Characterization of extracellular vitamin B12 producing Lactobacillus plantarum strains and assessment of the probiotic potentials

Accepted Manuscript Characterization of extracellular vitamin B12 producing Lactobacillus plantarum strains and assessment of the probiotic potentials Ping Li, Qing Gu, Lanlan Yang, Yue Yu, Yuejiao Wang PII: DOI: Reference:

S0308-8146(17)30815-4 http://dx.doi.org/10.1016/j.foodchem.2017.05.037 FOCH 21098

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

7 February 2016 3 May 2017 6 May 2017

Please cite this article as: Li, P., Gu, Q., Yang, L., Yu, Y., Wang, Y., Characterization of extracellular vitamin B12 producing Lactobacillus plantarum strains and assessment of the probiotic potentials, Food Chemistry (2017), doi: http://dx.doi.org/10.1016/j.foodchem.2017.05.037

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Characterization of extracellular vitamin B12 producing Lactobacillus plantarum strains and assessment of the probiotic potentials Running title: Extracellular vitamin B12 producing L. plantarum strains Ping Li, Qing Gu*, Lanlan Yang, Yue Yu, Yuejiao Wang Zhejiang Gongshang University, Key Laboratory for Food Microbial Technology of Zhejiang Province, Hangzhou 310018, China *Corresponding author. E-mail address: [email protected] (Q. Gu). [email protected] (P. Li). [email protected] (L. Yang). [email protected] (Y. Yu). [email protected] (J. Wang).

1

ABSTRACT We investigated extracellular vitamin B12-producing Lactobacillus strains and their characteristics in tolerance to environmental stresses, gastric acid and bile salts. Two isolates, Lactobacillus plantarum LZ95 and CY2, showed high extracellular B12 production, 98 ± 15 µg/L and 60 ± 9 µg/L respectively. Extracellular B12 from LZ95 were identified as adenosylcobalamin and methylcobalamin using a combination of solid phase extraction and reverse-phase HPLC, while that from CY2 was adenosylcobalamin. Both strains grew under environmental stresses, and LZ95 exhibited better tolerance to low temperature and high ethanol concentration. LZ95 also showed good viability when exposed to gastric acid (pH 2.0 and 3.0) and bile salts (0.3%) as well as good adhesion to Caco-2 cells. The viability of CY2 was significantly reduced under low pH and exposure to bile salt. Together, extracellular B12 producer LZ95 with good probiotic properties might be a candidate for in situ B12 fortification in the food industry. Keywords:

vitamin

B12,

extracellular,

fortification

2

Lactobacillus

plantarum,

probiotic,

Introduction Vitamin B12, also known as cobalamin, is a water-soluble vitamin that functions as a cofactor in fatty acid and amino acid metabolism, hemoglobin synthesis, and energy production as well as DNA synthesis and regulation. It is synthesized by some bacteria and archaea, and is concentrated in animal tissues but not in higher plants (Nielsen, Rasmussen, Andersen, Nexo, & Moestrup, 2012). Humans cannot produce vitamin B12 but have nutritional requirements for it, and they obtain vitamin B12 mainly from foods derived from animals, such as milk, meat and eggs. Deficiency of vitamin B12 is associated with hematological and neurological disorders, causing pernicious

anemia,

peripheral

neuritis,

anemia,

coronary

disease,

stroke,

hyperhomocysteinaemia and myocardial infarction (Pawlak, 2015). Strict vegetarians with low intakes of animal-source foods and elderly populations with certain gastric dysfunctions are at higher risk of developing B12 deficiency. B12 deficiency also occurs commonly in countries, such as India, due to lacto-vegetarianism and a scarcity of meat (Green, 2009; Pawlak, 2015; Watanabe, Yabuta, Tanioka, & Bito, 2013). B12-fortified foods and B12-containing dietary supplements have been considered to be good alternatives to prevent this deficiency in recent years (Watanabe, Yabuta, Tanioka, & Bito, 2013). However, most of the B12 for fortification are chemically synthetized, which is costly and may cause undesirable side effects. In comparison, use of vitamin-producing microorganisms for in situ fortification is a feasible and economical alternative, and it is less likely to cause side effects from elevated concentrations of vitamins. Fermentation with food-grade bacteria is a good strategy to improve the nutritional values and vitamin contents of food products. The generally recognized as safe (GRAS) status lactic acid bacteria (LAB), especially strains belonging to genus 3

