06165 • Publication Date (Web): 23 Feb 2016 Downloaded from http://pubs.acs.org on February 23, 2016

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Journal of Agricultural and Food Chemistry

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Screening of riboflavin producing lactobacilli by PCR based approach and

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microbiological assay

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Kiran Thakur , Sudhir Kumar Tomar*1 Biswajit Brahma2 and Sachinandan De2

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Dairy Microbiology Division, National Dairy Research Institute, Karnal, Haryana, India

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Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana, India

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Correspondent footnote:

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Dr. Sudhir Kumar Tomar

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Principal Scientist,

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Dairy Microbiology Division,

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National Dairy Research Institute,

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Karnal (Haryana)-132001, India.

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E mail: [email protected]

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Ph.: +91-184-2259196(O)

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FAX:0184-2250042

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ACS Paragon Plus Environment


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ABSTRACT

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Riboflavin has an important role in various cellular metabolic activities through its

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participation in oxidation–reduction reactions. In this study, as many as 60 lactobacilli were

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screened for the presence or/ absence of riboflavin biosynthesis genes and riboflavin

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production. Out of these, only 14 strains were able to grow in a commercial riboflavin-free

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medium. We observed that the presence of riboflavin biosynthesis genes is strain specific

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across different species of lactobacilli. The microbiological assay was found to be

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appreciably reproducible, sensitive, rapid, and inexpensive, and hence can be employed for

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screening the riboflavin producing strains. The study thus represents a convenient and

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efficient method for selection of novel riboflavin producers. These riboflavin+ strains so

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identified and characterized could be explored as potent candidates for the development of

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wide range of dairy as well as cereal based foods for the delivery of in situ riboflavin to the

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consumers.

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Key Words: Riboflavin, Lactic acid bacteria, biosynthesis and dairy food

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INTRODUCTION

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The microbial production of riboflavin is gaining importance as the consumers have become

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increasingly health conscious and aware of their nutritional requirements1. Being a precursor

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of the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), riboflavin plays

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a crucial role in metabolism, reproduction, lactation, and general wellness of human beings. It

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also helps other B vitamins to undergo the chemical changes. Riboflavin deficiency which is

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prevalent in both industrialized and developing countries2,3 leads to a host of health disorders

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e.g. loss of hair, reduction of growth rate, inflammation of the skin, sore throat, hyperemia,

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edema of oral and mucous membranes, cheilosis and glossitis4. It is also associated with

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vision deterioration, increased levels of homocysteine with consequent cardiac risk5,

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preeclampsia6, oxidative stress7, anaemia8, and changes in the brain glucose metabolism9. In

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the recent years this vitamin has been found to be effective in the treatment of migraine10,

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malaria11, Parkinson's disease12, headaches13, and

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role of antioxidant as a component of glutathione redox cycle7 and has been observed as an

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efficient adjuvant in many cancer cell lines and animal-based studies15.

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The vitamins producing ability of bacteria has attracted many research groups for humans

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lack the biosynthetic capacity for most of vitamins and hence, are dependent on dairy and

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cereal based food, plants, fungi, and bacteria for dietary supplementation16. Riboflavin has

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been produced industrially by chemical synthesis from ribose17, but in recent years, the

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biotechnological production has received more attention. The use of lactic acid bacteria

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(LAB) for this purpose sounds promising as these microorganisms are able to synthesize B-

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group vitamins during fermentation18-20. LAB besides their starter and probiotic nature, have

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been shown to be ideal cell factories as they are known to produce a range of metabolites that

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are collectively termed as ‘nutraceuticals’, which include B vitamins like riboflavin (B2),

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folate (B11) and cobalamine (B12), low calorie sugars like mannitol and sorbitol,

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exopolysaccharides, diacetyl and L-alanine21. According to Capozzi et al. (2012), the genetic

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information for riboflavin biosynthesis is a species and/or strain-specific trait in LAB22.

