Bacillus licheniformis Isolated from Traditional Korean Food

Nov 6, 2015 - Bacillus licheniformis Isolated from Traditional Korean Food Resources Enhances the Longevity of Caenorhabditis elegans through Serotoni...
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Bacillus licheniformis Isolated from Traditional Korean Food Resources Enhances the Longevity of Caenorhabditis elegans through Serotonin Signaling Mi Ri Park,† Sangnam Oh,*,†,¶ Seok Jun Son,† Dong-June Park,‡ Sejong Oh,§ Sae Hun Kim,∥ Do-Youn Jeong,⊥ Nam Su Oh,∇ Youngbok Lee,○ Minho Song,# and Younghoon Kim*,†,¶ †

BK21 Plus Graduate Program, Department of Animal Science and Institute Agricultural Science & Technology, Chonbuk National University, Jeonju 561-756, Korea ‡ Korea Food Research Institute, Seongnam-si, Gyeonggi-do 463-746, Korea § Division of Animal Science, Chonnam National University, Gwangju 500-757, Korea ∥ Division of Food Bioscience and Technology, College of Life Sciences and Biotechnology, Korea University, 136-701 Seoul, Korea ⊥ Microbial Institute for Fermentation Industry, Sunchang, Jeonbuk 595-804, Republic of Korea ∇ R&D Center, Seoul Dairy Cooperative, Ansan, Gyeonggi-do 425-839, South Korea ○ Department of Applied Chemistry, Hanyang University, ERICA Campus, Ansan, Gyeonggi-do 426-791, Korea # Department of Animal Science and Biotechnology, Chungnam National University, Daejeon 305-764, Korea S Supporting Information *

ABSTRACT: In this study, we investigated potentially probiotic Bacillus licheniformis strains isolated from traditional Korean food sources for ability to enhance longevity using the nematode Caenorhabditis elegans as a simple in vivo animal model. We first investigated whether B. licheniformis strains were capable of modulating the lifespan of C. elegans. Among the tested strains, preconditioning with four B. licheniformis strains significantly enhanced the longevity of C. elegans. Unexpectedly, plate counting and transmission electron microscopy (TEM) results indicated that B. licheniformis strains were not more highly attached to the C. elegans intestine compared with Escherichia coli OP50 or Lactobacillus rhamnosus GG controls. In addition, qRT-PCR and an aging assay with mutant worms showed that the conditioning of B. licheniformis strain 141 directly influenced genes associated with serotonin signaling in nematodes, including tph-1 (tryptophan hydroxylase), bas-1 (serotonin- and dopamine-synthetic aromatic amino acid decarboxylase), mod-1 (serotonin-gated chloride channel), ser-1, and ser-7 (serotonin receptors) during C. elegans aging. Our findings suggest that B. licheniformis strain 141, which is isolated from traditional Korean foods, is a probiotic generally recognized as safe (GRAS) strain that enhances the lifespan of C. elegans via host serotonin signaling. KEYWORDS: Caenorhabditis elegans, Bacillus licheniformis, serotonin signaling, antiaging, Korean traditional foods

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INTRODUCTION Traditional Korean foods have long been considered to promote health. Some fermentation products derived from traditional Korean foods are also known to act as anticarcinogenic and antioxidant agents.1 Typically, traditional Korean foods involve a dynamic community of microbes including fungi, bacteria, and yeast. Above all, Bacillus species play a major role in the fermentation processing of traditional Korean foods. Bacillus species are Gram-positive, endospore-forming bacteria that are best characterized by aerobic or facultative anaerobic metabolism, catalase production, and ubiquitous distribution in soil, dust, and aquatic environments.2,3 Moreover, B. subtilis and B. licheniformis include representative species that are “generally recognized as safe” (GRAS) in that they are active against many pathogens and free of toxins. These bacteria are also considered to be important producers of antimicrobial substances including bacteriocin.4,5 However, despite the various health-promoting effects of Bacillus species, the importance and industrial value of Bacillus spp. have been largely underestimated, and only a small number of food© XXXX American Chemical Society