Lactobacillus, are the best known probiotics and widely used in fermented foods (LeBlanc, Laino, del Valle, Vannini, van Sinderen, Taranto, et al., 2011; Taranto, Vera, Hugenholtz, De Valdez, & Sesma, 2003). Addition of folate-, riboflavin- and vitamin B12-producing LABs into fermented milk, yogurt, or soybean could potentially increase the vitamin concentrations (LeBlanc, Burgess, Sesma, Savoy de Giori, & van Sinderen, 2005; Mo, Kariluoto, Piironen, Zhu, Sanders, Vincken, et al., 2013). The advantage of in situ fortification therefore makes LABs as ideal candidates to supply vitamins to human hosts (Burgess, Smid, & van Sinderen, 2009; Gu, Zhang, Song, Li, & Zhu, 2015; Santos, Wegkamp, de Vos, Smid, & Hugenholtz, 2008). Lactic acid bacterial (LAB) strains have been found to be vitamin B12 producers (LeBlanc, Laino, del Valle, Vannini, van Sinderen, Taranto, et al., 2011; Taranto, Vera, Hugenholtz, De Valdez, & Sesma, 2003). Lactobacillus reuteri CRL1098, a strain isolated from sourdough, was the first B12-producer among LABs (Taranto, Vera, Hugenholtz, De Valdez, & Sesma, 2003). Vitamin B12 synthesized by Lactobacillus reuteri was confirmed to be biological active and capable of preventing diseases caused by vitamin B12 deficiency in both pregnant mice and the weaned young (Molina, Medici, Taranto, & Font de Valdez, 2009). Genetic evidence has shown that Lactobacillus reuteri CRL 1098 contains at least 30 genes in the cob-pdu gene cluster involved in the de novo synthesis of vitamin B12. In addition to CRL1098, other Lactobacillus reuteri strains were shown to produce B12, such as Lactobacillus reuteri JCM1112 (Santos, Wegkamp, de Vos, Smid, & Hugenholtz, 2008), DSM 20016 (Sriramulu, Liang, Hernandez-Romero, Raux-Deery, Lunsdorf, Parsons, et al., 2008), CRL 1324 and 1327 (LeBlanc, et al., 2011). We have isolated a B12-producing strain Lactobacillus reuteri ZJ03, the addition of which made the vitamin B12 content of the fermented soy-yogurt higher than other fermented soybean-based food (Gu, 4

Zhang, Song, Li, & Zhu, 2015). Recently, other strains in the genus Lactobacillus were shown to produce cobalamin-type compounds, including Lactobacillus coryniformis isolated from goat milk (Martin, Olivares, Marin, Xaus, Fernandez, & Rodriguez, 2005), Lactobacillus plantarum from kanjika or Japanese pickles (Madhu, Giribhattanavar, Narayan, & Prapulla, 2010; Masuda, Ide, Utsumi, Niiro, Shimamura, & Murata, 2012), Lactobacillus rossiae from sourdoughs (De Angelis, Bottacini, Fosso, Kelleher, Calasso, Di Cagno, et al., 2014), and Lactobacillus fermentum CFR 2195 from breast fed healthy infants’ fecal (Basavanna & Prapulla, 2013). Moreover, the genetic and biochemical data suggested that B12 biosynthesis genes likely have been spread to genus Lactobacillus. Lactobacillus buchneri, Lactobacillus hilgardii and Lactobacillus brevis also contain genes of the cob-pdu gene cluster (Capozzi, Russo, Duenas, Lopez, & Spano, 2012). Therefore, the study of various cobalamin-producing strains and species of LAB would be beneficial for not only the understanding on cobalamin production, but also the development of vitamin B12-containing fermented products. Extracellular production of vitamin B12 is considered a better alternative than the intracellular production for in situ fortification fermented foods, especially for dairy products. However, there were no reports on extracellular vitamin B12 production by Lactobacillus reuteri strains (LeBlanc, et al., 2011; Santos, Wegkamp, de Vos, Smid, & Hugenholtz, 2008; Taranto, Vera, Hugenholtz, De Valdez, & Sesma, 2003; Vannini, de Valdez, Taranto, & Sesma, 2008), and the extracellular vitamin B12 production by Lactobacillus coryniformis and Lactobacillus plantarum is relatively low (2 µg/L) (Masuda, Ide, Utsumi, Niiro, Shimamura, & Murata, 2012). The non-secretion property of Lactobacillus strains may limit their application in the food industry. In this study, we aim at isolating extracellular vitamin B12-producing 5

Lactobacillus strains from our lab stocks and evaluate their probiotic potentials for application in food industry. Two Lactobacillus plantarum strains, LZ95 originally from infant feces and CY2 from fresh milk, were identified to be capable of producing extracellular B12. Lactobacillus plantarum is a highly versatile lactic acid bacterium, with many excellent traits that meet the needs of industrial production, and some strains are marketed as starter cultures or probiotics (Siezen, Tzeneva, Castioni, Wels, Phan, Rademaker, et al., 2010). An ideal LAB strain with industrial potential should be able to survive harsh conditions, including the adverse conditions during food fermentation and the physical-chemical conditions in the gastrointestinal tract. Here, we measured the response of the extracellular vitamin B12-producing Lactobacillus plantarum strains to different environmental stresses (hot temperature, cold temperature, ethanol and NaCl); we also assayed their tolerance to gastric acid and bile salts, and their ability to adhere to the intestinal wall in vitro.

