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cider wine preparation using different yeast cultures


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cider wine preparation using honey and different yeast cultures (Saccharomyces cerevisiae and Saccharomyces uvarum), testing their efficiency based on alcohol production, in single and mixed cultures, …

cider wine preparation using honey and different yeast cultures (Saccharomyces cerevisiae and Saccharomyces uvarum), testing their efficiency based on alcohol production, in single and mixed cultures, fortification by honey to increase vit b content as well help in initial calrification, and testing antimicrobial character of cider types on E.coli

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  • 2. Project Details
    • Author’s name: Ritwik Bhattacharya
    • Department: Microbial and Food Technology
    • Class: M.Sc. (IV semester)
    • College: Dolphin (PG) college of life sciences (Affiliated to Punjabi University, Patiala)
    • Project guide: Dr. Geeta Mehra
    • Co-Guide: Mrs. Harbinder Kaur
  • 3. Abstract
    • The project discusses preparation of honey incorporated hard cider using pure and mixed cultures of Sacharomyces cerevsiae & Sacharomyces uvarum (NDRI, Karnal) , and testing their ethanol production efficiency. Physicochemical tests showed lower acidity (0.188 % malic acid) in mixed culture cider ( Sacharomyces cerevsiae + Sacharomyces uvarum ), lower total soluble solids (12 o Brix), but higher ethanol content (5.45%), than pure culture cider (SC-3.44%,SU-2.90%), using same cultures independently and that of pure apple juice. Sensory evaluation also confirmed that mixed culture cider had higher consumer acceptability (8.31 on overall acceptability) . The cider verities prepared were also tested for antimicrobial action on E . coli . The test gave clear indications on higher microbicidal effect of mixed culture cider, than pure culture cider based on diameter zone of inhibition on E . coli lawn {SC+SU(2.67cm)>SC(1cm)>SU(0.73cm)}. As an extension of the project , isolation of indigenous microflora responsible for natural fermentation in apple juice was done. Isolated colonies under microscope gave two types of colonies (white & fluorescent orange), with same morphology (gummy, oval, chain-like arrangement) . The isolated colonies thus showed yeast like characteristic .
    • Cider is the sweet juice of apples. It is typically a clear, golden drink, which can range in color from a pale yellow to a dark amber rose. It has a fruity flavor and a varying degree of taste from very sweet to tart. Sweet cider is the non-alcoholic versions of cider. Hard cider is the product that results when the juice is allowed to undergo fermentation.
    • Cider can be made from almost any type of apple. Apple varieties should be centered on the high-tannin 'bittersweet' and 'bittersharp' varieties (if low in tannin, these are correspondingly described as 'sweets' or 'sharps').Apples should have high percentage of tannin and have a great body.
    • Tannin is exemplified by the mouth-puckering taste of strong tea, or by the taste of a sloe - it can be both bitter and/or astringent ('hard' or 'soft'), depending on its chemical structure and molecular size.
  • 5.
    • In cider making, both tannin and acidity are needed in moderate amounts, as will appear later. The other major component was sugar to ferment into alcohol. Tannins (more than 0.05 percent) are responsible for the astringency, while pectins (0.25 to 0.75 %) are mostly responsible for the body or viscosity of cider.
    • Apples contain two primary enzymes: polyphenol oxidase and peroxidase. Apple juice cannot be sterilised by heating since the pectin esterase enzymes in the juice are destroyed by heat, thus the resulting cider will not be clear. Addition of sulphur dioxide is the most common way of controlling unwanted organisms.
    • The amount of sulphur dioxide needed depends on the pH of the juice. Between pH 3.0 to 3.3, 75 ppm is needed, between pH 3.3 and 3.5, 100 ppm is necessary and 150 ppm between 3.5 and 3.8 (Valentas et al., 1991).
    • Various indigenous yeast species in apple are responsible for the initial fermentation for enhanced flavour. Ripe apples have less than 500 yeast-like organisms per gram of sound fruit.
    • Main organisms are Aureobasidium pullulans, Rhodotorula spp., Torulopsis, Candida, Metschnikowia, and Kloeckera apiculata
    • The malo-lactic fermentation is carried out by non-slime forming strains of Leuconostoc mesenteroides , Lactobacillus collinoides and very rarely Pediococcus cerevisiae .
