Sporolactobacillus Inulinus Is a Spore-Forming Homo-Fermenting Lactic Acid Bacterium Described by Kitaharaand Suzuki (1)

J. Gen. Appl. Mierobiol., 33, 33-45 (1987)

MORPHOLOGICAL, BIOCHEMICAL, AND PHYSIOLOGICAL CHARACTERISTICS OF SPORE-FORMING LACTIC ACID BACTERIA

FUJITOSHI YANAGIDA, KEN-ICHIRO SUZUKI,* TAKICHI KANEKO,* MICHIO KOZAKI, AND KAZUO KOMAGATA**

Department of Agricultural Chemistry, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156, Japan *Japan Collection of Microorganisms, RIKEN, Wako, Saitama 351-01, Japan **Institute of Applied Microbiology, The University of Tokyo, Bunk vo-ku, Tokyo 113, Japan

(Received December 1, 1986)

One hundred and eighteen strains of spore-forming lactic acid bacteria, and one strain of Lactobacillus plan tarum were investigated with regard to morphological, biochemical, and physiological characteristics. These characteristics were analyzed numerically. The strains were separated into three clusters. Cluster 1 consists of 44 strains including Sporolactobacillus inulinus (2 strains), "Sporolactobacillus laevas" (15 strains), "Sporolacto- bacillus laevas var. intertnedius" (7 strains), "Sporolactobacillus racemicus" (7 strains), "Bacillus laevolacticus" (12 strains), and "Bacillus racetnilac- ticus" (1 strain). Cluster 2 consists of 70 strains: "B. laevolacticus" (57 strains), "B. racemilacticus" (2 strains), "S. racemicus" (8 strains), "S. laevas" (2 strains), and "S. laevas var. intertnedius" (1 strain). Cluster 3 comprises 3 strains including the type strain of Bacillus coagulans and one strain each of "B. laevolacticus" and "B. racemilacticus." One strain of "S. racemicus" and the type strain of Lactobacillus plantarum were not included in the above clusters. The clusters are considered to correspond respectively to the taxa Sporolactobacillus, "Bacillus laevolacticus," and Bacillus coagulans. Catalase activity and production of acid from lactose and melibiose were useful in differentiating the three clusters.

Sporolactobacillus inulinus is a spore-forming homo-fermenting lactic acid bacterium described by KITAHARAand SuzuKi (1). This species has characteristics intermediate between those of the genus Bacillus and the genus Lactobacillus. S. inulinus is catalase-negative and produces lactic acid as Lactobacillus species do. On

Address reprint requests to: Dr. M. Kozaki, Department of Agricultural Chemistry, Tokyo University of Agriculture, 1-1, Sakuragaoka 1-chome, Setagaya-ku, Tokyo 156, Japan. 33 34 YANAOIDA,Suzun, KANEco,KozAxl, and KoMAGATA VOL.33 the other hand, it is motile and forms endospores as Bacillus species do. The genus Sporolactobacillus was formerly described as a subgenus of the genus Lactobacillus (1). Later, it was described as an independent genus in the family Bacillaceae by KITAHARAand TOYOTA(2) and in Bergey's Manual of Determinative Bacteriology, 8th edition (3). NAKAYAMAand YANOSHI(4, 5), and AMEMIYAand NAKAYAMA(6) isolated a number of spore-forming, lactic acid-producing strains from soil in plant rhizo- spheres. Most of the strains produced the D(-) form of lactic acid and lacked catalase. Several strains produced the DL form of lactic acid and lacked catalase (5). These strains were considered to be the members of the genus Sporolactobacillus (5, 7). At the same time, NAKAYAMAand YANOSHI(4,8) also isolated a number of catalase-positive, spore-forming lactic acid bacteria, and classified them in the genus Bacillus (4, 7). To differentiate the two genera, NAKAYAMAand YANOSHI(8), using the catalase activity and the optical form of lactic acid produced, established two species, "Bacillus laevolacticus" and ``Bacillus racemilacticus" (7). Here we investigate the phenotypic characteristics of S. inulinus and some other spore-forming lactic acid bacteria. Some authentic strains of Bacillus coagulans and Lactobacillus plan tarum are also included for comparison. B. coagulans, a member of the genus Bacillus, produces lactic acid (9). L, plan tarum has meso-diamino- pimelic acid in the cell wall (10, 11). From the phenotypic viewpoint, these two species are considered to be most closely related to Sporolactobacillus in the two genera.

