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Α-Amylase and Β-Galactosidase Production on Potato Starch Waste by Lactococcus Lactis Subsp Lactis Isolated from Pickled Yam

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Α-Amylase and Β-Galactosidase Production on Potato Starch Waste by Lactococcus Lactis Subsp Lactis Isolated from Pickled Yam Journal of Scientific & Industrial Research Vol. 73, May 2014, pp. 324-330 α-amylase and β-galactosidase production on Potato starch waste by Lactococcus lactis subsp lactis isolated from pickled yam Seema Bhanwar and Abhijit Ganguli* Department of Biotechnology and Environmental Sciences Thapar University, Patiala-147004, India Received 18 January 2013; revised 25 December 2013; accepted 25 March 2014 Potato starch waste, a chips industry effluent, was used for the production of industrially important enzymes by an amylolytic strain of lactic acid bacteria, isolated from pickled yam & identified as Lactococcus lactis. The strain was observed to co-produce α-amylase and β-galactosidase. Potato starch waste was efficiently utilized (91.6%) along with high co-production of α-amylase and β-galactosidase. Optimum culture conditions favouring maximum production of these two hydrolases in MRS medium containing 2% potato starch were temperature 55°C, pH 7 and temperature 35°C, pH 5 for α-amylase (2.54 U/mL) and β-galactosidase (2.67 U/mL) respectively. In potato starch waste, Lactococcus lactis retained viability and could co-produce α-amylase (17.54 U/mL) and β-galactosidase (25.35 U/mL) at a temperature of 45ºC and pH 6.5 within a period of 48 hrs. Thus, the study suggests a potential applicability of the isolate for the production of industrially significant hydrolases from a cheap agro industrial source. Keywords: β-galactosidase, α-amylase, Lactococcus lactis, potato starch waste, co-production Introduction amylase application has expanded in many fields Microorganisms have been employed for the such as clinical, medicinal and analytical chemistry, production of industrially important enzymes over as well as their widespread application in starch decades. Among the group of hydrolases, two enzymes saccharification and in the textile, food, brewing and namely α-amylase and β-galactosidase are significantly distilling industries5. important enzymes with industrial applications13. Currently, various microbial amylases available Lactobacillus manihotivorans LMG18011using soluble commercially holds a broad spectrum of applications starch and food wastes as substrates19 and as compared to chemical hydrolysis of starch in starch L. amylovorus utilizing raw corn starch, rice starch processing industry; plant and animal α-amylases30. and wheat starch medium35 are few examples of A most recently isolated ALAB strain L. paracasei microorganisms utilizing cheaper substrates for B41 was the first amylolytic representative of L. casei enzyme production. Complete starch hydrolysis group23 among other amylase producing bacteria requires two kinds of enzyme activities: against such as Lb. plantarum and Lb. manihotivorans15,18, α-1,4 and α-1,6 glycosidic bonds. These two types isolated from cassava-based fermented products of activities can be shown by one enzyme and Lb. cellobiosus29, Lb. amylovorus16, and (amylopullulanase) or by two enzymes, such as Lb. amylolyticus4. The amylolytic lactic acid bacteria α-amylase and pullulanase type I27. α-Amylase (genera of Lactobacillus, Lactococcus, Streptococcus, (E.C.3.2.1.1) catalyzes the hydrolysis of internal Pediococcus, Carnobacterium, and Weissella) producing α-1,4-glycosidic linkages in starch and have starch-modifying enzymes have been summarized24. significant application in a wide number of industrial β-galactosidase (EC.3.2.1.23), most commonly processes such as food, fermentation, textile, paper, known as lactase, hydrolyses lactose into its detergent, and pharmaceutical industries9,11,26. Fungal monomers glucose and galactose. It has potential and bacterial amylases could be potentially useful applications in food processing industry and is in the pharmaceutical and fine-chemical industries. produced in a variety of sources, including plants, However, with the advances in biotechnology, the animals and microorganisms17,1,21. The dairy industry employs β-galactosidase enzyme produced by the —————— microorganisms belonging to genera of Lactobacillus *Author for correspondence 7,10,34 Email: [email protected] and Bifidobacterium . However, different GANGULI & BHANWAR: α-AMYLASE AND β-GALACTOSIDASE PRODUCTION ON POTATO STARCH 325 microorganisms differ in their respective USA) and potato starch (HiMedia, Mumbai, India) for physiological conditions for the optimal production of α-amylase production. Remazol Brilliant blue (RBB)- enzyme. Though β-galactosidase has been identified starch agar medium was prepared by the method of in a wide variety of fungal, yeast and bacterial Omemu et al. (2005). The isolates were plated on