And Sulfate Reduction (Sodium Molybdate) on Volatile Sulfur Pro Duction in Batch Cultures of Pig Cecal Bacteria

J Nutr Sci Vitaminol, 46, 193-198, 2000

Volatile Sulfur Production by Pig Cecal Bacteria in Batch Culture and Screening Inhibitors of Sulfate Reducing Bacteria

Tsutomu ARAKAWA,1,* YoshimiISHIKAWA2 and Kazunari USHIDA2

1Food Material Section, Central Laboratory, Lotte Co., Ltd., Urawa, Saitama 336-0027, Japan2 Laboratory of Animal Science,Kyoto Prefectural University, Kyoto 606-8522, Japan (Received December 27, 1999)

Summary We studied the effects of specific inhibitors of methanogenesis (2-bromo ethane sulfonate, BES) and sulfate reduction (sodium molybdate) on volatile sulfur pro duction in batch cultures of pig cecal bacteria. The volatile sulfur concentration in head space gas was determined by flame-photometric detector gas chromatography. BES stimu lated production of hydrogen sulfide (H2S) and methanethiol, and sodium molybdate com pletely inhibited the production of these volatile sulfur compounds. The results indicated that dissimilate sulfate reduction is mainly responsible for volatile sulfur production in the hindgut. Therefore the extracts of herbs, food colors, and aroma chemicals were tested for their inhibitory effects on H2S production by a dissimilatory sulfate-reducing bacteria, Desulfovibriodesulfuricans DSM642. H2S was measured by the chromatography of the head space gas, using a flame photometric detector. Of 306 herbal extracts tested, 69 extracts from 38 herbs inhibited H2S production at 1.0mg/mL. Sisymbrium officinale (hedge mus tard) was the most potent inhibitor. Six pigments inhibited H2S release. Erythrosine and rose Bengal showed inhibitory effects at 0.01mg/mL. Peppermint oil and 96 aroma chemi cals were assayed for their effects on H2S release. Thirty-two aroma chemicals suppressed H2S production at 0.1mg/mL, and camphene, 1-decanol, and 2-nonanone were effective at 0.01mg/mL. Key Words Desulfovibrio desulfuricans, inhibition of H2S production, herbs, pigments, aroma chemicals

Volatile sulfur compounds, such as methanethiol and tion in pure cultures. H2S, have an unpleasant odor and are fecal malodor ants (1). Hydrogen sulfide might also injure colonocytes MATERIALS AND METHODS (2) and be responsible for ulcerative colitis (3, 4) and Experiment 1; Effect of specific inhibitors of met hanogen colon cancer (5). Methanethiol produced from methio esis and sulfate reduction on volatile sulfur production. nine by intestinal bacteria may cause hepatic en Cecal contents were sampled from a mature crossbred cephalopathy in rats, dogs (6, 7), and cirrhosis patients. (Landlace•~Large White•~Duroc) pig with a cecal fistula Therefore it is important to develop ingestible agents to 1h after morning feeding. Judging from the kinetics of control intestinal sulfide generation in mammals, with digestion (10), the morning meal should not have minimal toxicity. Commercial nonspecific biocides are reached the cecum by this time. The pig was fed 250g usually used to control sulfide production by sulfate meat-bone meal, 250g alfalfa (Medicago sativa) meal, reducing bacteria (SRB) in industrial settings (8). For and 820g cracked maize at 10 a. m. and 6p. m. The example, molybdate suppresses sulfide production in daily diet was supplemented with NaCl, CaCO3, and a feces and digesta in vitro (9). Clinically, however, few vitamin-mineral premix (Kokin Kagaku Co., Osaka, drugs regulate SRB. Japan) (11). Water and a mineral block were always We first tried to identify the organisms responsible for available. volatile sulfur production in the hindgut. To do this, we The cecal contents were diluted to half strength with examined the effect of specific inhibitors of sulfate re MES (2-morpholinoethane sulfonic acid; 50mM pH duction and methanogenesis. These experiments sug 6.5) and homogenized in a blender under an N2 atmo gested the significance of sulfate-reducing bacteria in sphere. The sample was diluted to reduce the viscosity sulfide production in the large intestine. Therefore we and to facilitate complete mixing and dispensing. The used one such bacteria, Desulfovibriodesulfuricans, as a homogenate was squeezed through two layers of gauze. test organism to screen the inhibitory action of a range A portion (5mL) of the filtrate was incubated under N2 of herbs and food additives on bacterial sulfide produc in a 10mL vial tube with a butyl rubber septum for 24h at 37•Ž. Either a methanogenic inhibitor, 2-bro * To whom correspondence should be addressed . moethane sulfonic acid (BES) (12), or an inhibitor of