MATERIALSAND METHODS

Bacterial strains. The bacterial strains used in this study are listed'below. The names in quotation marks refer to taxa not on the Approved Lists of Bacterial Names (12) or have not been validly published. The superscript "T" shows the type strain of the species. The abbreviations for culture collections are as follows: ATCC, American Type Culture Collection, Rockville, Maryland, U.S.A.; IAM, Institute of Applied Microbiology, the University of Tokyo, Tokyo, Japan; JCM, Japan Collection of Microorganisms, RIKEN, Wako-shi, Saitama, Japan; NRIC, NODAI Research Institute Culture Collection, Tokyo University of Agriculture, Tokyo, Japan. The type strains of Sporolactobacillus inulinus NRIC 1133' (=ATCC 15538), Bacillus coagulans NRIC 1005' (= IAM 1115 = JCM 2257 =ACTT 7050), Lactobacillus plantarum NRIC 1067T(= ATCC 14917 = JCM 1149) were obtained from the NRIC Culture Collection. Sporolactobacillus inulinus NRIC 1134 (5-50- C), came from Prof. 0. Kandler (University of Munich, West Germany). A total of 116 strains of spore-forming lactic acid bacteria isolated by NAKAYAMAand YANOSHI(4,5) and AMEMIYAand NAKAYAMA(6) were obtained from Dr. 0. Nakayama, Yamanashi University and the TAM Culture Collection. In 1987 Taxonomy of Spore-forming Lactic Acid Bacteria 35 the list below, the strains are arranged according to the classification by NAKAYAMA(8). "Bacillus laevolacticus" Nakayama and Yanoshi (71 strains): M-1 (=IAM 12322 =ATCC 23493 =JCM 2514), M-4, M-7 (= IAM 12323 = ATCC 23494 =JCM 2515), M-8 (=IAM 12321 =ATCC 23492 =JCM 2513), M-21, M-22, M- 23, M-24, M-25, M-26, M-27, M-28, M-29, M-34, M-35, M-36, M-37, M-38, M- 40 (=IAM 12324 =ATCC 23495), M-41, M-42, M-43, M-44, M-48, M-49, M-52, M-53, M-55, M-57, M-58, M-63, M-65, M-66 (= IAM 12327), M-67, M-68 (= IAM 12328), M-69, M-70, M-71(= IAM 12329), M-72, M-73, M-75 (= IAM 12379), M- 76, M-77, M-78, M-79, M-80, M-81, M-82, M-83, M-89, M-90, M-91 (= IAM 12330), M-92, M-93, M-94, M-95, M-97, M-100, M-102, M-104 (=IAM 12331), M- 105 (=IAM 12326), M-106, M-107, M-108, M-109, M-110, M-111, M-119, M-120, M-121, and M-122. "Bacillus racemilacticus" Nakayama and Yanoshi (4 strains): M-5 ( = IAM 12319 =ATCC 23497 =JCM 2518), M-14 (=IAM 12318 =ATCC 23496 =JCM 2517), M-39 (=IAM 12320 =ATCC 23498), and M-64. "Sporolactobacillus laevas" Nakayama (17 strains): M-18, M-19, M-20, M-45 (= IAM 12382), M-46 (= IAM 12383), M-47, M-50, M-51, M-56, M-59, M-84, M- 87 (=IAM 12385), M-88 (=IAM 12386), M-101, M-114 (=IAM 12388), M-117, and M-118 (= IAM 12384). "Sporolactobacillus laevas var. intermedius" Nakayama (8 strains): M-54, M- 85, M-86 (=IAM 12384), M-96, M-98, M-99, M-103 (=IAM 12387), and M-113. "Sporolactobacillus racemicus" Nakayama and Yanoshi (16 strains): M-3, M- 10, M-11, M-12, M-13, M-16, M-17, M-30, M-31, M-32, M-33, M-60 (= IAM 12398), M-61, M-62, M-115, and M-116. Strain designations with "M" were those originally used by NAKAYAMAand YANOSHI(4, S), and NAKAYAMA(8). Cultivation media. All the strains were grown in GYP medium containing 2° glucose, 1 % yeast extract (Oriental Yeast Co., Tokyo, Japan), 1% peptone (Kyokuto Seiyaku Co., Tokyo, Japan), 100 sodium-acetate, and 0.5% (voljvol) salts solution (pH 6.8). The salts solution contained 4% Mg5O4 7H20, 0.2 MnSO4.4H20, 0.2% FeSO4 7H20, and 0.2°c NaCI. For solid medium, the above medium contained 2/ agar in addition. All organisms were grown at 30 C. Characterisation test. Gram staining was carried out by the method of HUCKERand CONN's modification (13). Motility was examined by the method of OKADAet al. (14) using the cells grown in GYP soft agar medium (GYP + 0.15 agar). For the catalase test, the cells grown on GYP agar slants for 5-7 days were dipped in a 3 ° o hydrogen peroxide solution, and bubbling was examined. The presence of spores was observed by microscopy and tested by survival after heating the cells at 80` C for 10 min. Growth in nutrient broth containing 100 meat extract, 0.500 peptone, and 0.3 sodium-chloride was determined after one week of incubation at 30°C. Effect of temperature on growth was trested in GYP broth at 15, 20, 25, 30, 35, 40, 45, and 36 YANAGIDA, SUZUKI, KANEKO, KOZAKI, and KOMAGATA VOL. 33

