Folia Mierobiol. 23, 82--83 (1978)

Activation of Proteolytic Activity of Lactobacillus casei by Nitrosoguanidine JASJIT SINGH and B. I~ANGANATHAN Department of Bacteriology, National Dairy Research Institute, Karnal (Haryana) India

Received May 15. 1977

Lactobacilli are known to bring about desired types of changes which include production of lactic acid, coagulation and partial degradation of casein. Although a number of workers have demonstrated the mutagenic action of N-methyl-N-nitro2Y-nitrosoguanidine (NTG) in a variety of microorganisms (Adelberg et al., 1965; KoniSkovs and Ms 1969; Kuila et al., 1971), very little information is available on the effectiveness of this mutagen for lactobacilli except in a recent report b y Singh (1974). In view of the major contribution of proteolysis to flavour production in yoghurt, butter and cheese, an a t t e m p t has been made to isolate mutants of Lactobacillus casei with increased proteolytic activity using nitrosoguanidine. A strain of Lactobacillus casei (RTS) maintained in the culture collection at the National Dairy Research Institute was used. Washed cells of the test culture were suspended in 0.02 M phosphate buffer (pH 7.0 and the cell density was adjusted to anoptical density of 0.55--0.60 (2. l0 s cells ml-1). A 5 ml aliquot of t o m a t o juice medium was inoculated with the test culture at 2~o level and incubated for 9 h at 37 ~ Solution of nitrosoguanidine (Koch-Light, England) was prepared fresh at a concentration of 2 mg ml-1 and filtered through a bacteriological filter. It was added to the inoculated broth-tubes to give a final concentration of 400 ~g m1-1 and samples were withdrawn at 10 min intervals up to a period of 100 min. After plating control and experimental samples on an enriched medium, devised b y Singh and l~anganathan (1974), survivors were isolated at random from Petri dishes showing 1.0--0.1~/o survival. The isolates were tested for proteolytic activity according to Hull's method (1947) and acid production b y titrating a known aliquot of the sample with 0.1 M N a O H . The percentage of survivors after exposure of Lactobacillus casei (RTS) to the action of nitrosoguanidine showed that NTG-treatment for 70 min gave less than 1 . 0 ~ survival and the mutants were isolated from such Petri plates. A marked increase in the proteolytic activity to the extent of 75% and 88% was noticed after first and second treatment with nitrosoguanidine, respectively. However, a downward trend was evident after the third treatment, although the range in proteolytie activity was higher as compared with the untreated parent culture (Table I). The m u t a n t strains did not show any further physiological variations even after prolonged maintainance and repeated subeulturing for six months. I t is of interest to note t h a t nitrosoguanidine had no effect on acid production by the culture after

1978

PROTEOLYTIC

ACTIVITY

O F L. C A S E I

$3

TABLE I. I n c r e a s e in p r o t e o l y t i e a c t i v i t y in L. easel (RTS) e.fter N T G t r e a t m e n t * NTG treatment

Survival

Untreated 1st

0.42

N u m b e r of s u r v i v o r s examined

Proteolytie activity mg tyrosine/g

Titratablr acidity %

Parent

0.350

0.90

25

0.610

o.95~ 0.85--1.05

2nd

0.75

25

0.655

0.89 0.87--0.91

3rd

2.50

25

0.522

0.85 0.80--0.90

* Cultures were g r o w n in r e c o n s t i t u t e d n o n f a t a v e r a g e of t h r e e trials.

m i l k a n d e x a m i n e d a f t e r 24 h. T h e results r e p r e s e n t a n

first, second and third treatments. Burrow et al. (1970) reported an increase in acid production by NTG t r e a t m e n t in Streptococcus diacetilactis, while Kuila et al. (1971) observed a decrease in the acid-producing ability by the same method of treatment. The proteolytie activity in NTG-treated Lactobacillus casei RTS appears to be independent of the acid-producing ability of the culture. REFERENCES

ADEI,BERG E . M., MANDEL M., CHEN G. C. C.: O p t i m a l c o n d i t i o n s for m u t a g e n e s i s b y N - m e t h y l - N - n i t r o N - n i t r o s o g u a n i d i n e in Escherichia coli K 12. Biochem. Biophys. Res. Cvmmun. 18, 788 (1965). BURROW C. D., SANDINE W . E., ELLIKER P. R., SP~.CKMA~ C.: C h a r a c t e r i z a t i o n o f diacetyl n e g a t i v e m u t a n t s of Streptococcus diacetilactis. J. Dairy Sci. 53, 121 (1970). HULL M. E. : S t u d i e s on m i l k proteins. II. Color,metric d e t e r m i n a t i o n o f t h e p a r t i a l h y d r o l y s i s o f t h o p r o t e i n s in milk. J. Dairy Sci. 30, 881 (1947). KONi~KOV$,-RADOCl~OVX M., M$-LEX I. : T h e m u t a g e n i c effect of n i t r o s o g u a n i d i n e o n Mycobacterium phlei P.A. Folia Microbiol. 14, 201 (1969). KUILA R. K . , •ANGANATHAN B., DU~TA S. M., LAX~INARAYANA H . : I n d u c t i o n o f m u t a t i o n in Streptococcus diacctilactis b y n i t r o s o g u a n i d i n e a n d u l t r a v i o l e t irradiation. J. Dairy Sci. 54, 331 (1971). SINaH JASJIT, RANGANATHA~- B.: Effect of s o m e c h e m i c a l m u t a g e n s on p r o t e o l y t i c a c t i v i t y in lacgobaeilli. Acta Microbiol. 6, 15 (1974). ~INGH JASJIT: I n d u c t i o n o f m u t a t i o n in laetobacilli w i t h p a r t i c u l a r reference to proteolytie a c t i v i t y . P h . D . Thesis, P u n j a b U n i v e r s i t y (1974).

Activation of proteolytic activity of Lactobacillus casei by nitrosoguanidine.

Folia Mierobiol. 23, 82--83 (1978) Activation of Proteolytic Activity of Lactobacillus casei by Nitrosoguanidine JASJIT SINGH and B. I~ANGANATHAN Dep...
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