2. Materials and methods 2.1 Isolation of vitamin B12-producing Lactobacillus strains and culture conditions A total of 31 LAB strains originally isolated from fermented vegetables, fresh milk or infant feces by Key Laboratory for Food Microbial Technology of Zhejiang Province were used in this study. Each strain was grown in MRS broth (Luqiao, China) for 24 h at 37 °C without shaking. 30 µL of each culture was then centrifuged at 5000 g for 5 min. The obtained cell pellets were washed twice with sterilized PBS (pH7.3) and resuspended in 30 µL PBS, which were subsequently inoculated into 5 mL of vitamin B12-free assay medium (Luqiao, China), grown at 37 °C overnight and transferred eight times. All tubes were wrapped with alumina foil during incubation. 6

Strains showing ~0.5 of OD600 after eight rounds of growth were preliminarily determined as vitamin B12 producers. The isolates were then identified by 16S rRNA gene sequencing with primers 27F

(5’-AGAGT

TTGATCCTGGCTCAG-3’

forward

primer)

and

1495R

(5’-CTACGGCTACCTTGTTACGA-3’, reverse primer) (Liu, Bao, Jirimutu, Qing, Siriguleng, Chen, et al., 2012). The phylogenetic tree was constructed by CLUSTAL W using the neighbor-joining method within the MEGA.6 package. 2.2 Determination of extracellular and intracellular vitamin B12 production Vitamin B12 production of the isolated Lactobacillus strains were determined by a microbiological assay as described by Taranto et al., with some modifications (Taranto, Vera, Hugenholtz, De Valdez, & Sesma, 2003). Cell extract (CE) prepared according to the protocol of Taranto et al. from the pellets, and the supernatants obtained after centrifugation, were collected for analyzing the intracellular and extracellular vitamin B12 production, respectively. Lactobacillus delbrueckii subsp. lactis ATCC 7830, an indicator strain that requires B12 for growth (Kelleher & Broin, 1991), was used to evaluate the cobalamin contents. Growth was determined by measuring OD620 after 20 h of incubation at 37 °C. 2.3 HPLC analysis of vitamin B12 To identify the forms of extracellular B12 produced by Lactobacillus plantarum LZ95 and CY2, cells were grown in 100 mL of MRS broth (Luqiao, China) for 24 h at 37 °C, and the supernatants were collected by centrifugation. To clean the supernatants, 75 mL of supernatants were loaded onto five C18 solid-phase extraction (SPE) cartridges, which have been activated with 3 mL of methanol and washed with 6 mL of ddH2O. After washed with 3 mL of ddH2O twice, vitamin B12 was eluted 7

with 3 mL of 100% methanol from each SPE column. 15 mL of eluents were collected, rotary evaporated at 20–25 °C and redissolved in 10 mL of ddH2O. For further cleaning, 10 mL of the sample was loaded onto a SPE column, washed twice with 6 mL of distilled water, eluted by 60% methanol, evaporated, dissolved in 500 µL of ddH2O, filtered by 0.22 µm membranes and stored in the dark until used. The entire procedure was carried out under alumina foil wrapping condition to reduce risk of cobalamin photo-degradation (Heudi, Kilinc, Fontannaz, & Marley, 2006). The pre-cleaned and concentrated samples were passed through a C18 column (4.6 mm × 250 mm, 5 µm, Waters) and analyzed by a reverse-phase (RP)-HPLC with Waters 2535 quaternary gradient module system and Waters 2998 photodiode array (PDA) detector. A flow of 0.5 mL/min of methanol with 0.1% formic acid (A) (Merck, Darmstadt, Germany) and ultra-purified water with 0.1% formic acid (B), which was degassed by an ultrasonic water bath (Sonorex TK 52, ultrasonic water bath, Bandelin Electronics, Berlin, Germany), was used as the mobile phase, and the gradient elution was programmed as follows: 0–2 min 20% A, 2–3 min 20–28% A, 3–11 min 28–35% A, 11–19 min 35–20% A, 20–22 min 100–100% A, 22–26 min 100–20% A, and 26–36 min 20% A (Gu, Zhang, Song, Li, & Zhu, 2015). The injection volume was 100 µL. UV-vis spectrums were scanned from 210 nm to 800 nm and diode array detection was carried at a wavelength of 361 nm. The retention times of authentic standards

of

cyanocobalamin

(CN-Cbl),

methylcobalamin

(Me-Cbl),

adenosylcobalamin (Ado-Cbl) and hydroxocobalamin (OH-Cbl) were also recorded. 2.4 Environmental stress tolerance assay Each single colony was inoculated in MRS broth for 24 h at 37 °C. 0.1 mL culture, in which the cells were at stationary phase, was transferred into 5 mL MRS

8

broth twice. Cells was collected by centrifugation (5000 g, 5 min), washed three times by PBS (pH7.0), resuspended in an equal volume of PBS and subsequently applied for environmental stress tolerance assay, simulated gastric juice tolerance assay and bile salt tolerance assay. The total viable count of the washed cell suspension was determined prior to the assays. For environmental stress tolerance assay, the growth of Lactobacilli isolates was observed at different incubation temperatures (15 °C, 25 °C, 37 °C, and 45 °C), various concentrations of NaCl (0, 3%, 6%, 8%, and 10%) and ethanol (0, 2.5%, 5%, 10%, and 15%). 30 µL cell in PBS was inoculated into 5 mL of MRS broth containing of salt or ethanol, and incubated at 37 °C. The growth was evaluated by measuring OD620 after 24 h of incubation without shaking. 2.5 Simulated gastric juice tolerance assay The tolerance of isolated Lactobacillus strains to simulated gastric juices was studied as described by Charteris et al. (Charteris, Kelly, Morelli, & Collins, 1998). Simulated gastric juices were prepared by suspending pepsin (Sigma-Aldrich) in PBS buffer to a final concentration of 0.3% (w/v), with pH 2.0, 3.0 and 4.0, respectively. 200 µL cell in PBS as mentioned in section 2.4 was mixed with 300 µL NaCl (0.5% w/v) and 1 mL of simulated gastric juice. An aliquot of 100 µL was removed after incubation at 37 °C for 0 h, 2 h and 4 h, and then plated on MRS plates with appropriate dilutions. The total viable counts of the strains were determined anaerobically after 48 h of incubation at 37 °C and the number of viable cells was expressed as log10 CFU/mL. 2.6 Bile salt tolerance assay The simulated small intestinal juices were prepared by suspending pancreatin 9