193 194 ARAKAWAT et al.

sulfate reduction, sodium molybdate (13), was added O2-free CO2 atmosphere. The culture medium consisted to cultures at 5, 10, and 20mM. Fermentation was of (g/L) K2HPO4 0.5, NH4Cl 1.0, Na25O4 1.0, CaCl2 stopped by the addition of 6N HCl (1mL), and 0.5mL of 2H2O 0.1, MgSO4•E7H2O 2.0, yeast extract 1.0, FeCl2 headspace gas was collected at the end of incubation. 0.1, resazurin 0.001, 70% dl-sodium lactate 3.2, Acid volatile sulfur compounds (AVS), such as hydrogen sodium thioglycolate 0.1, ascorbic acid 0.1, and sulfide, methanethiol, and dimethyl sulfide, were ana Na2CO3 0.2 (16). The pH of this medium was adjusted lyzed by an FPD-GC (flame photometric detector-gas to 6.8, using NaOH. Ten milliliters of this medium were chromatograph) (14), using gaseous materials as stan dispensed into a HUNGATE-type tube (Bellco Glass, Inc., dards. Since hydrogen sulfide was the only AVS de Vineland, NJ, USA) and used as the basal medium. tectable in trace amounts at the beginning of incuba Assay media were prepared by the addition of herb ex tion, AVS production was estimated by the concentra tracts or food pigments to the basal medium just before tion in the headspace gas at the end of incubation. inoculation. water extracts of herbs and food pigments There was a linear correlation of hydrogen sulfide con were resolved in H2O. Extracts of herbs obtained with centration between head space gas (ppm) and solution methanol, ethanol, aqueous methanol, or aqueous

(M) after HCl addition at least from 50mM to 5mM for ethanol, and aroma chemicals were resolved in aque hydrogen sulfide (HC=7,695.4•~SC-1,876.4) and di ous ethanol (final 1% v/w). methyl sulfide (HC=2,476.1•~SC+612.6), 500mM to D. desulfuricans DSM642 was precultured in the basal 20mM for methanethiol (HC=170.8•~SC+49.5), medium for 24h and inoculated (0.2mL) into assay where HC is a concentration of sulfide in headspace media (10mL). The incubation temperature was 37•Ž.

(ppm) and SC is a concentration in a culture medium The H2S concentration was measured by FPD gas chro (mM). The detected ranges of headspace AVS were over matography after 43h in the following manner. One 1,000ppm for hydrogen sulfide and about 100ppm milliliter of the headspace gas in the assay tube was an for methanethiol that were placed in these ranges (see alyzed with a Shimadzu GC-9A gas liquid chromato Fig. 1). Dimethyl sulfide was not detected within this range. Experiment 2: Screening inhibitors of H2S production. Materials tested: Four hundred and thirty products were evaluated for their inhibitory effects on sulfide production by D. desulfuricans, one of the major sulfate reducers in the hindgut (15). These included 306 herbal extracts, 27 pigments, and 97 aroma chemicals. The substances were tested at concentrations of 1 mg/mL, 0.1mg/mL or 0.01mg/mL. European herbs were obtained from KARIS-Seijo