    • Nitrogenous compounds released at the end of the yeast fermentation are: Pantothenic acid, riboflavin along with some phosphorus compounds (necessary for the malo-lactic fermentation ) ( Beech et al. , 1972 ).
  • 7. Health benefits of apple juice & cider wine
    • Apple juice has a significant concentration of phenolics thought to help protect from many diseases associated with aging, including heart disease and cancer. Aside from other obvious fruit vitamins like Vit C, apple juice also contains the mineral nutrient boron, which is thought to promote healthy bones.
    • Research suggests that drinking cider may be good for health. Cider is rich in antioxidants known as polyphenols which reduce chances of lung cancer. However, it can be very acidic and contain high sugar levels, which results in decay of tooth enamel . ( Macrae et al. , 1993; Bizeau et al., 1997 & Beech et al., 1993) .
    • Mother’s eating apples at pregnancy protect/develop child’s immunity to asthma in later life
  • 8.
    • The epidemiological studies of French population showed lower rate of cardiovascular diseases and death compared to those with normal diet without wine.
    • Antimicrobial activity-low concentration of ethanol (4-8%) inhibits the growth of mould, yeast and many bacteria.
    • Glucose tolerance factor chromium containing compound is synthesized by yeast and related into fermenting medium. It is useful in curing diabetes.
    • The presence of excessive ethyl alcohol in the diet might lead to insulin resisitance and glucose intolerance.
    • Both red and white wine enhance iron absorption from foods due to simple phenolics of wine.
    • Wine antioxidants help to prevent, though indirectly diabetes and associated visual loss.
    • Antioxidants protect against liver damage (
    • Production of honey based cider using lab scale fermenter using pure cultures ( Saccharomyces cerevisiae & Saccharomyces uvarum ) and their mixed form.
    • Testing efficiency of different pure & mixed cultures potency in production of alcohol (ethanol content).
    • Isolation of indigenous microflora from apple juice.
    • Observation of antimicrobial effect of cider on Escherichia coli.
    • 1. Raw material : (a)Apples Apples were procured from Reliance fresh stores, Mohali.
    • (b) Honey -Dabur honey was obtained from the local market.
    • 2. Pure cultures : Saccharomyces cerevisiae & Saccharomyces uvarum were procured from NDRI, Karnal
    • 3. Nutrients : Ammonium sulfate/ Thiamine (From College Laboratory).
    • 5. Culture medi ums : Potato dextrose agar/ broth, malt extract broth, nutrient agar (From College Laboratory).
    • 6. Reagents : Potassium Meta Bisulfite (KMS), gelatin, tannin, bentonite powder, agar-agar (From College Laboratory).
    • 7. Dye : 1% Lactophenol cotton blue (From College Laboratory).
    • 8. Instruments : Lab scale fermenter (5 litres), distillation apparatus, baume’s refractometer, laminar air flow chamber, autoclave, sterile glasswares & general lab apparatus.
    • Selection of apples : Apples were tested for juice content. Three principal varieties were selected: Kinnaur varieties, Shimla varieties & Kashmir varities.
    • Surface sterilization : Apples skin surface were sterilized using 0.5% H2O2, to destroy the exogenous contaminating microorganisms.
    • Juice Extraction: using juicer.
    • Clarification: (a) using honey: According to Kime et al. , 1982 , addition of honey (15%) at 75 o C was done. The juice was left for 24 hrs, for sedimentation of undissolved matter.
  • 13.
    • (b) using gelatin- tannin :
    • stock solutions: (i) Tannin: 9.5gm of tannin was dissolved in 1ltr water.
    • (ii) Gelatin: 21.25gm of gelatin was dissolved into 600-800ml cold water to form gel like mass. Solution was heated to boil.
    • Procedure : For every litre of juice: 10.6 ml of tannin + 17.63 ml gelatin was added. The sample was left for clarification under room temperature for 24 hours to obtain clarified juice.
