PREPARATION & PHYSICO-CHEMICAL ANALYSIS OF HONEY HARD CIDER

USING PURE & MIXED CULTURES(SACCHAROMYCES CEREVSIAE &

SACCHAROMYCES UVARUM)& ISOLATION OF INDIGENOUS MICROFLORA

FROM APPLE JUICE PRESENTED TO THE

FACULTY OF LIFE SCIENCESPUNJABI UNIVERSITY PATIALA

Project DetailsProject Details

Author’s name:Author’s name: Ritwik Bhattacharya Ritwik Bhattacharya

Department:Department: Microbial and Food Microbial and Food Technology Technology

Class:Class: M.Sc. M.Sc. (IV (IV semester) semester)

College:College: Dolphin (PG) Dolphin (PG) college of life sciences college of life sciences (Affiliated to Punjabi (Affiliated to Punjabi University, Patiala) University, Patiala)

Project guide:Project guide: Dr. Dr. Geeta Mehra Geeta Mehra

Co-Guide:Co-Guide: Mrs. Mrs. Harbinder Kaur Harbinder Kaur

AbstractAbstract The project discusses preparation of honey incorporated hard The project discusses preparation of honey incorporated hard

cider using pure and mixed cultures ofcider using pure and mixed cultures of Sacharomyces Sacharomyces cerevsiaecerevsiae & & Sacharomyces uvarumSacharomyces uvarum (NDRI, Karnal)(NDRI, Karnal), , and testing and testing their ethanol production efficiency. Physicochemical teststheir ethanol production efficiency. Physicochemical tests showed lower acidity (0.188 % malic acid) in mixed culture showed lower acidity (0.188 % malic acid) in mixed culture cider (cider (Sacharomyces cerevsiaeSacharomyces cerevsiae + + Sacharomyces uvarumSacharomyces uvarum), ), lower total soluble solids (12lower total soluble solids (12ooBrix), but higher ethanol content Brix), but higher ethanol content (5.45%), than pure culture cider (SC-3.44%,SU-2.90%), using (5.45%), than pure culture cider (SC-3.44%,SU-2.90%), using same cultures independently and that of pure apple juice. same cultures independently and that of pure apple juice. Sensory evaluationSensory evaluation also confirmed that mixed culture cider had also confirmed that mixed culture cider had higher consumer acceptability (8.31 on overall acceptability)higher consumer acceptability (8.31 on overall acceptability). . The cider verities prepared were also testedThe cider verities prepared were also tested for antimicrobial for antimicrobial action onaction on EE..colicoli. The test gave clear indications on higher . The test gave clear indications on higher microbicidal effect of mixed culture cider, than pure culture microbicidal effect of mixed culture cider, than pure culture cider based on diameter zone of inhibition on cider based on diameter zone of inhibition on EE..colicoli lawn lawn {SC+SU(2.67cm)>SC(1cm)>SU(0.73cm)}. As an extension of {SC+SU(2.67cm)>SC(1cm)>SU(0.73cm)}. As an extension of the projectthe project, , isolation of indigenous microflora responsible for isolation of indigenous microflora responsible for natural fermentation in apple juice was done. Isolated colonies natural fermentation in apple juice was done. Isolated colonies under microscope gave two types of colonies (white & under microscope gave two types of colonies (white & fluorescent orange), with same morphology (gummy, oval, fluorescent orange), with same morphology (gummy, oval, chain-like arrangement)chain-like arrangement).. The isolated colonies thus showed The isolated colonies thus showed yeast like characteristicyeast like characteristic..

INTRODUCTIONINTRODUCTION Cider is the sweet juice of apples. It is typically a Cider is the sweet juice of apples. It is typically a

clear, golden drink, which can range in color from a clear, golden drink, which can range in color from a pale yellow to a dark amber rose. It has a fruity flavor pale yellow to a dark amber rose. It has a fruity flavor and a varying degree of taste from very sweet to tart. and a varying degree of taste from very sweet to tart. Sweet cider is the non-alcoholic versions of cider. Sweet cider is the non-alcoholic versions of cider. Hard cider is the product that results when the juice Hard cider is the product that results when the juice is allowed to undergo fermentation. is allowed to undergo fermentation.

Cider can be made from almost any type of apple. Cider can be made from almost any type of apple. Apple varieties should be centered on the high-tannin Apple varieties should be centered on the high-tannin 'bittersweet' and 'bittersharp' varieties (if low in 'bittersweet' and 'bittersharp' varieties (if low in tannin, these are correspondingly described as tannin, these are correspondingly described as 'sweets' or 'sharps').Apples should have high 'sweets' or 'sharps').Apples should have high percentage of tannin and have a great body.percentage of tannin and have a great body.