related applications have been reported. Thus, widespread use of Bacillus spp. as potential probiotic agents will require further scientific evidence in order to firmly establish their health benefits. To this end, our group has been focused on the potentially probiotic activity of functional GRAS strains of Bacillus spp. (B. subtilis and B. licheniformis are representative species of GRAS) isolated from traditional Korean foods. We previously verified that the cell-free supernatants of potentially health-promoting Bacillus spp. have specific antibacterial activities against Gram-positive pathogens.1 Probiotic bacteria are living microorganisms that exert beneficial effects on human health when ingested in sufficient numbers.6 Probiotics have a variety of beneficial health effects including positive regulation of the intestinal microbiota, immunomodulation, and life span extension.7,8 Among these Received: August 4, 2015 Revised: November 6, 2015 Accepted: November 6, 2015

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DOI: 10.1021/acs.jafc.5b03730 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry properties, the antiaging effects of probiotics were first proposed by Metchnikoff, who reported that Bulgarian farmers consuming large quantities of fermented milk containing lactobacilli experienced promoted health and longevity.9 Unfortunately, studies on the antiaging impact of probiotics are limited because of a lack of suitable experimental models for determining host longevity. As an in vivo model system, the nematode species Caenorhabditis elegans has become a major host model organism due to its small size and transparent body. This animal does not present ethical issues, is inexpensive to maintain, and involves less time and labor for screening.10 Interestingly, the relationship between diet and longevity is an important consideration in the interpretation of assays of C. elegans life span involving modification of bacterial food source, and several studies have demonstrated that lactobacilli and bifidobacteria modify host defense and prolong the lifespan of nematodes.7,8,11,12 It is well-established that the interactions between C. elegans and bacteria with respect to aging and host longevity are complicated and multifactorial,13−15 among which the neurotransmitter serotonin has long been suggested to be an important food signal in C. elegans.16 The unifying characteristic of C. elegans serotonergic responses is the coupling of food perception to various endocrine outputs. Serotonin and dopamine signaling pathways modulate food-related behaviors and physiology in diverse mammals17 and C. elegans.18,19 Serotonin is also a dose-sensitive neurotransmitter that modulates a variety of biological processes and behaviors. However, the mechanisms by which serotonin signaling pathways function in the immune response of the C. elegans remain poorly understood. At present, there is a lack of consistency in the outcomes of clinical and animal studies on probiotics, and neither the contributing bacterial factors that determine probiotic function nor the possible underlying mechanisms of these beneficial effects have been clarified. Thus, the objective of this study was to evaluate the potential life-prolonging properties of various strains of B. licheniformis isolated from traditional Korean foods and to investigate possible underlying mechanisms of B. licheniformis-mediated antiaging effects involving serotonin signaling in probiotic GRAS strains.

