β-GALACTOSIDASE (β-GAL) FROM THE YEAST RHODOTORULA INGENIOSA AND ITS UTILIZATION IN ICE MILK PRODUCTION

EL-Sayed Eliwa¹, Mahmoud El-Hofi^2
1 Microbial Chemistry Department, National Research Centre, Dokki, Cairo, Egypt
² Dairy Science Department, National Research Centre, Dokki, Cairo, Egypt

ABSTRACT

Among of 100 of yeast isolated from Egyptian soils and tested for their β;-galactosidase (β-gal) activity, the highest producer was characterized and identified as Rhodotorula ingeniosa .This enzyme was found to be intracellulary produced, therefore permealization treatments to release the enzyme in a good yield was noticed when homogenization with sterile sea sand under cooling was employed as compared with chemical treatment. The enzyme was maximally produced when cellobiose at 1% final concentration and ammonium suphate at 1.5% final concentration were employed. The enzyme was partially purified with ammonium sulphate followed by Sephadex G-100 column chromatography. Some properties of the purified enzyme including the Effect of pHs, Effect of temperature Effect of reaction periods, and Effect of metal ions on the activity were estimated. On the other hand the use of this enzyme in ice milk production was studied. Different concentration of the enzyme was added to fresh pasteurized milk. Results clear indicate that the addition of the enzyme to ice milk mixes increased the sweetness although the organoletic properties of the resultant ice milk  was slightly decreased.

Key words: β-galactosidase, Rhodotorula ingeniosa, isolation, purification, ice milk production.

INTRODUCTION

β-galactosidase (β-gal) hydrolyses the β-1, 4-d-galactosidic linkage of lactose, as well as those of related chromogens, o-nitrophenyl- β-d-galactopyranoside (ONPG), o-nitrophenyl-β-D-galactopyranoside (PNPG) and  6-bromo-2-naphthyl-galactopyranoside  (BNG). This enzyme is widely distributed in nature being found in various types of microorganisms, plants and animal tissues. The enzyme β-gal has two main biotechnological uses in the diary industry, e.g. removal of lactose from milk for lactose-intolerant people and the production of galacto-oligosaccharides for use in probiotic foodstuffs Karasov et al. [11].

The present work aims to screen some yeast isolates isolated from Egyptian soils around milk factories for their ability to produce β-gal enzyme. In addition some nutritional requirements affecting β-gal production such as carbon and nitrogen sources were studied. Furthermore, some properties of the purified enzyme were estimated. In addition the applications of the purified β-gal were employed for ice milk production.

STUDY AREA

Media
The following media were used through this study: 1-Malt yeast extracts (MYE) [6],  2-Czapex Dox and 3-Yeast nitrogen base (YNB) (Difco Manual, 1953).

Microorganisms
Lactose utilizing yeasts were isolated from a variety of Egyptian soils obtained from various locations using Dox agar medium containing lactose as only source of carbon instead of its carbon (i.e. glucose). All these isolates were screened for their β-gal activity. In addition, the most active isolate was selected and identified according to Lodder [12] and Barnett and Pankhurst [3].

Enzyme assay and definition of units
β-gal activity was determined by the release of o-nitro phenol from a 1.5 mM solution (18 mg/ml stock solution) of O-nitrophenyl-β-D-galactopyranoside (ONPG; Sigma) in 0.1M sodium phosphate buffer (pH 6.0) at 65°C.  One unit of the enzyme activity is the amount of β-gal which forms 1 µ mole of ONP in 1 min. The specific activity is the number of units of enzyme per mg of protein.

Enzyme release treatment
β-gal formation by the selected strains of yeasts was found to be intracellulary produced. Therefore, at the end of the incubation period, Cells were harvested by centrifugation under cooling and the release of active enzyme was carried out by mechanical agitation of yeast cells biomass with sterile sea sand. In addition, enzyme release treatment was also carried out using toluene or by homogenization using Brown Melsungen AG cell homogenizer as described by Selim et al.[17].

