Ph.D in Biology Azizpour K.

Artemia and Aquatic Animals Research Institute, Urmia University, Urmia-IRAN

Biochemical characterization of lactic acid bacteria

isolated from fish

Summary

Lactic acid bacteria (LAB) were isolated from various samples of fresh and frozen fish and prawn. Thirteen species of Lactobacillus were identified among the 64% isolates. Among them, L. plantarum was the dominant species. The remaining 36% isolates of Lactobacillus could not be assigned to any species with the available taxonomic schemes.

Introduction

Lactic acid bacteria (LAB) are character­ized as Gram - positive, usually non-motile, non - sporulating bacteria that produce lactic acid as a major or sole product of fer­mentative metabolism. Ringoe and Gatesoupe have prepared a review of the LAB present in fish intestine [4]. Taxonomic studies on LAB from poikilo­thermic animals are rare [4].

The aim of the study was the isolation of LAB from fresh and frozen fish their classification based on the mor­phological and biochemical characteristics.

Materials and Methods

Isolation of LAB. Fresh fish (22 Spp), frozen fish (15 Spp), fresh and frozen prawn (5 Spp) were procured from retail markets and cold storage in Kochi, packed in iceboxes and transferred to the laboratory within 2 h. MRS agar and broth were used for enumeration and culture of LAB [2, 5]. The samples of fish (skin with muscle) and prawns (peeled) were homogenized in a raffinose, rhamnose, D-ribose, salicin, sor­bitol, sucrose, trehalose and D-xylose in MRS broth devoid of glucose and beef extract with chlorophenol red as indicator; production of acid and gas from 1 % glucose (MRS broth without beef extract); methyl red and Voges-Proskauer test in MRVP medium; H&L test in O/F medium; production of ammonia from arginine; nitrate reduction in nitrate broth; indole production in tryptone broth and growth on acetate agar stomacher blender using saline, serially diluted and pour plated on MRS plates. The MRS plates overlaid with MRS agar and incubated in 5 % CO2 at 37 DC for 48-72 h. Well - isolated colonies with typical charac­teristics namely pure white, small (2-3 mm diameter) with entire margins were picked from each plate and transferred to MRS broth.

Identification of the bacterial strains

The cultures were identified according to their morphological, cultural, physiological and biochemical characteristics [3, 6].

The used tests were: Gram reaction; production of catalase, cytochrome oxidase and hydrogen peroxide; growth at 15 DC and 45 DC in 1 week; acid production from carbohydrates (1 % w/v) - L-arabinose, cellobiose, D-fruc­tose, D-galactose, esculin, lactose, maltose, melezitose, melebiose, mannitol, D-mannose, raffinose, rhamnose, D-ribose, salicin, sor­bitol, sucrose, trehalose and D-xylose in MRS broth devoid of glucose and beef extract with chlorophenol red as indicator; production of acid and gas from 1 % glucose (MRS broth without beef extract); methyl red and Voges-Proskauer test in MRVP medium; H&L test in O/F medium; production of ammonia from arginine; nitrate reduction in nitrate broth; indole production in tryptone broth and growth on acetate agar.

Results and Discussion

The LAB isolates were classified into the genera Streptococcus, Leuconostoc, Pedio­coccus and Lactobacillus based on their morphology and biochemical characters [6]. We showed the distribution of different genera of LAB in fresh and frozen fish of the cultures, 60 % in fresh fish, and 80 % in frozen fish belonged to the genus Lactobacillus. The species identified showed above 80 % or more similarity to the ATCC type cultures. Only tests that gave reproducible results were included in the classification scheme. The species identified were L. plan­tarum (138 isolates), L. brevis (66), L. diver gens (28), L. gasseri (24), L. rhamnosus (21), L. fer­mentum (20), L. viridescens (10), L. farciminis (7), L. buchneri (7), L. acidophilus (5), L. alimentarius (4), L. animalis (4) and L. reuteri (2). A significant fact is that 217 cultures (36.2 %) were found to belong to the genus Lactobacillus but could not be assigned to any particular species by these characters.

However, the L. divergens cultures we have isolated grew well on acetate agar. Finding that Lactobacillus formed the major flora in fish substantiated the observations of several other workers [1].

References

1. Collins, M. D., J. A. E. Farrow, B. A. Phillips, S. Ferusu, D. Jones.,1987. Int. J. Syst. Bacterio/., 37,310 - 316.

2. Cone, O.K., 1982. J. Fish. Diseases, 5,479-485. 3. De Man., J. C. Rogosa, M. E. Sharpe, 1960. J. Appl. Bacteriol., 23, 130- 135.

3. Kandler, 0., N. Weiss, 1986. In: Bergey's Manual of Systematic Bacteriology, P. H. A. Sneath, N. S. Mair, M. E. Sharpe, J. G. Holt (Eds), Vol. 2, Baltimore: Williams and Wilkins, 1209 – 1234.

4. Ringoe, E., F. J. Gatesoupe, 1998. Aquaculture, 160,177 - 203.

5. Schroder, K., E. Clausen, A. M. Sandberg, J. Raa, 1979. In: Advances in Fish Science and Technology, J. J. Connel (Ed.), Farnham, England: Fishing Newsbook Ltd, 480-483.

6. Sharpe, M. E., T. F. Fryer, D. G. Smith, 1979. Identification of Lactic Acid Bacteria. In: Identification Methods for Microbiologists, E. M. Gibbs, F. A. Skinner (Eds), London: Academic Press, 233-259.