Tetragenococcus Halophilus in Soy Sauce Fermentation

Review

Diversity and Ecology of Salt Tolerant Lactic Acid Bacteria: Tetragenococcus halophilus in Soy Sauce Fermentation

Kinji UCHIDA

Culture Collection Center, Tokyo University of Agriculture 1-1-1 Sakuragaoka, Setagaya, Tokyo 156-8502, Japan

Soy Sauce (Shoyu) is one of the most representative Japanese traditional fermented foods and has recently become increasingly popular throughout the world. During the moromi-fermentation of soy sauce making processes, a group of lactic acid cocci known as Tetragenococcus halophilus proliferate in moromi-mash which contains a high concentration of sodium chloride, around 18% (w/v) , and produce nearly 1% (w/v) of L-lactic acid. In the early 1980's, a technique for discriminating individual strains was developed, and as a result the diversity in physiological properties among the natural flora of soy lactic acid bacteria has become well known. A wide variety of strains have been found based on physiological properties such as arginine degradation, aspartate decarboxylation, amine-formation from histidine, phenylalanine or tyrosine, consumption of citric or malic acids, and reduction of environmental red-ox potentials, besides in utilization of carbohydrates. Diversity among strains was also observed in their phage-susceptibility and plasmid-profiles. Many of these activities substantially affect the quality of the end products. Accordingly, strain-level control of the fermenting microbes is needed for preparation of high quality soy sauce. Significance of this diversity and possible mechanisms which might have produced it were also discussed from a microbial ecology perspective.

INTRODUCTION salt, through the following five main processes, the Soy Sauce (Shoyu) is one of the most representa- treatment of raw materials, koji production, moro- tive Japanese traditional fermented foods. Recently, mi fermentation, pressing and refining. The moromi it has become increasingly popular throughout the fermentation proceeds under the presence of a high world. Over one million kilo-liters of soy sauce is concentration of salt and takes for more than half produced annually in Japan, and up to one hundred a year. During the moromi period, high molecular thousand kilo-liters of Japanese-type soy sauce is weight substrates in the raw materials are digested produced a year outside Japan, i.e., U.S.A. , the by the enzymes produced by the koji mold. Protein Netherlands, and some other Asian countries. is hydrolyzed into amino-acids or small peptides, Soy sauce is made from soybeans, wheat and and starch is decomposed to glucose which is subse- quently converted into alcohol, lactic acid and other Paper presented in the symposium on "Traditional various flavor substances by the action of Fermented Foods: Microbial Ecology and Technol- fermenting-microorganisms. The concentration of ogy" held on August Pt, 1998, at Hokkaido Univer- sodium chloride in the moromi is usually around sity, Sapporo, Japan. 18% (w/v) , and the microbes involved must be To whom correspondence should be addressed Phone : +81-3-5477-2549 salt-tolerant i.e. a group of lactic acid bacteria Fax : +81-3-5477-2549 known as Tetragenococcus halophilus and some E-mail : [email protected] halophilic or halo-tolerant yeasts. These fermenting

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microbes are often added artificially as starters, Table 1 Diversity in sugar fermentation patterns but remain a part of the natural flora in many tra- of soy lactic acid bacteria ditional fermentation facilities. The role of lactic acid bacteria (soy LAB) in low- ering the pH of moromi has long been thought to be necessary prior to alcoholic fermentation by yeasts, but the physiological properties other than lactic acid fermentation were not known. In the early 1980's, a technique to discriminate individual strains of soy LAB was developee, and the result- ing diversity of physiological properties, which in- fluence the quality of the final product, have become known4.5. Here, the diversity in physiological or biochemical properties among natural flora of soy LAB will be discussed. The significance of the diversity and pos- *A : Arabinose sible mechanisms causing it will also be discussed , L : Lactose, B : Melibiose, from a microbial ecology perspective. S : Sorbitol, M : Mannitol