migraine prophylaxis14. It also plays the

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They have also reported that comparative genome analysis reveals that the ability to

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synthesize riboflavin is shared by several of the sequenced members of LAB although an

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interrupted riboflavin operon is sometimes observed in certain strains22. Recent studies have

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reported that the selection of riboflavin producing strains has potential food and dairy

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applications, for example, for the manufacture of riboflavin enriched dairy and cereal based

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products10. The metabolic exploitation of such virtuous microbes has a relevant importance in

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preparation of various fermented foods. Hence, LAB with riboflavin producing ability will

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serve as better inoculants in the fermented food industry as the foods can be naturally

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fortified with riboflavin. Till date, scanty information is available on microbiological

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screening of riboflavin production. The present study was designed to screen the lactobacilli

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for harbouring riboflavin biosynthesis genes by PCR based method followed by screening of

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the isolates for riboflavin production by using microbiological assay methods. The riboflavin

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producing lactobacilli were characterized for their potential to develop riboflavin enriched

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fermented foods in the future.

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MATERIALS AND METHODS

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Bacterial Strains and Growth Conditions. The lactobacilli used in this work were putative

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riboflavin producing Lactobacillus strains reported in our previous study23. All the strains

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stored previously at -80ºC in MRS supplemented with glycerol (20% v/v) were routinely

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cultured on de Man-Rogosa -Sharp (MRS) medium (Sigma- Aldrich (St. Louis MO USA) for

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this study.

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Polymerase chain reaction (PCR) based screening for riboflavin biosynthesis genes. The

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presence of genes involved in the biosynthesis of riboflavin such as ribG, ribB, ribA and ribH

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in lactobacilli was determined by PCR. The rib operon region was amplified by using primer

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pairs (Table 1) for the rapid selection of candidate riboflavin-producing strains in touchdown

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cycling parameters with a gradual decrease of annealing temperature from a higher annealing

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temperature to an optimised temperature and, finally 30 more cycles were performed in the

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lowest annealing temperature (Eppendorf Mastercycler, Hamburg, Germany) (Table 2). The

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DNA from strain Lactobacillus fermentum MTCC 8711 was used as a positive control.

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Lactobacillus fermentum IFO3956 (NC_010610) was considered for primer selection and

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primers were designed for PCR analysis using Oligo Analyzer 3.0 software (Integrated DNA

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Technologies, Inc.; www.idtdna.com) and their specificity was ensured in silico using Blast

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analysis. The purified amplified PCR products were sequenced and analysed using the

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Chromas software (version 1.45, http:/ /www.technelysium. com.au/chromas.html) and were

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further compared with known sequences using BLAST tool of the U.S. National Centre for

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Biotechnology Information (blast.ncbi.nlm.nih.gov).

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Selection of riboflavin-producing strains by Microbiological Assay. The assay was carried

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out as described by Salvetti et al., (2003)24. The riboflavin-free assay medium (RAM, Difco,

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Becton, Dickinson, and Co., Sparks, Maryland, USA) was used for this purpose. The assay

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was performed as per instructions of BD Difco for riboflavin assay. Briefly, riboflavin

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auxotroph (L. casei subsp. rhamnosus ATCC 7469) was inoculated into 10 ml of micro

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inoculum broth. Following incubation for 16-24 h at 35-37°C, the culture was centrifuged

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under aseptic conditions and the supernatant liquid was decanted. After washing 3 times with

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10 ml sterile 0.85% saline, the cells were re-suspended in 10 ml sterile 0.85% saline to get the

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cell count of riboflavin auxotroph strain equivalent to 1 x 10-2 cells/mL. This suspension was

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used to inoculate each of the assay tubes. RAM was used for both turbidimetric and agar

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diffusion method. Turbidimetric readings were made after 18-24 h incubation at 35-37°C,

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whereas, in case of agar diffusion assay, readings were taken after 72 h incubation at 35-

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37°C. Test cultures were grown in RAM broth. After 6 h at 37ºC, bacterial cells were pelleted

