Available online at www.ijpab.com Kaushal and Phutela Int. J. Pure App. Biosci. 5 (5): 264-271 (2017) ISSN: 2320 – 7051 DOI: http://dx.doi.org/10.18782/2320-7051.5773 ISSN: 2320 – 7051 Int. J. Pure App. Biosci. 5 (5): 264-271 (2017) Research Article Vinegar Production from Barley Malt using Immobilized Gluconobacter oxydans Naveet Kaushal1* and R. P. Phutela2 1Research Scholar, 2Senoir Microbiologist, Department of Microbiology, Punjab Agricultural University, Ludhiana *Corresponding Author E-mail: [email protected] Received: 23.09.2017 | Revised: 4.10.2017 | Accepted: 5.10.2017 ABSTRACT Cell immobilization comprises the retention of metabolically active cells inside a polymeric matrix. In this study, the production of barley malt vinegar using immobilized Gluconobacter oxydans cells has proposed as a new method for commercial houses. Barley malt wort was converted to ethanol by Saccharomyces cerevisiae strain 35 producing 5.2% (v/v) ethanol. This ethanol was used for vinegar production using encapsulated (calcium alginate and agar) cells of Gluconobacter oxydans NBRC 3432. The optimized conditions for malt vinegar production were designed by response surface methodology. Both of encapsulation materials were statistically similar for acetic acid production and produced total acidity upto 4.66% (w/v). By semi continuous fermentation, the time of acetic acid fermentation was reduced to 14 days compared to 28 days of batch fermentation. In terms of economy of time, calcium alginate column was proved to be the best fermentation column when compared to agar bead column with the fermentation efficiency of 85.5%. Key words: Barley malt, semi continuous, acetous fermentation, immobilization. INTRODUCTION sugar is approximately 40%, with the Vinegar is defined as “a liquid produced from remaining sugar metabolites either lost to raw material of agricultural origin, containing volatilization or converted into other starch and sugars by the process of double compounds19. Natural vinegar is a quality food fermentation, alcoholic and acetous and additive as it abounds in most of the essential contains a specified amount of acetic acid”. amino acids contributed from its raw materials Vinegar, a traditional acidic condiment, is as well as fermenting micro-organisms29. It is widely produced from rice, malt, apples, wine reported to possess ample medicinal values to and various other agricultural material5. cure aches and gastric troubles 42. Moreover, Vinegar production ranges from traditional people are unaware of the good properties of methods employing wood casks and surface natural vinegar and have inhibition including it culture to submerged fermentation in in their daily diet. acetator13. Acetic acid yield from fermented Cite this article: Kaushal, N. and Phutela, R.P., Vinegar Production from Barley Malt using immobilized Gluconobacter oxydans, Int. J. Pure App. Biosci. 5(5): 264-271 (2017). doi: http://dx.doi.org/10.18782/2320-7051.5773 Copyright © Sept.-Oct., 2017; IJPAB 264 Kaushal and Phutela Int. J. Pure App. Biosci. 5 (5): 264-271 (2017) ISSN: 2320 – 7051 Those who use vinegar, fall prey to using MATERIAL AND METHODS synthetic vinegar due to its low cost but high The yeast Saccharomyces cerevisiae strain 35, risk of harmful toxic components in it. Starter an isolate from brewery waste and the bacteria cultures are commonly used in the food Gluconobacter oxydans NBRC 3432 isolated fermentation industry to predict and safeguard from rotten grapes were used for the dual the product quality 30.These microbial starters alcoholic and acetic acid fermentation.Barley have a significant role in the fermentation malt was saccharified and wort was prepared process 2.Yeast inoculation has been used using the conditions standardized by response extensively in the food industry to obtain a surface methodology . Allowing dextrans product with a predictable quality, including in allowed to settle, the clarified wort was placed the production of beverages such as wine in 5 litre flasks each with 3 litre at 10.5°B and 14,11,18. In contrast, the use of acetic acid inoculated with 16–20 h grown culture of the bacteria (AAB) as inoculum in the vinegar yeast with viable cell population of 106 cfu industry has traditionally been limited to the ml–1. Samples were drawn every 12 h and °B use of the mother of vinegar or back slopping. determined until the fermentation stopped as In such cases, the produced vinegar is a result indicated by cessation of CO2 evolution. of competition between microorganisms Ethanol was determined by chemical oxidation present in the base wine, particularly, wild method6. The fermented wort, say malt wine AAB. The taxonomy of AAB has changed was allowed to settle for 2 days at 4°C and the significantly in the last years. Currently, apart supernatant was siphoned off. The ethanol from Acetobacter and Gluconobacter17 , other content of the wine was estimated to be 4.8 % genera are