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Utilization of sugars by Lactobacillus acidophilus strains
International Journal of Food Microbiology, 10 (1990) 51-58
51
Elsevier FOOD 00295
Utilization of sugars by Lactobacillus acidophilus strains D . S r i n i v a s a, B . K . M i t a l 1 a n d S . K . G a r g
2
1 Food Science and Technology Department, College of Agriculture, G.B, Pant University of Agriculture and Technology, Nainital, India, and e Department of Microbiology College of Basic Sciences & Humanities G.B. Pant University of Agriculture and Technology, Nainital, India
(Received 8 May 1989; accepted 9 August 1989)
Utilization of various carbohydrates viz., glucose, fructose, sucrose, lactose and galactose by Lactobacillus acidophilus strains was investigated in Lactobacillus Selection Broth. Maximum viable counts, acid production and sugar utilization by different test strains were in the order: glucose>/fructose > sucrose >t lactose > galactose. The generation time of the tested strains was shorter in glucose medium as compared to sucrose or lactose medium. Key words: Sugar fermentation; Acidophilus milk; Lactobacillus acidophilus
Introduction Ingestion of Lactobacillus acidophilus is considered beneficial in maintaining good health and correcting intestinal disorders (Sandine et al., 1972; Shahani and Chandan, 1979). This organism is capable of producing microbial inhibitors which prevent the growth of undesirable organisms in the intestine (Babel, 1977). It has been reported to lower the level of bacterial enzymes responsible for production of carcinogenic amines (Goldin and Gorbach, 1984) and to possess anti-cholesteremic activity (Gilliland et al., 1985). Since fermented milks are widely consumed, it is appropriate that milk should be used as carrier for this organism. The growth of an organism in a medium depends upon its ability to utilize the available fermentable sugars. The principal sugar in milk is lactose. L. acidophilus utilizes lactose but is unable to initiate rapid growth in milk. Presumably the inducible nature of fl-galactosidase required for hydrolysis of lactose is responsible for this effect (Kachhy et al., 1977; Tobe et al., 1981). Hence, possibilities of fortifying milk with other sugars and nutrients to promote rapid growth of this organism have been explored (Vedamuthu, 1974; Agrawal et al., 1986).
Correspondence address." B.K. Mital, Food Science and TechnologyDepartment, College of Agriculture, G.B. Pant University of Agriculture and Technology, Pantnagar-263 145, Nainital, India.
0168-1605/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
52 The present investigation was undertaken to study the utilization pattern of c o m m o n monosaccharides such as glucose, galactose, fructose and disaccharides such as sucrose and lactose by different strains of L. acidophilus.
Materials and Methods
Cultures Five strains of Lactobacillus acidophilus labelled Hansen, Base, 111 M L and Russian which were all obtained from National Dairy Research Institute, Karnal, India and L. acidophilus NRRL-B-629 respectively, which was obtained from Northern Regional Research Laboratory, Peoria, IL, U.S.A., were used in this study. Stock cultures were routinely maintained in reconstituted 10% N o n Fat Dry Milk by fortnightly transfers and held at 5 ° C between transfers.
Media and growth conditions Lactobacillus Selection Broth (LBS; BBL, 1973) without any sugar was used as the basal medium for growth and sugar utilization studies. Test sugars glucose, galactose, fructose, lactose and sucrose were added to the basal medium at 2% ( w / v ) and the contents were sterilized at 121°C for 15 rain. The test inoculum was prepared by subculturing the stock cultures in LBS broth 2 - 3 times at 37 ° C and daily intervals. The experimental media with test sugar were inoculated with test cultures (O.D. 0.50-0.60 at 660 nm) at 2% and incubated at 37 ° C. Viable counts were determined using LBS agar according to A P H A (1972) procedures. Generation time was determined according to the procedure described by Stanier et al. (1987).
Chemical analyses Per cent titratable acidity was determined as lactic acid by titrating 10-ml samples with 0.1 N N a O H to p H 8.4. Percent acidity was calculated by subtracting the initial titratable acidity values from the values obtained at test hour. The rate of acid production by different L. aeidophilus strains in various sugar media was calculated on the basis of acid produced over an 8-h period. The changes in p H were measured using a p H meter (Philips Model pp 9045). The Shaffer-Somogyi micro method was used for determinations when glucose, galactose, fructose and lactose were the test sugars (AOAC, 1980). A modified Lane and Eynon method as described by Ranganna (1977) was followed when sucrose was the test sugar.