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cultures were washed and resuspended with sterile M9 medium. Cells were then exposed to 100 °C for 30 min, washed with M9 medium, and deposited onto NGM plates in the same manner as with the live bacteria. C. elegans Nematode. C. elegans N2 Bristol wild-type and the mutant strains CF512 fer-15(b26)II;fem-1(hc17)IV ( fer-15;fem-1 worms), ser-1(ok345)X, tph-1(mg280) II, bas-1(ad446) III, and mod1(ok103) V were obtained from the Caenorhabditis Genetics Center (CGC, St. Paul, MN, USA).21,22 Of note, the fer-15;ferm-1 mutant animals are particularly suited for killing assay experiments as these worms are unable to produce progeny at 25 °C without altering the C. elegans phenotype.13 Worms were routinely maintained on nematodegrowth medium (NGM) agar using standard techniques23 and seeded with OP50, an internationally established feed. Synchronized L1 larvae were produced using sodium hypochlorite−sodium hydroxide solution, placed onto NGM plates seeded with OP50, and grown at a restrictive temperature (25 °C) in order to obtain sterile adults. Bacterial Choice Assay. The bacterial choice assay was performed as described previously, with some modifications.24 Briefly, bacteria were resuspended in M9 buffer, and 20 μL of each bacteria concentrate was spotted onto a 60 mm NGM agar plate and airdried at 20 °C. Adult nematodes were washed three times in M9 buffer, and 40 worms were placed near the center of an NGM plate, equidistant from the two bacteria sources. Nematodes were allowed to move freely for 2 h, and the number of animals on each bacterial lawn was scored. Nematodes outside the bacterial lawn were not counted, and assays were repeated at least three times. C. elegans Lifespan Assays. Nematode lifespans were determined using established methods.13 Briefly, 20 μL aliquots of concentrated bacteria were plated on 35 mm diameter NGM agar plates, and L4/ young adult stage N2 wild-type, fer-15;fem-1, tph-1, bas-1, mod-1, or ser-1 mutant nematodes were transferred to prepared NGM plates with a platinum wire. For each life span assay, 90 worms per bacterial species were assayed in three plates (30 worms/plate). The plates were incubated at 25 °C, and the numbers of live worms were counted every day. N2 wild-type nematodes were transferred to fresh plates daily during the progeny production period and every second to third day afterward, although they were monitored daily for dead animals. A worm was considered dead when it failed to respond to a gentle touch with a platinum wire pick. Bacterial Attachment Assay Using Plate Count and Transmission Electron Microscopy (TEM). Bacteria cell numbers in nematodes were determined according to an established method.8 After exposing the worms to each of the B. licheniformis lawns prepared on NGM agar plates containing nystatin suspension (50 unit/mL) for 24 h, 10 worms were picked randomly, washed twice in M9 buffer, and placed on a BHI agar plate containing 100 μg/mL kanamycin and streptomycin. Next, the worms were washed in 5 μL drops of 100 μg/ mL gentamycin on agar plates for 5 min to remove surface bacteria. The washed nematodes were lysed in M9 buffer with 1% Triton X-100 and mechanically disrupted using a mortar and pestle (Kontes, Vineland, NJ, USA). The worm lysates were serially diluted in M9 buffer and incubated overnight at 37 °C on LB plates. Colonies were quantified and used to calculate the number of bacteria per nematode. At the same time, bacterial attachment was visualized by TEM (HITACHI H-7650, Tokyo, Japan), as previously described.10 Animals were plated on 60 mm NGM plates seeded with B. licheniformis strains or control plates seeded with OP50 and LGG. For each observation, at least 10 cross sections were evaluated, and representative images were chosen. Quantitative Real-Time Polymerase Chain Reaction (qRTPCR) Analysis. Total RNA from worms was quickly isolated using TRIzol reagent (Invitrogen) and purified using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) including an on-column DNase digestion step with RNase-free DNase (Qiagen). Total RNA quality was tested using an Agilent 2100 bioanalyzer and stored at −80 °C prior to analysis. A total of 50 ng of total RNA was used for a qRTPCR using the SuperScript III Platinum SYBR Green One-Step qRTPCR Kit (Invitrogen, Carlsbad, CA, USA). qRT-PCR was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems,