I. Taxonomy of the soy LAB fermenting ability from the description of this spe- The presence of some tetrad-forming cocci in fer- cies (Pediococcus halophilus) in Bergey's Manual menting soy-moromi was first reported in 1907,' (1986) were observed. but due to the limited technology available 90 years The sugar fermenting property of each strain ago, details of this report cannot be confirmed. could be assayed with good experimental reproduci- After a series of studies on tetrad-forming cocci, a bility, and each strain was thought to be geneti- group of halophilic lactic acid coccus was described cally stable. Therefore, this trait was used as a as a new species, Pediococcus soyae, in 19587.8'. It strain-discriminating property for flora analysis, was soon reclassified as Pediococcus halophilus" and when no sophisticated genetic method was yet cited under this name in Bergey's Manual. In 1990, available. For practical purposes, a soy LAB flora- based on 16S rRNA sequence analysis, this species analyzing scheme was developed, in which 50-100 was transferred to a new genus and named random isolates from a moromi specimen were ex- Tetragenococcus halophilusi°'"). amined based on the fermentation of five sugars, and isolates sharing the same fermentation patterns II. Diversity in Physiological Activities of Soy LAB were tentatively treated as an identical strain. By 1. Sugar fermentation patterns and their applica- the use of this scheme, it was first demonstrated tion to flora analysis') that traditionally processed soy moromi usually Physiological heterogeneity of soy LAB was first harbored at least 10, presumably many more differ- demonstrated in sugar-fermentation studies. Over ent strains of soy LAB. 1500 isolates of soy LAB from various soy moromi Extensive studies on numerous soy LAB isolates samples were tested for their fermention ability on from a variety of naturally brewed soy moromi re- five selected sugars: arabinose, lactose, melibiose, vealed that they were very heterogeneous not only sorbitol and mannitol. According to the fermenta- in their sugar-fermenting abilities but also in other tion patterns, the isolates were separated into 28 physiological properties such as arginine-degra- types (Table 1). The use of 10 kinds of sugar in the dation, amino-acids decarboxylation, organic acids fermentation tests, enabling them to be separated metabolism and environment-reducing activity'''). into at least 67 types having distinct sugar- 2. Amino acid decomposition fermentation patterns. Some discrepancies in sugar The major flavoring ingredients of soy sauce are

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amino acids. In small scale brewing trials, 100 rep- of soy moromi as well as the final products. resentative isolates of soy LAB were tested for Decarboxylation of aspartic acid should be com- their reactions on amino acids. Some strains split parable to that of malic acid in wine making, arginine into ornithine and ammonia (the Arginine known as malo-lactic fermentation (MLF)12. (Fig. deiminase pathway) as known before but the other 1 ) The conversion of a sour amino acid (Asp) to strains did not. The other strains were found to a sweet amino acid (Ala) would make the taste of decarboxylate amino-acids, such as aspartic acid, the products milder as observed in MLF. Recently, histidine, phenylalanine or tyrosine, respectively. a 22kb plasmid encoding aspartate-decarboxylating The generation of two moles of ammonia from a trait was obtained from a Asp-decarboxylating mole of arginine, or the disappearance of a carbox- strain"). This plasmid was determined to contain ylic moiety from one mole of acidic or neutral both genes for an Asp-decarboxylase and an Asp- amino acid results either in an increase in environ- transfering protein according to sequence analysis mental pH or in the neutralization of the lactic of its 10 kb Sall fragment. acid formed. Because of the high concentration of Decarboxylations of His, Phe, and Tyr result in amino acids in moromi, compared with the lactic formation of their amines : histamine, 2-phenethy- acid produced, such reactions on amino acids by lamine and tyramine, respectively. such active strains will significantly affect the pH

Decarboxylation of L-Aspartate in Soy Moromi Fermentation

Malo-Lactic Fermentation(MLF) in Wine Making

Fig.1 L-Aspartate decarboxylation by soy lactic acid bacteria and malo-lactic fermentation in wine.

3. Organic acid metabolism as in MLF, whereas the amount of malic acid in Tetragenococcus halophilus is a homo-fermenter, soy moromi is not great enough to have a signifi- and usually forms nearly 2 moles of L-lactic acid cant effect. from 1 mole of glucose consumed. A few strains 4. Reducing activity form n-lactic acid. In soy moromi fermentation, a This species is known to be facultatively anaero- smaller amount of acetic acid is produced and the bic. Generally microbes have the ability to control ratio of acetic to lactic varies largely strain by the environmental red-ox potential to levels com- strain'''. Most strains can use or decompose citric patible with their survival. Among soy LAB, some acid which is derived from soybeans or formed by strains were found to substantially reduce the red- koji mold, into lactic and acetic acids, but some ox potential of their growing environment"). Other strains can not"). strains reduced the red-ox potential only weakly or Malic acid is decomposed by most parts strains not at all. When the reducing strain grew in soy

