Polysaccharide Formation by Spore-Bearing Lactic Acid Bacteria

J. Gen. App!. Microbiol., 26, 159-166 (1980)

POLYSACCHARIDE FORMATION BY SPORE-BEARING LACTIC ACID BACTERIA

YUMIKO AMEMIYA AND OOKI NAKAYAMA

Department of Engineering, Yamanashi University, Kofu, 400 Japan

(Received February 1, 1980)

A small amount of water-soluble fructan (F) was formed from sucrose by 71 strains of Bacillus laevolacticus (I) and some strains of Sporo- lactobacillus inulinus. A gelatinous mass of water insoluble glucan (G), together with F, was formed from sucrose by a few strains of I. Polysaccharide formation was not observed in such groups of spore- bearing lactic acid bacteria as Bacillus coagulans, Bacillus racemilacticus and racemic lactic acid-producing Sporolactobacillus. Aeration was not needed for the formation of either F or G. Yield of G was up to 25 from sucrose and 2 °o from yeast extract-peptone broth with 8 % sucrose. G was solubilized in 0.1 N sulfuric acid by heating at 100° for 20 min, and the solubilized G (SG) was partially hydrolyzed by dextranase. Molecular weights of SG and F were about 6.4 x 10~ and more than 2 x 107, respectively. Antitumor activity of both SG and F against mouse Sarcoma 180 was about 60%.

So called spore-bearing lactic acid bacteria, i.e., sporulating rode capable of homolactic acid fermentation, may be classified into the following five species in respect to the optical form of the excreted lactic acid and catalase activity: D(-) lactic acid-producing group catalase (+) Bacillus laei'olacticus NAKAYAMAet al. (1) (I) catalase (-) Sporolactobacillus inulinus KITAHARAet al. (2) (II) DL lactic acid-producing group catalase (+) Bacillus racemilacticus NAKAYAMAet al. (1) (III) catalase (-) unidentified Sporolactobacillus (3) (IV) L(±) lactic acid-producing strains catalase (-}-) Bacillus coagulans HAMMER(4,5) (V) We isolated many strains of these spore-bearing lactic acid bacteria, and found polysaccharide formation by the D(-) lactic acid producing group.

159 160 AMEMIYA and NAKAYAMA VOL. 26

MATERIALSAND METHODS Bacterial strains. Seventy-one strains of I, 16 strains of II, 4 strains of III, 25 strains of IV and 78 strains of V were used. Most strains were isolated from the soil of Japan and Southeast Asia, according to the method described in a pre- vious paper (1). Examination for polysaccharide production. Polysaccharide-producing strains were selected by observation of slime formation on the yeast extract-peptone- sucrose (0.5 %, 1 % and 2 %) agar slant, after incubation at 30° for several days. Conditions for polysaccharide production were determined by modifying the medium composition, inoculum and other factors. Preparation of polysaccharides. Water-soluble polysaccharide was prepared as follows : Yeast extract-peptone-sucrose (0.5, 1 and 2 %) broth was inoculated with Bacillus laevolacticus IAM 12326 which produces a water-soluble polysaccharide only, or IAM 12329 which produces both water-soluble and -insoluble polysaccharides, and incubated at 30° for several days. After removal of bacterial cells and water-insoluble polysaccharide from the culture broth, the water-soluble polysaccharide was precipitated by adding to the broth twice its volume of aceton and collected by centrifugation, and the precipitate was dissolved in water and lyophilized after repeated precipitation by aceton and dialysis against distilled water in cellophane tubing. Water-insoluble polysaccharide was prepared by the following procedures : Yeast extract-peptone-sucrose (0.5, 1 and 8 %) agar slants were inoculated with freshly germinated cells of Bacillus laevolacticus IAM 12329, and incubated at 30° for several days. An amorphous brittle mass of water-insoluble polysaccharide, in which bacterial cells were embedded, was formed. Yeast extract-peptone-sucrose (0.5, 1 and 8 %) broth was inoculated with the mass, incubated at 30° for several days until the mass grew larger, and the mass was scooped from the culture broth, and washed in running water overnight. The polysaccharide, which is not soluble in water and barely soluble in alkaline water, was solubilized

