Effect of casitone and fructose on the growth of Lactobacillus acidophilus and its survival during storage

International Journal of Food Microbiology" 21 (1094) 271-276 ~ 1994 Elsevier Science B.V. All rights reserved 0168-1605/94/$07.00

271

FOOD 675

Short Communication

Effect of casitone and fructose on the growth of Lactobacillus acidophilus and its survival during storage * S.N. S a x e n a a, B.K. M i t a l a a n d S.K. G a r g b Department of ~ Food Science and Technology and b Microbiology, G.B. Pant Unirersity of Agriculture and Technology, Pantnagar, U.P., India (Received 29 June 1993; accepted 7 October 1993)

Supplementation of milk with combination of casitone (0.5% (w/w)) and fructose (0.5% (w/w)) resulted in greater acid production, higher viable counts, increased sugar utilization and shorter generation time for the L. acidophilus strains tested. The experimental product prepared by using these additives contained 1.5-2.0-fold more viable L. acidophilus than the control (no additives) throughout 21 days of storage. Further, both control and experimental products remained acceptable throughout the storage period. However, the former was rated superior in flavor, whereas the latter exhibited better texture. Key words: Acidophilus milk; Casitone; Lactobacillus acidophilus

Introduction

Intake of Lactobacilhts acidophilus may impart health benefits. The benefits claimed include: control of colon cancer and serum cholesterol, antagonistic action against intestinal and food-borne pathogens, and stabilization of intestinal microflora (Gilliland, 1989; Mital and Garg, 1992). Since milk is an essential component of our diet, it can serve as an excellent medium for intake of this organism. However, L. acidophih~s grows slowly in milk and attempts have been made to promote its growth by fortifying milk with tomato juice (Miller and Puhan, 1981; Babu et al., 1992), casein peptone (Miller and Puhan, 1981), whey protein (Marshall et al., 1982), sucrose (Agrawal et al., 1986), papya pulp (Kumar et al., 1989), and manganese and magnesium ions (Ahmed et al., 1990). Products have also been developed by combined use of L. acidophilus and other lactic acid bacteria (Speck, 1980). Correspondence address: Dr B.K. Mital, Professor and Head, Department of Food Science and Technology, G.B. Pant University of Agriculture and Technology, Pantnagar-263 145, U.P., India. * Research paper No. 7665 through Director, Experiment Station, G.B. Pant University. of Agriculture and Technology, Pantnagar, India. SSDI O I 6 8 - 1 6 0 5 ( 9 3 ) E O 0 9 1 - 5

Casein hydrolysis products have been found to stimulate the growth of yoghurt cultures (Desmazeaud and Hermier, 1972; Cousins and Marth, 1977). Fortification of milk with simple fermentable sugars has also been suggested to support the growth of L. acidophih~s (Srinivas et al., 1990). The present investigation was undertaken to study the effect of fortifying milk with casitone, a casein hydrolysate, and fructose, individually and in combination, on the growth of L. acidophilus and its survival during storage.

Materials and Methods

Cttltttres Lactobacillus acidophilus strains 1899 and 301 obtained from the National Dairy Research Institute, Karnal, India and L. acidophihts NCFM provided by Dr T.R. Klaenhammer, Department of Food Science, North Carolina State University, Raleigh, USA, were used. The cultures were maintained in sterilized litmus milk by transfers every 2 weeks and held at 5°C between transfers. The cultures were sub-cultured in sterile litmus milk 2-3 times prior to use.

Add#ices Twenty grams of casitone (Difco) were dissolved in 100 ml glass distilled water, boiled, cooled, filtered through activated charcoal and sterilized at 121°C for 15 rain. The levels of casitone tested ranged from 0.25 to 1.5% (w/w) final concentration. Twenty grams of fructose was dissolved in 100 ml glass distilled water and sterilized at 115°C for 10 min. Fructose was tested for its effect at 0.5% (w/w) and 1.0% (w/w) final concentrations.

Acidophilus milk preparation Fresh milk sterilized at 115°C for 10 min was used to prepare the acidophilus milk. It was fortified aseptically with desired level(s) of the additive(s) singly or in combination, inoculated with 16-18 h old test culture at a rate of approx. 1% (cell density 1.2 × 10 s to 7.1 x 108 c f u / m l determined as described below) and incubated at 37°C. For acceptability trials, milk sweetened with 7% (w/w) cane sugar was used to prepare acidophilus milk. For storage studies, inoculum rate used was 3% (w/w). The experimental medium was incubated at 37°C until 0.8% titratable acidity was attained.