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moromi, partial suppression of the browning reac- notable that the frequencies of each fermentation tions was observed. The reddish-brown color of soy pattern were not as one-sided as first speculated. sauce is formed by the browning reaction between The highest frequency observed for a pattern amino acids and sugars. The color could therefore amounted only to 14.1%. In other words, no over- be controlled, to some extent, strains with greater whelming majority exists in the fermentation types. reducing properties. This is very surprising considering that the soy The source of the reducing activity is not pre- LAB have survived countless generations in same cisely known , but reducing strains were found to environment and yet retain this level of diversity. have a NADH-dehydrogenase, for which the optimal In The five test sugars, only arabinose is a known electron acceptors were quinones, such as menadione component of soy moromi, and the others: lactose, or a -naphtoquinonei7). Reduction with NADH of melibiose, sorbitol and mannitol have not been de- some quinone-like intermediates in the browning re- tected. At the stage of lactic acid fermentation, soy actions might be responsible for the lightening of moromi contains nearly 10 % glucose. Under the the moromi color. system of so called catabolite control, the ability 5. Other variations among strains to ferment sugars other than glucose have not A number of bacteriophages infecting soy LAB played any vital role in their survival and most of have been found'''. Their host range is nearly strain- the naturally introduced genetic variations in sugar specific, and the phage-susceptibility of individual metabolism might accumulate as they have, on the strains was found to differ strain by strain. Thus genes of the present population. so called phage-typing of strains of soy LAB be- The possible mechanisms which led to such ge- came feasible. netic diversity, include spontaneous mutations, It has also been shown that strains of this species transformation (conjugal transfer), transduction, harbor a variety of plasmids and plasmid patterns and gene-destruction by transposons, prophages or of the individual strains differ largely among other insertions. Indeed, a number of plasmids espe- strains'''. Most of the plasmids are cryptic and the cially those of smaller sizes were found in traits encoded are not known. T.halophilus isolatee, and conjugal transfer of an antibiotic-resistant plasmid from Ent erococcus III. Significance of the Diversity in Soy Lactic Acid fecalis was also observed2" . The existence of a Bacteria prophage in T. halophilus cells has not been re- Despite all these diversities among the strains of ported so far, but it could reasonably be supposed soy LAB mentioned above, it was reported recently taking into account the occurrence of virulent that all of the 46 isolates of salt-tolerant lactic phages infecting this species. cocci obtained from soy moromi and other origins Genetic studies on soy LAB are still very meager showed very high DNA-DNA homology to the type to date, and much researches will be needed before strain of Tetragenococcus halophilus"). This indi- detailed speculations can be made. cates that they can be classified within a single spe- In contrast to the diversities found in sugar- cies. Sequence data on 16S rRNA of some fermentation, variations in amino-acid conversion representative strains also revealed their among soy LAB have a substantial impact on their phylogenetic uniformity. It is therefore likely that, viability, and on product quality, since amino acids the diversity in biochemical or physiological proper- are the major ingredients of soy sauce. ties observed among populations of soy LAB are Some strains of soy LAB degrade arginine variations occurring within a species. through the Arg-deiminase pathway. This results in The diversity among the strains of Tetrageno- formation of 1 mole of ornithine, 2 moles of am- coccus halophilus is most evident in their sugar fer- monia and 1 mole of ATP. For a bacterial strain, mentation. Twenty-eight out of the theoretically possession of an additional energy-supplying system possible 32 types were obtained from the fermenta- other than the glycolysis pathway is of vital impor- tion tests using five sugars on 1586 isolates. It is tance especially under the unfavorable conditions

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such as low temperature, low pH, and high salt mentioned before, a plasmid encoding the aspartate- concentrations, as found in soy moromi. It has been decarboxylating trait has recently been reported'', observed that the population of Arg-degrading but other genetic factors controlling these activities strains was significantly larger in low-temperature are not yet known. processed moromi than in moromi processed at a normal temperature. On the other hand, from the decarboxylations of aspartic acid, histidine, phenylalanine or tyrosine, no substrate-level energy is generated. Until several years ago, the advantage of the decarboxylating trait for a strain was thought to be only in coun- tering environmental pH changes. But recently, it became known that accumulation of proton motive force generated by a set of reactions: CI entry of a carboxyl-group bearing precursor into the cell, ® in- tracellular decarboxylation and (D exit of the decarboxylated product, could promote ATP synthe- sis by a FfiF, type proton-ATPase (Proton-motive metabolic cycle) (Fig.3). Therefore, the decarboxylation of amino acids would assist energetically the growth or other biological activities of the decarboxylating strains, in addition to prevent a Fig. 2 Time course of oxidation-reduction poten- decrease in environmental pH. The decarboxylation tials of soy moromi inoculated with reducing (0) would no doubt be advantageous for positive or non-reducing (S) strains of soy lactic acid bacteria (From Kanbe & Uchida, Nippon Nogeikagaku strains in competition with negative strains. As Kaishi, 58, 487).

Fig. 3 A proton-motive metabolic cycle associated with aspartate decarboxylation (From Abe et al., J. Biol. Chem. 271, 3079).