Results
Fig. 1 shows the percent acidity developed upon fermentation of test sugars by different L. acidophilus strains. The ranges (in percentage) of acid production for various strains after 8 h at 3 7 ° C were: 0.39-0.47 in glucose; 0.35-0.45 in fructose; 0.10-0.24 in sucrose; 0.08-0.20 in lactose; and 0.01-0.03 in galactose medium.
53 [] [] [] [] []
Galactose Lactose Sucrose Fructose Glucose
0.5
04
q
i~ 03
~- 0.2 iv 123 0.1
0 __
g'7111f :: Russian
Base
111 ml
Hansen
I
NRRL- B - 6 2 9
LQctobociHus clcidophilus strains Fig. 1. Acid production after 8 h at 3 7 ° C by different strains of Lactobacillus acidophilus in various sugar media.
Maximum acid production was observed in media containing glucose and fructose, and minimum production in the galactose-containing medium. The acid production rates for different strains were (/~mol/s): 1.50-1.83 in glucose, 1.45-1.75 in fructose, 0.39-0.93 in sucrose, 0.31-0.86 in lactose and 0.02-0.12, in galactose-containing medium (Table I). L..acidophilus-R generally exhibited higher acid production than other strains under study. The pH values for all five strains after 8 h were: 5.15-5.25 for glucose; 5.17-5.28 for fructose; 5.20-5.35 for sucrose; 5.22-5.40 for lactose; and 5.30-5.47 for galactose-containing medium. The utilization of different sugars by various strains is depicted in Fig. 2. For glucose, fructose, sucrose, lactose and galactose it was in the range of 73-84%,
TABLE I Rate of acid production by L. acidophilus strains in Lactobacillus Selection Broth (BBL) as influenced by different sugars Strain
R Base 111 ML Hansen NRRL-B-629
Rate of acid production (/~ m o l / s )
a
Glucose
Fructose
Sucrose
Lactose
Galactose
1.83 1.76 1.68 1.58 1.50
1.74 1.66 1.54 1.50 1.35
0.93 0.77 0.58 0.50 0.39
0.86 0.66 0.54 0.39 0.31
0.12 0.08 0.06 0.04 0.02
Determined as lactic acid.
54 [] [] [] [] []
100
Galactose Lactose Sucrose Fructose Glucose
Bo
6O
4o b 2o
Russian
V'/AIII~ Base Loctobocillus
111 ml ociciophiius
Hansen stroin
NRRL-B- 629
Fig. 2. Sugar utilization pattern by different strains of Lactobacillus acidophilus in Lactobacillus Selection Broth (BBL) after 8 h.
TABLE I1 Rate of sugar utilization by L. acidophilus strains in Lactobacillus Selection Broth (BBL) Strain
R Base 111 ML Hansen NRRL-B-629
Rate of utilization ( m g / m i n ) Glucose
Fructose
Sucrose
Lactose
Galactose
3.25 3.28 3.14 3.04 2.81
3.20 3.16 3.06 2.98 2.78
2.60 2.18 1.41 1.05 0.85
2.30 1.83 1.35 0.91 0.70
0.14 0.12 0.10 0.08 0.06
TABLE III Viable counts of different L. acidophilus strains in Lactobacillus Selection Broth (BBL) containing different sugars after 8 h incubation at 37 o C. Strain
R Base 111 ML Hansen N RRL-B-629
Viable counts (1 × 107/ml) Glucose
Sucrose
Lactose
40.00 17.00 14.00 4.00 2.20
14.00 5.30 4.90 1.10 0.81
5.70 1.70 2.19 0.64 0.45
55 TABLE IV Generation time at 37 °C for Lactobacillus acidophilus strains in Lactobacillus Selection Broth (BBL) containing different sugars
R Base 111 ML Hansen NRRL-B-629
Glucose
Sucrose
Lactose
86.3 90.2 105.6 106.2 107.0
113.0 130.4 141.9 161.3 173.1
151.0 161.2 189.5 198.5 208.1
71-83%, 22-68%, 18-62% and 2-4% of the initial sugar concentration, respectively, over 8 h. As is indicated by acid production, maximum utilization was noted for glucose and fructose and minimum utilization for galactose. The sugar utilization rates for all five strains were ( m g / m i n ) : 2.81-3.28 for glucose, 2.78-3.20 for fructose, 0.85-2.60 for sucrose, 0.70-2.30 for lactose and 0.06-0.14 for galactose (Table II). The results obtained for acid production and sugar utilization showed a more or less similar pattern for glucose and fructose and were lower for galactose. Therefore, for viable counts, glucose, sucrose and lactose were selected. The viable counts of all five strains at the end of 8 h at 37 ° C are shown in Table III and the generation time of the five strains are shown in Table IV. It is seen that the order of growth rate of strains under study was: L. acidophilus-R > Base > 111 M L > Hansen > N R R L - B 629.