Tannin is exemplified by the mouth-puckering taste Tannin is exemplified by the mouth-puckering taste of strong tea, or by the taste of a sloe - it can be of strong tea, or by the taste of a sloe - it can be both bitter and/or astringent ('hard' or 'soft'), both bitter and/or astringent ('hard' or 'soft'), depending on its chemical structure and molecular depending on its chemical structure and molecular size.size.

In cider making, both tannin and acidity are In cider making, both tannin and acidity are needed in moderate amounts, as will appear needed in moderate amounts, as will appear later. The other major component was sugar to later. The other major component was sugar to ferment into alcohol. ferment into alcohol. Tannins (more than 0.05 Tannins (more than 0.05 percent) are responsible for the astringency, percent) are responsible for the astringency, while pectins (0.25 to 0.75 %) are mostly while pectins (0.25 to 0.75 %) are mostly responsible for the body or viscosity of cider.responsible for the body or viscosity of cider.

Apples contain two primary enzymes: polyphenol Apples contain two primary enzymes: polyphenol oxidase and peroxidase. oxidase and peroxidase. Apple juice cannot be Apple juice cannot be sterilised by heating since the pectin esterase sterilised by heating since the pectin esterase enzymes in the juice are destroyed by heat, thus enzymes in the juice are destroyed by heat, thus the resulting cider will not be clear. Addition of the resulting cider will not be clear. Addition of sulphur dioxide is the most common way of sulphur dioxide is the most common way of controlling unwanted organisms. controlling unwanted organisms.

The amount of sulphur dioxide needed depends The amount of sulphur dioxide needed depends on the pH of the juice. Between pH 3.0 to 3.3, 75 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 ppm is needed, between pH 3.3 and 3.5, 100 ppm is necessary and 150 ppm between 3.5 and 3.8is necessary and 150 ppm between 3.5 and 3.8 (Valentas (Valentas et al.,et al., 1991). 1991).

INDIGENOUS INDIGENOUS MICROORGANISMSMICROORGANISMS

Various indigenous yeast species in apple are Various indigenous yeast species in apple are responsible for the initial fermentation for enhanced responsible for the initial fermentation for enhanced flavour. Ripe apples have less than 500 yeast-like flavour. Ripe apples have less than 500 yeast-like organisms per gram of sound fruit. organisms per gram of sound fruit.

Main organisms are Main organisms are Aureobasidium pullulans, Aureobasidium pullulans, Rhodotorula spp., Torulopsis, Candida, Rhodotorula spp., Torulopsis, Candida, Metschnikowia,Metschnikowia, and and Kloeckera apiculataKloeckera apiculata

The malo-lactic fermentation is carried out by non-The malo-lactic fermentation is carried out by non-slime forming strains of slime forming strains of Leuconostoc Leuconostoc mesenteroidesmesenteroides, , Lactobacillus collinoidesLactobacillus collinoides and very and very rarely rarely Pediococcus cerevisiaePediococcus cerevisiae. .

Nitrogenous compounds released at the end of the Nitrogenous compounds released at the end of the yeast fermentation are: Pantothenic acid, riboflavin yeast fermentation are: Pantothenic acid, riboflavin along with some phosphorus compounds (necessary along with some phosphorus compounds (necessary for the malo-lactic fermentation ) for the malo-lactic fermentation ) ((Beech Beech et al.et al., , 19721972).).

Health benefits of apple Health benefits of apple juice & cider winejuice & cider wine

Apple juice has a significant concentration of Apple juice has a significant concentration of phenolics thought to help protect from many diseases phenolics thought to help protect from many diseases associated with aging, including heart disease and associated with aging, including heart disease and cancer. Aside from other obvious fruit vitamins like cancer. Aside from other obvious fruit vitamins like Vit C, apple juice also contains the mineral nutrient Vit C, apple juice also contains the mineral nutrient boron, which is thought to promote healthy bones.boron, which is thought to promote healthy bones.

Research suggests that drinking cider may be good Research suggests that drinking cider may be good for health. Cider is rich in antioxidants known as for health. Cider is rich in antioxidants known as polyphenols which reduce chances of lung cancer. polyphenols which reduce chances of lung cancer. However, it can be very acidic and contain high sugar However, it can be very acidic and contain high sugar levels, which results in decay of tooth enamellevels, which results in decay of tooth enamel. . ((Macrae Macrae et al.et al., 1993; Bizeau et al., 1997 & , 1993; Bizeau et al., 1997 & Beech et al., 1993)Beech et al., 1993)..

Mother’s eating apples at pregnancy Mother’s eating apples at pregnancy protect/develop child’s immunity to asthma in later protect/develop child’s immunity to asthma in later lifelife

The epidemiological studies of French population The epidemiological studies of French population showed lower rate of cardiovascular diseases and showed lower rate of cardiovascular diseases and death compared to those with normal diet without death compared to those with normal diet without wine.wine.