    • Sulphiting: Potasium Meta bisulphite (KMS) is added to juice which is then broken down to produce sulfur dioxide, which in turn kills bacteria but not the yeasts (indigenous).
  • 14. SO 2 addition required in apple juice according to pH 150 3.5 - 3.8 100 3.3 - 3.5 75 3.0 - 3.3 Addition required (mg/l) pH
  • 15.
    • Fortification :According to Lea et al. , (1995) & Amerine et al ., (1972) , Ammonium sulfate was added as a yeast nutrient for providing additional nitrogen in growth medium, at a concentration of 250 ppm.
    • Fermentation:
    Culture revival Culture propagation Staining Culture inoculation
  • 16.
    • Filtration: using filter paper.
    • Pasteurization: Filtered wine was pasteurized at 65-68 o C for 30 seconds to prevent further fermentation by yeasts (Chavan et al., 2008 ).
    • Final clarification: (a) using Bentonite-Agar suspension: Final clarification was done using bentonite: agar-agar mixture (1:1). To dissolve 1.8 gm agar-agar, 100 ml distilled water is needed, therefore 1.8 gm of bentonite was taken along with same amount of agar-agar, and dissolved in 100 ml distilled water to prepare a suspension. The suspension was added to the cider at 0.015% to obtain clear cider (Ranganna, 1986 ).
    • (b) using honey: 2.5% honey and distilled water (1:1) mixture was added to cider to clarify remaining clouding and hazes from cider. The suspension was kept for 24 hours, sediment observed, was then filtered using sterile filter paper (Kime et al. , 1982) .
  • 17.  
  • 18.
    • Pure culture isolation:
    Fermentation by indigenous microflora Spreading Streaking Staining Preservation of culture in glycerol broth
  • 19.
    • Microbiological analysis:(APHA, 1992)
    • Total plate count
    • Yeast and mold
    • Physico-chemical analysis of cider samples :
    • TSS (b) pH (c) Titrable Acidity
    • (d) Sugars (reducing & total sugar percentage by Lane and Eyon volumetric method, AOAC , 1984 )
    • Antimicrobial effect of cider on Escherichia coli
    Preparation of nutrient agar plates Inoculation/ spreading Measurement of zone of inhibition
  • 20.
    • Ethanol content estimation: ( Caputi et al., 1968)
    Preparation of Standard stock ethanol solution Preparation of potassium dichromate solution Preparation of standard curve Sample of alcoholic products/cider
    • Sensory analysis: by 9 different
    • panel members based on the
    • following criteria
    1 Dislike extremely 2 Dislike very much 3 Dislike moderately 4 Dislike slightly 5 Neither like nor dislike 6 Like slightly 7 Like moderately 8 Like very much 9 Like extremely Scores Quality
    • Selection of apples :
    • Among the three varities of apple available, selection was done on basis of maximum pulp less juice production. It was observed that, shimla apples gave highest percentage of juice (around 44%)
    4.5 ml 4.4 ml 4.2 ml small Shimla 3 3.5 ml 3.3 ml 3.2 ml large Kinnaur 2 2.4 ml 2.3 ml 2.4 ml large Kashmir 1 Observation 3 Observation 2 Observation 1 Juice content (per 10 ml) Size Type S. no
  • 22.  
  • 23.
    • Physico-chemical tests of apple juice:
    10.5263 Total Invert sugar (%) 5.7142 Reducing sugar (%) 0.799 %( as malic acid) Acidity (%) 3.5 pH 22 o Brix TSS (°Brix) value Parameters
  • 24.
    • Isolation of indigenous micro-flora from apple juice:
    • Unpasteurized apple juice showed presence of oval chain, gummy colonies, which were typically yeast like, and also observed to be responsible for fermentation. Two principal types of colonies with similar texture, microscopic morphology but different colour were observed, and specifically isolated.
    gummy Oval – spherical, in chains Fluroscent orange 2. gummy Oval, in chains White 1. Texture Microscopic morphology Colony colour S. No.
  • 25. Plate 3: White, gummy Plate 4: Fluoroscent orange, gummy
  • 26.
    • Analysis of cider samples:
    2.67 1 0.73 ANTIMICROBIAL EFFECT (diameter, zone of inhibition E . coli , cm) 8. 5.45 3.44 2.90  ETHANOL CONTENT % 7. 15 X 10 3 c.f.u./ml  7 X 10 4 c.f.u./ml  9 X 10 3 c.f.u./ml 3 X 10 4 c.f.u./ml 3 X 10 3 c.f.u./ml  10 X 10 4 c.f.u./ml  YEAST & MOULD (10 -2 dilution) (10 -3 dilution) 6. 2 X 10 3 c.f.u/ml  5 X 10 4 c.f.u./ml  8 X 10 3 c.f.u./ml 6 X 10 4 c.f.u./ml 1 X 10 3 c.f.u. /ml  0c.f.u/ml T.P.C (10 -2 dilution) (10 -3 dilution) 5. 0.188  0.1038 0.1316  ACIDITY (%) 4. 3.2 3.4 3.3 pH 3. 11.62 12.8 12.34 TOTAL SUGAR (%) 2. 13 16 14 TSS ( o Brix) 1. S. cerevsiae + S. uvarum Sacharomyces cerevsiae Sacharomyces uvarum CHARACTERESTICS Sl.No
  • 27.  
  • 28. Honey Hard Cider Samples (SU, A & SC) Sample code (Abbreviations) : SC- Sacharomyces cerevsiae (pure culture) SU- Sacharomyces uvarum (pure culture) A- Sacharomyces cerevsiae + Sacharomyces uvarum (mixed culture: 1:1)
  • 29.
    • Alcohol content of different samples:
    • The standard curve was plotted using the optical density determined using a spectrophotometer of various ethanol concentrations (0.5%-5.5%), standardized before using an appropriate blank. Later on the OD of cider wine samples (SC, SU & A), were also measured similarly, and extrapolated on the graph drawn above, to find the corresponding ethanol concentration (%) by drawing a perpendicular to X-axis.
    OD standard curve 0.642 5.5 11 0.578 5.0 10 0.510 4.5 9 0.484 4.0 8 0.422 3.5 7 0.372 3.0 6 0.310 2.5 5 0.258 2.0 4 0.208 1.5 3 0.152 1.0 2 0.064 0.5 1 Optical density (600nm) Sample ethanol conc. (%) S. No.
  • 30.  
  • 31. OD of cider samples 5.45 0.636 A 3 2.9 0.360 SU 2 3.44 0.415 SC 1 Ethanol conc(%) Optical density (600nm) Sample name S. No.
  • 32.
    • Antimicrobial effect of cider on E . coli:
    • The zone of inhibition of the E . coli lawn on the nutrient agar petriplates was measured using a centimeter scale. Values obtained, demonstrate the higher microbicidal activity of cider, due to the production of alcohol during fermentation, as well as the additional antimicrobial properties contributed by the honey incorporated in the cider, as a sugar source.SU was shown to have the lowest average diameter zone of inhibition, followed by SC and finally the mixed culture cider (A), has the highest diameter. This is because, the combined effect of pure cultures ( Sacharomyces cerevsiae & Sacharomyces uvarum) , produced higher ethanol content.
  • 33. 2.67 2.7 2.7 2.6 A 3 0.73 0.8 0.7 0.7 SU 2 1 1.1 0.9 1.0 SC 1 3 rd 2 nd 1 st Average (cm) Diameter of Zone of inhibition (cm) Sample name/code Sl.No.
  • 34. Zone of inhibition by cider samples (SC, SU & A) on E.coli inoculated nutrient agar petriplates Plate 5: Zone of inhibition by SC & SU Plate 6: Zone of Inhibition by SC & A
    • According to the various microbiological, physico-chemical, sensory and biochemical tests performed, as a part of this project, it was observed that, hard honey ciders prepared by using mixed cultures, were more superior (ethanol content, sensory evaluation , antimicrobial effect on E . coli ), than ciders prepared using pure cultures ( Sacharomyces cerevsiae & Sacharomyces uvarum ).
    • Cider prepared by mixed culture had 13°Brix TSS, 11.62% total sugars, 3.2 pH, 0.188% acidity and 5.54% ethanol content. It was also found that mixed culture cider had a higher antibacterial effect on E.coli (diameter of zone of inhibition is 2.67 cm) than pure culture ciders, which can be regarded as an indication to higher alcohol production as well as it may be hypothesized that lactic acid is produced in a higher proportion by malo-lactic fermentation in the mentioned cider sample.
    • Amerine, M.A., Berg, H.W.,Kunkee, R.E., Ough, C.S., Singleton, W.L. & Webb,A.D.(1980). The technology of wine making (4th edn). AVI Pub Comp., Westport Connecticut.USA.p.p.523-547.
    • Beech, F.W., (1972a). English cidermaking-Technology, microbiology & biochemistry. In Progress in Industrial Microbiology. Pp. 133-213. Edited by D.J.D. Rockenhall, London, Churchill Livingstone
    • Beech, F.W., (1993). Yeasts in cider making. In The Yeasts, 2nd edn. Vol 5, Yeast Technology. pp. 169-213. Edited by A.H. Rose & J. S. Harrison. London: Academic press
    • Carr, J.G. (1987). Microbiology of wines & cider. In Essays in agricultural & Food Microbiology.
    • Chavan, U.D.(2008). Fruit based fermented beverages. In Beverages and Food World.
    • Choi, L.H. & Nielsen, S. (2005). The efefects of thermal & non thermal processing methods on apple cider quality & consumer acceptability. In J Food Quality 28(1).p.p. 13-29.
    • Deal, J.(1976). Making cider. In Amateur Wine Makers Pub.
    • Dold et al.(1937). Antibacterial properties of honey-role oh inhibine.
    • Grafton, G.
  • 37.
    • Karwanna & Pradit et al.(1976). Wine fermentation with mixed yeast cultures. In Dept of Viticulture & Enology, UCD.
    • Kime, R.W. & Romulus, N.Y.(1982). Clarification of fruit juice with honey. Assignee : Cornell Research Foundation, Ithaca, N.Y. U.S. Patent: 4327115.
    • Konowalchuk, J. & Speirs, J.I.(1978). Antiviral effects of apple beverages. In Appl Environ Microbiol 36(6).p.p. 798-801.
    • Lea, A.G.H (2007). The Craft Cider Revival- some technical considerations. In SWEA.
    • Lea, A.G.H. & Piggot J. (1995). Fermented Beverage Production (2nd edn). In .
    • Morrissey, W.F., Davenport, B., Querol, A. & Dobson, A.D.W. (2004). The role of indigenous yeasts in traditional irish cider fermentations. In J. Appl Microbial 97. pp. 647-655.
    • National Honey Board. In
    • NACM (1998). Code of practice for the production of cider & perry. National Association of Cidermakers. London WC2.
    • Predominance of Saccharomyces uvarum during spontaneous alcoholic fermentation for three consecutive years in an Alsatian winery.(2004). In J Appl Microbiol.
    • Pooley, M. & Lomax, J.(1999). Real cider making-on small scale. In Nexus Special Interests, Kent.
  • 38.
    • Roozen,J.P. & Buren,J.P.V(1979). Sensory analysis of bitterness in apple wine. In Inst J Food Sci Tech 14(3).p.p. 315-320.
    • Uljas, H. E., Schaffner, D.W., Duff, S., Zhao, L.& Ingham, S.C. (2001). Modeling of combined processing steps for reducing E.coli O157:H7 population in apple cider. In Appl Environ Microbiol 67(1).p.p 133-141.
    • Valles, B. S., Bedrinana, R.P., Tascon, N.F., Simon, A.Q. & Madrera, R.R.( 2007). Yeast species associated with the spontaneous fermentation of cider. In Food Microbiol 24(1). p.p. 25-31.
  • 39.
    • I would like to extend my word of thanks to my guide, faculty of my department as well as Dr.Dadhich & Mr. Saurav Gupta (Dept of Microbiology).
    • I am very greatful to my parents who provided me with immense moral support throughout the project tenure.
    • I would like to thank my classmates for their great cooperation.
    • I would like thank my sisters for providing me insights during frustrating pahses of this journey.
  • 40. CHEERS TO ALL!!