Screening and characterization of β-Galactosidase producing Lactobacillus sp. isolated from dairy samples

 

Nivetha. A, Mohanasrinivasan*

School of Bio Sciences and Technology, VIT University, Vellore, Tamil Nadu

 

ABSTRACT:

Lactose intolerance in humans is the major concern in current scenario. β-Galactosidase has tremendous potential in the preparations of lactose free product development. The present research was carried out to screen β-GalactosidaseproducingLactic acid bacteria (LAB) from milk and milk products such asdonkey milk, Curd and yogurt. Based on the morphological characteristics, about 70 bacterial colonies were selected. Out of these, 5 isolates from donkey milk, 7 from curd and 3 from yogurt sample were found positive for β-Galactosidase production using X-Gal method. Potent strains were identified by determining the enzyme activity using ONPG assay.The maximum enzyme activity was found in isolate no. VITDM15 (Donkey milk) with of 375.21 AU/ml followed by 235.36 AU/ml from VITCD01(Curd) and 142.35 AU/ml from VITYT13(Yogurt) respectively. Hence these strains can be exploited for lactose free product development.

 

 

 

INTRODUCTION:

β-D-galactosidase also known as lactase (EC 3.2.1.23) is an enzyme which catalyzes the hydrolysis of lactose, the main carbohydrate present in milk, to monosaccharide glucose and galactose¹ which gets absorbed across the intestinal epithelium and has a potential importance in the dairy industry. β-gal also hydrolyzes the D-galactosyl residues in polymers, oligosaccharide and secondary metabolites. β-gal belongs to sub-families 1, 2, 35 and 42 of GH-A superfamily of glycoside hydrolases². β-gal is a well known biocatalyst which catalyzes hydrolytic and transgalactosylation reactions. In some cases it takes part in the production of prebiotic galacto-oligosaccharide (GOS), which are synthesized due to its associative transglycosylase activity³. β-galactosidase has two enzymatic activities: on one hand, it cleaves β-glycosidic bond between galactose and its organic residues and also cleaves cellobiose, cellotriose, cellotetrose and cellulose.

 

On the other hand, it catalyzes the transgalactosylation of lactose to allolactose⁴. There are of two types of lactases, neutral and acidic, based on their optimum pH for enzyme activity. β-gal is an important enzyme in food processing and pharmaceutical industries. The nutritional value of lactose is limited because a large portion of population in the world lacks this enzyme and cannot utilize lactose, as a result of which they develop lactose maldigestion or intolerance ⁵. Moreover lactose is a hygroscopic sugar and has a strong tendency to absorb flavors and odors that causes many defects in refrigerated foods such as crystallization in dairy products, development of sandy or gritty texture and deposit formation⁶.This however creates a potential application of β-gal. Enzymatic hydrolysis of lactose has beneficial effects on assimilation of foods containing lactose for lactose intolerant population, as well as technological and environmental advantages for industrial applications. The enzyme is industrially important because it can be used to prevent lactose crystallization, to increase the solubility of milk product and solve problems associated with whey utilization and disposal, which would lead to environmental pollution⁷. Furthermore, the hydrolyzed milk can be applied in the manufacturing of caramelized milk to avoid crystallization during the storage caused by the low solubility of lactose. In addition, the transgalactosylation property of β-gal has prominent medical applications such as treatment of disorders and development of digestive supplements. It also has potential applications in bioremediation, biosensor and diagnosis⁸.

 

The present research was carried out to screen the efficient lactic acid bacteria (LAB) for β-Galactosidse production.

 

MATERIALS AND METHODS:

Ortho Nitro phenol (ONP), Ortho Nitro Phenyl Galactopyranoside (ONPG), (5-Bromo-4-Chloro-3-indoxy-β-D-galactopyranoside) X-Gal and De Man Rogosa Sharpe (MRS) were purchased from Himedia. β-Galactosidase from E.Coli (standard enzyme) was procured from Sigma Aldrich. All the reagents used were analytical grade.