Antimicrobial activity-low concentration of ethanol (4-Antimicrobial activity-low concentration of ethanol (4-8%) inhibits the growth of mould, yeast and many 8%) inhibits the growth of mould, yeast and many bacteria.bacteria.

Glucose tolerance factor chromium containing Glucose tolerance factor chromium containing compound is synthesized by yeast and related into compound is synthesized by yeast and related into fermenting medium. It is useful in curing diabetes.fermenting medium. It is useful in curing diabetes.

The presence of excessive ethyl alcohol in the diet The presence of excessive ethyl alcohol in the diet might lead to insulin resisitance and glucose might lead to insulin resisitance and glucose intolerance.intolerance.

Both red and white wine enhance iron absorption Both red and white wine enhance iron absorption from foods due to simple phenolics of wine.from foods due to simple phenolics of wine.

Wine antioxidants help to prevent, though indirectly Wine antioxidants help to prevent, though indirectly diabetes and associated visual loss.diabetes and associated visual loss.

Antioxidants protect against liver damage Antioxidants protect against liver damage (http://winesandbenefits.com).(http://winesandbenefits.com).

OBJECTIVESOBJECTIVES Production of honey based cider using lab Production of honey based cider using lab

scale fermenter using pure cultures scale fermenter using pure cultures ((Saccharomyces cerevisiae & Saccharomyces cerevisiae & Saccharomyces uvarumSaccharomyces uvarum) and their mixed ) and their mixed form.form.

Testing efficiency of different pure & Testing efficiency of different pure & mixed cultures potency in production of mixed cultures potency in production of alcohol (ethanol content).alcohol (ethanol content).

Isolation of indigenous microflora from Isolation of indigenous microflora from apple juice.apple juice.

Observation of antimicrobial effect of cider Observation of antimicrobial effect of cider on on EscherichiaEscherichia coli.coli.

MATERIALS & MATERIALS & INSTRUMENTSINSTRUMENTS1. 1. Raw materialRaw material: (a)Apples Apples were procured from : (a)Apples Apples were procured from

Reliance fresh stores, Mohali. Reliance fresh stores, Mohali. (b) Honey -Dabur honey was obtained from the local market.(b) Honey -Dabur honey was obtained from the local market.2. 2. Pure culturesPure cultures: : Saccharomyces cerevisiae Saccharomyces cerevisiae & & SaccharomycesSaccharomyces

uvarumuvarum were procured from NDRI, Karnal were procured from NDRI, Karnal3. 3. NutrientsNutrients: Ammonium sulfate/ Thiamine (From College : Ammonium sulfate/ Thiamine (From College

Laboratory).Laboratory).5. 5. Culture mediCulture mediumsums: Potato dextrose agar/ broth, malt extract : Potato dextrose agar/ broth, malt extract

broth, nutrient agar (From College Laboratory). broth, nutrient agar (From College Laboratory). 6. 6. ReagentsReagents: Potassium Meta Bisulfite (KMS), gelatin, tannin, : Potassium Meta Bisulfite (KMS), gelatin, tannin,

bentonite powder, agar-agar (From College Laboratory).bentonite powder, agar-agar (From College Laboratory).7. 7. DyeDye: 1% Lactophenol cotton blue (From College Laboratory).: 1% Lactophenol cotton blue (From College Laboratory).8. 8. InstrumentsInstruments: Lab scale fermenter (5 litres), distillation : Lab scale fermenter (5 litres), distillation

apparatus, baume’s refractometer, laminar air flow chamber, apparatus, baume’s refractometer, laminar air flow chamber, autoclave, sterile glasswares & general lab apparatus. autoclave, sterile glasswares & general lab apparatus.

SELECTION OF APPLES

SURFACE STERILIZATION

EXTRACTION OF JUICE

CLARIFICATION (addition of honey)

SULPHITING

INITIAL TESTS

FORTIFICATION

FERMENTATION

FILTERATION & PASTEURIZATION

PURE CULTURE ISOLATION

STAINING

STREAKING

PRESERVATION IN GLYCEROL BROTH

CLARIFICATION / CENTRIFUGING

CIDER

CHECKING ANTIMICROBIAL ACTIVITY IN E.coli. FINAL ANALYSIS

PURE CULTURE PROCUREMENT

REVIVAL OF LYOPHILIZED CULTURE

INOCULATION

FLOWCHART

METHODOLOGYMETHODOLOGY Selection of apples Selection of apples : Apples were tested : Apples were tested

for juice content. Three principal varieties for juice content. Three principal varieties were selected: Kinnaur varieties, Shimla were selected: Kinnaur varieties, Shimla varieties & Kashmir varities.varieties & Kashmir varities.

Surface sterilizationSurface sterilization: Apples skin surface : Apples skin surface were sterilized using 0.5% H2O2, to were sterilized using 0.5% H2O2, to destroy the exogenous contaminating destroy the exogenous contaminating microorganisms.microorganisms.