classified as AAB. Different (v/v). It was inoculated with the entrapped vinegars show different AAB profiles; cells of G.oxydans . For entrapment, a cell nevertheless, species of the genera paste (36 h grown cells of bacterial culture Acetobacter38,16,Gluconobacter34,37 and with cell count 3 x 10 6 cells /ml) was used. Gluconacetobacter7,13. Acetobacter aceti is a Bead Preparation bacterium frequently used in the vinegar a) Ca –alginate beads industry36 because it immediately starts the A 4% (w/v) sodium alginate solution were fermentation process; however, when acetic mixed and extruded through a syringe into 0.2 acid concentrations exceed 5 %, other bacterial M CaCl2 (anhydrous) solution to form the species take over the process22. On the other calcium alginate beads. The mixture was hand, Gluconobacter gives a distinctive allowed to rest for 24 h and the beads were flavour to vinegar and can oxidize ethanol to harvested by filteration acetic acid under acidic conditions34.Cell b) Agar beads immobilization can be defined as the physical A 2% solution of gum was prepared, sterilized confinement of intact cells to a defined space and cooled to 45°C and bacterial paste was to preserve the metabolic or catalytic activity. added to it. The molten preparation was Immobilization mimics the enclosure or cell dropped into ice-cold vegetable oil to produce aggregation that normally occurs when an emulsion that was cooled to 5°C with gentle microorganisms grow in natural environments, stirring. The mixture was allowed to rest for with the benefit of compartmentalizing the 24 h and the beads were harvested by immobilized cell4. The present study was filtration. The excess of oil was removed by undertaken to develop an efficient process five washings with distilled water. using free cells of Saccharomyces cerevisiae Viable cells: The viable cells (cfu/ml) for the for alcoholic fermentation and immobilized total number of bacterial cell inside the beads cells of Gluconobacter oxydans for acetic acid were counted at time zero (when the malt wine fermentation using two inert materials for was inoculated) and every 24 h until the end of vinegar production from barley malt. vinegar production . Ten beads (approx. 0.5 ml Copyright © Sept.-Oct., 2017; IJPAB 265 Kaushal and Phutela Int. J. Pure App. Biosci. 5 (5): 264-271 (2017) ISSN: 2320 – 7051 of carrier beads) were placed on a glass filter and 30 min and the recovered volume of the to drain the solution. The beads were then wort was 2.7 l representing 90% recovery. The transferred to a 5ml burette containing 3.5 ml total and the reducing sugars in the wort were of sterile distilled water. The liquid height was 9.8 and 8.8 % respectively. The alcohol recorded to determine the increase in volume content of the malt wine at the end of due to the beads. The beads were then crushed fermentation was 5.2% (v/v) with the initial in sterilized water using a glass stick to total acidity of 0.48 % (w/v (Table 1) recover the immobilized cells. The total The bacteria laiden beads of sodium number of Gluconobacter oxydans cells alginate and agar using 400 ml malt wine (cfu/ml) was determined by inoculating this supplemented with 5% mother vinegar, served cell suspension on GYC agar containing (in as acetous fermentation medium .Whereas %): yeast extract 1, d-glucose 10, calcium control run used 5% (v/v) inoculum of 36 h carbonate 2 and agar 2 (Merck) for 24–48 h at old free cell culture of Gluconobacter oxydans 30 °C. Chromatographic analyses: Volatile in submerged fermentation . The immobilized compounds in the vinegar were analyzed cells used 250 beads packed in a vertical directly, according to using a gas column (glass burrette with 3cm diameter) chromatography system (GC-17A; Shimadzu) through which malt wine was allowed to with a flame ionization detector (GC–FID) percolate at 5ml/min to constitute a flow cycle fitted with DB Wax silica capillary column of 40 min. A temperature of 30 °C was (30 m×0.25 μm×0.25 mm i.d.; J&W Scientific, maintained uniformly for optimum acetous Folsom, CA, USA) 39. fermentation. The acidity (both total and Scanning microscopy volatile ) levels were estimated every alternate Bacterial cell pellet (from one ml of the culture day and this practice continued by the course broth) was processed for scanning electron of fermentation until the total acidity reached microscopy (SEM) for the record of shape, 4.4% which was supposed to match 4.0% size and arrangement of cells. Conventional volatile acidity in general1. method was followed for the sample The fermentation efficiency of acetic preparation 32. acid production was calculated as: Statistical analysis Volatile acidity % (w/v)/ {alcohol (%, All the experiments of alcoholic and acetic acid v/v)x1.304}x100 fermentation were carried out in quadruplicate. Alcohol (%,v/v) = Actual ethanol The results of quadruplicate were filled in (%,v/v) x 0.8 random block design of G Stat software8.