 

Sample collection and isolation of lactic acid bacteria:

Milk and milk products such as donkey milk, curd and yogurt were aseptically collected in cold condition from Vellore district, Tamilnadu and microbiologically processed. Samples were serially diluted up to 10^-8 in sterile normal saline. Pour plated on De Man Rogosa Sharpe (MRS) agar platesand incubated for 48 hours at 30°C. Isolates with colony morphology similar to Lactobacillus were selected and sub cultured in MRS medium to obtain pure culture. Pure culture strains were numbered isolates from donkey milk VITDM01 to VITDM026, curd VITCD01 to VITCD025 and yogurt VITYT01 to VITYT 019 and maintained at -20°C in MRS broth with 20% glycerol and enriched in MRS broth by incubating at 37°C for 24 h for future study. These cultures were retrieved twice in MRS broth before the experiment^9-10.

 

Preliminary Characterization and screening for β-Galactosidase production:

The isolates were initially characterized for its Grams reaction and Catalase test. Positive isolates were screened for β-Galactosidase production using X-Gal (5-Bromo-4-Chloro-3-indoxy-β-D-galactopyranoside) method. Single colony was streaked on MRS agar plate containing 20µl of X-Gal (20mg/ml of DMSO) on its surface. The plates were incubated at 30°C for 48h.After incubation the plates were noted for green colored colonies, which indicates the β-Galactosidase production¹¹.

 

Production and extraction of intracellular β-Galactosidase:

The positive strains from X-Gal assay were grown in MRS broth and the overnight culture was centrifuged at 12,000 RPM for 20mins at 4°C. Obtained culture pellet (100µl) is mixed with 900µl of Z-Buffer (Na₂HPO₄, NaH₂PO₄, KCl, MgSO₄, β-Mercaptoethanol) and disrupted by the ultrasonication treatment using the sonics – ultra cell, UK with a ½ diameter tapped biohorn that delivers ultrasonic sound at frequency of 20KHz. Sonication process was done at constant acoustic power,constant duty cycle of 20W and 50% respectively. The samples were kept in icebath to avoid the denaturaion of protein during lysis process¹².

 

Enzyme activity determination by ONPG assay:

To the disrupted cells 100µl of chloroform, 50µl of 1:9 ratio of Toluene: Acetone and 200µl of ONPG (4mg/ml of Z-Buffer) was added and incubated at 37°C for 15mins. The yellow color was observed which determines the presence of enzyme activity. β-Galactosidase enzyme activity was determined by measuring the release of O-nitrophenol from ONPG (ABR-2013) and the reaction stopped by 100µl of Sodium carbonate (100g/l). The Optical density value at 420 and 550nm were noted for each isolates and β-Galactosidase activity was calculated. 1 unit of β-Galactosidase is defined as the amount which hydrolyses 1 µmol of ONPG to O-nitrophenol and D-galactose per min per cell^13-17. The isolates were screened for an effective enzyme production which is further selected for the optimization of its enzyme production in different culture condition.

 

β- Galactosidase Units = 1000 x OD420nm/ (t x v x OD600nm ).

 

Biochemical characterization of the positive isolates:

Positive isolates were selected and subjected for biochemical characterization. Pure culture of strains were tested for Gram staining, catalase test, citrate utilization, Triple Sugar Ion (TSI), Mannitol salt agar test, Indole test, MR-VP test, Nitrate reductase test, sugar fermentation¹⁵. Culture and biochemical characterization of these isolates were compared with data from Bergey’s manual of determinative bacteriological¹⁶

 

RESULTS AND DISCUSSION:

Isolation of LAB:

Based on colony morphology a total of 70 bacterial isolatesin which 25 from Curd sampledesignated as VITCD01to VITCD025, 26 from donkey milkdesignated as VITDM01 to VITDM026 and 19 from yogurt designated as VITYT01 to VITYT019 were isolated Shown in Table 1. For preliminary characterization the isolates were subjected for gram’s reaction and catalase test.5 Isolates from donkey milk, 7 isolates from curd and 3 from yogurt samplewere found to be gram positive Bacilli and catalase negative (Figure 1). These isolates were subjected forX-Gal assay for the β-Galactosidase activity.