Juice Extraction: Juice Extraction: using juicer.using juicer. Clarification: Clarification: (a)(a) using honey:using honey: According According

to to Kime Kime et al.et al., , 19821982, addition of honey , addition of honey (15%) at 75(15%) at 75ooC was done. The juice was left C was done. The juice was left for 24 hrs, for sedimentation of undissolved for 24 hrs, for sedimentation of undissolved matter.matter.

(b) (b) using gelatin- tanninusing gelatin- tannin: :

stock solutions: (i) Tannin: 9.5gm of tannin was stock solutions: (i) Tannin: 9.5gm of tannin was dissolved in 1ltr water.dissolved in 1ltr water.

(ii) Gelatin: 21.25gm of gelatin was dissolved into 600-(ii) Gelatin: 21.25gm of gelatin was dissolved into 600-800ml cold water to form gel like mass. Solution 800ml cold water to form gel like mass. Solution was heated to boil.was heated to boil.

ProcedureProcedure: : For every litre of juice: 10.6 ml of tannin + For every litre of juice: 10.6 ml of tannin + 17.63 ml gelatin was added. The sample was left for 17.63 ml gelatin was added. The sample was left for clarification under room temperature for 24 hours clarification under room temperature for 24 hours to obtain clarified juice.to obtain clarified juice.

Sulphiting:Sulphiting: Potasium Meta bisulphite (KMS) is Potasium Meta bisulphite (KMS) is added to juice which is then broken down to added to juice which is then broken down to produce sulfur dioxide, which in turn kills bacteria produce sulfur dioxide, which in turn kills bacteria but not the yeasts (indigenous). but not the yeasts (indigenous).

SOSO22 addition required in apple juice addition required in apple juice

according to pHaccording to pH

pHpH Addition required Addition required (mg/l)(mg/l)

3.0 - 3.0 - 3.3 3.3

75 75

3.3 - 3.5 3.3 - 3.5 100 100

3.5 - 3.8 3.5 - 3.8 150 150

FortificationFortification:According to :According to Lea Lea et al.et al., (1995) & , (1995) & Amerine Amerine et alet al., (1972)., (1972), Ammonium sulfate was , Ammonium sulfate was added as a yeast nutrient for providing additional added as a yeast nutrient for providing additional nitrogen in growth medium, at a concentration of nitrogen in growth medium, at a concentration of 250 ppm.250 ppm.

Fermentation:Fermentation: Culture revival

Culture propagation

Staining

Culture inoculation

Filtration: Filtration: using filter paper.using filter paper. Pasteurization: Pasteurization: Filtered wine was pasteurized at 65-Filtered wine was pasteurized at 65-

6868ooC for 30 seconds to prevent further fermentation by C for 30 seconds to prevent further fermentation by yeasts (Chavan yeasts (Chavan et al., 2008et al., 2008).).

Final clarification: Final clarification: (a)(a) using Bentonite-Agar using Bentonite-Agar suspension: suspension: Final clarification was done using Final clarification was done using bentonite: agar-agar mixture (1:1). To dissolve 1.8 gm bentonite: agar-agar mixture (1:1). To dissolve 1.8 gm agar-agar, 100 ml distilled water is needed, therefore agar-agar, 100 ml distilled water is needed, therefore 1.8 gm of bentonite was taken along with same amount 1.8 gm of bentonite was taken along with same amount of agar-agar, and dissolved in 100 ml distilled water to of agar-agar, and dissolved in 100 ml distilled water to prepare a suspension. The suspension was added to prepare a suspension. The suspension was added to the cider at 0.015% to obtain clear cider the cider at 0.015% to obtain clear cider (Ranganna, (Ranganna, 19861986). ).

(b) (b) using honey: using honey: 2.5% honey and distilled water (1:1) 2.5% honey and distilled water (1:1) mixture was added to cider to clarify remaining mixture was added to cider to clarify remaining clouding and hazes from cider. The suspension was clouding and hazes from cider. The suspension was kept for 24 hours, sediment observed, was then filtered kept for 24 hours, sediment observed, was then filtered using sterile filter paper using sterile filter paper (Kime (Kime et al.et al., 1982), 1982)..