(Tokyo, Japan). Oriental herbs were purchased from Uchida-Wakanyaku (Tokyo, Japan). The 69 ground ma terials were extracted with water, 50% ethanol, and ethanol; another 72 materials were extracted with water, aqueous ethanol, ethanol, aqueous methanol, or methanol by refluxing for 2h at 40-60•Ž, The extracts thus obtained were concentrated under vacuum and lyophilized to dry powder. In total, 306 extracts ob tained from 141 samples derived from 126 herbs (Table 1) were evaluated for inhibitory effects on sulfide pro duction by SRB. Food pigments were purchased from Tokyo Kasei

(Tokyo, Japan). These included: amaranth, erythrosine, allura red AC, new coccine, phloxine, rose bengal, acid red, tartrazine, sunset yellow FCF, fast green FCF, bril liant blue FCF, indigocarmine, ponceau 3R, ponceau SX, ponceau R, eosin, naphthol yellow S, light green SF yellowish, orange I, orange II, martins yellow, uranin, guinea green B, brilliant milling green, azure blue VX, and acid violet 6B. All the aroma chemicals were Fig, 1. Effects of 2-bromoethane sulfonate (BES) and reagent grade. sodium molybdate on acid volatile sulfur production Bacterial strain and culture condition: Desulfovibrio by pig cecal bacteria in batch culture. n=3, Bars with desulfuricans DSM642 was obtained from Deutsche different superscripts differ significantly (p<0.05). Sammlung von Mikroorganismen and Zellkulturen Student's t-test was applied separately to the values for (Braunschweig, Germany). BES-treatments and MoO42- treatments because of the The following manipulations were all done under an large variations between the two. Inhibition against Volatile Sulfide Production by SRB 195

Table 1. Inhibitory activity of herbal extracts on H2S production by D. desulfuricans. 196 ARAKAWA T et al.

Table 1. Continued.

n=3. A: aqueous extract, 50: 50% ethanol extract. E: ethanol extract, 30: 30% ethanol extract. M: methanol extract, +: % inhibition _??_50,-: % inhibition <50, ND: not determined. Herbs showing no inhibitory activity in H2S production by D. desulfuricans: Achras zapota, Agaricus bisporus, Agrimonia eupa torla, Alpinla oxyphylla, Amomum tsao-ko, Apocynum venetum, Artemisia vulgaris, Aspalathus linearis, Asperula odorata, Boesenbergiapandurata, Boswellia carterli, Buddleia officinalis, Camellia sinensis (Green, black, and Oolong tea), Capsella bursa pastoris, Carthamus tinctorius, Carom carvi, Centaurea cyanus, Chaenomelessinensis, Cichoriumintybus, Cinnamomumcamphora, Cinnamomum cassia, Citrus paradisi, Citrus tachibana, Citrus unshu, Cleyera ochnacea, Crocus sativus, Curcuma zedoaria, Cymbopogoncitratus, Daphne genkwa,Dyera costulata, Elettaria cardamomum Maton, Eriobotryta japonica, Eucalyptus citriodora, Eucalyptus globulus, Euphoria longana, Euphrasia rostkoviana, Foeniculum vulgare, Forsythia suspensa, Fragaria vesca, Fragaria ananassa, Garcinia mangostana, Hibiscus rosa-sinensis, Hypericum erectum, Illicium verum, Juniperus communis, Lavandula angus tifolia, Lindera umbellata, Lonicera japonica, Magnolia obovata, Matricaria recutita, Mentha pulegium, Monarda didyma, Myristica fragrans, Nandina domestica, Nelumbo nucifera, Origanum vulgare, Piper nigrum, Plantago asiatica, Platycodon grandiflorum, Plectranthus japonicus, Polygonatumfalcatum, Poncirus trifoliata, Prunus persica, Punica granatum, Raphanus sativa, Rosa canina, Rosa rugosa, Rosa spp., Rosmarinus officinalis, Rubus fruitcosus, Rubus suavissimus, Rubus chingii, Salvia officinalis,Sasa veitchii, Schizonepeta tenuifolia, Scutellaria baicalensis, Sparganium stoloniferum, Styrax benzoin, Tabebuia avellanedae, Tagetes patula, Taraxacum sect, Theobromacacao, Thuja orientalis, Trachycarpusfortunei, Vacciniumcorymbosum, Viola odorata, Zizyphusjujuba.