Discussion In general, the order of sugar utilization by L. acidophilus strains tested were: glucose >/fructose > sucrose >i lactose > galactose. Since L. acidophilus is a homofermentative organism, it utilizes glucose through the Embden Meyerhof Parnas (EMP) pathway. However, the pathway followed for the metabolism of fructose depends on whether this sugar is supplied exogeneously or is a product of sucrose hydrolysis. When the latter is the case, fructose is phosphorylated to fructose 6-phosphate by sucrose induced o-phosphofructokinase (E.C.2.7.1.4) (Doelle, 1975). In contrast, if fructose is supplied exogeneously, it enters the cell as fructose 1-phosphate through the D-fructose 1-phosphotransferase system which is induced by fructose (Gottschalk, 1986). Another inducible enzyme, D-fructose 1-phosphate kinase (Hansen and Anderson, 1966), then phosphorylates fructose 1-phosphate to fructose 1,6-diphosphate which then enters the EMP pathway. This mechanism has been demonstrated in E. coli (Fraenkel, 1968). Presumably the same mechanism for fructose metabolism is operative in L acidophilus when this sugar is supplied exogeneously. This may lead to a faster utilization of glucose than fructose. It
56 implies that the enzymes required for glucose metabolism are constitutive in L. acidophilus whereas fructose metabolism requires induction of certain enzymes in the initial stages before the sugars enter the EMP pathway. Lactobacilli metabolize galactose through the Leloir pathway (Eskin et al., 1971; Gottschalk, 1986). In this, galactokinase catalyses the phosphorylation of galactose to galactose 1-phosphate which then reacts with U D P G (uridine diphosphate glucose) to form UDP-galactose and glucose 1-phosphate. This reaction is mediated by galactose 1-phosphate uridyl transferase. Conversion of UDP-galactose to U D P glucose is mediated by uridine diphosphogalactose 4-epimerase. in the presence of the enzyme phosphoglucomutase glucose 1-phosphate is converted to glucose 6phosphate which then enters the EMP pathway. The galactokinase, transferase and epimerase involved in the Leloir pathway are inducible and synthesized coordinately in response to galactose (Stanier et al., 1987). In the present investigation, only 2-4% utilization of galactose by different strains of L. acidophilus was observed. Such an insignificant utilization of galactose could be attributed to intricacies in the induction of enzymes involved in the Leloir pathway. The L. acidophilus strains tested appeared to have a slightly faster utilization of sucrose than lactose. This difference could be attributed to the activities of fl-galactosidase and /3-fructofuranosidase which are required for the hydrolysis of lactose and sucrose, respectively. Kachhy et al. (1977) found that /3-galactosidase is an inducible enzyme in L. acidophilus whereas/3-fructofuranosidase has been reported to be a constitutive enzyme in this organism (Mital et al., 1973). Complexities involved in metabolism of galactose, a constituent sugar of lactose, may play a role in the slower utilization of lactose. Agrawal et al. (1986) reported that addition of sucrose to skim milk enhanced acid production by L. acidophilus. The sugar present in the medium significantly influenced the generation time of the organism. All the L. acidophilus strains tested exhibited shorter generation times in medium containing glucose, followed by sucrose and lactose. The generation time of the test strains ranged from 86.3 to 107.0 min when glucose was provided as the energy source. Angles and Marth (1971) have reported a more or less similar range (82.2-112.8 min) of generation time for different lactobacilli in Elliker broth. In conclusion, the results obtained in the present investigation indicate that addition of glucose or fructose to milk will stimulate the growth and enhance acid production by L. acidophilus.
