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Study on Isolation and Partial Purification of Lactase (galactosidase) Enzyme from Lactobacillus Bacteria Isolated from Yogurt

N. H. M. R.Mozumder
Department of Food Science and Nutrition, Hajee Mohammad Danesh Science and Technology University, Dinajpur-5200, Bangladesh

M. Akhtaruzzaman
Institute of Nutrition and Food Science, University of Dhaka, Dhaka-1000, Bangladesh

M. A. Bakr
Diet Solution Limited, Dhanmondi, Dhaka-1209, Bangladesh

Fatema-Tuj-Zohra
Nestle Nutrition Department, Nestle Bangladesh Limited, Dhaka, Bangladesh

Abstract/ Executive Summary:

Lactase has many applications in dairy industry including for the treatment of lactose intolerance. The present study was conducted to identify the activity of lactase enzyme produced by Lactobacillus bacteria isolated from yogurts available in Dhaka city. The strains were identified to be gram positive, catalase negative, fermentative and lactase producer when cultured on selective MRS agar media by using standard bacteriological procedures and techniques. The study revealed that enzymes produced by lactobacilli were capable to produce glucose from substrate lactose in lactose modified media using lactase assay Kit Glu IB and their highest protein concentration (17.25 mg/ml) was observed in the supernatant of culture media isolated from L. lactis. Highest total activity (850.69 U/l) and specific activity (50.04 U/mg) of lactase enzyme was observed in the strain of L. bulgaricus. The crude extract which showed highest activity was further purified by ammonium sulphate precipitation followed by anion exchange column chromatography (DEAE cellulose). Final specific activity and fold purification of lactase enzyme reached to 62.80 U/mg and 1.47 respectively. The highest physic-chemical properties (Effect of pH and temperature) of lactase enzyme were observed at pH 6.0 which was 43.98 U/mg of protein and at 70°c temperature which was 111.11 U/mg of protein.

Keywords: Galactosidase; Specific activity; DEAE cellulose; Fold purification; Yogurt.

Location: Department of Food Science and Nutrition, Hajee Mohammad Danesh Science and Technology University, Dinajpur

Start Date:

End Date:

Program Area: Quality and Nutrition

Commodity: Yogurt

Objectives:

An attempt has been made for the production of lactase enzyme from yogurt since yogurt is one of the popular fermented milk products which are a sub continental equivalent of curd.

Methodology:

Chemicals, reagents and bacteriological media: The Bovine Serum Albumin (BSA), Folin Ciocalteau Reagent (E. Merck Germany), MRS (Hi Media Laboratory Pvt. Ltd, India) were purchased and collected from local scientific store except Glu IB kit (Wako Japan) and DEAE cellulose (Wako Japan). All other reagents such as maleate buffer, lactose solution, Tris buffer and glucose standard solution were available in the laboratory of institute of Nutrition and Food Science, Dhaka University. Sampling and analysis of organolaptic characteristics of yogurt: A total number of ten traditionally manufactured popular yogurt samples (JGS: Jadab Gosh and Sons, BS: Bikrampur Sweets, CM: Commilla Mistanno Bhander, MS: Muslim Sweets, AD: Aftab Dhai, TMG: Tripty Misty, RM: Rajshahi Misty Ghar, HD: Haque Dhai, BD: Bogra Dhai Bhander, MCG: Mohon Chan & Grand Sons) were collected from different sweetmeat shops of Dhaka City and their organolaptic characters viz., color flavor, consistency taste and pH were analyzed through sensory evaluation. Isolation and identification of lactobacillus bacteria: The isolation of genus of Lactobacillus sp. was done on de Man Rogosa and Sharrp agar (MRS agar, Hi-media, M-369). Clear and discrete colonies of presumptive Lactobacillus that isolated by repeated pour plate and spread plate techniques using selective M.R.S. agar at pH of 6.2-6.6 were used for pure culture and preserved in refrigerator at 4-7ºC for sub culturing. The presumptive lactobacilli isolates were characterized and identified on the basis of cultural growth (shape, edge, elevation, opacity, consistency), cellular morphology (gram reaction, motility, shape) biochemical (catalase test, acid production test from different sugars, NH3 production from Arginine for, CO₂ gas from glucose in Gibson’s semi solid Tomato Juice Medium) and physiological characteristics; growth at different temperature and pH. Enzyme preparation: For the production of lactase, presumptive lactobacilli was grown in MRS broth (100ml) with 0.1 ml of culture inoculums and incubated at 37^oC for 24- 48 hours. After incubation cells were removed by centrifugation at 12,000X g RPM for 20 minutes (Kokusan-H-200NR centrifuge machine, Japan) below the temperature of 4ºC. The cell free supernatants (crude enzyme) containing intracellular materials were stored in the freezer and used for enzymatic assay and partial purification. Determination of concentration of extracellular soluble protein: Protein concentration was determined with the method of Lowry et al., using Bovine Serum Albumin (BSA) as the standard and optical density of the reaction mixture was measured at 660nm by the spectrophotometer. The amount of the soluble protein was calculated from the standard curve as mg of protein per ml of test samples. Assay of lactase enzyme: catalytic and specific activity determination: The lactase activity was measured as a total lactase by incubating with lactose followed by the measurement of the glucose liberated with a glucose oxidase reagent (GOD> 2400 U/L, POD 1100 U/L) served from Glu IB kit (Wako Japan) providing glucose oxidase , peroxidase and buffer. A 20μl of lactose solution (56m.mol/l) in maleic acid was mixed with 40μl of supernatant and incubated at 37ºC for 60 minutes and finally added with 3ml of glucose reagent and again incubated for 30 minutes and liberated glucose was measured by spectro-photometrically at 510 nm against blank. Partial purification of lactase enzyme: For this purpose, the clarified supernatant obtained from first centrifugation of culture media brought to 80% ammonium sulfate precipitation in a refrigerated condition and dialyzed by dissolving in 10mM Tris- HCL (pH 7.0) buffer for 24 hours and finally centrifuged at 15000 g X rpm for 20 minute (Kokusan-H-200NR centrifuge machine, Japan). All subsequent steps involving ammonium sulfate were carried out at 4^oC. The obtained pellet was further purified by an ion exchange column chromatography. An ion exchange chromatography was performed on column using a DEAE cellulose bed and equilibrated with buffer 10mM Tris-HCL; the target enzyme was eluted by a linear gradient of 250mM NaCl in 10 mM Tris-HCl buffer. The fractions which showed highest activity were pooled for lactase activity at different pH and temperatures. Effect of pH and temperature on enzyme activity: The effect of pH on lactase enzyme was determined at optimum temperature (37⁰C) in five buffer system by incubating the enzyme in water bath for 1 hour and finally by the measurement of glucose liberated with a glucose standard reagent (Glu IB Kit) as described by earlier study.

Source of Information: J. Sci. Res. 4 (1), 239-249 (2012)

Article Link (Online Journal):

Funding Source:

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Achievement/Findings:

This paper reports the isolation and partial-purification of lactase enzyme from Lactobacillus strains isolated from yogurt. Once the strains had been isolated it was characterized by various tests and screened for efficient lactase enzyme producer. The enzyme is active at high temperature up to 70^oC and showed highest activity at pH 6.0 which indicates its high level of thermostability. The maximum total activity of enzyme was recorded as 850.69U/l. The purification fold was 1.47 after column chromatography. After considering the all findings it might be concluded that the enzyme produced in this work was lactase that showed a consistent with results obtained from previous study.

Knowledge Management: Journal