Recently, two articles dealing with the phenotype isolates from Chinese- and Japanese-type soy mash diversity in T. halophilus were published. R ling in Indonesia, and found that populations in the and Verseveld2a characterized 69 T. halophilus Japanese-type were more heterogeneous regarding

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sugar-utilization. Finger-printings of cellular pro- S.: The phylogeny of Aerococcus and Pediococcus tein showed that all the isolates belonged to one as determined by 165 rRNA sequence analysis: Description of Tetragenococcus gen. nov. FEMS species: T. halophilus. They also studied genetic re- Microbial. Lett. , 70, 255-262 (1990). latedness by RAPD analysis, and speculated on the 11) IUMS Committee: Validation of the publication of new names and new combinations previously ef- reasons for the development of heterogeneous popu- fectively published outside the IJSB. List No. 44. lations. More recently, Giirtler et al." reported hit. J. Syst. Bacterial. , 43, 188-189 (1993) 12) Uchida, K.: Trends in preparation and uses of physiological, diversity among strains of T. fermented and acid-hydrolyzed soy sauce. In "Proceedings of the World Congress on V halophilus, e. g. salt-tolerance, pH-dependency, abil- egetable ity to reduce nitrite, or response to oxygen expo- Protein Utilization in Human Foods and Animal Feedstuffs, Singapore, 1988". ed. Applewhite, T. sure, with special reference to their application to H., American Oil Chemists' Society (1989). protein-rich foods. 13) Higuchi, T., Uchida, K. and Abe, K.: Aspartate Accumulation of the studies in this field will con- decarboxylation encoded on the plasmid in the soy sauce lactic acid bacterium, Tetragenococcus tribute to a better understanding of the ecological halophila D10. Biosci.Biotchnol.Biochem., 62, relationships in soy LAB. 1601-1603 (1998). 14) Kanbe, C. and Uchida, K.: Diversity in the me- All these microbial diversities associated with soy tabolism of organic acids by Pediococcus moromi fermentation are likely to affect the qual- halophilus. Agric. Biol. Chem., 46, 2357-2359 ity of moromi and accordingly that of the end (1982). 15) Kanbe, C. and Uchida, K.: Citrate metabolism by products. Strain level control of the fermenting mi- Pediococcus halophilus. Appl. Enuir. Microbial., crobes will thus be needed for fine control of soy 53, 1257-1262 (1987). 16) Kanbe, C. and Uchida, K. Reducing activity of sauce quality. This should also be applicable in the soy pediococci (Pediococcus halophilus) and near future to other traditional fermented foods. oxidation-reduction potentials of soy sauce mash. Nippon Nogeihagaku Kaishi (in Japanese), 58, 487-490 (1984). References 17) Kanbe, C. and Uchida, K.: NADH dehydrogenase 1) Yokotsuka, T.: Soy sauce biochemistry. Adv. activity of Pediococcus halophilus as a factor'de- Food Research, 30, 195-282 (1986). termining its reducing force. Agric. Biol. Chem., 2) Fukushima, D.: Industrialization of fermented soy 51, 507-514 (1987). sauce production centering around Japanese 18) Uchida, K. and Kanbe, C.: Occurrence of shoyu. In "Industrialization of indigenous fer- bacteriophages lytic for Pediococcus halophilus, a mented foods" ed. Steinkraus, K. H., Marcel halophilic lactic acid bacterium, in soy sauce fer- Dekker, Inc. , New York, (1989). mentation. J. Gen. Appl. Microbial., 39, 429-437 3) Uchida, K.: Multiplicity in soy pediococci carbo- (1993). hydrate fermentation and its application for 19) Kayahara, H., Yasuhira, H. and Sekiguchi, J.: analysis of their flora. J. Gen. Appl. Microbial., Isolation and classification of Pediococcus 28, 215-223 (1982). halophilus plasmids. Agric. Biol. Chem., 53, 3039- 4) Uchida, K.: Heterogeneity of soy lactic acid bac- 3041 (1989). teria and their use in soy sauce brewing. Nippon 20) Hanagata, H., Shida, 0., Tagami, H., Takagi, H. Jozo Kyokaishi (in Japanese), 77, 740-742 (1982). and Kadowaki, K.: unpublished results. (cf. 5) Uchida, K. and Kanbe, C.: Studies on the physio- Nippon Nogeikagaku Kaishi, 71, supplement: 1997 logical diversity of soy pediococci and their ap- Ann. Meeting Abstr. 215) plications. Nippon Shoyu Kenkyusho Zasshi (in 21) Toda, A., Kayahara, H., Yasuhira, H., and Japanese), 13, 251-258 (1987). Sekiguchi, J.: Conjugal transfer of pIP501 from 6) Saito, K.: Mikrobiologische Studien uber die Enterococcus faecalis to Pediococcus halophilus. Soyabereitung. Centralbl. f. Bakt. Abt. II, 17, 152- Agric. Biol. Chem., 53, 3317-3318 (1989). 161 (1907). 22) Abe, K., Hayashi, H. and Maloney, P. C.: Ex- 7) Sakaguchi, K.: Studies on the activities of bacte- change of aspartate and alanine — Mechanism for ria in soy sauce brewing III. Taxonomic studies development of a proton-motive force in bacteria. on Pediococcus soyae nov. sp., the soy sauce lac- J: Biol. 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