(Sigma-Aldrich) into sterile saline to a final concentration of 1 g/L and adjusting the pH to 8.0, with or without 0.3% bile salts (Sigma-Aldrich) (Charteris, Kelly, Morelli, & Collins, 1998). 200 µL of cell suspension in PBS was diluted with 300 µL NaCl (0.5% w/v) and 1mL of simulated small intestinal juices at 37 °C. As described in previous section, at regular time intervals (0 h, 2 h and 4 h), the residual viability was determined by the count plate method. 2.7 In vitro adhesion assays The adherence of the isolates was examined using human colon adenocarcinoma cell line Caco-2, as described by Pisano et al., with some modifications (Pisano, Viale, Conti, Fadda, Deplano, Melis, et al., 2014). Briefly, cells were grown in DMEM medium (Thermo Fisher scientific) containing 1 mM sodium pyruvate, 25 mM glucose and supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 1% non-essential amino acid preparation, 100 U/mL penicillin and 100 µg/mL streptomycin. For the adherence assays, Caco-2 cells were cultured to confluence in 2 ml of medium without antibiotics. Approximately 10 days post-confluence, 1 mL of the medium was replaced with 1 mL of Lactobacillus cells suspension (2 × 108 CFU/mL). After incubation at 37 °C in 5% CO2 for 1.5 h, the monolayer was washed five times with sterile PBS, and then cells with adherent bacteria were harvested with Triton-100X, centrifuged at 10,000 g for 5 min, suspended in PBS, serially diluted, and finally inoculated on MRS plates. The viable counts of the bacteria were determined after 48 h of incubation at 37 °C and the adhesion rate was calculated. 2.8 Statistical analysis The experiments were performed in triplicates and the values were indicated as 10

the mean ± SD. Student’s t test was used for statistical analysis.

3. Results and discussion 3.1 Isolation of vitamin B12-producing Lactobacillus strains 18 of the 31 LAB strains from our lab stocks showed the growth in the vitamin B12-free assay medium after eight rounds of transfer and thus were preliminarily defined as vitamin B12 producers. The vitamin B12-producing strain Lactobacillus reuteri ZJ03 was used as a positive control (Gu, Zhang, Song, Li, & Zhu, 2015). 16S rDNA nucleotide sequences of the strains were amplified by PCR and the species were

initially

determined

by

the

BLAST

program

on

NCBI

(http://www.ncbi.nlm.nih.gov/). 7 of the 18 LAB strains were identified as Lactobacillus strains and displayed a similarity of ≥ 99% to type strains deposited in NCBI. As shown in Table 1, six of them (ZJ5, ZJ316, LZ95, CY2, CY3 and ZJ008) are Lactobacillus plantarum, and one is Lactobacillus casei (LZ54). This is the first report of a Lactobacillus casei strain to produce vitamin B12, although members of the genus Lactobacillus have been reported as vitamin B12 producers, including Lactobacillus reuteri (Gu, Zhang, Song, Li, & Zhu, 2015; Santos, et al., 2008; Santos, Wegkamp, de Vos, Smid, & Hugenholtz, 2008; Taranto, Vera, Hugenholtz, De Valdez, & Sesma, 2003; Vannini, de Valdez, Taranto, & Sesma, 2008), Lactobacillus coryniformis (Martin, Olivares, Marin, Xaus, Fernandez, & Rodriguez, 2005), Lactobacillus plantarum (Madhu, Giribhattanavar, Narayan, & Prapulla, 2010; Masuda, Ide, Utsumi, Niiro, Shimamura, & Murata, 2012), Lactobacillus fermentum (Basavanna & Prapulla, 2013), and Lactobacillus rossiae (De Angelis, et al., 2014). The vitamin B12 production property of the isolates is likely attributed to the presence 11

of B12 biosynthetic gene cluster which encodes the enzymes required for the synthesis of this vitamin (Santos, et al., 2008). Whole genome sequencing would help to verify this hypothesis. Both Lactobacillus plantarum and Lactobacillus casei are the most common organisms in gastrointestinal tract of humans and have the potential to function as probiotics (Hosseini Nezhad, Hussain, & Britz, 2015; Siezen, et al., 2010). Thus, these Lactobacillus plantarum and Lactobacillus casei isolates may provide benefits for food industry. 3.2 Vitamin B12 production of the Lactobacillus strains Vitamin B12 content of the seven isolates was evaluated by microbiological assay (Taranto, Vera, Hugenholtz, De Valdez, & Sesma, 2003). As shown in Table 1, the reference strain Lactobacillus reuteri ZJ03 produced 174 µg/L of vitamin B12 in MRS broth intracellularly, which was about three times as that in the non-optimized soy-yogurt (68.10 µg/L), and about the same as that in the glycerol and fructose co-fermentated soy-yogurt (180.30 µg/L) (Gu, Zhang, Song, Li, & Zhu, 2015). Intracellular B12 production by the seven isolates varied from 19 µg/L to 996 µg/L. Three bacteriocin-synthesizing strains identified in our lab, ZJ316, ZJ5 and ZJ008 (Song, Zhu, & Gu, 2014; Suo, Yin, Wang, Lou, Song, Wang, et al., 2012; Zhu, Zhao, Sun, & Gu, 2014), produced less vitamin B12. Nevertheless, the four newly identified Lactobacillus strains LZ54, LZ95, CY2 and CY3 synthesized more vitamin B12 than Lactobacillus reuteri ZJ03. Two strains, LZ95 originally from infant feces and CY2 from fresh milk, showed the highest intracellular B12 production, at 996 µg/L and 565 µg/L, which were about 5.7 and 3.2 folds of the control respectively. Phylogenetic analysis demonstrated that both strains were closely related to the strain Lactobacillus plantarum WCFS1 (Fig. 1 and Table 1). Moreover, B12 production by Lactobacillus casei LZ54 (295 µg/L) is about 1.7 fold as the reference strain. Therefore, these four 12

newly identified isolates might be better candidates for soy-yogurt fermentation than Lactobacillus reuteri ZJ03. Intracellular nature of the vitamin is a major stumbling block for the application of their in situ fortification, therefore it is important to screen and characterize extracellular B12 producers. Few researches have been reported the extracellular B12 production in Lactobacillus. Masuda et al. investigated the extracellular B12 production of LAB isolates from nukazuke firstly (Masuda, Ide, Utsumi, Niiro, Shimamura, & Murata, 2012). Two strains, Lactobacillus coryniformis CN-229 and Lactobacillus plantarum CN-225, showed the highest extracellular B12 production level among the examined LABs, ~2 µg/L. In our study, it is noteworthy that the Lactobacillus plantarum LZ95 and CY2 showed extracellular B12 yields of 98 µg/L and 60 µg/L respectively, about 46 and 30 folds of that from the strain reported by Masuda et al. (Masuda, Ide, Utsumi, Niiro, Shimamura, & Murata, 2012). However, the extracellular B12 was only 8.9% and 9.6% of the total B12 content in LZ95 and CY2, further investigation is required to improve the extracellular B12 production. Metabolic engineering allows increased extracellular vitamin production by Lactobacillus not only through overexpressing the genes involved in the biosynthesis, but also via overexpressing genes involved in the biosynthesis pathway of related metabolites (LeBlanc, et al., 2011; Sybesma, Starrenburg, Kleerebezem, Mierau, de Vos, & Hugenholtz, 2003; Wegkamp, van Oorschot, de Vos, & Smid, 2007). Overexpression of two folate biosynthesis genes, folK and folE, in Lactobacillus lactis, resulted in 10-fold increase of extracellular folate production (Sybesma, Starrenburg, Kleerebezem, Mierau, de Vos, & Hugenholtz, 2003). Wegkamp et al. reported overproduction of pABA led to a relatively high secretion of folate (Wegkamp, van Oorschot, de Vos, & Smid, 2007). However, no studies have been 13

conducted to improve vitamin B12 production by metabolic engineering strategies in Lactobacillus strains so far. The biosynthetic pathways for the vitamin B12 has been reported (Burgess, Smid, & van Sinderen, 2009), moreover, Lactobacillus reuteri has been shown to encode all the genes necessary for vitamin B12 synthesis (Santos, et al., 2008), which provide major advantages for metabolic engineering strategies to improve extracellular B12 production by Lactobacillus reuteri. Meanwhile, the increased availability of genome sequences and the development of novel engineering tools for Lactobacillus would also make metabolic engineering a good strategy to improve extracellular B12 production (Capozzi, Russo, Duenas, Lopez, & Spano, 2012). 3.3. RP-HPLC analysis of vitamin B12 To confirm the extracellular B12 production and identify the forms of B12 produced by Lactobacillus plantarum LZ95 and CY2, RP-HPLC analyses were performed (Fig. 2). Fig. 2A shows the UV-Vis spectrum of vitamin B12 standards, with the identical peaks at ~361 nm and 550 nm. We confirmed the presence of B12 in the supernatants of LZ95 (Fig. 2A) and CY2 (Fig. 2B) based on the similarity of the UV-Vis spectra. Fig. 2C and Fig. 2D indicate the HPLC chromatograms of supernatants and standards. The retention times (RTs) of CN-Cbl, Ado-Cbl, OH-Cbl and Me-Cbl were 5.491 min, 8.484 min, 13.120 min and 17.215 min, respectively (Fig. 2C). The supernatant of Lactobacillus plantarum LZ95 showed two peaks with RTs of 8.501 and 17.095 min (Fig. 2C), indicating that the samples contained Ado-Cbl and Me-Cbl. Fig. 2D shows a single peak with RT of 8.528 min, indicating the presence of Ado-Cbl in the supernatant from Lactobacillus plantarum CY2. Taken together, both Lactobacillus plantarum LZ95 and CY2 strains contained the naturally occurring

14

vitamin B12 compounds (Nielsen, Rasmussen, Andersen, Nexo, & Moestrup, 2012), which might be suitable for diary industry application. 3.4 Tolerance of the Lactobacillus isolates to NaCl, temperature and ethanol An ideal strain with industrial potential should resist harsh conditions during the food fermentation process, such as extreme temperatures, ethanol and osmotic stresses. We focused on the two strains with extracellular B12 production (Lactobacillus plantarum LZ95 and CY2) and a strain with the third highest vitamin B12 production amongst the isolates (Lactobacillus plantarum CY3, Table 1), and examined their tolerance to different incubation temperatures, various concentrations of NaCl and ethanol. Growth of each strain (OD620 > 0.1) was observed after 24 h of incubation (Masuda, Ide, Utsumi, Niiro, Shimamura, & Murata, 2012). Fig. 3A shows that all strains grew at cold (15 °C) and hot (45 °C) temperatures, although not as good as that at 25 °C and 37 °C. Lactobacillus plantarum LZ95 showed a better growth than the other two strains at 15 °C and 25 °C. OD620 value of Lactobacillus plantarum LZ95 was ~0.5 at 15 °C after 24 h (Fig. 3A) and reached almost 5.0 after 48 h of incubation (data not shown). The bacterial capability to grow at low temperature is a good characteristic for dairy industry. Masuda et al. reported few LAB strains grew at 45 °C (Masuda, Ide, Utsumi, Niiro, Shimamura, & Murata, 2012), interestingly the three isolates in our study showed growth at 45 °C with an OD620 value of about 1.5 (Fig. 3A). The capability of growth at 45 °C allows high temperature fermentation, which is very important for the brewing industry (O'Sullivan & Condon, 1997). Moreover, the bacterial capability to grow at high concentrations of ethanol is necessary in the dairy and vine industries. Most of LAB strains in Masuda et al. study showed tolerance to 5% of ethanol, only 3 of 74 grew in broth with 15% of ethanol (Masuda, Ide, Utsumi, Niiro, Shimamura, & Murata, 2012). Our three Lactobacillus 15

plantarum isolates exhibited the ability to grow in 15% of ethanol (Fig. 3B). OD620 values of Lactobacillus plantarum LZ95 and CY3 in 2.5%, 5%, 10% and 15% of ethanol were ~100%, 80%, 50% and 25% as that in 0% ethanol respectively, indicating that these two strains had good tolerance to 5% of ethanol but the tolerant ability reduced with the increase of ethanol concentration. The tolerance of Lactobacillus plantarum CY2 to ethanol was not as good as Lactobacillus plantarum LZ95 and CY3 (Fig. 3B). Similar to the most LAB strains reported by Masuda et al. (Masuda, Ide, Utsumi, Niiro, Shimamura, & Murata, 2012), our isolates showed growth in high osmotic concentration of NaCl, at 6%, not 8% or 10% (Fig. 3C). High osmotolerance would be a prerequisite of LAB strains for their commercial application. Because when lactic acid is produced by the strain during fermentation, alkali would subsequently be applied to prevent an excessive reduction in pH, which would result in the conversion of free acid into its salt form and thus increasing the osmotic pressure on the bacterial cells (Mohd Adnan & Tan, 2007). Taken together, the three Lactobacillus plantarum isolates had the ability to survive the examined harsh conditions and Lactobacillus plantarum LZ95 showed the best tolerance to environmental stresses among the three Lactobacillus strains. 3.5 Tolerance of the Lactobacillus isolates to simulated gastric juices Because of the low pH and the antimicrobial action of pepsin, gastric juice in stomach is considered as one of the first major physiological challenges for probiotic strains (Charteris, Kelly, Morelli, & Collins, 1998; Rupa & Mine, 2012). Therefore, tolerance to acidic conditions is an important criterion for potential probiotic isolates. The probiotic bacteria have to survive the transit through the upper gastrointestinal tract, where the pH generally ranges from 2.5 to 3.5, and remain alive in the stomach for 2 - 4 hours or more before they were moved to the intestinal tract. LABs showed 16

variable resistance to acidic conditions, and this characteristic is strain specific. A range of pH values, from pH 2.0 to 4.0, has been used to screen the acid tolerance of LABs in vitro (Charteris, Kelly, Morelli, & Collins, 1998). Our study evaluated the effect of different pHs (2.0, 3.0 and 4.0) of simulated gastric juices on the viability of the three isolates during 4h of simulated gastric transit. There was no significant loss of viability for the three strains after 2 h of incubation at different pHs (Table 2). Lactobacillus plantarum LZ95 and CY3 maintained the viability even after 4h of incubation, in contrast, CY2 showed a significantly reduced viability at pH 2.0 and pH 3.0, which lost about 60 % viability after 4 h of simulated gastric transit. Previous studies have reported that most of the Lactobacillus spp. exhibited good survival at pH 3.0, and showed lower viability when exposed to pH 2.0 (Wang, Lin, Ng, & Shyu, 2010). Thus, Lactobacillus plantarum LZ95 and CY3 strains with high acid tolerance are likely to show better gastric survival. 3.6 Tolerance of the Lactobacillus isolates to small intestinal juices (including bile salt) Because of the presence of bile salts and pancreatin in small intestine, survival in small intestine is considered as another challenge for probiotic bacteria (Rupa & Mine, 2012). In general, small intestinal transit tolerance is essential for colonization and metabolic activity of bacteria in the host’s intestine. The transit time of food through the small intestine is normally 1–4 hours. Bile salts influence the intestinal microflora through its antimicrobial activities (Charteris, Kelly, Morelli, & Collins, 1998). A concentration of 0.15–0.3% of bile salt has been recommended as a suitable concentration for selection for probiotic bacteria. Table 3 shows the results of bile tolerance of the Lactobacilli isolates assayed. The three strains maintained the same viability during the 4 h of simulated small intestinal transit in the absence of bile salts 17

(Table 3). In the presence of 0.3% bile salts, LZ95 and CY3 demonstrated high levels of small intestinal transit tolerance, with no loss of viability after incubation in simulated small intestinal juices for 4 h, however, the strain CY2 showed a significant reduction in viable cell counts (Table 3). Therefore, Lactobacillus plantarum LZ95 and CY3 survived better in small intestine than CY2. Bile salt resistance of some strains is related to the enzyme activity of bile salt hydrolase (BSH) which helps to hydrolyze conjugated bile, reducing its toxicity. BSH activity has most frequently been found in microorganisms isolated from intestines or feces (Tanaka, Doesburg, Iwasaki, & Mierau, 1999), which could explain the high bile tolerance of the infant feces original strain Lactobacillus plantarum LZ95. 3.7 Adhesion properties of Lactobacillus isolates The adhesion capacity of the Lactobacillus isolates to the intestinal mucosa is a prerequisite for their colonization and retention in the host’s intestine, and is believed to be one of the most essential features of the probiotics for health benefits (Lavilla-Lerma, Perez-Pulido, Martinez-Bueno, Maqueda, & Valdivia, 2013; Rupa & Mine, 2012). The human colon adenocarcinoma cell line Caco-2, which displays many features of the intestinal epithelial cells, is a widely used model to evaluate the adhesion ability of bacteria in vitro. The adhesive abilities of LABs, including Lactobacillus plantarum strains, to Caco-2 cells varied as reported, and this feature is strain-specific (Lavilla-Lerma, Perez-Pulido, Martinez-Bueno, Maqueda, & Valdivia, 2013; Pisano, et al., 2014; Ramos, Thorsen, Schwan, & Jespersen, 2013). Amongst the Lactobacillus plantarum, a strain from farmhouse goat's milk cheeses exhibited a highest Caco-2 adherence rate of 36% (Lavilla-Lerma, Perez-Pulido, Martinez-Bueno, Maqueda, & Valdivia, 2013), while an isolate from Brazilian food products only showed 1.6% of adhesion (Ramos, Thorsen, Schwan, & Jespersen, 2013). The three 18

Lactobacillus plantarum isolates LZ95, CY2 and CY3 from our lab were strongly adhesive to Caco-2 cells, with the adhesion rates of 19.3 ± 0.4%, 17.2 ± 0.8% and 22.3 ± 1.4%, respectively (Table 3), indicating all these three isolates may be good in vivo colonizers in small intestinal tract. Although the in vitro adhesion assay of bacterial cannot fully recapitulate the complex conditions in the human intestinal tract, it provides important information for understanding the mechanisms involved in the adhesion process and for selection of probiotic candidates.

4. Conclusions Vitamin B12 is vital for metabolic function and health in humans. The advantage of in situ fortification makes lactic acid bacteria as ideal candidates for supplying vitamin to human hosts. However, intracellular nature of vitamin B12 might be a major stumbling block for their application. In this study, we isolated extracellular vitamin B12-producing, as potential B12 in situ suppliers for food industry. 18 of the 31 LABs showed growth in the vitamin B12-free medium and 7 of them were identified as Lactobacillus, using 16S rDNA sequencing. Among the 7 Lactobacillus strains, two isolates, LZ95 originally from infant feces and CY2 originally from fresh milk, were identified as Lactobacillus plantarum that were capable of producing high level of extracellular vitamin B12. The extracellular vitamin B12 from LZ95 was identified as Ado-Cbl and Me-Cbl by HPLC, and that from CY2 was Me-Cbl. To evaluate their probiotic potentials, we further tested the microbes for their in vitro resistance to harsh conditions (extreme temperatures, ethanol and osmotic stresses), tolerance to gastric acid and bile salts, and ability to adhere to the intestinal wall. LZ95 showed high tolerance to cold temperature (15 °C), hot temperature (45 °C), high

19

concentration of ethanol (15%) and high osmotic stress (6% NaCl), as well as to gastric acid (strain remained unaffected at pH 2.0 after 4 h of incubation) and bile salts (0.3%, 4 h). LZ95 also exhibited good adhesion to Caco-2 cells. On the other hand, the tolerance of CY2 to cold temperature and high concentration of ethanol was not as good as LZ95, and its viability was significantly affected by the 4 h simulated gastric transit (pH 2.0 and pH 3.0) or small intestinal transit, losing about 60% of its viability. In conclusion, Lactobacillus plantarum LZ95 with high extracellular B12 production and good probiotic properties might be a good candidate for in situ vitamin B12 fortification. However, in vivo experiments are needed to further investigate the strain safety status and its potential benefits to health.