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by centrifugation at 3800g for 10 min. The cell suspension of test cultures was then diluted

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with sterile 0.85% saline, to a turbidity of 35-40% transmittance when read on the

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spectrophotometer at 660 nm. RAM broth tubes were inoculated with 100µL of auxotroph

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culture (OD 0.2) and 1.0 ml of supernatant of test cultures was added. The tubes were

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covered with foil paper to avoid the direct exposure to light. After 12 h at 37ºC, tubes were

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analyzed for the turbidity due to increase in growth density of riboflavin auxotroph. The

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riboflavin concentration was measured from standard curve prepared using known

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concentrations of riboflavin.

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The inocula of auxotroph and test cultures were used for agar diffusion assay, to seed RAM

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plates with 6 spots (10 µL) of test cultures around each spot of riboflavin auxotroph (10 µL).

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The plates were wrapped in foil paper to avoid the direct exposure to light. After 18 h at

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37ºC, plates were analysed for growth of riboflavin auxotroph and kept for 24h at 37ºC. The

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strains which did not grow in riboflavin-free medium were not used in further studies.

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Quantitative analysis of riboflavin25.

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The determination of riboflavin concentration was carried out with a HPLC equipped with

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fluorescence detector (Shimadzu Shimadzu RID10A, Japan) and the excitation and emission

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wavelengths were 445 and 530 nm, respectively. The chromatography was carried out on

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C18 reversed phase column. Mobile phase was an isocratic system consisting of a mixture of

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water methanol- acetic acid (68:32:0.1v/v). All solvents used in the mobile phase were HPLC

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grade from Merck Pvt. Ltd. (Mumbai, India) and degassed with helium gas for 10 min. A

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C18 column Pursuit (XRs 5 C18, 150 mm × 4.6 mm, Varian) was used. Standard curve was

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realized with different dilutions of commercial riboflavin (Sigma- Aldrich (St. Louis MO

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USA)). A volume of 10 ml of inoculated medium was acidified to pH 3 with aqueous glacial

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acetic acid (1:1v/v) added drop wise with constant stirring and then centrifuged at 15,000g

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force for 15 min. The supernatant was transferred to 25 ml volumetric flask and the sediment

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was washed twice with 5.0 ml of 2% acetic acid solutions and centrifuged at 15,000g force

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for 15 min. After removal of cells by centrifugation (5,000g, 15 min), the clear broth was

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passed through a 0.22 µm membrane filter and riboflavin was analysed directly by HPLC.

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The identification of the peak in the unknown sample was made through the comparison of

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retention times of the reference standards.

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RESULTS AND DISCUSSION

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Screening of riboflavin producing isolates by PCR based method. A total of 59 isolates

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were screened by PCR for the presence of the riboflavin operon using (4) primers set RibF

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and RibR. Among 59 isolates, 19 isolates (L. fermentum, L. plantarum, L. mucosae, L. casei)

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were found positive after this preliminary PCR-based screening, suggesting possible rib

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operon conservation (Figure 1).

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In the present study, it was observed that among the tested species, fermentum, plantarum,

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delrueckii subsp. bulgaricus and mucosae possessed complete rib structural genes with

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specific amplification products Primer1 Rib1(990bp), Primer2 Rib2 (856bp), Primer3 Rib3

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(914bp) and Primer4 Rib4 (793bp) encoding RibH, RibA, RibB and RibG respectively. The

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remaining 22 isolates have shown incomplete Rib structural genes (Figure 2) and rest of the

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isolates were found devoid of Rib structural genes (supplementary data Figure 2, 3). It was

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also reported that when the Rib 4 was absent in the isolates, it was more likely that the operon

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was incomplete (Figure 2 and (supplementary data Figure 2, 3). This was in the support of

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previously reported bioinformatics analysis which had shown that when LAB contain an

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incomplete rib operon, the first genes are absent from the genome26. The presence of

