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 pro- duction of lactic acid, coagulation and partial degradation of casein. Although a number of workers have demonstrated the mutagenic action of N-methyl-N-ni t ro- 2Y-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 by Singh (1974). In view of the major contribution of proteolysis to flavour production in yoghurt, but ter and cheese, an a t t empt has been made to isolate mutants of Lactobacillus casei with increased proteolytic activity using nitroso- guanidine.

A strain of Lactobacillus casei (RTS) maintained in the culture collection at the National Dairy Research Inst i tute 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 tomato 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. I t was added to the inoculated broth- tubes to give a final concentration of 400 ~g m1-1 and samples were withdrawn at 10 min intervals up to a period of 100 min.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 by Singh and l~anganathan (1974), survivors were isolated at random from Petri dishes show- ing 1.0--0.1~/o survival. The isolates were tested for proteolytic act ivi ty according to Hull 's method (1947) and acid production by t i trating a known aliquot of the sample with 0.1 M NaOH.

The percentage of survivors after exposure of Lactobacillus casei (RTS) to the action of nitrosoguanidine showed that NTG-trea tment for 70 min gave less than 1 .0~ survival and the mutants were isolated from such Petri plates. A marked increase in the proteolytic act ivi ty to the extent of 75% and 88% was noticed after first and second t reatment with nitrosoguanidine, respectively. However, a down- ward t rend was evident after the third t reatment , although the range in proteolytie act ivi ty was higher as compared with the untreated parent culture (Table I). The mutant strains did not show any further physiological variations even after pro- longed maintainance and repeated subeulturing for six months. I t is of interest to note tha t nitrosoguanidine had no effect on acid production by the culture after

1978 P R O T E O L Y T I C A C T I V I T Y OF L. C A S E I $3

TABLE I. Inc rease in p ro teo ly t i e ac t iv i ty in L. easel (RTS) e.fter N T G t r e a t m e n t *

N T G Surv iva l N u m b e r of su rv ivo r s P ro teo ly t i e a c t i v i t y T i t r a t ab l r t r e a t m e n t e x a m i n e d m g ty ros ine /g ac id i ty %

0.90 U n t r e a t e d

1st

2nd

3rd

0.42

0.75

2.50

P a r e n t

25

25

25

0.350

0.610

0.655

0.522

o.95~ 0 .85- -1 .05

0.89 0 .87- -0 .91

0.85 0 .80 - -0 .90

* Cul tures were g rown in r e cons t i t u t ed n o n f a t mi lk a n d e x a m i n e d a f t e r 24 h. T h e resul ts r epresen t a n average of th ree tr ials .

first, second and third treatments. Burrow et al. (1970) reported an increase in acid production by NTG t rea tment in Streptococcus diacetilactis, while Kuila et al. (1971) observed a decrease in the acid-producing ability by the same method of t reatment . The proteolytie activi ty in NTG-treated Lactobacillus casei RTS appears to be in- dependent 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 condi t ions for m u t a g e n e s i s by 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.: Charac te r i za t ion o f diacetyl nega t ive m u - t a n t s of Streptococcus diacetilactis. J. Dairy Sci. 53, 121 (1970).

HULL M. E. : S tudies on mi lk proteins . I I . Color ,metr ic d e t e r m i n a t i o n o f t he pa r t i a l hydro lys i s o f t ho p ro te ins in milk. J. Dairy Sci. 30, 881 (1947).

KONi~KOV$,-RADOCl~OVX M., M$-LEX I. : The m u t a g e n i c effect of n i t r o soguan id ine on 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 Strepto- coccus diacctilactis b y n i t rosoguan id ine a n d u l t r av io le t i r radia t ion. J. Dairy Sci. 54, 331 (1971).

SINaH JASJIT, RANGANATHA~- B.: Effect of some chemica l m u t a g e n s on p ro teo ly t i c ac t iv i ty 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 laetobaci l l i w i th pa r t i cu la r reference to proteoly t ie ac t iv i ty . Ph .D . Thesis , P u n j a b U n i v e r s i t y (1974).