Analytical determinations Per cent titratable acidity was determined according to the method of Atherton and Newlander (1977). pH was measured using EC Digital pH meter (Electronic Corporation of India Ltd, New Delhi, Model No. pH 5652). The fat content in milk was measured by the Gerber method (Atherton and Newlander, 1977), whereas a modified Gerber method, as described by Putambekar (1968), was used for determining fat in fermented milk samples. Moisture, ash and protein ( N × 6.38) in the samples were determined according to AOAC (1975) procedures, A modi-

273

fled Lane and Eynon method, as described by Ranganna (1986). was used to estimate total sugars in the samples. Soluble nitrogen in the samples was determined by the Kosikowski method (1966). Viable counts were determined according to APHA (1972) using Elliker agar (Elliker e t a [ . , 1956). Duplicate plates of appropriate dilutions were prepared and incubated at 37°C for 24-48 h. Generation time of the test organism was calculated according to Stanier et al. (1987).

Storage studies Acidophilus milk samples prepared with and without additives (0.5% (w/w) casitone plus 0.5% (w/w) fructose) were stored at 4_+ I°C for 21 days. Samples were drawn at predetermined intervals and analysed for changes in chemical, microbial and organo[eptical characteristics.

Organoleptic eca&ation The effect of different casitone concentrations on flavor of acidophilus milk was determined by Ranking test (Jellinek, 1985). The data were analysed using standard tables (Kramer et al., 1974). Sweetened acidophilus milk products, prepared by using L. acidophilus with and without additives, were evaluated for their acceptability using a paired comparison test (Ranganna, 1986). The acceptability of the product during storage was rated on a Hedonic scale using numerical values ranging from 1 to 9, where 1 represented 'dislike extremely' and 9 represented 'like extremely' (Larmond, 1977).

Statistical analysis The data were analysed statistically according to the procedures described by Miller and Freund (1987).

Results and Discussion

Preliminary investigation showed no appreciable increase in acid production by different L. acidophilus strains when 0.25% (w/w) casitone was added to milk. In comparison, acid production significantly increased when 0.5% (w/w) and higher concentrations were used. However, casitone addition above this (0.5%) level adversely affected the flavor of the product. Therefore, further studies were conducted using 0.5% (w/w) casitone. Preliminary results also revealed no significant effect of fructose addition on acid production both at 0.5 and 1.0% (w/w) levels, whereas the combination of 0.5% fructose and 0.5% casitone significantly enhanced acid production as compared to the control and 0.5% (w/w) casitone alone. Table 1 shows the effect of casitone (0.5% (w/w)) and fructose (0.5% (w/w)) on acid production singly and in combination. The combined use of additives showed greater acid production than observed when they were used alone for L. acidophilus 1899 and NCFM strains. Lactobacillus acidophilus 301 was only slightly affected. Addition of 0.5% fructose plus 0.5% casitone also significantly enhanced

274 TABLE I Effect of casitone and fructose single and in combination on acid production by different L. acidophilus strains a.b Strains of

Control

0.5% fructose

0.5% casitone

0.5% casitone + 0.5% fructose

301 % Titratable acidity pH Acid production rate ( # M / s )

1.20 + 0.07 3.98 ± 0.05 1.76 ± 0.13

1.16 _ 0.04 3.94 _ 0.01 1.70 ± 0.08

1.42 ± 0.05 3.9l _±0.0t 2.16 ± 0.10

1.39 ±_0.08 3.97 ± 0.02 2.10 ± 0.15

1899 % Titratable acidity pH Acid production rate ( u M / s )

0.44 ___0.02 5.38 ± 0.06 0.46 ± 0.04

0.42 ± 0.03 5.20 ± 0.11 0.42 +_0.05

0.67 ± 0.08 5.05 ± 0.09 0.79 ± 0.15

0.79 ± 0.07 4.61 _+0.12 1.02 ± 0.14

NCFM % Titratable acidity pH Acid production rate (~,M/s)

0.80 ± 0.06 4.54 ± 0.07 1.12+_0.12

0.79 ± 0.06 4.43 ± 0.12 1.10±0,12

0.98 ± 0.08 4.32 ± 0. i0 1.39+_0.15

1.29 ± 0.06 3.86 _ 0.05 2.00±0.11

L. acidophilus

a Values are mean + SD for 16 h fermentation at 37°C. b Initial % titratable acidity (% TA) and pH of milk were 0.28-0.30 and 5.7-5.9, respectively.