graph equipped with a glass column (5m•~3mm i. d.) Table 2. Inhibitory effects of food pigments on H2S packed with 20% dioctyl phthalate on Chromosorb W production by D. desulfuricans. AW-DMCS (60-80mesh) (Chromatotec Co., Tokyo,

Japan). The amount of H2S was calculated from a stan dard curve plotted in advance. The percent inhibition of H2S release is expressed as (C-S)/C•~100, where S and C are the amounts of H2S in the headspace gas of cul ture tubes with or without test samples, respectively.

RESULTS AND DISCUSSION

Effects of BES and sodium molybdate on AVS production

(Fig. 1) In our cultures, only hydrogen sulfide was detectable in trace amounts at the beginning of incubation, and H2S was the most abundant AVS, followed by n=3. methanethiol at the end of incubation. Trace amounts of dimethyl sulfide were detected, but these were below the quantitative range of the analysis system. competes with methanogenesis for the reducing equiva H2S is produced from dissimilate sulfate reduction by lents (15, 18, 19). In this study, the inhibition of H2S

SRB (15). This compound can also be produced from production by molybdate and enhancement by BES the sulfur-containing amino acids L-methionine and L suggested that dissimilatory sulfate reduction predomi cysteine (17). However, the relative contributions of sul nates over catabolism of sulfur amino acids in bacterial fate reduction and ƒÁ- or ƒÀ-elimination of sulfur-con H2S production in pig large intestine. taining amino acids to sulfide production in the mam Molybdate also inhibits methanethiol production malian large intestine is unknown. Sulfate reduction (Fig. 1). Methanethiol is produced by ƒ¿-ƒÁ elimination Inhibition against Volatile Sulfide Production by SRB 197

of L-methionine by L-methionine ƒÁ lyase [EC 4. 4. 1. 11]. Table 3. Inhibitory effects of peppermint oil and The inhibitory effect of molybdate on this enzyme is flavoring compounds on H2S production by D. desulfu unknown. ricans. Our results suggest that SRB may be the most impor tant target organisms for the control of AVS production in the large intestine, although the involvement of SRB in methanethiol production is still controversial. Screening for inhibitors of SRB H2S production Seventy extracts from 38herbs inhibited H2S produc tion by D. desulfuricans DSM642 at 1mg/mL or 0.1 mg/mL (Table 1). The most effective extracts were pre pared from herbs belonging to the Compositae (4spp.), Labiatae (16spp.), Myrtaceae (2spp.), or Zingiberaceae (5