 

FIGURE 1:- Biochemical characterization – Gram staining - Gram positive Bacilli selected for further study.

 

Table 1: Characterization of isolates based on their morphology

Origin	Isolate no.	Form	Elevation	Margin	Gram staining	Shape	Catalase test
	VITDM1	Circular	Flat	Entire	Positive	Cocci (Chain)	Negative
	VITDM2	Circular	Flat	Entire	Positive	Cocci (Chain)	Negative
	VITDM3	Circular	Raised	Entire	Positive	Cocci (Chain)	Negative
	VITDM4	Circular	Raised	Entire	Positive	Bacilli (Short rod)	Negative
	VITDM5	Irregular	Raised	Entire	Positive	Cocci (Tetrads)	Negative
	VITDM6	Circular	Convex	Entire	Positive	Bacilli (Short rod)	Negative
	VITDM7	Circular	Raised	Entire	Positive		Negative
	VITDM8	Circular	Raised	Entire	Positive	Cocci (Tetrads)	Negative
	VITDM9	Circular	Pulvinate	Entire	Positive	Cocci (Chain)	Negative
	VITDM10	Circular	Raised	Entire	Positive	Cocci (Chain)	Negative
Donkey Milk (26)	VITDM11	Irregular	Raised	Entire	Positive	Cocci (Tetrads)	Negative
	VITDM12	Irregular	Raised	Entire	Positive	Cocci (Tetrads)	Negative
	VITDM13	Circular	Raised	Entire	Positive	Cocci (Tetrads)	Negative
	VITDM14	Circular	Umbonate	Entire	Positive	Cocci (Chain)	Negative
	VITDM15	Circular	Raised	Entire	Positive	Bacilli (Short rod)	Negative
	VITDM16	Circular	Raised	Entire	Positive	Bacilli (Short rod)	Negative
	VITDM17	Circular	Raised	Entire	Positive	Cocci (Chain)	Negative
	VITDM18	Circular	Raised	Entire	Positive	Cocci (Tetrads)	Negative
	VITDM19	Circular	Raised	Entire	Positive	Cocci (Tetrads)	Negative
	VITDM20	Circular	Raised	Entire	Positive	Cocci (Chain)	Negative
	VITDM21	Circular	Pulvinate	Entire	Positive	Bacilli (Short rod)	Negative
	VITDM22	Irregular	Raised	Entire	Positive	Bacilli (Short rod)	Negative
	VITDM23	Circular	Raised	Entire	Positive	Bacilli (Short rod)	Negative
	VITDM24	Circular	Flat	Entire	Positive	Bacilli (Short rod)	Negative
	VITDM25	Circular	Flat	Entire	Positive	Cocci (Chain)	Negative
	VITDM26	Circular	Pulvinate	Entire	Positive	Cocci (Chain)	Negative
	VITCD27	Circular	Flat	Entire	Positive	Bacilli (Long rod)	Negative
	VITCD28	Circular	Raised	Entire	Positive	Bacilli (Long rod)	Negative
	VITCD29	Circular	Raised	Entire	Positive	Bacilli (Long rod)	Negative
	VITCD30	Circular	Convex	Entire	Positive	Cocci (Chain)	Negative
	VITCD31	Irregular	Umbonate	Entire	Positive	Cocci (Tetrads)	Negative
	VITCD32	Irregular	Pulvinate	Entire	Positive	Cocci (Chain)	Negative
	VITCD33	Circular	Convex	Entire	Positive	Cocci (Tetrads)	Negative