Pure culture isolation:Pure culture isolation: Fermentation by indigenous microflora

Spreading

Streaking

Staining

Preservation of culture in glycerol broth

Microbiological analysis:(APHA, 1992)Microbiological analysis:(APHA, 1992)

(a)(a) Total plate countTotal plate count

(b)(b) Yeast and moldYeast and mold Physico-chemical analysisPhysico-chemical analysis of cider samplesof cider samples::(a)(a) TSSTSS (b) pH (c) Titrable (b) pH (c) Titrable

AcidityAcidity

(d) Sugars (reducing & total sugar percentage by Lane (d) Sugars (reducing & total sugar percentage by Lane

and Eyon volumetric method, and Eyon volumetric method, AOAC , 1984AOAC , 1984)) Antimicrobial effectAntimicrobial effect of cider on of cider on EscherichiaEscherichia

colicoli Preparation of nutrient agar plates Inoculation/ spreading

Measurement of zone of inhibition

Ethanol content Ethanol content estimation: estimation: ((Caputi et Caputi et al., 1968)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

QualityQuality ScoresScores

Like extremelyLike extremely 99

Like very muchLike very much 88

Like moderatelyLike moderately 77

Like slightlyLike slightly 66

Neither like nor Neither like nor dislikedislike

55

Dislike slightlyDislike slightly 44

Dislike Dislike moderatelymoderately

33

Dislike very muchDislike very much 22

Dislike extremelyDislike extremely 11

RESULTS & DISCUSSIONRESULTS & DISCUSSION Selection of Selection of

applesapples::

Among the three Among the three varities of apple varities of apple available, selection available, selection was done on basis of was done on basis of maximum pulp less maximum pulp less juice production. It juice production. It was observed that, was observed that, shimla apples gave shimla apples gave highest percentage highest percentage of juice (around of juice (around

44%)44%)

S.S.nono

TypeType SizeSize Juice content (per 10 ml)Juice content (per 10 ml)

Observation Observation 11

Observation Observation 22

Observation Observation 33

11 Kashmir Kashmir largelarge 2.4 ml2.4 ml 2.3 ml2.3 ml 2.4 ml2.4 ml

22 Kinnaur Kinnaur largelarge 3.2 ml3.2 ml 3.3 ml3.3 ml 3.5 ml3.5 ml

33 Shimla Shimla smalsmalll

4.2 ml4.2 ml 4.4 ml4.4 ml 4.5 ml4.5 ml

Figure 4.1:Juice content (ml)

2.4

3.5

4.5

0

1

2

3

4

5

Kashmir Kinnaur shimla

Apple varities

Ju

ice

co

nte

nt

(ml)

Juice content (ml)

Physico-chemical tests of apple juice:Physico-chemical tests of apple juice:

ParametersParameters valuevalue

TSS (°Brix)TSS (°Brix) 22 22 oo Brix Brix

pHpH 3.53.5

Acidity (%)Acidity (%) 0.799 %( as malic acid)0.799 %( as malic acid)

Reducing sugar (%)Reducing sugar (%) 5.71425.7142

Total Invert sugar (%)Total Invert sugar (%) 10.526310.5263

Isolation of Isolation of indigenous micro-indigenous micro-flora from apple flora from apple juice:juice:

Unpasteurized apple Unpasteurized apple juice showed juice showed presence of oval presence of oval chain, gummy chain, gummy colonies, which were colonies, which were typically yeast like, typically yeast like, and also observed to and also observed to be responsible for be responsible for fermentation.fermentation. Two Two principal types of principal types of colonies with similar colonies with similar texture, microscopic texture, microscopic morphology but morphology but different colour were different colour were observed, and observed, and specifically isolated.specifically isolated.

S.S.No.No.

Colony Colony colourcolour

MicroscopicMicroscopic morphologymorphology

TextureTexture

1.1.

WhiteWhite Oval,Oval, in chainsin chains

gummygummy

2.2.

FluroscentFluroscentorangeorange

Oval –Oval –spherical, spherical, in chainsin chains

gummygummy

Plate 3: White, gummy

Plate 4: Fluoroscent orange, gummy

Analysis of cider samples:Analysis of cider samples:

