Biotechnology Letters Vol i0 No 6 427-430 (1988) Received May 5

PREPARATION OF LACTOSE FREE MILK BY FERMENTATION USING.

IMMOBILIZED SACCHAROMYCES FRAGILIS

B.Y.K. RAO, 5.5. GODBOLE and 5.F. DwSOUZA *

Food Technology & Enzyme Engineering Division Bhabha Atomic Research Centre, Bombay 400 085, India

SUMMARY

Saccharomyces fraQilis cells (40% w/v) were immobilized in 2% Ca-alginate and were used in a batch process for the removal of lactose from milk by fermentation. Immobilized cells (10 g) could completely desugaratelOOmLof milk in 3.5 h. The immobilized ptepa. ration was used repeatedly in 15 batches without decrease in the activity.

INTRODUCTION

Preparation of lactose free milk has recently gained importance due to increased incidence of lactose intolerance in some population (Gekas and Lopez-Leiva, 1985). A number of immobilized B-galactosl- dase systems have been investigated for hydrolysis of lactose in milk (Kaul et el., 1984; Gekas and Lopez-Leiva, 1985; D'Souza st al. 1985; Kaul et al. 1986). Alternative methods currently being investigated have focussed attention on the removal of lactose from mllk by fermen- tation with free ceils of ~. fragilis (Eldesten et al. 1979; Godbcle et al. 1988). Milk obtained by fermentation is totally sugar free with no major alterations in the nutritive value (Godbole st el. 1988) and could not only be consumed by lactose Intolerants but also by diabetic and galactosemlc patients.

The large scale use of this technology, however has been hampered due to various reasons. The complete removal of lactose by free ceil fermentation needs about 17 h (Godbole et al. 1988) and milk being a rich nutrient medium and a perishable commodity is easily amenable to microbial contamination. This necessitates the maintenance of stringent sterility conditions during the removal of lactose from milk by free ceil fermentation. Moreover, usage of yeast cells in free form imparts an yeasty odour to the milk. The present paper dilineates a novel method for the rapid removal of lactose from milk uslng~, fra~ilis immobilized in Ca-alglnate.

MATERIALS AND METHODS

The raw buffalo milk obtained from a local dairy was skimmed by centrifugatlon at 7DDO g for 20 min. The skimmed mllk (I.8% fat content) was boiled for 5 min and was used as such in these studies.

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~. fraoilis NCIR 3217 was obtained from National Chemical Labo- ratory, Puns, India. The organism was maintained by weekly transfer on 2~ agar slants containing yeast extract, 0.3~| malt extract, 0.3~! peptone, 0.5~ and lactose, 1~. The cells were grown aerobically at 30=C for 24 h in a liquid medium containing yeast extract, 0.15%; (NH4)2SO 4, 0.1~; KH2P04, 0.I~ and lactose, 2~. The initial pH of the medium was adjusted to 3.5. The cells mere harvested by centrifuga- tion and washed with cold saline. The amount of cells used in these studies has been indicated in terms of met weight of packed cells.

The yeast cells were immobilized in 2~ Ca-alginate using the standard techniques (Kierstan and Bucke, 1977). Yeast ce l ls (40% w/v) were mixed with 2% aqueous solut ion of Na-alginate and the mixture was extruded through a syringe needle in to 0.1 M CaC12. The beads were allowed to cure for 3 h in CaC12 solut ion, rinsed w i th water and used for the desugaration studies. The diameter of the beads obtained was about 3mm.

For the removal of lactose by fermentation, Ca-alginate beads containing 10 g of immobilized yeast ce l ls were s t i r red gently with 100 mL milk using an over-head s t i r r e r . At the end of fermentation the beads were retr ieved by f i l t r a t i o n using a sieve, washed with water and cured in 0. t M CaC12 for 30 mtn. The beads were then rinsed with water and transferred to a fresh batch of skimmed milk. When not in use the beads were stored in 0.1 M CaC12 at 4~ Reducing sugars were estimated using d i n i t r osa l i c yc l i c acid reagent (M i l l e r , 1959).