	VITCD34	Circular	Convex	Entire	Positive	Cocci (Chain)	Negative
	VITCD35	Circular	Convex	Entire	Positive	Cocci (Chain)	Negative
Curd (25)	VITCD36	Circular	Raised	Entire	Positive	Cocci (Chain)	Negative
	VITCD37	Circular	Pulvinate	Entire	Positive	Bacilli (Short rod)	Negative
	VITCD38	Circular	Flat	Entire	Positive	Cocci (Tetrads)	Negative
	VITCD39	Circular	Flat	Entire	Positive	Cocci (Tetrads)	Negative
	VITCD40	Circular	Raised	Entire	Positive	Cocci (Chain)	Negative
	VITCD41	Circular	Flat	Entire	Positive	Cocci (Tetrads)	Negative
	VITCD42	Circular	Raised	Entire	Positive	Cocci (Chain)	Negative
	VITCD43	Irregular	Raised	Entire	Positive	Cocci (Tetrads)	Negative
	VITCD44	Circular	Raised	Entire	Positive	Cocci (Tetrads)	Negative
	VITCD45	Circular	Raised	Entire	Positive	Bacilli (Short rod)	Negative
	VITCD46	Circular	Flat	Entire	Positive	Cocci (Chain)	Negative
	VITCD47	Circular	Raised	Entire	Positive	Cocci (Chain)	Negative
	VITCD48	Irregular	Raised	Entire	Positive	Cocci (Tetrads)	Negative
	VITCD49	Irregular	Flat	Entire	Positive	Cocci (Chain)	Negative
	VITCD50	Irregular	Flat	Entire	Positive	Bacilli (Short rod)	Negative
	VITCD51	Circular	Flat	Entire	Positive	Bacilli (Short rod)	Negative
	VITYT52	Circular	Raised	Entire	Positive	Cocci (Chain)	Negative
	VITYT53	Circular	Raised	Entire	Positive	Cocci (Chain)	Negative
	VITYT54	Circular	Raised	Entire	Positive	Cocci (Chain)	Negative
	VITYT55	Circular	Raised	Entire	Positive	Cocci (Tetrads)	Negative
	VITYT56	Circular	Raised	Entire	Positive	Cocci (Tetrads)	Negative
	VITYT57	Irregular	Flat	Entire	Positive	Bacilli (Short rod)	Negative
	VITYT58	Irregular	Flat	Entire	Positive	Bacilli (Short rod)	Negative
Yogurt (19)	VITYT59	Irregular	Flat	Entire	Positive	Cocci (Chain)	Negative
	VITYT60	Circular	Raised	Entire	Positive	Cocci(Chain)	Negative
	VITYT61	Circular	Raised	Entire	Positive	Cocci (Tetrads)	Negative
	VITYT62	Circular	Raised	Entire	Positive	Cocci (Chain)	Negative
	VITYT63	Irregular	Flat	Entire	Positive	Cocci (Tetrads)	Negative
	VITYT64	Irregular	Flat	Entire	Positive	Bacilli (Short rod)	Negative
	VITYT65	Irregular	Flat	Entire	Positive	Cocci (Chain)	Negative
	VITYT66	Circular	Flat	Entire	Positive	Cocci (Chain)	Negative
	VITYT67	Circular	Flat	Entire	Positive	Cocci (Tetrads)	Negative
	VITYT68	Circular	Raised	Entire	Positive	Cocci (Tetrads)	Negative
	VITYT69	Circular	Raised	Entire	Positive	Cocci (Chain)	Negative
	VITYT70	Circular	Raised	Entire	Positive	Cocci (Tetrads)	Negative