v i a b l e c o u n t s a n d s u g a r u t i l i z a t i o n by d i f f e r e n t L. acidophilus s t r a i n s as c o m p a r e d t o t h e c o n t r o l ( T a b l e II). In t h e p r e s e n t i n v e s t i g a t i o n , d i f f e r e n t L. acidophilus s t r a i n s e x h i b i t e d a g e n e r a t i o n t i m e in t h e r a n g e o f 1 0 1 - 1 3 0 rain, H i g h e r v a l u e s r a n g i n g f r o m 130 to 200 rain f o r d i f f e r e n t L. acidophilus s t r a i n s h a v e b e e n r e p o r t e d ( K u m a r e t al., 1989; A h m e d e t al., 1990; S r i n i v a s e t al., 1990). A d d i t i o n o f a c o m b i n a t i o n o f 0 . 5 % f r u c t o s e a n d 0 . 5 % c a s i t o n e in air s i g n i f i c a n t l y r e d u c e d t h e generation time of the strains included.

TABLE II Effect of addition of 0.5% (w/w) casitone plus 0.5% (w/w) fructose on the growth characteristics of different L. acidophilus strains a Strain

301 1899 NCFM

Viable counts ( x 107 cfu/ml) % sugar utilization b

Generation time (min)

Control

Experimental

Control

Experimental

Control

Experimental

62 ll 44

110 58 t 10

19.4 12.6 15.2

29.3 14.7 26.9

103 130 101

91 77 80

a Values are average of three trials for 16 h fermentation at 37°C. b Percentage of total sugar consumed.

275 S t o r a g e s t u d i e s were c o n d u c t e d over a p e r i o d o f 21 days using L. acidophihts N C F M . D u r i n g s t o r a g e d e c r e a s e in the viable c o u n t s a n d i n c r e a s e in % t i t r a t a b l e acidity, soluble n i t r o g e n a n d % s u g a r u t i l i z a t i o n with b o t h e x p e r i m e n t a l a n d c o n t r o l p r o d u c t s w e r e o b s e r v e d ( d a t a not shown). T h e e x p e r i m e n t a l p r o d u c t was f o u n d to c o n t a i n a slightly h i g h e r n u m b e r o f viable L. acidophilus at any given time t h r o u g h o u t the s t o r a g e p e r i o d t h a n the c o n t r o l ( d a t a not shown). Both the c o n t r o l a n d e x p e r i m e n t a l p r o d u c t s w e r e f o u n d to be a c c e p t a b l e by the o r g a n o l e p t i c e v a l u a t i o n s , b u t the f o r m e r was r a t e d s u p e r i o r in flavor, w h e r e a s the l a t t e r e x h i b i t e d s u p e r i o r texture. S w e e t e n e d a c i d o p h i l u s milks p r e p a r e d with a n d w i t h o u t additives s h o w e d a l m o s t the s a m e c o m p o s i t i o n , i.e., a r o u n d (in % ( w / w ) ) m o i s t u r e 80.0, p r o t e i n 4.0, fat 4.25, s u g a r 10.0 (results not shown). B o t h the c o n t r o l a n d e x p e r i m e n t a l p r o d u c t s w e r e e v a l u a t e d for t h e i r a c c e p t a b i l ity by a p a i r e d c o m p a r i s o n test. T h e results s h o w e d that six p a n e l i s t s d e t e c t e d d i f f e r e n c e in color, 11 in t e x t u r e b u t all 13 in flavor o f the p r o d u c t s . T h e c h i - s q u a r e test r e v e a l e d t h a t the d i f f e r e n c e in c o l o r o f the c o n t r o l a n d e x p e r i m e n t a l p r o d u c t s was statistically insignificant ( P < 0.05) in c o n t r a s t with the significant d i f f e r e n c e in t e x t u r e a n d flavor. F u r t h e r , the p a n e l i s t s r a t e d the e x p e r i m e n t a l p r o d u c t s u p e r i o r in texture but i n f e r i o r to the c o n t r o l in flavor. T h i s i n f e r i o r rating c o u l d be a s c r i b e d to the use of c a s i t o n e , a p a n c r e a t i c digest of casein. H o w e v e r , this d e f e c t c o u l d be o v e r c o m e by the use o f b l a n d s o u r c e s of casein h y d r o l y s a t e p r o d u c t s a n d flavor m a s k i n g agents.

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