The milk was spray dried using Anhydro (Copenhagen DerwnarkType Lab, 5-1) spray d r ie r with i n l e t temperature of 1800C and out le t temperature of 100~ with a spindle drive of50,O00 rpm.

RESULTS AND DISCUSSION

The kinetics of sugar removal from milk by immobilized cells is shown in Fig. 1. More than 90% of the sugar could be removed in the first batch in about 6 h. The immobilized cells were retrieved, washed and transferred to a fresh batch of milk (100 mL). The effici- ency of lactose fermentation was found to increase steadily upto 4 batches of reuse. More than 95% of the sugar was removed in 3.5 h in the 4th batch after which it remained constant (Fig. 1). The immo- bilized cells were repeatedly used in 15 batches carried out over a period of ? days without loss in efficiency or change in the mechanical stability of the beads (Fig. 2).

The time required for sugar removal from milk could be conside- rably reduced by changing the ratio of milk to immobilized yeast cells (Fig. 3). Thus more than 95% of the sugar could be removed in just 2 h when immobilized yeast cells (10 @) were treated with 50 mL of milk.

Removal of lactose from milk was carried out at natural pH of milk. The initial pH of milk which was 6.5 was reduced sharply to 5.7 after I h of fermentation, after which only a marginal change in pH was observed (Fig. 4). The pH of the milk could, however, be brought back to 6.5 by the addition of NaOH before spray drying or heating to prevent coagulation.

428

ol

Q

W

3

2

1--

0 60 120 180 240 300 360 TIME (rain)

Fig.1. Kinetics of sugar removal using immobilized ~. fraqilis cells. Ca-alginate beads contain- ing 10 g yeast cells were used in 100 mL of milk (-X-) first; (-o-) second; -A-) third and ( - (~) fourth batch.

LLI

p.- 2

0

\ -- X~X

\ .,,X X--X--,X- X - x -X ..X. X - X ~ x

I I I I /, 8 12 16

BATCH NO.

Fig.2. Reuse efficiency of immo- bilized ~. fraQilis. Total time required for 95% of sugar removal has been indicated in this figure. The details as stated in the text.

5

4

~3 t~

,e,

1

60 120 180 240 300 TIME (rain)

Fig.3. Effect of volume of milk on the kinetics of sugar removal using immobilized ~. fragilis. Ca-alginate beads containing 10 9 of yeast cells were used in these studies. (-X-) 150 mL; ( -0- ) 100 mL; (-A-) 50 mL mi lk .

7"0

zo. 6"0

&C

0T I l I I I 60 120 180 2&0 300

TIME (rain)

Fig.4. Effect of fermentation time on the pH of milk. Ca- alginate beads containing 10 g of yeast ceils were used in 100 mL of milk.

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The immobilized whole cells have gained considerable importance during the past few years for a wide variety of chemical transforma- tion (OWSouza and Nadkarni, 1980! Mattiasson, 1983; O'Souza et el., 1986; Tampion and Tampion, 1987). Among these the viable yeast cells have been mainly investigated for the preparation of fuel ethanol and fermented beverages (Godia et el., 1987). The studies presented in this paper have shown the feasibility of using immobilized viable cells of ~. fraQilis for the efficient removal of lactose from milk by fermentation.

Immobilized viable cells have a number of advantages in hetero- 9enous catalysis. High cell densities could be retained as compared to traditional free cell system, thus leading to faster reaction rates. Also the risk of contamination of the final product with yeast could be minimised even while using high cell densities. These advantages are of considerable significance while treating perishable items like milk. Unlike the free cell fermentation methods investi- gated for this purpose (Godbole et al., 1988) the use of immobilized technique has the unique advantage of rapidly removing sugars from milk which is known for its limited shelf-life. This technique not only helps in the reuse of cells, but also confers the flexibility of altering the fermentation time by appropriately selecting the cell density and milk volume. The fermented milk obtained had an alcoholic odour which was eliminated on spray drying. The yeasty odour however was negligible as compared to milk obtained by free cell fermentation (Godbole et el., 1988). Thus this technique can find potentials in obtaining sugar free milk.

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