Fig. 1. Glucan produced by B. laevolacticus IAM 12329. (A) Sucrose broth culture with masses of glucan. (B) Drained raw glucan. (C) Solubilized and lyophilized glucan. 1980 Spore-Bearing Lactic Acid Bacteria 161 by partial hydrolysis in 0.1 N sulfuric acid held at 100° for 20 min. The solution was centrifuged to remove bacterial cells, dialysed against distilled water and lyophilized. Samples used for physico-chemical examination were further purified by successive chromatography on a DEAE-cellulose column and Sephadex G- 200 gel column filtration. Investigation of polysaccharides. Constituent sugars were determined by thin layer chromatography (TLC) on a chrystalline cellulose plate and gas-liquid chromatography (Hitachi 163, FID) of trimethylsilyl derivatives, after hydrolysis by 0.3 N HCI at 85° for 10 min for soluble polysaccharide, or by methanolic 1 N HCl at 85° for 3 hr for solubilized water-insoluble polysaccharide. Yields of reducing sugars were estimated by SoMOCYI-NELSON'Smethod. Solvents for TLC were as follows : a) n-propanol-ethylacetate-H2O, 14 : 2 : 7, b) n-butanol-AcOH-H20, 60:15: 25, c) n-butanol-ethanol-H20, 4:1: 5 (upper layer) and d) phenol-1 % NH4OH, 4 : 1; ratios are v/v. Conditions for gas-liquid chromatography were as follows : Glass column 5 mm x 1 m, Chromosorb WHP 60/80 mesh, 1.5% OV-1 or OV-17, column temperature 140°, injection temperature 190°: flow rate N2 20 ml/min, H2 50 ml/min, air 100 ml/min, range 10, attenuation 32, injection volume 0.1,u1, trimethylsilyl derivative, determination: SENNELLO'smethod. Enzymatic analysis was performed by determination of reducing sugars after incubation with glycosidases at the optimum pH and temperature for 24 hr. Mole- cular weight was roughly determined by gel filtration through Sepharose CL-2B for water-soluble polysaccharide and Sephadex G-200 for solubilized water-insoluble polysaccharide. The following instruments were used for determination of the other items: Perkin Elmer-240 elemental analyzer, Yanagimoto MP-S2 melting point meter, Hitachi PO-B polarimeter, Hitachi EPI-S2 infrared spectrometer and Ostwald type viscosimeter. Toxicity and antitumor activity of polysaccharides. Five-week-old ICR mice obtained from Charles River Breeding Laboratories, Inc. were used after feed- ing for 4 days. Sarcoma 180 (2 x 106 cells/0.25 ml) in ascites was injected sub- cutaneously in the groin of each mouse, and dosal polysaccharide dissolved in 0.25 ml of saline was injected intraperitoneally every day for 10 days. After 5 weeks, the inhibition ratio was calculated from the weights of tumors from experimental (T) and control (C) mice according to the following equation: Inhibition ratio=(C-T) x 100/C.

RESULTS

Screening of polysaccharide producing strains Polysaccharide production seems to be restricted to D(- ) lactic acid-producing strains as shown in Tables 1 and 2. 162 AMEMIYA and NAKAYAMA VOL. 26

Table 1. Polysaccharide-producing strains of spore-bearing lactic acid bacteria.