Enzyme purification
Partial purification of the crude enzyme was carried out using different concentrations of ammonium sulphate. Active fraction obtained was loaded onto a column of Sephadex G-100.  Elution was carried out by 0.05 M phosphate buffer pH 6.8. Tubes containing the most active sample were collected and used for further studies.

Protein determination
Protein was determined by the method of Lowry et al. [13] using bovine serum albumin as standard.

Milk and skim milk powder
Fresh Buffalo's milk and skim milk powder were obtained from the Research Institute of Animal Production, Agricultural Research Center, Ministry of Agriculture, Dokki, Giza. Egypt. Sucrose and vanilla was obtained from local market. Gelatin was obtained from BDH Chemicals Ltd Pool England.

Ice milk preparation
The Ice milk was made as described by Ismail et al. [9]. Ice milk mixture consisted of 3% fat, 12% skimmed milk powder, 12% sucrose, vanilla and 0.5% gelatin. Before addition of mixes, levels of extracts of β-gal used were 12.3 (TI), 24.6 (TII) and 36.9 (TIII) units of β-gal/ml pasteurized milk, were added and incubated for 2 hr at 40°C. The treated milk was refrigerated at 4°C overnight. A control without β-gal was treated similarly.

Chemical analysis of Ice milk
In ice milk samples, lactose content was measured according to  Barrent and Abd El-Tawab [1]. Specific gravity was determined by means of a pycnometer bottle. Weight per gallon was calculated in pounds according to Bruke [2] by multiplying the specific gravity of ice milk by the factor 8.34. Overrun was determined by the method mentioned by Sommer [18].

Organoleptic properties
Ice milk samples from all treatments were scored for flavors, body & texture and appearance by regular score panels chosen from the staff members of N.R.C., according to the score card given by Nelson and Trout [16].

RESULTS AND DISCUSSION

1-Screening test
Results in (Table 1) Data clear indicate that the isolate no 45 supported highest production of β-gal. Furthermore, 24 hrs incubation period was optima for maximum production of its enzyme although, the growth was not so high. Thereafter some decrease of enzyme formation was noticed at longer incubation (150, 141 and 132 u/ml).

2-Identification of yeast isolate
Taxonomical studies on the most promising isolate No.45 indicate that these isolate could be identified as Rhodotorula ingeniosa

3-Enzyme Release treatments
The industrial development of microbial intracellular enzymes has been hampered by the difficult and expense of releasing active enzymes from cells with a good yield. Results of chemical or physical treatments are shown (Tables 2, 3 and 4). Data obtained clear indicate that grounding yeast cells biomass with sterile sea sand in mortar under cooling of cell was the most efficient. The highest β-gal release was detected after 30 min of grounding (150 unit/ml). Furthermore, the use of longer grounding times was not so useful.

4-Effect of different carbon sources on the enzyme production
Twelve of different carbon source each was tested separately in medium. Results (Table 5) shows that both growth and enzyme were maximally produced in the presence of cellobiose as only source of carbon (188 unit/ml and 4.5 OD 600 with 41.8 relative activity). On the other hand, lactose was correlated with a high amount of β-gal enzyme production although the growth was not high (145 unit/ml and 2.1 OD 600 with relative activity 69.5). Data (Table 5) clear indicate the constitutive synthesis of this enzyme by the strain under study. Furthermore, According to relative activity (R.A), lactose was used in medium with different concentrations (0.5-3.0%) a notable increase of enzyme activity was correlated with the increase of lactose concentration reaching its maximum value (200 unit/ml) when 1% final concentration was tested. Followed by a notable decrease was noticed at higher concentration (Fig. 1). Many authors used lactose for the production of β-gal Manera et al. [15] and Grosova et al. [7].

5-Effect of different nitrogen source
Fifteen of different organic or inorganic nitrogen sources were tested separately in medium in such amount that the final concentration of N-base was not changed as that in the basal medium. Results in Table 6 clear indicate that ammonium sulphate was the most suitable for highest β-gal enzyme production and relative activity (190, 65.5) followed by ammonium triphosphate (130 and 41.9, respectively). This data are in agreement with the data obtained by Manera et al. [15] who used   ammonium sulphate for β-gal production and studied its concentration effect on the production process.