Table I. Biochemical and physiological 1987 Taxonomy of Spore-forming Lactic Acid Bacteria 37 38 YANAGIDA, SUZUKI, KANEKO, KOZAKI, and KOMAGATA VOL. 33

Table 1. 1987 Taxonomy of Spore-forming Lactic Aci d Bacteria 39 40 YANAGIDA SUZUKI, KANEKO, KOZAKI, and KOMAGATA VOL. 33

Table 1. 1987 Taxonomy of Spore-forming Lactic Acid Bacteria 41 42 YANAGIDA, SuzuKI, KANEKO, KOZAKI, and KOMAGATA VOL. 33

50°C. The effect of NaCI concentration on growth was determined in GYP broth with different concentrations of sodium chloride (l, 2, 3, 4, 5, 7, and l0%). The litmus milk reaction was investigated for acidification, coagulation, liquefaction, and reduction of an indicator. Acid production from carbohydrates was examined by the method of MITSUOKA(15), modified by using liquid media without L-cystein-HC1 •H2O. The optical forms of lactic acid were determined by the method of OKADAand KozAKI (16). Numerical analysis. Similarity values were calculated using the formula of JACCARD(17). Dendrograms were obtained by the arithmetic average linkage- clustering method (18), using 44 phenotypic characters of 119 strains. A FACOM M380 computer (Fujitsu Co., Tokyo, Japan) was used for the calculation. For the result of optical forms, D( -) and D+ DLlactic acids were described as " + ," and the others as "- ."

RESULTS

The morphological, biochemical, and physiological characteristics of the strain studied are shown in Table 1. The strains are arranged according to the result of numerical analysis. Details of the clusters will be described below.

Morphological characteristics All of the strains studied were gram-positive rods. Strains named "B. laevolacticus" and "B. racemilacticus" except for "B. laevolacticus" M-22 produced endospores which are resistant to 80'C for 10 min. Cells of "S. laevas, " "S. laevas var. intermedius," and the "S. racemicus" strains were also heat-resistant except for "S . racemicus" M-3, "S. racemicus" M-11, "S. racemicus" M-33, and "S. laevas var. intermedius" M-113. Motility appeared in 108 strains but not in 8 strains of NAKAYAMAand YANOSHI(4, 5), and AMEMIYAand NAKAYAMA(6).

Catalase activity Seventy-five strains named "B. laevolacticus" and "B. racemilacticus" by NAKAYAMAand YANOSHI(4,5) and AMEMIYAand NAKAYAMA(6) showed catalase activity except for six strains of "B. laevolacticus" (M-43, M-70, M-102, M-104, M- 108, and M-122). Some strains exhibited catalase activity in the cells grown on solid medium but not in the cells grown in liquid medium. However, the activity of these strains was not so strong as that of B. coagulans strains. The strains "B. laevolacticus" M-27 and "B. racemilacticus" M-39 exhibited strong catalase activity. Most of the Sporolactobacillus strains were catalase negative, even when the cells grew on solid medium. The strains "S. racemicus" M-31, "S. laevas" M-117, and "S. laevas" M-118 showed a positive reaction for catalase. 0 0

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Determination of optical form of lactic acid "B , laevolacticus" (69 strains) except strains M-27 and M-106 produced D(-) or D+ DL lactic acid. Strains of "B. racemilacticus" produced D+ DL lactic acid but strain M-39 produced L+ DL isomer. Both "S. laevas" (16 strains) and "S. laevas var. intermedius" (6 strains) produced D+ DL lactic acid. "S. racemicus" (7 strains) produced DLlactic acid, and "S. racemicus" (9 strains) produced D+ DLisomer. The optical forms of lactic acid produced by several strains do not coincided with those determined by NAKAYAMAand YANOSxI(4, 5), and by AMEMIYAand NAKAYAMA(6).