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riboflavin biosynthetic genes and their biosynthetic ability was not found to be conserved

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within all examined LAB genomes as it was depicted through insilico analysis

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(Supplementary data Figure 1) as demonstrated by homology searches. The presence or

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absence of the rib biosynthetic genes does not appear to be linked to whether the LAB in

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question is a hetero- or homofermentative species, whether they are pathogenic species or

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phylogenetically closely related26. In LAB, the riboflavin synthesis genes form a single

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operon with the same order of genes: riboflavin-specific deaminase and reductase or ribG,

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riboflavin synthase alpha subunit or ribB, a bifunctional enzyme which also catalyses the

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formation of 3,4-dihydroxy-2-butanone 4-phosphate from ribulose 5-phosphate or ribA, and

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riboflavin synthase beta subunit or ribH22. The genetic information for riboflavin biosynthesis

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is species and/or strain-specific traits in LAB. Comparative genome analysis suggests that the

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ability to synthesize riboflavin is shared by several of the sequenced members of LAB

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although an interrupted rib operon is sometimes observed in certain strains. The lack of

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genetic information is usually associated with the inability to produce riboflavin in LAB. For

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example, the sequenced genome of L. plantarum strain WCFS1 contains an incomplete rib

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operon, which is devoid of the entire ribG and part of the ribB gene27. Burges et al28, seem to

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suggest that some species have lost the entire rib operon (or never possessed it), whereas in

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some cases, such as L. plantarum, only part of the operon appears to have been lost. As

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expected, this strain was not unable to grow in the absence of riboflavin28.

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However, several selected strains of L. plantarum harbour the whole rib operon and are able

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to synthesize riboflavin. L. plantarum NCDO 175228, and the recently sequenced L.

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plantarum JDMI and L. plantarum strains isolated from cereals-derived products11 are good

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examples. The presumed riboflavin biosynthesis genes of L. lactis subsp. cremoris MG1363

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were identified by homology with the characterized Bacillus subtilis rib genes and the

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annotated Lactococcus. lactis IL-1403 rib genes29,30.These homologies were used to design

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primers for NZ9000 in order to obtain and determine the sequence of the entire rib operon.

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The two Lc. lactis subspecies, as well as Leuconostoc mesenteroides and Pediococcus

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pentosaceus ATCC 25745, harbour a complete set of riboflavin biosynthetic genes in the

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same order as in B. subtilis2. Also, both Streptococcus pneumoniae strains TIGR4 and R6 and

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Streptococcus agalactiae 2603V/R contain the full complement of similarly organized

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riboflavin biosynthesis genes, in contrast to other streptococcal species (S. thermophiles

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LMD-9 and S. mitis) which do not appear to contain such homologues. In S. pneumoniae and

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Lc. lactis IL-1403 structurally conserved RFN elements have been previously identified2.

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This region has also been identified in other LAB containing the complete rib operon. Valle

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et al., (2014) have reported the presence of complete riboflavin operon in L. plantarum

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CRL725 while evaluating over 179 strains of LAB to increase the riboflavin levels in

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soymilk31. Identification of genes involved in riboflavin biosynthesis was carried only in L.

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plantarum CRL 725 using primers designed based on conserved sequences of previously

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sequenced LAB. The PCR products were sequenced (data not shown) and in this way it was

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possible to confirm that L. plantarum CRL 725 possesses all the genes that encode enzymes

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involved in different steps in the biosynthesis of riboflavin.

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Microbiological assay method and HPLC. In one study24, a number of probiotic

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formulations has been analysed for riboflavin secretion during growth by using riboflavin

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deficient medium (RAM) plates, indicating that each of the strains synthesized riboflavin for

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their own need24. Most of the rib+ isolates were found to grow in the absence of riboflavin,

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indicating a functional riboflavin biosynthetic capability.

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These isolates were grown in RAM and, after the entry in the stationary phase, the

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supernatant was analysed for riboflavin secretion into medium. The microbiological method

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available for determining riboflavin concentration is based on the use of Lc. casei ATCC

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7469. The sensitive growth response of this auxotroph to riboflavin was used to develop one

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of the first microbiological assays for a vitamin32 and is based on the presence of an efficient

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transport system that allows the uptake of exogenous riboflavin. Riboflavin uptake inversely

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correlates with the riboflavin concentration present during cell growth and increases in

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riboflavin requiring mutants33. This organism is highly sensitive to variations in the riboflavin

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concentration added to the growth medium34, thus being suitable for accurate determinations

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of riboflavin amounts in various samples by turbidimetric and acidimetric assays35. In our

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study, initially, all the 30 isolates were inoculated into RAM medium which is devoid of

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riboflavin to screen the isolates for functional riboflavin operon. It was observed that isolates

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possessing incomplete operon were not able to survive in the medium conditions as reported

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earlier31. Most of the bacteria require riboflavin for their growth36. Out of 19 isolates carrying

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the complete rib operon, 14 isolates were found to grow well in the absence of riboflavin,

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indicating a functional riboflavin biosynthetic capability. These isolates were further selected

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for turbidimetric assay and agar diffusion assay. The rest of the isolates which did not survive

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in RAM indicating non-functional riboflavin operon were not carried further. We have used

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turbidimetric assay to evaluate the ATCC7469 growth response to increasing concentrations

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of exogenous vitamin riboflavin. For turbidimetric assay, the supernatant of the isolates, (L.

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fermentum KTLF1, L. plantarum KTLP13, L. fermentum KTF3 L. fermentum KTF2, L.

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plantarum KTP11, L. fermentum KTLF25 and L. delbrueckii. subsp. bulgaricus KTB10)

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showed significant increase in the growth density of riboflavin auxotroph (in terms of OD of

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cells) (Figure 3.1). To evaluate the riboflavin secretion, the test isolates were grown on RAM

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plates in proximity of an ATCC7469 spot inoculum. For agar diffusion assay, it was observed

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that the growth of riboflavin auxotroph (white sport in the middle) started after 18h in the

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presence of riboflavin producing isolates (yellowish spots) (Figure 3.2).

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In the present study, the isolates KTLF1, KTLP13 and KTLF3 were able to grow well on

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RAM agar as well as supported the growth of riboflavin auxotroph strain. The isolate KTLF5

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was not able to sustain the growth of riboflavin auxotroph since the riboflavin produced by

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this isolate is less than 0.5mg/l23. The chromatogram of test isolates showed different peaks

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corresponding to different components present in sample as per procedure described by

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Ashoor et al., (1983)25. The peak at retention time of 5.49 ±0.4 corresponds to riboflavin

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(Figure 4). The average values of riboflavin produced by KTLF1 and KTP13 were 0.75mg/L

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and 0.65mg/L respectively.

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As there is no method available which is completely effective, study was aimed to compare

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the actual presence of all 4 riboflavin genes vis-a-vis production of the vitamin. This study

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highlights the microbial diversity of riboflavin producing strains isolated from various

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sources. The findings suggest that the lactobacilli isolated from human faeces and fermented

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bamboo shoots have shown maximum riboflavin production. The incomplete riboflavin

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operon was observed in most of the isolates of dairy origin. The data generated in this study

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suggest that isolates of human and plant origins were more likely to possess complete

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riboflavin operon. The bioavailability of riboflavin produced by these isolates can be

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evaluated in the suitable animal models by inducing riboflavin deficiency markers. The

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putative riboflavin producing isolates have shown appreciable probiotic attributes37 which

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can be an economically feasible biotechnological strategy that could be easily adopted by the

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food industry to develop novel riboflavin bio-enriched functional foods with enhanced

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consumer appeal.

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There is no data available till date through which the authors can justify the reason why the

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strains isolated from human faeces and fermented bamboo shoots are prolific riboflavin

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producers.

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Conflict of Interest

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Authors declare no conflict of interest.

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Acknowledgments

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The authors thankfully acknowledge the support extended by Indian Council of Agricultural

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Research (ICAR), New Delhi and the Director, National Dairy Research Institute (NDRI

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Karnal, India).

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Supporting Information

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Figure 1. 1.8% Agarose Gel electrophoresis showing the amplification of Riboflavin operon

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in Lactobacillus fermentum MTC8711 (Positive control), C= Lactobacillus casei 1407

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(Negative control) and Lactobacillus fermentum (k1). Figure 2.

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electrophoresis showing the presence/ absence of riboflavin biosynthesis genes in different

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strains of Lactobacilli as designated with ABCDEF. Figure 3.

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electrophoresis showing the presence/ absence of riboflavin biosynthesis genes in different

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strains of Lactobacilli. Figure 4. 1.8% Agarose Gel electrophoresis showing the presence/

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absence of riboflavin biosynthesis genes in different strains of Lactobacilli. This material is

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available free of charge via the Internet at http://pubs.acs.org.

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1.8% Agarose Gel

1.8% Agarose Gel


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17) Kurth, R.; Paust, J.; Hanlein, W. Riboflavin. pp. 521–530. Ullmann's Encyclopedia Industrial Chemistry. 1996, Weinheim, Germany: Wiley-VCH.

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Fares, C.; Spano, G. Biotechnological production of vitamin B2-enriched bread and

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pasta. J. Agric. Food Chem. 2011, 59, 8013–8020.

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19) Laino, J. E.; Juarez del Valle, M.; Savoy de Giori, G.; LeBlanc, J. G. Development of

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bacteria. LWT — Food Sci Technol, 2012, 54, 1–5.

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20) Vaesken, S. M.; Aperte, A. E.; Moreiras, V. G. Vitamin food fortification today. Food Nutr Res, 2012, 56: 5459. 21) Stiles, M. E. Biopreservation by lactic acid bacteria. Antonie van Leeuwenhoek . 2004, 70, 331–345.

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producing B-group vitamins: a great potential for functional cereals products. Appl

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Microbiol Biotechnol. 2012, 96, 1383-1394.

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Ehrlich, Sorokin. A. The complete genome sequence of the lactic acid bacterium

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34) Freed, M. Methods of Vitamin Assay ed. The Association of Vitamin Chemists Inc.New York: Interscience Publishers, 1966. 35) AOAC. Official Methods of Analysis, 13th edn. pp. 760–766. Washington, DC: Association of Official Analytical Chemists, 1980. 36) Koser, S. A. Vitamin requirements of bacteria and yeasts. Charles C. Thomas, Springfield, IL, 1968. 37) Thakur, K.; Tomar, S. K .Invitro study of Riboflavin producing lactobacilli a potential Probiotic. LWT-Food science and technology, (2015)(In press)


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Legends to Tables

468

Table 1: Primers used for amplification of riboflavin biosynthesis genes in Lactobacilli

469

Table 2: Touch down PCR conditions for amplification of riboflavin biosynthesis genes

470

Table 3: The riboflavin producing species identified in the present study

471 472

Legends to Figures

473 474 475 476

Figure 1: Agarose gel (1.5%) electrophoresis showing amplified PCR products obtained by 4 rib primers set (Lane 1=1kb ladder, Lane2= KTLP (Primer1), Lane3= KTLP (Primer2), Lane 4= KTLP (Primer3), Lane 5= KTLP (Primer4), Lane 6=1kb ladder

477

Figure 2: Presence /absence of rib genes among various Lactobacillus isolates (Presence of

478

box represents the presence of genes)

479 480 481 482 483

Figure 3.1: Graphical representation of comparison of increase in the growth of riboflavin Auxotroph in terms of the OD values in the presence of supernatant of riboflavin producing Lactobacillus isolates. Different superscript (a-f) bearing isolates differ significantly at P < 0.05. Superscript marked by same letter are not different at P < 0.05.