 

X-Gal Assay (Enzyme activity):

On X-Gal plates green color isolated colonieswere observed which indicates the presence of β-Galactosidase enzyme producing bacteria. Totally 15 strains (VITDM4, VITDM6, VITDM15, VITDM23, VITDM24, VITCD27, VITCD28, VITCD29, VITCD37, VITCD45, VITCD50, VITCD51, VITYT57, VITYT58, VITYT64) were showed positive for X-Gal assay (Figure 2).Positive strains were subjected for ONPG assay to know the effectiveness of enzyme activity. The result detected by X-Gal method were confirmed by previous work.

 

 

FIGURE 2:- Blue color colony indicates the presence of β-Galactosidase by X-Gal assay.

ONPG assay:

All the 15 strains carried out for ONPG assay. The formation of yellow color designates the production of β-Galactosidase enzyme. The maximum activity 375.21 U/min/cell were noted from isolate no. VITCD27 obtained from curd followed by 235.96 U/min/cell from isolate No.VITDM15obtained from donkey milk and 142.35 U/min/Cell isolated from yogurt sample respectively (Table 2&Figure 3)(Al-jazairi et al., 2014 and Sumit Sharma et al., 2014).The three potent strains which exhibitedmaximum enzyme activity were characterized for biochemical characterization and different culture condition (Temperature, pH, Carbon source, different Solvents andmetal ions).

 

 

 

 

 

 

 

 

 

Table 2: β- galactosidase activity in the best fifteen bacterial isolates.

Origin	Isolate No.	ONPG
	VITDM4	115.23
	VITDM6	104.52
Donkey	VITDM15	235.96
	VITDM23	132.5
	VITDM24	112.69
	VITCD27	375.21
	VITCD28	115.63
	VITCD29	125.72
Curd	VITCD37	111.32
	VITCD45	132.21
	VITCD50	115.28
	VITCD51	128.92
	VITYT57	124.52
Yogurt	VITYT58	113.56
	VITYT64	142.35

 

β- galactosidase activity was measured using ONPG and calculated as follows: Units = 1000 *OD420/ Volume (3 ml) *time (1 min) *OD600. We select VITDM15, VITCD27 and VITYT64 for further study.

 

 

Figure 3:- Observation of yellow color for presence of enzyme by ONPG (fifteen isolates).

 

Biochemical characterization for potent strain:

For Biochemical characterization, Indole, MR, VP, Oxidase, TSI, Nitrate reductase, citrate utilization, mannitol salt, were performed. Sugar fermentation was performed to examine whether the isolates can ferment glucose, lactose, galactose, sucrose, maltose, mannitol, ribose, xylose, rhamnose, threlose and fructose. The results described in table 3 show that all isolates were able to ferment given sugars (Table 3).

 

 

 

 

 

 

 

 

 

 

Table 3: Biochemical characterization for potent isolates

Test parameters	VITDM15	VITCD01	VITYT13
Indole test	NEG	NEG	NEG
Simmon's citrate slant test	NEG	NEG	NEG
Methyl red (MR) test	NEG	NEG	NEG
VogesProskauer (VP) test	NEG	NEG	NEG
Oxidase test	NEG	NEG	NEG
Triple sugar iron (TSI) test	POS	POS	POS
Mannitol salt agar test	NEG	NEG	NEG
Nitrate Reductase test	POS	POS	NEG
Glucose fermentation	POS	POS	POS
Lactose fermentation	POS	POS	POS
Galactose fermentation	POS	POS	POS

NEG– Negative; POS-Positive

 

 

 

 

 

CONCLUSION:

From the results obtained in the study, it was concluded that Lactobacillus sp., (VITDM15, VITCD01, VITYT13) can efficiently produce β-galactosidase enzyme. Thus can be used in the commercial production of lactase as it is considered safe to use as a probiotic microorganism and it also gives high enzyme yield. Moreover, the use of lactase can reduce the amount of lactose present in whey which causes environmental problem when discharged in large quantities. As Lactobacillus sp., represents as a potential source of lactase it can be used for the treatment of milk to reduce their lactose content. The hydrolysis of lactose in dairy products by lactase can help lactose maldigesters, improves solubility and lack of sweetness in milk products.

 

ACKNOWLEDGEMENTS:

The authors are thankful to the Management, VIT University, Vellore for providing the facilities.

 

REFERENCES:

1.        Kader ASSA, Dosouky MA, Abouwarda A, All SMA, Osman MI. Characterization of partially purified β-galactosidase from Bacillus subtilis. J. Appl. Sci. Res. 2012; vol. 8, pp. 2379-85.