MATERIALS AND METHODS

Bacterial Strains and Culture Conditions. B. licheniformis was isolated from traditional Korean foods including doenjang, cheongkookjang, kochujang, and kanjang in Sunchang (Jeollabuk-Do, Korea). All bacillus strains were cultured in Luria−Bertani (LB) broth (BD Biosciences, Sparks, MD, USA) at 30 °C for 24 h with shaking (200 rpm). Escherichia coli strain OP50 (referred to as OP50), a standard food for nematodes, was grown in LB broth at 37 °C for 24 h with shaking (225 rpm).13 The Lactobacillus rhamnosus strain GG (referred as LGG), which is one of the most widely recognized probiotics that is highly attached to the intestinal tract and extensively used in the food and pharmaceutical industries,20 was cultured in De Man, Rogosa, and Sharpe (MRS) broth at 37 °C for 24 h. For long-term storage, cultures were maintained at −80 °C with 15% glycerol in cryoprotectant. All strains were subcultured two times prior to experimental analysis. To prepare live bacteria lawns for C. elegans feeding, bacteria were harvested by centrifugation at 13,000 rpm for 1 min, washed twice in sterile M9 buffer, and centrifuged at 13,000 rpm for 1 min to remove the supernatant. Next, the bacteria were adjusted to a final concentration of 2.5 mg (wet weight) per μL in M9 buffer, which was subsequently used as a concentrated bacteria supply. In addition, to employ heat-inactivated cells of B. licheniformis strains, overnight B

DOI: 10.1021/acs.jafc.5b03730 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Foster City, CA, USA). Primers, which are listed below, were designed using Primer3Input Software (v0.4.0). Relative expression levels were calculated using the 2−ΔΔCt method. The control gene snb-125 (forward 5′-CCGGATAAGACCATCTTGACG-3′ and reverse 5′-GACGACTTCATCAACCTGAGC-3′) was used to normalize gene expression data. The following primers were used to confirm sample genotypes: tph-1 (forward 5′-AAGAGGCCCAGCAGAAACTC-3′ and reverse 5′-ATGGAACGGGAGTTGTTGAG-3′), bas-1 (forward 5′GGCTGCTGAATTCTCCAAGT-3′ and reverse 5′-CTTCTCATTATCCGCGTTGG-3′), mod-1 (forward 5′-GAAGCAACGGGTATGCAA-3′ and reverse 5′-CCGTTTCGATGAAGTGATCC-3′), ser-1 (forward 5′-AAGAGCCAGTCGCCAGAAC-3′ and reverse 5′-GTGGTTGATGCCTCTGTCGT-3′), and ser-7 (forward 5′-TGCTAGCACTGTGGTTAGGC-3′ and reverse 5′-GTAGCACAGCGACAAGCAAG-3′). Statistical Analysis. C. elegans survival was analyzed using the Kaplan−Meier method, and differences were determined using the log-rank test (STATA6; STATA, College Station, TX, USA). Student’s t test was performed to determine statistical differences in CFU count for determining bacterial abundance. All data represent the results of three independent replicates. A p value of 0.05 in all replicate experiments was considered reflective of a significant difference from the control.