484 485 486

Figure 3.2: Turbidimetric assay method to observe the increase in the growth of riboflavin Auxotroph in the presence of supernatant of riboflavin producing Lactobacillus isolates

487 488 489 490 491

Figure 3.3: Agar Diffusion assay method to observe the growth of riboflavin auxotroph (Middle spot) in the presence of supernatant of riboflavin producing Lactobacillus isolates spotted around the auxotroph. The number 1, 2, 3, 4 and 5 corresponds to KTF1, KTF5, KTP13, KTP16 and KTF2.

492 493 494

Figure 4: A typical HPLC profile of 2 prolific riboflavin producing isolates in Riboflavin free medium

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Page 16 of 25

Table 1

Sr. No.

Gene

1 Rib H

2 Rib A 3 Rib B 4 Rib G

Primer Sequence (5’-3’)

Tm

Product

(ºC)

size

F’ CGGAGTGGCCGGTTTTGCTTTGG

61

R’ GGGGACCCGACCACGACTAC

60

F’

60

GTGAAGCACTCGGAGTGGACCC

R’ CACCCAGACCGTTACCAACCTG

59

F’ GGTTTCTAAGTTGACTAGGTCGCCAAC

60

R’ GACCGCTTTGTCTTCACCGATGCTTC

61

F’ GCCCGGTTGGTGGTGACTAACCA

61

R’ GGAGGTTGGTTCCCACTCACCTATG

61

Table 2


990bp

856 bp

914bp

793bp

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Step No.

Process

Temp/Time

1

Initial Denaturation

95ºC (2-3mins)

2

Denaturation

95ºC (30sec)

3

Annealing

64ºC (25sec)

4

Extension

72ºC (30sec)

5

Repeat steps 2 to 4 (for 6 cycles). Decrease annealing temperature by 1 ºC after each cycle

6

Denaturation

95ºC (30sec)

7

Annealing

59ºC (35sec)

8

Extension

72ºC (30sec)

9

Repeat steps 6 to 8 ( for 25 cycles)

10

Extension

72ºC (4 min)

11

Holding

4ºC

Table 3

Strain designation K1 K2 K3 K4 K5 K6 K7 K8 K9 K10 K11 K12 K13 K14 K15 K16

Species Lactobacillus fermentum Lactobacillus plantarum Lactobacillus plantarum Lactobacillus plantarum Lactobacillus fermentum Lactobacillus del. bulgaricus Lactobacillus brevis Lactobacillus fermentum Lactobacillus plantarum Lactobacillus reuteri Lactobacillus fermentum Lactobacillus mucosae Lactobacillus fermentum Lactobacillus plantarum Lactobacillus del. bulgaricus Lactobacillus fermentum (MTCC8711)



K17 K18 K19 K20 K21 K22 K23 K24 K25 K26 K27 K28 K29 K30 K31 K32 K33 K34 K35 K36 K37 K38 K39 K40 K41 K42

Lactobacillus casei (MTCC1407) Lactobacillus fermentum Lactobacillus fermentum Lactobacillus plantarum Lactobacillus acidophilus Lactobacillus acidophilus Lactobacillus acidophilus Lactobacillus fermentum Lactobacillus fermentum Lactobacillus plantarum Lactobacillus casei Lactobacillus casei Lactobacillus plantarum Lactobacillus ramnosus Lactobacillus plantarum Lactobacillus casei Lactobacillus acidophilus Lactobacillus casei Lactobacillus plantarum Lactobacillus ramnosus Lactobacillus casei Lactobacillus plantarum Lactobacillus plantarum Lactobacillus casei Lactobacillus plantarum Lactobacillus ramnosus


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Figure 1



Figure 2


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Figure 3.1

Figure 3.2



Figure 3.3


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Figure 4




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TOC Graphic


Screening of Riboflavin-Producing Lactobacilli by a Polymerase

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