References 1 Agrawal, V., Usha, M.S. and Mital, B.K. (1986) Preparation and evaluation of acidophilus milk. Asian J. Dairy Res. 5, 33-38. Angles, A.G. and Marth, E.H. (1971) Growth and activity of lactic acid bacteria in soy milk. J. Milk Food Technol. 34, 30-36. AOAC (1980) Official Methods of Analysis, 13th 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.
57 Babel, F.J. (1977) Antibiosis by lactic culture bacteria. J. Dairy Sci. 60, 815-821. BBL (1973) manual of products and laboratory procedures. BBL, Benton Dickson and Co., Cickeysville, MD. Docile, H.W. (1975) Bacterial Metabolism, 2nd edn. Academic Press, New York, NY. Eskin, N.A.M., Handerson, H.M. and Townsend, R.J. (1971) Biochemistry of foods. Academic Press, New York, NY. pp. 153-217. Fraenkel, D.G. (1968) The phosphoenol pyruvate-initiated pathway of fructose metabolism in E. coil J. Biol. Chem. 243, 6458-6463. Gilliland, S.E., Nelson, C.R. and Maxwell, C. (1985) Assimilation of cholesterol by Lactobacillus acidophilus. Appl. Environ. Microbiol. 49, 377-381. Goldin, B.R. and Gorbach, S.L. (1984) The effect of milk and Lactobacillus feeding on human intestinal bacterial enzyme activity. Am. J Clin. Nutr. 39, 756-761. Gottschalk, G. (1986) Bacterial Metabolism, 2nd edn. Springer-Verlag, New York, NY. pp. 96-103. Hansen, T.E. and Anderson, R.L. (1966) D-fructose-l-phosphate kinase, a new enzyme instrumental in metabolism of D-fructose. J. Biol. Chem. 241, 1644-1645. Kachhy, A.N., Modi, V.V. and Vaidya, N.C. (1977) Induction of fl-galactosidase in Lactobacillus acidophilus. Indian J. Exp. Biol. 15, 271-273. Mital, B.K., Shallenberger, R.S. and Steinkraus, K.H. (1973) Alpha-galactosidase activity of lactobacilli. J, Appl. Microbiol. 26, 783-788. Ranganna, S. (1977) Manual of Analysis of Fruit and Vegetable products. Tara McGraw Hill Publ. Comp., New Delhi, India. Sandine, W.E., Muralidhara, K.S., Elliker, P.R. and England, D.C. (1972) Lactic acid bacteria in food and health: A review with special reference to enteropathogenic Escherichia coil as well as certain enteric diseases and their treatment with antibiotics and lactobacilli. J. Milk Food Technol. 35, 691-702. Shahani, K.M. and Chandan, R.C. (1979) Nutritional and healthful aspects of cultured and culture containing dairy products. J. Dairy Sci. 62, 1685-1694. Stanier, R.Y., Ingraham, J.L., Wheelis, M.L. and Painter , P.R. (1987) General Microbiology. 5th edn. MacMillan Education Ltd., London, pp. 183-195. Tobe, T., Tomita, Y., Itoh, T. and Adachi, S. (1981) fl-galactosidases of lactic acid bacteria. Characterization of oligosaccharides formed during hydrolysis of lactose. J. Dairy Sci. 54, 185-192. Vedamuthu, E.R. (1974) Culture for butter milk, sour cream and yoghurt with special comments on acidophihis yoghurt. Cult. Dairy Prod. J. 9, 16-21.