cultures, Streptococcus thermophilus and Bacillus MRS-RBB starch agar plate and incubated at 37oC for stearothermophilus can be considered as potential 24hrs. The starch utilization was monitored by bacterial sources21. For instance, β-galactosidase the disappearance of the blue color of the medium based medical and industrial applications include based on the intensity of amylase production. cleavage of blood group A and B glycotopes, The screened isolates were further monitored for biosensor for specific lactose determination in milk their β-galactosidase production on MRS agar and disease diagnosis, treatment of lactose supplemented with 30μg ml-1 of 5-bromo-4-chloro-3- malsorption, production of lactose hydrolysed milk3. indoyl-β-D-galactopyranoside (X-gal) and incubated Lactic acid bacteria have been exploited for at 37°C for 24 hrs. production of both these enzymes separately, but very Biochemical analysis few studies have investigated the production of both One isolate capable of hydrolyzing starch these enzymes simultaneously13. maximally was identified by its colony morphology, Thus, the main focus of the study was to optimize gram-staining, and biochemical tests (catalase test, the culture conditions such as temperature and pH for carbohydrate fermentation etc.), as well as by 16S co-production of industrially important α-amylase and 33 rRNA gene sequencing . β-galactosidase from economically viable sources like potato starch waste of potato based snack food Genetic Identification industry. Genomic DNA of the strain was isolated by usimg QIAamp DNA Mini kit as explained (Qiagen, Materials and Methods Valencia, CA, USA). Bacteria-specific universal primers used for amplification of 16S rRNA gene were the Chemicals forward primer 27F (5’-AGAGTTTGATCATGGCTC-3’) Potato starch was purchased from HiMedia, and the reverse primer 1327R (5’-CTAGCGATTCCG Mumbai, India and potato starch waste water was a ACTTCA-3’) 33. The 16S rRNA gene was amplified kind of a gift from local potato chips industry (Patiala, in 35 cycles with a Gene Amp PCR System 2400 Punjab, India). All other reagents and chemicals were (Perkin Elmer, Waltham, MA, USA). The thermal purchased from HiMedia (India) or Sigma (USA). program consisted of one cycle at 94ºC 4 min, Modified MRS was prepared containing starch in 35 cycles of 94ºC 40s, 46ºC 40s, 72ºC 2 min, final place of carbon source at 2% concentration. one cycle of 72ºC 15s and stored at 4ºC. A 100-bp Potato Starch Waste water DNA ladder was used as the molecular marker The compositional analysis of potato starch waste (Fermentas). PCR products were purified using water including reducing sugar13, starch8, pH, total QIA quick PCR purification kit (Qiagen, Valencia, solids, and chemical oxygen demand (COD), CA, USA) and sequenced from both ends with an Biochemical oxygen demand (BOD) and total ABI3700 DNA sequencer (Applied Biosystems, nitrogen was done as per APHA standard methods Foster City, CA, USA) using the same oligonucleotide (2005) for water and wastewater2 before and after primers used for PCR. The sequenced 16S rDNA analysis. sequences for the bacterial isolates were analyzed to detect the presence of possible chimeric artefacts and Screening of Bacterial isolates compared with the similar gene sequences. Six strains of lactic acid bacteria, isolated from pickled yam and other fermented foods were Enzyme production screened4 based on their starch utilizing potential. The identified strain was grown in MRS medium Further, screening was based on the selection of α- and inoculated with 1% (v/v) (105 CFU/mL) amylase and β-galactosidase producing lactic acid overnight grown culture and incubated at 30ºC for bacteria. The starch utilizing strains were screened on 24 h. The carbon source in the medium was modified MRS medium by using Remazol Brilliant substituted with 0.5, 1, 2 and 3% (w/v) potato starch. Blue (RBB) R salt (Acros Organics, New Jersey, Aliquots were removed at regular intervals for biomass 326 J SCI IND RES VOL 73 MAY 2014 and enzyme activity determination. The enzyme Statistical analysis co-production was estimated in Potato starch waste All the experiments were performed in triplicate. thereafter. Error bars on graphs show the standard deviation. The data were analyzed by analysis of variance Enzyme assay (ANOVA). α-Amylase production was studied on modified medium of MRS containing 20 g/l of potato starch Results source. 1% bacterial isolate was inoculated in respective modified MRS medium at pH 7.0 for Characterization & Composition of Potato starch waste 24 hrs. Aliquots were withdrawn periodically and The potato wastewater, with characteristics as centrifuged at 8000 rpm for 5 min and supernatant shown in Table 1, used in this investigation was was analyzed for enzyme activity. The extracellular collected from Potato Chips Industry, Patiala, Punjab, Amylase activity was assayed by measurement of the India, The maximum
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