Other phenotypic characters All the strains studied grew at 20 to 40`'C. About 40°c of "B. laevolacticus" and "B. racemilacticus" strains and 85% of "S. laevas," "S. laevas var. intermedius," and "S. racemicus" strains grew at 15°C. "B. laevolacticus" and "B. racemilacticus" grew weakly in nutrient broth, whereas all strains of "S. laevas," "S. laevas var. intermedius," and "S. racemicus" did not grow in the medium. Most of the Bacillus strains studied exhibited acidification, reduction of the indicator, and coagulation of litmus milk, whereas only acidification was observed in Sporolactobacillus strains. The Sporolactobacillus strains generally tolerated higher concentrations of sodium chloride (5-7/) than the Bacillus strains (3-4%). All the strains studied produced acid from glucose, fructose, and mannose.

Numerical analysis Figure 1 shows a dendrogram drawn on the basis of the above phenotypic data. Spore-forming lactic acid bacteria were divided into three clusters. The members of each cluster are shown in Table 1. Cluster 1 comprised 44 strains: S. inulinus (2 strains), "S. laevas" (15 strains), "S . laevas var. intermedius" (7 strains), "S. racemicus" (7 strains), "B. laevolacticus" (12 strains), and "B. racemilacticus" (l strain). These strains produced D + DL lactic acid, and did not produce acid from lactose and melibiose. Furthermore, 33 out of 44 strains of cluster 1 were catalase negative. Cluster 2 comprised 70 strains: "B. laevolacticus" (57 strains), "B. racemilacticus" (2 strains), "S. racemicus" (8 strains), "S. laevas" (2 strains), and "S. laevas var. intermedius" (1 strain). This cluster was characterized by the ability to produce acid from lactose and melibiose. Six strains of "S. racemicus" in cluster 2 produced DLlactic acid and other strains produced D + DL lactic acid. Furthermore, 57 out of the 70 strains were catalase positive. Cluster 3 comprised three strains: B, coagulans NRIC 1005T, "B. laevolacticus" M-27, and "B. racemilacticus" M-39. They are matched at 80°c of similarity. These strains produced the L( +) isomer of lactic acid and grew at 50°C. Strains M-27 and M-39 were identified as B. coagulans. L. plantarum NRIC 1067Tand "S. racemicus" M-3 were not included in any of the three clusters. These strains produced the DL form of lactic acid and lacked catalase. 44 YANAGIDA, SUZUKI, KANEKO, KOZAKI, and KOMAGATA VOL. 33

DISCUSSION

Numerical analysis of the phenotypic data indicated that the strains studied formed two large clusters which were independent of the strains of B. coagulans and L. plantarum. These two clusters seem to correspond to the genus Sporolactobacillus and the "B. laevolacticus" species. The genus Sporolactobacillus was originally characterized by a negative reaction in catalase activity (1, 2). The concept for differentiation of the genera Sporolactobacillus from Bacillus by NAKAYAMAand YANOSHI(4, 5) is generally supported by the present results. However, clear differentiation of the two clusters cannot be completed using catalase activity only. In cluster 1, '75°c of the strains were catalase negative. In cluster 2, 8l° of the strains were catalase positive. The combination of catalase activity and acid production from lactose and melibiose strongly support the numerical clustering of the strains. Strains positive in catalase and in acid production from lactose and melibiose were found only in cluster 2. Strains which did not have these three characteristics were exclusively in cluster 1. So the two clusters can be distinguished best by the production of acid from lactose and melibiose. Among the strains positive in acid production from at least one of the two sugars, 92°c of the strains are included in cluster 2. Ninety percent of the strains that did not produce acid from either lactose or melibiose belonged to cluster 1. Strains of cluster 3 show strong catalase activity and produce L( + )lactic acid. Cluster 3 includes the type strain of B. coagulans and corresponds to that species. On the other hand, the optical form of lactic acid, which NAKAYAMAand YANOSHI(4, S) regarded as the specific criterion of these bacteria, was not effective for differentiation. Therefore, DNA-DNA homology studies are required not only for the differentiation of these strains at a species level, but also for evaluation of the phenotypic characteristics. Wethank Dr. 0. Nakayama,Yamanashi University, Kofu, for kindlysupplying cultures and also for valuablecomments. We also thank Dr. K. Yamasato,Institute of AppliedMicrobiology, The Universityof Tokyo,Tokyo, for supplyingcultures. REFERENCES

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