51
Elsevier FOOD 00295
Utilization of sugars by Lactobacillus acidophilus strains D . S r i n i v a s a, B . K . M i t a l 1 a n d S . K . G a r g
2
1 Food Science and Technology Department, College of Agriculture, G.B, Pant University of Agriculture and Technology, Nainital, India, and e Department of Microbiology College of Basic Sciences & Humanities G.B. Pant University of Agriculture and Technology, Nainital, India
(Received 8 May 1989; accepted 9 August 1989)
Utilization of various carbohydrates viz., glucose, fructose, sucrose, lactose and galactose by Lactobacillus acidophilus strains was investigated in Lactobacillus Selection Broth. Maximum viable counts, acid production and sugar utilization by different test strains were in the order: glucose>/fructose > sucrose >t lactose > galactose. The generation time of the tested strains was shorter in glucose medium as compared to sucrose or lactose medium. Key words: Sugar fermentation; Acidophilus milk; Lactobacillus acidophilus
Introduction Ingestion of Lactobacillus acidophilus is considered beneficial in maintaining good health and correcting intestinal disorders (Sandine et al., 1972; Shahani and Chandan, 1979). This organism is capable of producing microbial inhibitors which prevent the growth of undesirable organisms in the intestine (Babel, 1977). It has been reported to lower the level of bacterial enzymes responsible for production of carcinogenic amines (Goldin and Gorbach, 1984) and to possess anti-cholesteremic activity (Gilliland et al., 1985). Since fermented milks are widely consumed, it is appropriate that milk should be used as carrier for this organism. The growth of an organism in a medium depends upon its ability to utilize the available fermentable sugars. The principal sugar in milk is lactose. L. acidophilus utilizes lactose but is unable to initiate rapid growth in milk. Presumably the inducible nature of fl-galactosidase required for hydrolysis of lactose is responsible for this effect (Kachhy et al., 1977; Tobe et al., 1981). Hence, possibilities of fortifying milk with other sugars and nutrients to promote rapid growth of this organism have been explored (Vedamuthu, 1974; Agrawal et al., 1986).
Correspondence address." B.K. Mital, Food Science and TechnologyDepartment, College of Agriculture, G.B. Pant University of Agriculture and Technology, Pantnagar-263 145, Nainital, India.
0168-1605/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
52 The present investigation was undertaken to study the utilization pattern of c o m m o n monosaccharides such as glucose, galactose, fructose and disaccharides such as sucrose and lactose by different strains of L. acidophilus.
Materials and Methods
Cultures Five strains of Lactobacillus acidophilus labelled Hansen, Base, 111 M L and Russian which were all obtained from National Dairy Research Institute, Karnal, India and L. acidophilus NRRL-B-629 respectively, which was obtained from Northern Regional Research Laboratory, Peoria, IL, U.S.A., were used in this study. Stock cultures were routinely maintained in reconstituted 10% N o n Fat Dry Milk by fortnightly transfers and held at 5 ° C between transfers.
Media and growth conditions Lactobacillus Selection Broth (LBS; BBL, 1973) without any sugar was used as the basal medium for growth and sugar utilization studies. Test sugars glucose, galactose, fructose, lactose and sucrose were added to the basal medium at 2% ( w / v ) and the contents were sterilized at 121°C for 15 rain. The test inoculum was prepared by subculturing the stock cultures in LBS broth 2 - 3 times at 37 ° C and daily intervals. The experimental media with test sugar were inoculated with test cultures (O.D. 0.50-0.60 at 660 nm) at 2% and incubated at 37 ° C. Viable counts were determined using LBS agar according to A P H A (1972) procedures. Generation time was determined according to the procedure described by Stanier et al. (1987).
Chemical analyses Per cent titratable acidity was determined as lactic acid by titrating 10-ml samples with 0.1 N N a O H to p H 8.4. Percent acidity was calculated by subtracting the initial titratable acidity values from the values obtained at test hour. The rate of acid production by different L. aeidophilus strains in various sugar media was calculated on the basis of acid produced over an 8-h period. The changes in p H were measured using a p H meter (Philips Model pp 9045). The Shaffer-Somogyi micro method was used for determinations when glucose, galactose, fructose and lactose were the test sugars (AOAC, 1980). A modified Lane and Eynon method as described by Ranganna (1977) was followed when sucrose was the test sugar.
Results
Fig. 1 shows the percent acidity developed upon fermentation of test sugars by different L. acidophilus strains. The ranges (in percentage) of acid production for various strains after 8 h at 3 7 ° C were: 0.39-0.47 in glucose; 0.35-0.45 in fructose; 0.10-0.24 in sucrose; 0.08-0.20 in lactose; and 0.01-0.03 in galactose medium.
53 [] [] [] [] []
Galactose Lactose Sucrose Fructose Glucose
0.5
04
q
i~ 03
~- 0.2 iv 123 0.1
0 __
g'7111f :: Russian
Base
111 ml
Hansen
I
NRRL- B - 6 2 9
LQctobociHus clcidophilus strains Fig. 1. Acid production after 8 h at 3 7 ° C by different strains of Lactobacillus acidophilus in various sugar media.
Maximum acid production was observed in media containing glucose and fructose, and minimum production in the galactose-containing medium. The acid production rates for different strains were (/~mol/s): 1.50-1.83 in glucose, 1.45-1.75 in fructose, 0.39-0.93 in sucrose, 0.31-0.86 in lactose and 0.02-0.12, in galactose-containing medium (Table I). L..acidophilus-R generally exhibited higher acid production than other strains under study. The pH values for all five strains after 8 h were: 5.15-5.25 for glucose; 5.17-5.28 for fructose; 5.20-5.35 for sucrose; 5.22-5.40 for lactose; and 5.30-5.47 for galactose-containing medium. The utilization of different sugars by various strains is depicted in Fig. 2. For glucose, fructose, sucrose, lactose and galactose it was in the range of 73-84%,
TABLE I Rate of acid production by L. acidophilus strains in Lactobacillus Selection Broth (BBL) as influenced by different sugars Strain
R Base 111 ML Hansen NRRL-B-629
Rate of acid production (/~ m o l / s )
a
Glucose
Fructose
Sucrose
Lactose
Galactose
1.83 1.76 1.68 1.58 1.50
1.74 1.66 1.54 1.50 1.35
0.93 0.77 0.58 0.50 0.39
0.86 0.66 0.54 0.39 0.31
0.12 0.08 0.06 0.04 0.02
Determined as lactic acid.
54 [] [] [] [] []
100
Galactose Lactose Sucrose Fructose Glucose
Bo
6O
4o b 2o
Russian
V'/AIII~ Base Loctobocillus
111 ml ociciophiius
Hansen stroin
NRRL-B- 629
Fig. 2. Sugar utilization pattern by different strains of Lactobacillus acidophilus in Lactobacillus Selection Broth (BBL) after 8 h.
TABLE I1 Rate of sugar utilization by L. acidophilus strains in Lactobacillus Selection Broth (BBL) Strain
R Base 111 ML Hansen NRRL-B-629
Rate of utilization ( m g / m i n ) Glucose
Fructose
Sucrose
Lactose
Galactose
3.25 3.28 3.14 3.04 2.81
3.20 3.16 3.06 2.98 2.78
2.60 2.18 1.41 1.05 0.85
2.30 1.83 1.35 0.91 0.70
0.14 0.12 0.10 0.08 0.06
TABLE III Viable counts of different L. acidophilus strains in Lactobacillus Selection Broth (BBL) containing different sugars after 8 h incubation at 37 o C. Strain
R Base 111 ML Hansen N RRL-B-629
Viable counts (1 × 107/ml) Glucose
Sucrose
Lactose
40.00 17.00 14.00 4.00 2.20
14.00 5.30 4.90 1.10 0.81
5.70 1.70 2.19 0.64 0.45
55 TABLE IV Generation time at 37 °C for Lactobacillus acidophilus strains in Lactobacillus Selection Broth (BBL) containing different sugars
R Base 111 ML Hansen NRRL-B-629
Glucose
Sucrose
Lactose
86.3 90.2 105.6 106.2 107.0
113.0 130.4 141.9 161.3 173.1
151.0 161.2 189.5 198.5 208.1
71-83%, 22-68%, 18-62% and 2-4% of the initial sugar concentration, respectively, over 8 h. As is indicated by acid production, maximum utilization was noted for glucose and fructose and minimum utilization for galactose. The sugar utilization rates for all five strains were ( m g / m i n ) : 2.81-3.28 for glucose, 2.78-3.20 for fructose, 0.85-2.60 for sucrose, 0.70-2.30 for lactose and 0.06-0.14 for galactose (Table II). The results obtained for acid production and sugar utilization showed a more or less similar pattern for glucose and fructose and were lower for galactose. Therefore, for viable counts, glucose, sucrose and lactose were selected. The viable counts of all five strains at the end of 8 h at 37 ° C are shown in Table III and the generation time of the five strains are shown in Table IV. It is seen that the order of growth rate of strains under study was: L. acidophilus-R > Base > 111 M L > Hansen > N R R L - B 629.