Sl.NoSl.No CHARACTERESTICSCHARACTERESTICS SacharomycesSacharomyces uvarumuvarum

SacharomycesSacharomyces cerevsiaecerevsiae

S. cerevsiaeS. cerevsiae + + S. uvarumS. uvarum

1.1. TSS (TSS (oo Brix) Brix) 1414 1616 1313

2.2. TOTAL SUGAR (%)TOTAL SUGAR (%) 12.3412.34 12.812.8 11.6211.62

3.3. pHpH 3.33.3 3.43.4 3.23.2

4.4. ACIDITY (%)ACIDITY (%) 0.1316 0.1316  0.10380.1038 0.188 0.188 

5.5. T.P.CT.P.C(10(10-2-2 dilution) dilution)(10(10-3 -3 dilution)dilution)

1 X 101 X 1033c.f.u. /ml c.f.u. /ml 0c.f.u/ml0c.f.u/ml

8 X 108 X 1033c.f.u./mlc.f.u./ml6 X 106 X 1044c.f.u./mlc.f.u./ml

2 X 102 X 1033c.f.u/ml c.f.u/ml 5 X 105 X 1044c.f.u./ml c.f.u./ml 

6.6. YEAST & MOULDYEAST & MOULD(10(10-2-2 dilution) dilution)(10(10-3 -3 dilution)dilution)

3 X 103 X 1033c.f.u./ml c.f.u./ml 10 X 10 X

101044c.f.u./ml c.f.u./ml 

9 X 109 X 1033c.f.u./mlc.f.u./ml3 X 103 X 1044c.f.u./mlc.f.u./ml

15 X 1015 X 1033c.f.u./ml c.f.u./ml 7 X 107 X 1044c.f.u./ml c.f.u./ml 

7.7. ETHANOL CONTENT ETHANOL CONTENT %%

2.90 2.90  3.443.44 5.455.45

8.8. ANTIMICROBIAL ANTIMICROBIAL EFFECTEFFECT

(diameter, zone of (diameter, zone of inhibitioninhibition

EE..colicoli, cm), cm)

0.730.73 11 2.672.67

Figure 4.2:Physico-chemical properties of cider samples

12.812.34

11.62

16

14

13

0.1038 0.1316 0.188

3.4 3.3 3.2

0

2

4

6

8

10

12

14

16

18

SC SU A

Cider samples Total sugar TSSAcidity pH

Honey Hard Cider Samples (SU, A & SC)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)

Alcohol content of Alcohol content of different samples:different samples:

The standard curve was The standard curve was plotted using the optical plotted using the optical density determined using a density determined using a spectrophotometer of spectrophotometer of various ethanol various ethanol concentrations (0.5%-5.5%), concentrations (0.5%-5.5%), standardized before using standardized before using

an appropriate blank.an appropriate blank. Later Later on the OD of cider wine on the OD of cider wine samples (SC, SU & A), were samples (SC, SU & A), were also measured similarly, and also measured similarly, and extrapolated on the graph extrapolated on the graph drawn above, to find the drawn above, to find the corresponding ethanol corresponding ethanol concentration (%) by concentration (%) by drawing a perpendicular to drawing a perpendicular to X-axis.X-axis.

S. S. No.No.

Sample ethanol conc. Sample ethanol conc. (%)(%)

Optical densityOptical density(600nm)(600nm)

11 0.50.5 0.0640.064

22 1.01.0 0.1520.152

33 1.51.5 0.2080.208

44 2.02.0 0.2580.258

55 2.52.5 0.3100.310

66 3.03.0 0.3720.372

77 3.53.5 0.4220.422

88 4.04.0 0.4840.484

99 4.54.5 0.5100.510

1010 5.05.0 0.5780.578

1111 5.55.5 0.6420.642

OD standard curve

Figure 4.4:Ethanol Concentration (%)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5

Ethanol Conc (%)

Op

tical

Den

sit

y (

nm

)

OD of cider samplesOD of cider samples S. S. No.No.

Sample Sample namename

Optical Optical densitydensity

(600nm)(600nm)

EthanolEthanol conc(%)conc(%)

11 SCSC 0.4150.415 3.443.44

22 SUSU 0.3600.360 2.92.9

33 AA 0.6360.636 5.455.45

Figure 4.5:Ethanol Concentration (%) of cider samples

0.415

0.36

0.636

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

SC (3.44) SU (2.90) A (5.45) Ethanol Conc.(%)

OD

(600 n

m)

Antimicrobial effect of cider on Antimicrobial effect of cider on EE..coli:coli:

The zone of inhibition of the The zone of inhibition of the EE..colicoli lawn on the lawn on the nutrient agar petriplates was measured using a nutrient agar petriplates was measured using a centimeter scale. Values obtained, demonstrate the centimeter scale. Values obtained, demonstrate the higher microbicidal activity of cider, due to the higher microbicidal activity of cider, due to the production of alcohol during fermentation, as well production of alcohol during fermentation, as well as the additional antimicrobial properties as the additional antimicrobial properties contributed by the honey incorporated in the cider, contributed by the honey incorporated in the cider, as a sugar source.SU was shown to have the lowest as a sugar source.SU was shown to have the lowest average diameter zone of inhibition, followed by SC average diameter zone of inhibition, followed by SC and finally the mixed culture cider (A), has the and finally the mixed culture cider (A), has the highest diameter. This is because, the combined highest diameter. This is because, the combined effect of pure cultures (effect of pure cultures (SacharomycesSacharomyces cerevsiaecerevsiae & & SacharomycesSacharomyces uvarum)uvarum), produced higher ethanol , produced higher ethanol content.content.