differences between OP50 and B. licheniformis plates for any of the criteria evaluated (data now shown). Exposure to a particular food plays a significant role in shaping the pattern of food intake by altering the subsequent consumption of the food; thus, these results indicate that B. licheniformis are nonpathogenic bacteria and an attractive nutritional source for C. elegans. B. licheniformis Enhance the Lifespan of C. elegans. To date, determining the ability of dietary probiotic GRAS bacterium to enhance longevity has been limited due to a lack of suitable experimental animal models. To explore the lifespan-prolonging effect of bacillus strains in C. elegans, young adult worms were exposed to plates containing one of six different bacillus strains. As a control, we used OP50, which is a normal feeding bacterium for C. elegans fer-15;fem-1 mutants. In this assay, we identified four potential probiotic GRAS strains of B. licheniformis in the nematode intestine that significantly prolonged the lifespan of nematodes by approximately 45% compared to the OP50 control (Figure 2). Among the bacillus strains tested, strain 141 had the most profound activity in terms of extending the C. elegans lifespan (p = 0.0095 compared with worms feeding on OP50). In addition, we confirmed the lifespan-prolonging activity of strain 141 in N2 wild-type nematodes (p < 0.05 for strain 141-conditioned nematodes compared with worms feeding on OP50) (Figure 5A), which led us to conclude that the selected B. licheniformis significantly influenced the longevity of C. elegans in vivo. In addition, we employed B. subtilis ATCC 6633 as similar GRAS bacillus species for the C. elegans lifespan assay. In a previous report, worms grown on soil with B. subtilis exhibited a longer lifespan compared to those grown with OP50 or other bacterial species.28 Unexpectedly, B. subtilis ATCC 6633 did not influence the lifespan of C. elegans (Figure 2). Consistent with these results, B. licheniformis strains 144 and 155 had no influence on the longevity of nematodes compared with B. licheniformis strains 141, 143, 147, and 156. Thus, we consider the prolongation of the C. elegans lifespan via conditioning with GRAS bacilli to be strain-specific. Importantly, similar results in which C. elegans fed selected bifidobacteria or lactobacilli increased their average lifespan by 17% to 33% were obtained by Ikeda et al.7 Taken together, our results indicate that the conditioning of specific strains of B. licheniformis enhances the lifespan of C. elegans. B. licheniformis Does Not Attach to the Intestinal Tract of C. elegans. C. elegans is an accepted in vivo model to study bacteria−host interactions in the gut because the structure of its intestinal cells is similar to that of humans.8 Given that the attachment of probiotic GRAS strains to the intestinal mucus is considered a prerequisite for profitable colonization, we investigated the ability of the bacterial strains to attach to the C. elegans intestinal tract. Unexpectedly, none of the tested bacillus strains exhibited good attachment, with the results from all strains appearing similar to the negative control OP50 at 2 CFU/mL per worm, with no statistical differences between groups (Figure 3A). In contrast, the L. rhamnosus strain GG was highly colonized in the gut environment,20 exhibiting greater attachment in the lumen of intestines compared with OP50 or B. licheniformis-fed C. elegans. We next used TEM to visualize and verify our findings. Transmission electron microscopy of the worms allowed for higher-resolution images of the lumen in the C. elegans intestine (Figure 3B). Consistent with the CFU results, worms that were conditioned with bacillus strains or OP50 did not exhibit

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RESULTS AND DISCUSSION B. licheniformis Serve as a Good Nutritional Source for C. elegans Nematodes. Olfactory chemotaxis toward foodassociated odors is a robust behavior, and odors are an innate behavior that are highly reproducible among animals.26 It was recently reported that C. elegans modify their olfactory preferences after exposure to pathogenic bacteria, avoiding odors from pathogens and demonstrating attraction to odors from nonpathogenic bacteria.24 In this way, the chemosensory system of C. elegans can modulate the lifespan of animals in adulthood.27 Thus, we evaluated the bacilli bacterial preferences of C. elegans using a binary choice assay with OP50 as a normal feeding control strain. In our previous report, we showed that six strains of B. licheniformis possess potential probiotic activities including antimicrobial activity and immune enhancement activity.1 The approach of the animals over 2 h was dominated by olfactory preferences for volatile odors released by the bacilli. As expected, we found that there was no significant difference in the preference of C. elegans for strains of B. licheniformis and OP50 (Figure 1). Moreover, we determined C. elegans movement, body size, and brood size on either OP50 or B. licheniformis plates. Overall, we could not identify any

Figure 1. Olfactory preferences after 2 h of exposure to L. rhamnosus strain GG and B. licheniformis (blue bars) compared with the normal feed source E. coli OP50 (orange bars). Data are the mean ± SD of three independent experiments. Preferences did not depend on the species or genus of tested bacteria. C

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Figure 2. Preconditioning with six B. licheniformis strains prolonged the lifespan of C. elegans nematodes. Survival statistics: p < 0.05 for strain 141-, 143-, 147-, and 156-conditioned nematodes compared with worms feeding on E. coli OP50 and B. subtilis ATCC 6633 control strains.