6-β-gal Purification
The obtained results given in Tables 7 and 8 showed that the best recovery (27.5%) with the highest specific activity (550 unit/ml) and purification fold (3.6) were obtained when 40% ammonium sulphate saturation was employed. The use of ammonium sulphate for β-gal enzyme precipitation was mentioned and used by  Gul-Guven et al. [8] Active fraction obtained was passed though Sephadex-G100 column chromatography (Fig. 2). Results shows that β-gal activity was only detected in one peak located in between fraction numbers 22 and 27.

7-Enzymatic properties
1-Effect of pHs values on the enzyme activity
β;-gal activity was determined under various pH values from 3-7 adjusted using citrate phosphate buffers. Results in Fig 3. shows that the enzyme activity was increased with the increase of pH value reaching its maximum at pH 4.5 (3.18 u/ml) followed by a notable decrease when higher pH values were employed.

2-Effect of temperature on the enzyme activity
The effect of temperature on β-gal activity was carried out by incubation the reaction mixture at various temperatures (30°C–90°C) for 10 min. Results obtained (Table 4) shows that 60°C was optima for β-gal enzyme activity. Therefore a notable decrease of the enzyme activity at higher temperature was noticed.

3-Effect of reaction periods on the enzyme activity
Pure enzyme was incubated with the substrate ONPG under optimized assay conditions for different time from 5 min and 25 min. Result (Fig. 5) showed that the release of ONP from ONPG was increased with the increase of time up to 20 min. After which no more ONP was released.

8-Effect of β-gal on milk lactose content and ice milk
Table 9 represents the data recorded as a response for adding different levels of β-gal to fresh pasteurized milk. Also, these data reveal that the higher the amount of β-gal TIII (33.6 Units/ml), the more hydrolyzed lactose (3.67%). Some references showed that increasing enzyme concentration increased the rate of hydrolysis as reported by Ismail et al. [9] and El-Hofi [4].

Data in Table 9 show the specific gravity, weight per gallon and overrun. The results indicated that both specific gravity and weight per gallon increased with the Increase of β-gal units. It could also be noticed that ice milk mixes had higher values of specific gravity and weight per gallon than that of the resultant ice milk. It was due to incorporation of air by whipping during the freezing process. In addition Table 12 showed that increasing β-gal decreased the overrun values. This can be attributed to the decrease in the amount of incorporation air. This is in full agreement with that of Ismail et al. [10].

9-Organoleptic properties
The results indicated that the average score points of TII and TIII decreased as the level of β-gal increased except TI (Table 10). The highest value is recorded with TI this may be due to the increase in the sweet taste based on the hydrolysis of lactose by the β-gal, such as taste may be more preferable by the Egyptian consumer. The average score points of body and texture decrease by increasing the level of added β-gal, being 32.0, 26.6 and 25.0 points for treatments containing 12.3,  24.6 and 36.9 units of β-gal/ml pasteurized milk, respectively,  while it was 30.0 for the control (Table 10). This mainly due to the increase in the values of viscosity, also to the decrease in the overrun for all β-gal treatments [5].

From these results it could be said that the addition of β-gal to ice milk mixes increased the sweetness but properties of body and texture and appearance of the resultant ice milk slightly decrease. These results are similar to those reported by Lindamood et al. [14]. Ismail et al. [9] also found that using of β-gal isolated from Bacillus species in ice milk increased the sweetness and sensory properties were affected.

CONCLUSIONS

The results of this study indicate that yeast Rhodotorula ingeniosa produces β-gal which can be purified by sequential use of ammonium sulfate precipitation,  gel filtration chromatography on Sephadex G-200, kinetics, properties and β-gal enzyme could be applied in manufacture of ice cream at the level of 33.6 units/ml pasteurized milk.

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Accepted for print: 18.01.2010

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