Discussion In general, the order of sugar utilization by L. acidophilus strains tested were: glucose >/fructose > sucrose >i lactose > galactose. Since L. acidophilus is a homofermentative organism, it utilizes glucose through the Embden Meyerhof Parnas (EMP) pathway. However, the pathway followed for the metabolism of fructose depends on whether this sugar is supplied exogeneously or is a product of sucrose hydrolysis. When the latter is the case, fructose is phosphorylated to fructose 6-phosphate by sucrose induced o-phosphofructokinase (E.C.2.7.1.4) (Doelle, 1975). In contrast, if fructose is supplied exogeneously, it enters the cell as fructose 1-phosphate through the D-fructose 1-phosphotransferase system which is induced by fructose (Gottschalk, 1986). Another inducible enzyme, D-fructose 1-phosphate kinase (Hansen and Anderson, 1966), then phosphorylates fructose 1-phosphate to fructose 1,6-diphosphate which then enters the EMP pathway. This mechanism has been demonstrated in E. coli (Fraenkel, 1968). Presumably the same mechanism for fructose metabolism is operative in L acidophilus when this sugar is supplied exogeneously. This may lead to a faster utilization of glucose than fructose. It
56 implies that the enzymes required for glucose metabolism are constitutive in L. acidophilus whereas fructose metabolism requires induction of certain enzymes in the initial stages before the sugars enter the EMP pathway. Lactobacilli metabolize galactose through the Leloir pathway (Eskin et al., 1971; Gottschalk, 1986). In this, galactokinase catalyses the phosphorylation of galactose to galactose 1-phosphate which then reacts with U D P G (uridine diphosphate glucose) to form UDP-galactose and glucose 1-phosphate. This reaction is mediated by galactose 1-phosphate uridyl transferase. Conversion of UDP-galactose to U D P glucose is mediated by uridine diphosphogalactose 4-epimerase. in the presence of the enzyme phosphoglucomutase glucose 1-phosphate is converted to glucose 6phosphate which then enters the EMP pathway. The galactokinase, transferase and epimerase involved in the Leloir pathway are inducible and synthesized coordinately in response to galactose (Stanier et al., 1987). In the present investigation, only 2-4% utilization of galactose by different strains of L. acidophilus was observed. Such an insignificant utilization of galactose could be attributed to intricacies in the induction of enzymes involved in the Leloir pathway. The L. acidophilus strains tested appeared to have a slightly faster utilization of sucrose than lactose. This difference could be attributed to the activities of fl-galactosidase and /3-fructofuranosidase which are required for the hydrolysis of lactose and sucrose, respectively. Kachhy et al. (1977) found that /3-galactosidase is an inducible enzyme in L. acidophilus whereas/3-fructofuranosidase has been reported to be a constitutive enzyme in this organism (Mital et al., 1973). Complexities involved in metabolism of galactose, a constituent sugar of lactose, may play a role in the slower utilization of lactose. Agrawal et al. (1986) reported that addition of sucrose to skim milk enhanced acid production by L. acidophilus. The sugar present in the medium significantly influenced the generation time of the organism. All the L. acidophilus strains tested exhibited shorter generation times in medium containing glucose, followed by sucrose and lactose. The generation time of the test strains ranged from 86.3 to 107.0 min when glucose was provided as the energy source. Angles and Marth (1971) have reported a more or less similar range (82.2-112.8 min) of generation time for different lactobacilli in Elliker broth. In conclusion, the results obtained in the present investigation indicate that addition of glucose or fructose to milk will stimulate the growth and enhance acid production by L. acidophilus.
References 1 Agrawal, V., Usha, M.S. and Mital, B.K. (1986) Preparation and evaluation of acidophilus milk. Asian J. Dairy Res. 5, 33-38. Angles, A.G. and Marth, E.H. (1971) Growth and activity of lactic acid bacteria in soy milk. J. Milk Food Technol. 34, 30-36. AOAC (1980) Official Methods of Analysis, 13th 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.
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