Figure 3. Attachment of B. licheniformis in C. elegans gut. Plate counting (A) and transmission electron microscopy (TEM) images (B) indicated that B. licheniformis strains were not attached to the C. elegans intestine compared with E. coli OP50 or L. rhamnosus strain GG controls. The scale bar represents 2 μm.

lacks the ability to attach to the C. elegans intestine, stimulates an immune response following infection with a foodborne pathogen.13 Taken together, the plate counting and TEM images indicated that B. licheniformis strains were not attached to the C. elegans intestine, and unknown components of CWMs produced from bacillus cells, rather than intact cells, may influence the enhanced longevity of C. elegans. In addition, the enhancement of C. elegans longevity by conditioning with B. licheniformis may be mediated by bacterial features (PAMPs/ MAMPs). Thus, further studies will be required to identify which PAMPs/MAMPs are directly involved in B. licheniformismediated host longevity. Bacillus Spp. Regulate Serotonin Signaling Gene Transcription. It was recently reported that neuronal signaling pathways may be involved in C. elegans longevity, and that serotonin in particular plays a role in modulating behavior that has been established largely through the analysis of mutants for

bacterial attachment of the intestinal tract. However, consistent with plate counting, numerous large pockets of undigested bacteria were visible in the intestinal lumen of positive control nematodes pre-exposed to L. rhamnosus strain GG. Interestingly, it has been established that some cell wall molecules (CWMs) may be key players as critical ligands for interacting with the intestinal environment of the host. Of note, a number of bacteria share common features such as pathogen/ microbe-associated molecular patterns (PAMPs/MAMPs) including peptidoglycan, lipoteichoic acid, and polysaccharides.29,30 In the present study, we confirmed that conditioning with heat-inactivated B. licheniformis cells also significantly influenced worm lifespan extension (Figure S1). Thus, it is possible that uncharacterized CWMs may be released from B. licheniformis cells by C. elegans ingestion, and that specific PAMPs/MAMPs in uncharacterized CWMs act to prolong the longevity of C. elegans. This hypothesis was also supported by our previous finding that the L. acidophilus strain NCFM, which D

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Figure 4. B. licheniformis strain 141 regulates serotonin signaling genes. qRT-PCR analysis evaluating the impact of conditioning on serotonin-related genes. Transcript levels were measured in young adult fer-15;fem-1 worms conditioned with B. licheniformis strain 141 for 6, 9, or 12 days (old animal) compared with those conditioned for 1 day (young animal).

Figure 5. tph-1, bas-1, mod-1, and ser-1 are required for the regulation of aging and immunity by B. licheniformis strain 141. Solid killing of C. elegans strain N2 wild-type worms (A) and tph-1 (B), bas-1 (C), ser-1 (D), or mod-1 (E) loss-of function mutants [survival statics: wild-type N2 (p = 0.0376 worms conditioned with strain 141 compared to OP50), tph-1 (p = 0.4145 worms conditioned with strain 141 compared to OP50), bas-1 (p = 0.8133 worms conditioned with strain 141 compared to OP50), ser-1 (p = 0.8547 worms conditioned with strain 141 compared to OP50), and mod1 (p = 0.0960 worms conditioned with strain 141 compared to OP50)].

sustaining a high serotonin level.32 Moreover, emerging research suggests that bacteria in the gastrointestinal tract can communicate with the central nervous system, even in the absence of an immune response.33 Next, we speculated that gut immunity might be more efficient after conditioning with probiotic B. licheniformis that upregulate specific immune factors associated with the C. elegans neurotransmitter

serotonin signaling including tph-1 (encodes tryptophan hydroxylase), bas-1 (encodes serotonin- and dopaminesynthetic aromatic amino acid decarboxylase), mod-1 (encodes serotonin-gated chloride channel), and ser-1 (encodes serotonin receptors).15,31 Notably, serotonin level significantly decreases with age in C. elegans, and the diet of C. elegans influences healthy behaviors in aged worms at least partially by E