2.        Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombart V&Henrissat B. The carbohydrate-active enzymes database (CAZy): an expert resource for glycogenomics, Nucleic Acids Res. 2009;vol.37, pp. 233–238.

3.        Mahoney R.R. Enzymes exogenous to milk in dairy β-D galactosidase, Encyclopedia of Dairy sciences, Academic Press, London. 2003; vol. 2, pp 907-914.

4.        Juers DH, Matthews BW and Huber R. LacZ- β-galactosidase: Structure and function of an enzyme of historical and molecular biological importance, Protein Sci. 2003; vol.21(12), pp. 1792-1807.

5.        Vasiljevic T, Jelen P. Lactose hydrolysis in milk as affected by neutralizers used for the preparation of crude _-galactosidase extracts from Lactobacillusbulgaricus11842, Innovative Food Science & Emerging Technologies. 2002; vol. 3, pp. 365-370.

6.        Carrara CR and Rubiolo AC. Immobilization of β- galactosidase on chitosan, Biotechnology Progress, 1994; vol. 10, no.2, pp. 220–224.

7.        Mlichova Z, Rosenberg M. Current trends of β-galactosidase application in food technology, J. Food Nutr. Res. 2006; vol. 45, pp. 47-54.

8.        Asraf SS, Gunasekaran P. Current trends of β-galactosidase research and application, Cur. Res. Tech. Edu. Top Appl. Microbiol Microbial Biotech. Formatex Res. Center. 2010; vol. 2, pp. 880-890.

9.        Mathara, JM, Schillinger U, Kutima PM, Mbugua SK and Holzapfel NH. Isolation , identification and characterization of dominant microorganisms of kulenaoto: the Maasal tradition fermented milk in Kenya. Int. J. Food Microbiol. 2004; 94:269-279.

10.     De Man JC, and Sharpe ME. A medium for the cultivation of Lactobacillus. J. Appl. Bacteriology, 1960; 23:130-135.

11.     Vinderola CG, and Reinheimer JA, Enumeration of Lactobacillus casei in the presence of L. Acidophilus, bifidobacteria and lactic acid starter bacteria in fermented dairy products. International Dairy Journal. 2000; 10: 271-275.

12.     Choonia HS, Lele SS. β-Galactosidase release kinetics during ultrasonic disruption of Lactobacillus acidophilus isolated from fermented Eleusine coracana. Food Bio. Prod. Process. 2011; vol. 89, pp. 288–293.

13.     Al-jazari M, Abou-ghorra S, Bakri Y and Mustafa M. Optimization of β-galactosidase production by response surface methodology using locally isolated Kluyveromyces marxianus. International Food Research Journal. 2015; 22(4): 1361-1367.

14.     Abhijit Chowdhury MD, Nur Hossain, Nure Jannatul Mostazir, Md Fakruddin, Md. Morsaline Billah and Monzur Morshed Ahmed. Screening of Lactobacillus spp. From Buffalo Yoghurt for Probiotic and Antibacterial Activity. Journal of Bacteriology & Parasitology. 2012; 3.8

15.     Graciela F, De V and María PT. Food Microbiology Protocols. (J.F.T. Spencer and A.L.R.D. Spencer, Ed.), Totowa, New Jersey: Humana Press Inc., chap. 21 (Probiotic Properties of Lactobacilli), 2001; pp: 173-181.

16.     D H Bergey; John G Holt. Bergey's manual of determinative bacteriology. Baltimore : Williams & Wilkins. 1994;

17.     Sumit Sharma and Priyanka Singh. Isolation and Characterization of - Galactosidase Enzyme Producing Microbe and Optimization of its Enzyme Activity under different culture condition. Int.J.Curr.Microbiol.App.Sci. 2014; 3(7) 148-155.

 

 

 

Accepted on 21.09.2017           © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(5):1778-1783.