spp.). A. milefolium, A, schoenoprasum, A. oficinarum, A. graveolens, A. dracunculus, A. princeps, C. arabica, C. aro matica, C. Tonga, C, coccineum, E. cardamomum, Mag, of ficinalis, Mel. officinalis, M. rubra, M. communis, N. cataria, P. guajava, R, ideaeus, S. hortensis, and Z. officinale showed inhibitory effects at 1.0mg/mL, but not at 0.1 mg/mL. A. decumbens, G. hederacea, H. lupulus, H. of fici nalis, L. japonicus, L. cardiaca, L. Iucidus, M. vulgare, M. piperita, M. spicata, O. basilicum, O. majarana, P. cablin, P. frutescens, P. crispum, S. rebaudiana, Sis. officinale, and Sym. officinale inhibited sulfide production at both 0.1 and 1.0mg/mL. The inhibitory effect of extracts differed for some herbs. Aqueous extracts of A. schoenoprasum, H. offci nalis, P. frutescens, and L. Iucidus had higher inhibitory effects than ethanol extracts did. A. decumbens showed the opposite tendency. At 0.01mg/mL, only the methanol extract of Sis, of fi cinale showed an inhibitory effect. Some of the herbs tested have antimicrobial activity. For example, Sym, officinale is active against Gram-neg ative and positive bacteria (20), where triterpenes are active components (21). Hop bitter resins are bacterici dal and bacteriolytic (22). The antimicrobial activity of essential oils, including basil, hyssop, parsley, patchouli, peppermint, and spearmint oil, is known (23, 24). In this study we extracted essential oils from M, piperita and M. spicata plants with n-hexane before preparing aqueous extracts. Therefore the components in the aqueous Mentha extracts inhibiting sulfide production by D. desulfuricans should be constituents other than es Compounds showing no inhibitory activity in H2S pro duction by D. desulfuricans: p-anisaldehyde, 1-carvone, sential oil components. caryophyllene, caryophyllene oxide, 1,8-cineole, cinnamyl The pigments eosin, orange I, martius yellow, ery alcohol, citronellyl acetate, ƒ¿-copaene, dimethylbenzyl throsine, phloxine, and rose bengal had an inhibitory carbinol, 2-ethylfuran, farnesene, farnesol, geranyl ace effect at 0.1mg/mL. Eosin, orange I, and martius yel tone, 3-heptanol, 3-heptanone, cis-3-hexenol, cis-3-hex low are not authorized as food additives in Japan. The enyl acetate, traps-2-hexenol, hexyl alcohol, hexyl alde hyde, ƒÀ-ionone, isoamyl alcohol, isoamyl isovalerate, iso effects of the remaining pigments were examined at pulegol, isovaleric acid, isovaleraldehyde, isomenthone, lower concentrations (Table 2). Erythrosine and rose linalool, linalool oxide, menthone, l-menthyl acetate, men bengal showed inhibitory effects at 0.01mg/mL. thyl valerate, 4-methoxybenzyl alcohol, methyl benzoate, Thirty seven of 97 aroma chemicals reduced H2S pro 3-methylcyclohexanone, 2-methyl-4-phenyl-2-butanol, duction at 0.1mg/mL (Table 3). Camphene, 1-decanol, mint lactone, myrcene, neomenthyl acetate, nerolidol, oci and 2-nonanone were the most potent inhibitors. men, 1-octen-3-ol, 1-octen-3-yl acetate, 3-octyl acetate, cis-2-pentenol, phenethyl alcohol, 2-phenoxyethanol, dl-1 In this study, H2S production was measured as the phenylethyl alcohol, phenylethylene glycol, phenylethyl H2S concentration in headspace gas from cultures of an n-valerate, 2-phenyl-2-propanol, 3-phenyl-l-propanol, a intestinal SRB strain, D. desulfuricans DSM642. This in pinene, piperitol, piperitone, pulegone, terpinen-4-ol, terpi vestigation demonstrated that some plant extracts, food neol, tetrahydrolinalool and vanillin. 198 ARAKAWAT et al.