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serotonin. To address this possibility, we evaluated the expression of genes in response to probiotic GRAS strain B. licheniformis feeding of animals as a functional marker of aging, including the following key factors of neurotransmitter serotonin signaling: tph-1, bas-1, mod-1, ser-1, and ser-7. Specifically, to determine if the lifespan-extending factor of B. licheniformis strains involves serotonin signaling, we investigated the transcription of these genes in C. elegans nematodes exposed to strain 141 for 1 day (young animal) or 6, 9, or 12 days (old animal) using quantitative real-time PCR (qRT-PCR) (Figure 4). Consistent with our hypothesis, the expression levels of the four serotonin-associated genes were significantly reduced in nematodes fed OP50 according to age. Contrary to normal fed OP50, conditioning with strain 141 strongly impacted the transcription of tph-1, bas-1, mod-1, ser-1, and ser-7 genes. Notably, the repressed transcriptional levels of bas1 and ser-7 were dramatically recovered by the presence of strain 141 compared with OP50. Together, these results indicate that conditioning with probiotic GRAS strain 141 specifically stimulated the transcription of serotonin signaling genes associated with nematode responses to aging. The Nematode Host Response to Conditioning with Strain 141 Involves the TPH-1, BAS-1, MOD-1, and SER-1 Signaling Pathways. Our results above support the hypothesis that the TPH-1, BAS-1, MOD-1, and SER-1 signaling pathways are involved in antiaging and immune stimulation through conditioning with strain 141. In order to further investigate this hypothesis, we carried out killing assays with loss-of-function mutants or N2 wild-type worms to confirm that the TPH-1, BAS-1, MOD-1, and SER-1 signaling pathways are directly involved in C. elegans after conditioning with strain 141. The results of this assay were consistent with qRT-PCR results (Figure 4). Specifically, worms lacking tph-1, bas-1, mod-1, or ser-1 were still susceptible to aging (Figure 5) even though they were conditioned with strain 141. Conversely, strain 141 had no effect on the lifespans of worms with deleted tph-1, bas-1, mod-1, or ser-1 genes, whereas the N2 wild-type exhibited significantly extended lifespans upon feeding with strain 141 (p < 0.0001) (Figure 5). Taken together, these results indicate that the serotonin signaling pathways have an important role in determining the lifespan of C. elegans with respect to probiotic GRAS strain 141. In agreement with our findings, Song and challengers reported that C. elegans detect bacteria using a pair of sensory cells called ADF neurons, which subsequently release the neurotransmitter serotonin upon bacteria detection.34 Importantly, recent evidence suggests that various factors from the intestine, including those mediated by gut microbiota, participate in and influence memory formation, affective behaviors, and decision-making processes.35 Among these factors, serotonin has been strongly implicated in the relay of the influence of gut microbiota to the brain. Taken together, the transcriptional regulation of serotonin-related signaling after exposure to B. licheniformis strain 141 provides a specific signal in gut environments that may modify various behaviors including aging (Figures 4 and 5). Our results also suggest that probiotic GRAS strains of B. licheniformis isolated from traditional Korean foods enhance the lifespan of C. elegans, and that B. licheniformis-mediated serotonin signaling as a potential therapeutic target is worth investigating further for prevention of age-related health claims.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b03730. C. elegans enhanced lifespan results (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(S.O.) E-mail: [email protected]. *(Y.K.) Tel: +82-63-270-2606. Fax: +82-63-270-2612. E-mail: [email protected]. Author Contributions ¶

These authors contributed equally to this study.

Funding

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2014R1A1A1006872) and by the High Value-Added Food Technology Development Program of the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry, and Fisheries (iPET), Ministry for Food, Agriculture, Forestry, and Fisheries of the Republic of Korea (111137-03-3-SB010). Notes

The authors declare no competing financial interest.

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DOI: 10.1021/acs.jafc.5b03730 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jafc.5b03730 J. Agric. Food Chem. XXXX, XXX, XXX−XXX