Table 2. List of polysaccharide producing strains. 1980 Spore-Bearing Lactic Acid Bacteria 163

Conditions for polysaccharide production The following sugars and sugar derivatives were not useful in polysaccharide production: L-arabinose, D-xylose, D-ribose, D-mannose, D-glucose, D-galactose, D-fructose, maltose, cellobiose, lactose, trehalose, a-methylglucoside, mannitol, sorbitol and Na-gluconate. Polysaccharide production was supported only by sucrose. Optimum concentrations of sucrose were about 2 and 8 % for the production of water-soluble polysaccharide by IAM 12326 and water-insoluble polysaccharide by IAM 12329, respectively. Aeration was not needed for production of either polysaccharide. Although special care for preparation of the inoculum was not necessary for the production of water-soluble polysaccharide, water-insoluble polysaccharide was produced only when sucrose medium was inoculated with the primary culture started from spores, or with masses of water-insoluble polysaccharide in which live bacterial cells were embedded. In the latter case, the masses grew gradually, until the flask was almost filled with large amorphous masses of gelatinous matter.

Yield of polysaccharides A small percentage of the consumed sucrose was converted into water-soluble polysaccharide in both sole production and production together with that of water- insoluble polysaccharide. On the other hand, yield of the water-insoluble polysaccharide was up to 2500 from used sucrose or 20 g/l from 8 % sucrose broth.

Characteristics of polysaccharides Yields of reducing sugars by acid hydrolysis of water-soluble and solubilized water-insoluble polysaccharides were 96.3 % and 97.2 %, respectively, and hydroly- sates gave only one spot corresponding to fructose or glucose on each TLC plate, and one peak corresponding to TMS-fructose or two peaks corresponding to TMS- a and i3-glucoses on gas chromatogram, respectively. Therefore, the water- soluble and water-insoluble polysaccharides produced by B. laevolacticus were fructan and glucan, respectively. As shown in Table 3, the jS-fructofuranoside configuration seems not to be the main part of the fructan. As generally known, /3-2, 1 and /3-2, 6 configura- tions are commonly distributed among fructans from higher plants and bacteria, respectively, but we have not yet obtained enough information to discuss the configuration of the fructan from B. laevolacticus and S. inulinus. The a-l, 6 configuration is marked in the solubilized water-insoluble polysaccharide, which is somewhat similar to dextran from Leuconostoc mesenteroides in specific rotation and profile of the infrared spectrum shown is Figs. 2-4, but is different in its low specific viscosity in spite of its relatively high molecular weight.

Toxicity and antitumor activity of polysaccharides Neither polysaccharide showed any acute toxicity after daily intraperitoneal injection of 50 mg/kg into the experimental mice. As shown in Table 4, anti- 164 AMEMIYA and NAKAYAMA VOL. 26

Table 3. Characteristics of polysaccharides produced by spore-bearing lactic acid bacteria.

Fig. 2. Infrared spectrum of fructan produced by B. laevolacticus IAM 12326 (0.3 % per KBr tablet). 1980 Spore-Bearing Lactic Acid Bacteria 165

Fig. 3. Infrared spectrum of solu bilized glucan produced by B, laei'olacticus TAM 12329 (0.3% per K Br tablet).

Fig. 4. Infrared spectrum of dextran from Leuconostoc mesenteroides as control (Dextran "Wako", Av. m. w. 100,000-200,000, 0.3 % per KBr tablet).

tumor activity of the polysaccharides did not show any striking values, but was of about the same order as that of ordinary biological polymers. 166 AMEMIYA and NAKAYAMA VOL. 26

Table 4. Antitumor activity of polysaccharides produced by spore-bearing lactic acid bacteria.

DISCUSSION

As generally known, glucan- and fructan-producing strains are widely distributed among lactic acid bacteria and bacilli, respectively. It is interesting from the philogenetic point of view that certain strains of spore-bearing lactic acid bacteria produce both types of polysaccharides. Solubilized glucan from B. laevolacticus is an attractive product, because it is easily produced and purified in high yield, and because it shows the peculiar characteristics of high solubility and low viscosity in spite of its high molecular weight.

We thank the Sanraku Ocean Company and Dr. Y. Inui, who kindly carried out animal tests, and the Yakult Company and Dr. M. Shirota, who supported this work in part.

REFERENCES

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