Sl.No.Sl.No. Sample Sample name/codename/code

Diameter of Diameter of Zone of Zone of inhibition inhibition

(cm)(cm)

AverageAverage(cm) (cm)

11stst 22ndnd 33rdrd

11 SCSC 1.01.0 0.90.9 1.11.1 11

22 SUSU 0.70.7 0.70.7 0.80.8 0.730.73

33 AA 2.62.6 2.72.7 2.72.7 2.672.67

Figure 4.6: ANTIMICROBIAL EFFECT OF CIDER (E.coli)

1

0.73

2.67

0

0.5

1

1.5

2

2.5

3

SC SU A Types of cider

Dia

meter o

f z

on

e o

f in

hib

itio

n 9

cm

)

Zone of inhibition by cider samples (SC, SU & A) Zone of inhibition by cider samples (SC, SU & A) on on E.coliE.coli inoculated nutrient agar petriplates inoculated nutrient agar petriplates

Plate 5: Zone of inhibition by SC & SU

Plate 6: Zone of Inhibition by SC & A

SUMMARY & SUMMARY & CONCLUSIONCONCLUSION

According to the various microbiological, physico-According to the various microbiological, physico-chemical, sensory and biochemical tests performed, as chemical, sensory and biochemical tests performed, as a part of this project, it was observed that, hard honey a part of this project, it was observed that, hard honey ciders prepared by using mixed cultures, were more ciders prepared by using mixed cultures, were more superior (ethanol content, sensory evaluation , superior (ethanol content, sensory evaluation , antimicrobial effect on antimicrobial effect on EE..colicoli), than ciders prepared ), than ciders prepared using pure cultures (using pure cultures (SacharomycesSacharomyces cerevsiaecerevsiae & & SacharomycesSacharomyces uvarumuvarum). ).

Cider prepared by mixed culture had 13°Brix TSS, Cider prepared by mixed culture had 13°Brix TSS, 11.62% total sugars, 3.2 pH, 0.188% acidity and 5.54% 11.62% total sugars, 3.2 pH, 0.188% acidity and 5.54% ethanol content. It was also found that mixed culture ethanol content. It was also found that mixed culture cider had a higher antibacterial effect on E.coli cider had a higher antibacterial effect on E.coli (diameter of zone of inhibition is 2.67 cm) than pure (diameter of zone of inhibition is 2.67 cm) than pure culture ciders, which can be regarded as an indication culture ciders, which can be regarded as an indication to higher alcohol production as well as it may be to higher alcohol production as well as it may be hypothesized that lactic acid is produced in a higher hypothesized that lactic acid is produced in a higher proportion by malo-lactic fermentation in the proportion by malo-lactic fermentation in the mentioned cider sample.mentioned cider sample.

REFERENCESREFERENCES Amerine, M.A., Berg, H.W.,Kunkee, R.E., Ough, C.S., Amerine, M.A., Berg, H.W.,Kunkee, R.E., Ough, C.S.,

Singleton, W.L. & Webb,A.D.(1980). The technology of wine Singleton, W.L. & Webb,A.D.(1980). The technology of wine making (4th edn). AVI Pub Comp., Westport making (4th edn). AVI Pub Comp., Westport Connecticut.USA.p.p.523-547.Connecticut.USA.p.p.523-547.

Beech, F.W., (1972a). English cidermaking-Technology, Beech, F.W., (1972a). English cidermaking-Technology, microbiology & biochemistry. In Progress in Industrial microbiology & biochemistry. In Progress in Industrial Microbiology. Pp. 133-213. Edited by D.J.D. Rockenhall, Microbiology. Pp. 133-213. Edited by D.J.D. Rockenhall, London, Churchill LivingstoneLondon, Churchill Livingstone

Beech, F.W., (1993). Yeasts in cider making. In The Yeasts, Beech, F.W., (1993). Yeasts in cider making. In The Yeasts, 2nd edn. Vol 5, Yeast Technology. pp. 169-213. Edited by 2nd edn. Vol 5, Yeast Technology. pp. 169-213. Edited by A.H. Rose & J. S. Harrison. London: Academic pressA.H. Rose & J. S. Harrison. London: Academic press

Carr, J.G. (1987). Microbiology of wines & cider. In Essays in Carr, J.G. (1987). Microbiology of wines & cider. In Essays in agricultural & Food Microbiology.agricultural & Food Microbiology.

Chavan, U.D.(2008). Fruit based fermented beverages. In Chavan, U.D.(2008). Fruit based fermented beverages. In Beverages and Food World.Beverages and Food World.

Choi, L.H. & Nielsen, S. (2005). The efefects of thermal & non Choi, L.H. & Nielsen, S. (2005). The efefects of thermal & non thermal processing methods on apple cider quality & thermal processing methods on apple cider quality & consumer acceptability. In J Food Quality 28(1).p.p. 13-29.consumer acceptability. In J Food Quality 28(1).p.p. 13-29.