pigments, and aroma substances inhibited H2S produc 1341-1350. tion by this strain. If 20mg/d of a material within the 11) The Ministry of Agriculture, Forestry and Fisheries. ileal fluid (1-2L per day) was transported to the human 1998. Japanese Feeding Standard for Swine, Central large intestine intact, its concentration might be 0.01 Association of Livestock Industry, Tokyo. - 0.02mg/mL, which is sufficient to reduce H2S produc 12) Miller V, Blaut M, Gottschalk G. 1993. Bioenergetics of tion by SRB. The ingestion of a few grams of edible methanogenesis. In: Methanogenesis (Ferry JG, ed), p 361-406. Chapman & Hall, NY. plants such as A. decumbens, H. lupulus, H. officinalis,M. 13) Barton LL, Tomei FA. 1995. Characteristics and activi piperita, M, spicata, O. basilicum, O, majarana, P. ties of sulfate reducing bacteria. In: Sulfate Reducing frutescens, P. crispum, S. rebaudiana, and Sis. officinale Bacteria (Barton LL,ed), p 1-32. Plenum, NY. could affect H2S concentration in the hindgut. 14) Ushida K, Fukusada S, Kojima Y, 1998. Effect of pH on dissimilatory sulfate reduction by porcine cecal mi REFERENCES croflora. ArnimSci Technol69: 571-575. 1) Moore JG, Jessop LD, Osborne DN. 1987. Gas-chro 15) Gibson GR. 1990. Physiology and ecology of the sul matographic and mass-spectrometric analysis of the phate-reducing bacteria. J Appl Bacteriol 69: 769-797. odor ofhuman feces.Gastroenterology 93: 1321-1329. 16) DSM-Deutsche Sammlung von Mikroorganismen und 2) RoedigerWE, Duncan A, Kapaniris O,Millard S. 1993. Zellkulturen GmbH. 1993. DSM Catalogue of Strains, Reducingsulfur compoundsof the colonimpair colono Fifth ed, p 357. cyte nutrition: implications for ulcerative colitis. 17) Persson S, Edlund M-B, Claesson R, 1990. The forma Gastroenterology104: 802-809. tion of hydrogen sulfide and methylmercaptan by oral 3) RoedigerWEW, Moore J, BabidgeW. 1997. Colonicsul bacteria. Oral Microbiol Immunol 5: 195-201. fide in pathogenesis and treatment of ulcerative colitis. 18) Butine TJ, Leedle JA. 1989. Enumeration of selected DigDis Sci42: 1571-1579. anaerobic bacterial groups in cecal and colonic con 4) Florin THJ,Gibson GR, NealeJH, CummingsJH. 1990. tents of growing-finishing pigs. Appl Environ Microbiol A role for sulfate for reducing bacteria in ulcerativecoli 55: 1112-1116. tis? Gastroenterology98: Al70. 19) Ushida K, Ohashi Y, Tokura M, Miyazaki K, Kojima Y. 5) Moore JW, Babidge W, Millard S, Roediger W. 1997. 1995. Sulphate reduction and methanogenesis in the Effectof sulphide on short chain acyl-CoAmetabolism ovine rumen and porcine caecum: a comparison of two in rat colonocytes.Gut 41: 77-81. microbial ecosystems. Dtsch Tierrztl Wochenschr 102: 6) Merino GE,Jetzer T, DoizakiWM, Najarian JS. 1975. 154-156. Methionine-inducedhepatic coma in dogs. Am J Surg 20) Izzo AA, Di Carlo G, Biscardi D, De Fusco R, Mascolo N, 130: 41-46. Borrelli F, Capasso F, Fasulo MP, Autore G. 1995. 7) Blom HJ, Chamuleau RA, Rothuizen J, Deutz NE, Biological screening of Italian medicinal plants for anti Tangerman A. 1990. Methanethiol metabolismand its bacterial activity. Phytother Res 9: 281-286. role in the pathogenesis of hepatic encephalopathy in 21) Ahmad VU, Noorwala M, Mohammad FV, Aftab K. rats and dogs. Hepatology11: 682-689. 1993. Triterpene saponins from roots of Symphytum of 8) Jack TR, Westlake WS. 1995. Control in industrial ficirnale.Fitoterapia 64: 478-479. settings. In: Biotechnology Handbooks. 8 Sulfate 22) Schmalreck AF, Teuber M, Reininger W, Hartl A. 1974. Reducing Bacteria (Barton LL, ed), p 265-292. Structural features determining the antibiotic potencies Plenum, New York. of natural and synthetic hop bitter resins, their precur 9) Gibson GR, Cummings JH, Macfarlane GT. 1988. sors and derivatives. Can JMicrobiol 21: 205-212. Competitionfor hydrogen between sulphate-reducing 23) Morris JA, Khettry A, Seitz EW. 1979. Antimicrobial ac bacteria and methanogenic bacteria from the human tivity of aroma chemicals and essential oils. J Am Oil large intestine. J ApplBacteriol 65: 241-247. Chem Soc 56: 595-603. 10) ClemensET, Stevens CE,Southworth M. 1975. Site of 24) Deans SG, Ritchie G. 1987. Antibacterial properties of organic acid production and patterns of digesta move plant essential oils. Int J Food Microbiol 5: 165-180. ment in the gastrointestinal tract of swine. J Nutr 105: