hUernational Jounml of Fot~l MicrohiohL~,9; 17 (1992)67-71} ~ 1992 Elsevier Science Publishers B.V. All rights reserved 0168-1605/92/$05.1}{)
67
FOOD {}{}542
Effect of tomato juice addition on the growth and activity of Lactobacillus acidophilus V.
Babu ~, B.K. Mital ~ and S.K. Garg b
" Department of I"¢~1 .~t'it,tltv and Technoloh~' and h l)t'lu~rtment of Microbiolo,~.', G.B. Pant Unirer.~ityof Agrit'tdtllre and Tt,t'hllohl~,. Pantnagar. hulia (Received I December 1991:accepted 17 July i'~2)
Addition of tomato juice to skimmed milk stimulated the growth of l.act(d~willns a('idoldlilus. It resulted in higher viable counts, shorter generation time and improvedsugar utilization with more acid produced and lower pH fi)r the strains tested. Varying results were obtained for the five strains examined. Key words: Lactobacillus achlold:ilu.s: Tomato juice
Introduction Lactobaciilus acidophilus is claimed to possess anti-cholesterolemic (Gilliland and Walker, 1990) and anti-carcinogenic (Goldin and Gorbach, 1984) properties and exerts antagonistic action against intestinal and food borne pathogens (Gilliland and Speck, 1977). Therefore, consumption of products containing L. acidophilus is considered beneficial. Since this organism also possesses the ability to colonize the intestine (Hood and Zottola, 1988), such benefits can be maintained over a longer period as compared to other organisms used for fermented dairy products. L. acidophilus grows slowly in milk and attempts have been made to stimulate the growth of L. acidophilus by enriching milk with additives such as whey proteins (Marshall et al., 1982), sucrose (Agrawal et al., 1986), papaya pulp (Kumar et ai., 1989), and magnesium and manganese ions (Ahmed et al., 1990). Tomato juice is a rich source of simple sugars, minerals and vitamins B and its use has often been suggested to manufacture acceptable acidophilus milk products (e.g. Miller and Puhan, 1980). The present investigation was, therefore, undertaken to study the effect of tomato juice addition on the growth and activity of different strains of L. acidophilus in skimmed milk. Correspondence address: Dr. B.K. Mital. Department of Food Science & Technology. G.B. Pant University of Agriculture and Technology,Pantnagar-263 145, India.
Materials and Methods
Five strains of L. acidophilus were included: L. acidophilus '111 ML', 'Russian', 'Hanson' and 'Base', obtained from National Dairy Institute, Karnal (India) and L. acidophihls - 'Isolate' from Culture Collection of the Department were used in the present study. Stock cultures were maintained in skimmed milk at 5°C with transfers to fresh sterilized skimmed milk every 2 weeks. The test cultures were subcultured 2-3 times in skimmed milk at daily intervals prior to use. Fresh, ripe and sound tomatoes were washed with water, sliced into small pieces and heated at 90°C for 5 rain to facilitate pulping and to inactivate pectin methyl esterase. The tomatoes were then cooled to 40°C, blended for about 30 s, sieved through 20 mesh sieve and centrifuged at 7000 rpm for 10-15 min. The clear juice was sterilized at 1210C for 15 min. Fresh skimmed milk was obtained from Livestock Research Center at the University and was sterilized at 115°C for 15 min. It was then aseptically supplemented with sterilized tomato juice at 3% and 6% (v/v) levels. The medium equilibrated to the incubation temperature and was inoculated with a 16-18 h test culture at the rate of 1% (cell density 6.2-6.8 × 11)8 ml-~) and incubated at 370C. Samples were drawn at selected intervals and analyzed for various parameters. Modified Lane and Eynon method as described by Ranganna 11986)was used to determine total and reducing sugars in the samples. Viable counts were estimated according to APHA 11972) method using Lactobacillus Selection (LBS) agar. The samples were plated by pour plate method in duplicate and incubated at 370C for 24-48 h. Generation time of the organisms was calculated. Titratable acidity was determined according to APHA 11972) procedure and changes in pH were recorded using Toshniwal (New Delhi, India) digital pH meter. Manganese was determined according to AOAC (1975) procedures. Magnesium was precipitated as magnesium ammonium phosphate and estimated coiorimetrically at 6611 nm (Ranganna, 1986).
Results and Discussion
Tomato juice used in the present investigation contained 6.1% (w/w) total sugars, 9.0 mg magnesium/100 g and 0.15 mg manganese/l(~) g. The reducing sugars (2.3% (w/w)) made up about 40% of the total sugars.. Addition of tomato juice to skimmed milk exerted a stimulatory effect on the growth of L. acidophilus strains tested (Table !). However, the magnitude of this effect varied among different strains. Among the levels of tomato juice tested greater stimulatory effect was observed at 6% level of addition than at 3%. All the L. acidophih~s strains showed higher viable counts and greater acid production with lower pH in tomato juice added skimmed milk than in the control (Table 1). Tomato juice addition also resulted in enhanced sugar utilization (data not shown) and shortening of generation time of the strains tested (data not shown). The generation time of different strains in skimmed milk (control) was in the range of
40.0 18.0 2.0 !.8 5.7
,,.: lOS/ml
0.23-0.25 1.38 (I.30 0.44 0.31 11.29
6.30-6.50 4.00 5.611 5.40 5.80 5.90
70.0 32.11 3.3 2.5 ! 1.0
× IO~/ml 0.25-0.27 i .66 (I.58 0.51 0.35 (I.38
¢~ TA
Viable counts
pH
Skimmed milk + 3r/~ tomato juice
(~ T A h
Skimmed milk (control)
viable counts
a A v e r a g e o f two determinations. b f~ T A = c~ titratable acidity.
None ' Rus,:ian' "Base" ' I 11 M L' 'Hansen" • Isolate'
Strain
Effect of tomato juice on growth characteristics of L. acidophilus after 16 h at 37~C "
TABLE I
6.15-6.4(I 3.60 4.90 5.20 5.50 5.60
pH
80.0 38.0 5.7 6.7 2(1.(1
x I(J'~/ml (I.27-(I.29 1.86 0.67 0.56 0.50 0.47
r~ TA
Skimmed milk + 65:; tomato juice Viable counts
6.05-6.2(I 3.50 4.511 4.80 5.20 5.40
pH
117-209 min which d e c r e a s e d by 8 - 4 6 min a n d 1 1 - 5 9 min u p o n a d d i t i o n o f t o m a t o juice at 3 % ( w / w ) a n d 6 % ( w / w ) levels, respectively at 37°C ( d a t a not shown). T h e s t i m u l a t o r y effect o f t o m a t o j u i c e on t h e g r o w t h o f L. acidophilus strains as o b s e r v e d in this investigation m a y b e a t t r i b u t e d to g r e a t e r availability o f s i m p l e s u g a r s mainly glucose a n d f r u c t o s e a n d m i n e r a l s , i.e. m a g n e s i u m a n d m a n g a n e s e . B o t h t h e s e s u g a r s have b e e n r e p o r t e d to p r o m o t e t h e g r o w t h o f L. acidophilus (Srinivas et al., 1990). A h m e d et al. ( 1 9 9 0 ) d e m o n s t r a t e d t h a t s u p p l e m e n t a t i o n o f milk with 19.72 m g m a g n e s i u m i o n / I o r with 11.39 m g m a n g a n e s e i o n / i p r o m o t e d g r o w t h o f L. acidophilus.
References Agrawal, V.. Usha. M.S. and Mital, B,K. (1986) Preparation and evaluation of acidophilus milk. Asian
J. Dairy Res. 5, 33-38. Ahmed. B.. Milal, B.K. and Garg, S.K. (1990) Effect of magnesium and manganese ions on the growth of Lactobacillus acidophilus. J. Ftx~d Sci. Technol. 27, 228-229. AOAC (19751 Official Methods of Analysis, 12th edn. Association of Official Analytical Chemists. Washington DC. APHA (1972) Standard Methods for the Examination of Dairy Products, 13th edn. American Health Association, New York, NY. Gilliland, S.E. and Speck, M.L. (19771 Antagonistic action of Lactobacillusacidophiht~ toward intestinal and foodborne pathogens in associative cultures. J. Food Prot. 40, 820-823. Gilliland, S,E. and Walker, D.K. (19901 Factors to consider when selecting a culture of Lacwbacillus acidophiht~ as a dietary adjunct to produce a hypocholesterolemic effect in humans. J. Dairy Sci. 73, 905-91 I. Goldin, B.R. and Gorbach, S.L. (19841 The effect of milk and Lactobacillus acidophiht~ feeding on human intestinal enzyme activity. Am. J. Clin. Nutr. 39, 756-761. Hood, S.K. and Zottola. E.A. (1988) Effect of low pH on the ability of Lactobacillus acidopl:ih~ to survive and adhere to human intestinal cells. J. Food Sci. 53, 1514-1516. Kumar, M., Mital. B.K. and Garg, S.K. (19891 Effect of papaya pulp addition on the growth of Lactobacillus acidophihes. J, Food Safety 10, 63-73. Marshall, V.M.. Cole. W.M. and Vega, J.R. (19821 A yoghurt-like product made by fermenting ultra filtered milk containing elevated whey proteins with Lactobacillus acidophilus. J. Dairy Res. 49, 665-670. Miller, B. and Puhan. Z. (1980) M(iglichkeiten zur Verkiirzung der Fermentation bei Acidophilus-Milch. Sehweig. Milchw. Forschung. 9, 49-56. Ranganna. S. (19861 Proximate constituents. In: Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edn., McGraw-Hill Publishing Company Ltd., New Delhi, pp. i-30. Srinivas, D., Mital, B.K. and Garg, S.K. (19901 Utilization of sugars by Lactobacillusacidophilus strains. Int. J. Food Microbiol. 10, 51-58.
67
FOOD {}{}542
Effect of tomato juice addition on the growth and activity of Lactobacillus acidophilus V.
Babu ~, B.K. Mital ~ and S.K. Garg b
" Department of I"¢~1 .~t'it,tltv and Technoloh~' and h l)t'lu~rtment of Microbiolo,~.', G.B. Pant Unirer.~ityof Agrit'tdtllre and Tt,t'hllohl~,. Pantnagar. hulia (Received I December 1991:accepted 17 July i'~2)
Addition of tomato juice to skimmed milk stimulated the growth of l.act(d~willns a('idoldlilus. It resulted in higher viable counts, shorter generation time and improvedsugar utilization with more acid produced and lower pH fi)r the strains tested. Varying results were obtained for the five strains examined. Key words: Lactobacillus achlold:ilu.s: Tomato juice
Introduction Lactobaciilus acidophilus is claimed to possess anti-cholesterolemic (Gilliland and Walker, 1990) and anti-carcinogenic (Goldin and Gorbach, 1984) properties and exerts antagonistic action against intestinal and food borne pathogens (Gilliland and Speck, 1977). Therefore, consumption of products containing L. acidophilus is considered beneficial. Since this organism also possesses the ability to colonize the intestine (Hood and Zottola, 1988), such benefits can be maintained over a longer period as compared to other organisms used for fermented dairy products. L. acidophilus grows slowly in milk and attempts have been made to stimulate the growth of L. acidophilus by enriching milk with additives such as whey proteins (Marshall et al., 1982), sucrose (Agrawal et al., 1986), papaya pulp (Kumar et ai., 1989), and magnesium and manganese ions (Ahmed et al., 1990). Tomato juice is a rich source of simple sugars, minerals and vitamins B and its use has often been suggested to manufacture acceptable acidophilus milk products (e.g. Miller and Puhan, 1980). The present investigation was, therefore, undertaken to study the effect of tomato juice addition on the growth and activity of different strains of L. acidophilus in skimmed milk. Correspondence address: Dr. B.K. Mital. Department of Food Science & Technology. G.B. Pant University of Agriculture and Technology,Pantnagar-263 145, India.
Materials and Methods
Five strains of L. acidophilus were included: L. acidophilus '111 ML', 'Russian', 'Hanson' and 'Base', obtained from National Dairy Institute, Karnal (India) and L. acidophihls - 'Isolate' from Culture Collection of the Department were used in the present study. Stock cultures were maintained in skimmed milk at 5°C with transfers to fresh sterilized skimmed milk every 2 weeks. The test cultures were subcultured 2-3 times in skimmed milk at daily intervals prior to use. Fresh, ripe and sound tomatoes were washed with water, sliced into small pieces and heated at 90°C for 5 rain to facilitate pulping and to inactivate pectin methyl esterase. The tomatoes were then cooled to 40°C, blended for about 30 s, sieved through 20 mesh sieve and centrifuged at 7000 rpm for 10-15 min. The clear juice was sterilized at 1210C for 15 min. Fresh skimmed milk was obtained from Livestock Research Center at the University and was sterilized at 115°C for 15 min. It was then aseptically supplemented with sterilized tomato juice at 3% and 6% (v/v) levels. The medium equilibrated to the incubation temperature and was inoculated with a 16-18 h test culture at the rate of 1% (cell density 6.2-6.8 × 11)8 ml-~) and incubated at 370C. Samples were drawn at selected intervals and analyzed for various parameters. Modified Lane and Eynon method as described by Ranganna 11986)was used to determine total and reducing sugars in the samples. Viable counts were estimated according to APHA 11972) method using Lactobacillus Selection (LBS) agar. The samples were plated by pour plate method in duplicate and incubated at 370C for 24-48 h. Generation time of the organisms was calculated. Titratable acidity was determined according to APHA 11972) procedure and changes in pH were recorded using Toshniwal (New Delhi, India) digital pH meter. Manganese was determined according to AOAC (1975) procedures. Magnesium was precipitated as magnesium ammonium phosphate and estimated coiorimetrically at 6611 nm (Ranganna, 1986).
Results and Discussion
Tomato juice used in the present investigation contained 6.1% (w/w) total sugars, 9.0 mg magnesium/100 g and 0.15 mg manganese/l(~) g. The reducing sugars (2.3% (w/w)) made up about 40% of the total sugars.. Addition of tomato juice to skimmed milk exerted a stimulatory effect on the growth of L. acidophilus strains tested (Table !). However, the magnitude of this effect varied among different strains. Among the levels of tomato juice tested greater stimulatory effect was observed at 6% level of addition than at 3%. All the L. acidophih~s strains showed higher viable counts and greater acid production with lower pH in tomato juice added skimmed milk than in the control (Table 1). Tomato juice addition also resulted in enhanced sugar utilization (data not shown) and shortening of generation time of the strains tested (data not shown). The generation time of different strains in skimmed milk (control) was in the range of
40.0 18.0 2.0 !.8 5.7
,,.: lOS/ml
0.23-0.25 1.38 (I.30 0.44 0.31 11.29
6.30-6.50 4.00 5.611 5.40 5.80 5.90
70.0 32.11 3.3 2.5 ! 1.0
× IO~/ml 0.25-0.27 i .66 (I.58 0.51 0.35 (I.38
¢~ TA
Viable counts
pH
Skimmed milk + 3r/~ tomato juice
(~ T A h
Skimmed milk (control)
viable counts
a A v e r a g e o f two determinations. b f~ T A = c~ titratable acidity.
None ' Rus,:ian' "Base" ' I 11 M L' 'Hansen" • Isolate'
Strain
Effect of tomato juice on growth characteristics of L. acidophilus after 16 h at 37~C "
TABLE I
6.15-6.4(I 3.60 4.90 5.20 5.50 5.60
pH
80.0 38.0 5.7 6.7 2(1.(1
x I(J'~/ml (I.27-(I.29 1.86 0.67 0.56 0.50 0.47
r~ TA
Skimmed milk + 65:; tomato juice Viable counts
6.05-6.2(I 3.50 4.511 4.80 5.20 5.40
pH
117-209 min which d e c r e a s e d by 8 - 4 6 min a n d 1 1 - 5 9 min u p o n a d d i t i o n o f t o m a t o juice at 3 % ( w / w ) a n d 6 % ( w / w ) levels, respectively at 37°C ( d a t a not shown). T h e s t i m u l a t o r y effect o f t o m a t o j u i c e on t h e g r o w t h o f L. acidophilus strains as o b s e r v e d in this investigation m a y b e a t t r i b u t e d to g r e a t e r availability o f s i m p l e s u g a r s mainly glucose a n d f r u c t o s e a n d m i n e r a l s , i.e. m a g n e s i u m a n d m a n g a n e s e . B o t h t h e s e s u g a r s have b e e n r e p o r t e d to p r o m o t e t h e g r o w t h o f L. acidophilus (Srinivas et al., 1990). A h m e d et al. ( 1 9 9 0 ) d e m o n s t r a t e d t h a t s u p p l e m e n t a t i o n o f milk with 19.72 m g m a g n e s i u m i o n / I o r with 11.39 m g m a n g a n e s e i o n / i p r o m o t e d g r o w t h o f L. acidophilus.
References Agrawal, V.. Usha. M.S. and Mital, B,K. (1986) Preparation and evaluation of acidophilus milk. Asian
J. Dairy Res. 5, 33-38. Ahmed. B.. Milal, B.K. and Garg, S.K. (1990) Effect of magnesium and manganese ions on the growth of Lactobacillus acidophilus. J. Ftx~d Sci. Technol. 27, 228-229. AOAC (19751 Official Methods of Analysis, 12th edn. Association of Official Analytical Chemists. Washington DC. APHA (1972) Standard Methods for the Examination of Dairy Products, 13th edn. American Health Association, New York, NY. Gilliland, S.E. and Speck, M.L. (19771 Antagonistic action of Lactobacillusacidophiht~ toward intestinal and foodborne pathogens in associative cultures. J. Food Prot. 40, 820-823. Gilliland, S,E. and Walker, D.K. (19901 Factors to consider when selecting a culture of Lacwbacillus acidophiht~ as a dietary adjunct to produce a hypocholesterolemic effect in humans. J. Dairy Sci. 73, 905-91 I. Goldin, B.R. and Gorbach, S.L. (19841 The effect of milk and Lactobacillus acidophiht~ feeding on human intestinal enzyme activity. Am. J. Clin. Nutr. 39, 756-761. Hood, S.K. and Zottola. E.A. (1988) Effect of low pH on the ability of Lactobacillus acidopl:ih~ to survive and adhere to human intestinal cells. J. Food Sci. 53, 1514-1516. Kumar, M., Mital. B.K. and Garg, S.K. (19891 Effect of papaya pulp addition on the growth of Lactobacillus acidophihes. J, Food Safety 10, 63-73. Marshall, V.M.. Cole. W.M. and Vega, J.R. (19821 A yoghurt-like product made by fermenting ultra filtered milk containing elevated whey proteins with Lactobacillus acidophilus. J. Dairy Res. 49, 665-670. Miller, B. and Puhan. Z. (1980) M(iglichkeiten zur Verkiirzung der Fermentation bei Acidophilus-Milch. Sehweig. Milchw. Forschung. 9, 49-56. Ranganna. S. (19861 Proximate constituents. In: Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edn., McGraw-Hill Publishing Company Ltd., New Delhi, pp. i-30. Srinivas, D., Mital, B.K. and Garg, S.K. (19901 Utilization of sugars by Lactobacillusacidophilus strains. Int. J. Food Microbiol. 10, 51-58.