Deal, J.(1976). Making cider. In Amateur Wine Makers Pub.Deal, J.(1976). Making cider. In Amateur Wine Makers Pub. Dold et al.(1937). Antibacterial properties of honey-role oh Dold et al.(1937). Antibacterial properties of honey-role oh

inhibine.inhibine. Grafton, G. www.realciderperry.orgGrafton, G. www.realciderperry.org

Karwanna & Pradit et al.(1976). Wine fermentation with mixed Karwanna & Pradit et al.(1976). Wine fermentation with mixed yeast cultures. In Dept of Viticulture & Enology, UCD.yeast cultures. In Dept of Viticulture & Enology, UCD.

Kime, R.W. & Romulus, N.Y.(1982). Clarification of fruit juice with Kime, R.W. & Romulus, N.Y.(1982). Clarification of fruit juice with honey. Assignee : Cornell Research Foundation, Ithaca, N.Y. U.S. honey. Assignee : Cornell Research Foundation, Ithaca, N.Y. U.S. Patent: 4327115.Patent: 4327115.

Konowalchuk, J. & Speirs, J.I.(1978). Antiviral effects of apple Konowalchuk, J. & Speirs, J.I.(1978). Antiviral effects of apple beverages. In Appl Environ Microbiol 36(6).p.p. 798-801.beverages. In Appl Environ Microbiol 36(6).p.p. 798-801.

Lea, A.G.H (2007). The Craft Cider Revival- some technical Lea, A.G.H (2007). The Craft Cider Revival- some technical considerations. In SWEA.considerations. In SWEA.

Lea, A.G.H. & Piggot J. (1995). Fermented Beverage Production Lea, A.G.H. & Piggot J. (1995). Fermented Beverage Production (2nd edn). In (2nd edn). In www.wkap.nl/prod/b/0-306-47706-8www.wkap.nl/prod/b/0-306-47706-8..

Morrissey, W.F., Davenport, B., Querol, A. & Dobson, A.D.W. Morrissey, W.F., Davenport, B., Querol, A. & Dobson, A.D.W. (2004). The role of indigenous yeasts in traditional irish cider (2004). The role of indigenous yeasts in traditional irish cider fermentations. In J. Appl Microbial 97. pp. 647-655.fermentations. In J. Appl Microbial 97. pp. 647-655.

National Honey Board. In www.nhb.org.p.p-1-8.National Honey Board. In www.nhb.org.p.p-1-8. NACM (1998). Code of practice for the production of cider & perry. NACM (1998). Code of practice for the production of cider & perry.

National Association of Cidermakers. London WC2.National Association of Cidermakers. London WC2. Predominance of Saccharomyces uvarum during spontaneous Predominance of Saccharomyces uvarum during spontaneous

alcoholic fermentation for three consecutive years in an Alsatian alcoholic fermentation for three consecutive years in an Alsatian winery.(2004). In J Appl Microbiol.winery.(2004). In J Appl Microbiol.

Pooley, M. & Lomax, J.(1999). Real cider making-on small scale. In Pooley, M. & Lomax, J.(1999). Real cider making-on small scale. In Nexus Special Interests, Kent. Nexus Special Interests, Kent.

Roozen,J.P. & Buren,J.P.V(1979). Sensory analysis of 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. bitterness in apple wine. In Inst J Food Sci Tech 14(3).p.p. 315-320.315-320.

Uljas, H.Uljas, H.E., Schaffner, D.W., Duff, S., Zhao, L.& Ingham, E., Schaffner, D.W., Duff, S., Zhao, L.& Ingham, S.C. (2001). Modeling of combined processing steps for S.C. (2001). Modeling of combined processing steps for reducing E.coli O157:H7 population in apple cider. In Appl reducing E.coli O157:H7 population in apple cider. In Appl Environ Microbiol 67(1).p.p 133-141.Environ Microbiol 67(1).p.p 133-141.

Valles, B.Valles, B.S., Bedrinana, R.P., Tascon, N.F., Simon, A.Q. & S., Bedrinana, R.P., Tascon, N.F., Simon, A.Q. & Madrera, R.R.( 2007). Yeast species associated with the Madrera, R.R.( 2007). Yeast species associated with the spontaneous fermentation of cider. In Food Microbiol 24(1). spontaneous fermentation of cider. In Food Microbiol 24(1). p.p. 25-31.p.p. 25-31.

I would like to extend my word of thanks to my guide, I would like to extend my word of thanks to my guide, faculty of my department as well as Dr.Dadhich & Mr. faculty of my department as well as Dr.Dadhich & Mr. Saurav Gupta (Dept of Microbiology).Saurav Gupta (Dept of Microbiology).

I am very greatful to my parents who provided me I am very greatful to my parents who provided me with immense moral support throughout the project with immense moral support throughout the project tenure.tenure.

I would like to thank my classmates for their great I would like to thank my classmates for their great cooperation.cooperation.

I would like thank my sisters for providing me insights I would like thank my sisters for providing me insights during frustrating pahses of this journey.during frustrating pahses of this journey.