Acknowledgements This project was funded by the National Key Research and Development Program of China (2016YFD0400400), the National Science Foundation of China (31601449), the Major Science and Technology Project of Zhejiang Province (2015C02039, 2015C02022), the International Science and Technology Cooperation Program of China (2013DFA32330), the Natural Science Foundation of Zhejiang Province (LY16C200002) and the Food Science and Engineering-the most important discipline of Zhejiang Province (2017SIAR202).

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Figure captions Fig. 1. Phylogenetic of Lactobacillus isolates LZ95 and CY2. Fig. 2. RP-HPLC chromatogram of extracellular B12 produced by Lactobacillus plantarum LZ95 and CY2. (A) UV-vis spectrum of the supernatant from LZ95 and the spectrum of standards (inset). (B) UV-vis spectrum of the supernatant from CY2. (C) Chromatogram recorded from the RP-HPLC analysis of the supernatant from LZ95. Chromatogram of the standards CN-Cbl, Ado-Cbl, OH-Cbl and Me-Cbl were shown (inset). (D) Chromatogram recorded from the RP-HPLC analysis of the supernatant from CY2. mAU, milli-absorbance units. Fig. 3. Tolerance of Lactobacillus plantarum LZ95, CY2 and CY3 strains to different environmental stresses. (A) temperature (15 °C, 25 °C, 37 °C and 45 °C), (B) ethanol (0, 2.5%, 5%, 10% and 15%) and (C) NaCl (0, 3%, 6%, 8% and 10%). The growth was evaluated by measuring OD620 after 24 h of incubation without shaking. *P<0.05, **P <0.01.

27

28

29

30

Table 1 Vitamin B12 production of Lactobacillus isolates. Strain

Original source

Intracellular Vitamin

Extracellular Vitamin

Identification

B12 (µg/L)

B12 (µg/L)

by 16S rRNA

ZJ03

fermented mustard

174 ± 18

nd

L. reuteri

ZJ5

fermented mustard

41 ± 9

nd

L. pantarum

ZJ316

infant feces

160 ± 23

nd

L. plantarum

LZ54

infant feces

295 ± 42

nd

L. casei

LZ95

infant feces

996 ± 37

98 ± 15

L. plantarum

CY2

fresh milk

565 ± 51

60 ± 9

L. plantarum

CY3

fresh milk

400 ± 67

nd

L. plantarum

ZJ008

fresh milk

19 ± 7

nd

L. plantarum

nd, not detected.

31

Table 2 Tolerance of the Lactobacillus plantarum strains to simulated gastric juices (pH 2.0, 3.0, 4.0). Strain

LZ95

CY2

CY3

pH of simulated gastric juices

Time 0

2h

4h

pH 2.0

8.11 ± 0.10

8.10 ± 0.02

8.06 ± 0.04

pH 3.0

8.14 ± 0.16

8.07 ± 0.16

8.07 ± 0.09

pH 4.0

8.10 ± 0.08

8.07 ± 0.08

8.07 ± 0.07

pH 2.0

8.84 ± 0.10

8.68 ± 0.02

8.36 ± 0.04**

pH 3.0

8.82 ± 0.01

8.77 ± 0.07

8.36 ± 0.07**

pH 4.0

8.88 ± 0.10

8.73 ± 0.03

8.71 ± 0.04

pH 2.0

8.44 ± 0.13

8.37 ± 0.02

8.24 ± 0.16

pH 3.0

8.43 ± 0.04

8.44 ± 0.06

8.48 ± 0.01

pH 4.0

8.47±0.01

8.45 ± 0.05

8.45 ± 0.08

Values represent the log10 transformation of viable counts. *P<0.05, **P <0.01.

32

Table 3 Survival of the Lactobacillus plantarum strains in simulated small intestinal juices and their capacity to adhere to Caco-2 cells. Strain

Viable count (log 10 CFU/mL) during simulated small intestinal transit Absence of bile salts 0

2h

Adhesion

In the presence of 0.3% bile salt 4h

0

2h

to Caco-2 cells (%)

4h

LZ95

8.11 ± 0.01

8.10 ± 0.04

8.10 ± 0.05

8.15 ± 0.03

8.04 ± 0.07

8.04 ± 0.03

19.3±0.4

CY2

8.34 ± 0.14

8.36 ± 0.28

8.27 ± 0.09

8.32 ± 0.01

8.08 ± 0.03*

7.92 ± 0.01**

17.2±0.8

CY3

8.46 ± 0.05

8.44 ± 0.02

8.48 ± 0.05

8.49 ± 0.03

8.44 ± 0.13

8.53 ± 0.02

22.3±1.4

Values represent the log10 transformation of viable counts. *P<0.05, **P <0.01.

33

Research highlights ►Two L.plantarum strains with high extracellular vitamin B12 yields were isolated. ►The extracellular B12 from L.plantarum LZ95 were identified as Ado-Cbl and Me-Cbl. ► LZ95 showed good tolerance to environmental stresses, low pH and bile salts. ► LZ95 exhibited great adhesion to Caco-2 cells. ► LZ95 might be a potential candidate for in situ B12 fortification in food industry.

34