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ABSTRACT

Author’s name: Ritwik Bhattacharya Project guide: Dr. Geeta Mehra

Department: Microbial and Food Technology: Co-Guide: Mrs. Harbinder Kaur

Class: M.Sc. (IV semester) College: Dolphin (PG) college of life sciences (Affiliated to Punjabi University, Patiala)

Title: Preparation and physico-chemical analysis of honey hard cider using pure and mixed cultures (Sacharomyces cerevsiae &

Sacharomyces uvarum), Isolation of indigenous micro flora from apple juice and observation of antimicrobial (E.coli) activity of cider

in different prepared cider varieties.

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 (12oBrix), 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.

INTRODUCTION

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. Shimla apples of kinnaur region

have been selected for the project. (Macrae et al, 1993)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)Inactivation of E.coli in fermenting cider is

attributed to the combined effects of pH & ethanol. (Wilson et al., 1996). Clarification of juice and cider was done using, different methods

viz, honey clarification, gelatin tannin clarification but bentonite agar-agar clarification was found to be most effective.( Rangana, 2001).

Honey imparts a sweet, smooth, mellow taste and pleasant floral scent to hard cider. It also increases the alcohol content of the cider by

increasing the specific amount of sugars available for fermentation. Honey naturally raises the specific gravity of pressed apple juice and

imparts a smooth honey taste to hard cider. The specific gravity of fresh pressed apple juice is approximately 0.050. At this level,

fermentation of the sugars present produces a hard cider with an alcohol content of 6 percent. However, contamination by yeast and

bacteria is still possible. Therefore, many manufacturers raise the specific gravity of their product to create a hard cider with a higher

alcohol content and longer shelf life. (National honey board).

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 g of sound fruit. The main organisms are Aureobasidium pullulans, Rhodotorula spp., Torulopsis, Candida,

Metschnikowia, and Kloeckera apiculata. Externally added yeast used for the project are: Saccharomyces cerevisiae & Saccharomyces

uvarum. The malo-lactic fermentation is carried out by non-slime forming strains of Leuconostoc mesenteroides, Lactobacillus collinoides

and very rarely Pediococcus cerevisiae. 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)

MATERIALS & METHODS

1. Raw material: Apples Apples were procured from Reliance fresh stores, in Mohali. Standard laboratory reagents were available from

college, Honey -Dabur honey was obtained from the local market, Pure cultures: Saccharomyces cerevisiae & Saccharomyces uvarum

were procured from NDRI, karnal, Nutrients : ammonium sulfate/ thiamine (college laboratory), Culture mediums: potato dextrose agar/

broth, malt extract broth, nutrient agar. (College laboratory), Reagents: Potassium meta bisulfite (kms), gelatin, tannin, bentonite powder,

agar-agar. (College laboratory), Dye: 1% lactophenol cotton blue (college laboratory), Instruments: lab scale fermenter (5 litres),

distillation apparatus, baume’s refractometer, laminar air flow chamber, autoclave, sterile glasswares & general lab appararus (college

laboratory).

Figure 2.1 Preparation of apple honey cider

2.2 Pre-Treatment of apples

2.2.1 Selection of apples Apples were tested for juice content. Three principal varieties were selected were : Kinnaur varieties, Shimla

varieties & Kashmir varities.

2.2.2 Surface sterilizationApples skin surface were sterilized using 0.5% H2O2, to destroy the exogenous contaminating microorganisms,

during harvesting handling and storage. 0.5 ml juice was dissolved in 100 ml distilled water to prepare the solution.

2.2.3 Juice Extraction: Juice extraction was done using juicer. Initially the apples were cut into small pieces for easy handling.

SURFACE STERILIZATION

EXTRACTION OF JUICE

SELECTION OF APPLES

PURE CULTURE ISOLATION

2.2.4 Clarification:

2.2.4.1 Clarification using honey: According to Kime et al, 1982, addition of honey (15%) at 75oC was done. The juice is left for 24 hrs,

for sedimentation of undissolved matter.

2.2.4.2 Clarification using gelatin- tannin According to Ranganna, 1991, gelatin-tannin clarification was carried out for better results.

Tannin: 9.5gm of tannin was dissolved in 1ltr water. Gelatin: 21.25gm of gelatin was dissolved into 600-800ml cold water to form gel

like mass. Solution was heated to boil. Volume was made up to 1000ml using water or ethanol (if storage is intended for few days). For

every litre of juice: 10.6 ml of tannin + 17.63ml gelatin was added. The sample was left for clarification under room temperature for 24

hours to obtain clarified juice.

2.2.5 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). Addition of sulphur dioxide was the most common way of controlling unwanted organisms.

Table 3.2.5: SO2 addition required in apple juice according to pH

pH Addition required (mg/l)

3.0 - 3.3 75

3.3 - 3.5 100

3.5 - 3.8 150

The amount of sulphur dioxide needed depends on the pH of the juice. Between pH 3.0 to 3.3, 75 ppm was needed, between pH 3.3 and

3.5 100 ppm was necessary and 150 ppm between 3.5 and 3.8.

2.2.6 Fortification : According to Lea et al, 1995, Ammonium sulfate is added as a yeast nutrient for providing additional nitrogen in

growth medium, at a concentration of 250 ppm. Therefore per liter of juice 0.25 gm of ammonium sulphate was added.3.2.7

Fermentation:

2.2.7.1 Pure culture revival

2.2.8 Culture inoculation: 5% culture w/w was added to apple juice and fermentation carried out under controlled conditions in lab scale

bench top (10 liter) fermenter for 2 days. Each culture is added in one bottle of apple juice. All tests to be carried out aseptically with

autoclaved utensils and media, to remove risk of contamination of cider. The fermented cider is filtered from the debris and sediment

using filter paper . Filtered wine was pasteurized at 65-68oC for 30 seconds to prevent further fermentation by yeasts. (Chavan et al)

2.2.9 Final clarification:

2.2.9.1 Clarification using Bentonite-Agar suspension: Final clarification was done using bentonite: agar-agar mixture (1:1). To dissolve

1.8gm agar-agar, 100ml distilled water is needed, therefore 1.8gm of bentonite was taken along with same amount of agar-agar, and

dissolved in 100ml distilled water to prepare a suspension. The suspension was added to the cider at 0.015% to obtain clear cider.

(Ranganna, 2001)

2.2.9.2 Clarification 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 (Wime et al., 1982).

2.3 Physico-chemical tests of juice and cider

TSS was recorded by Baume’s refractometer calibrated at 20oC. pH was Recorded by digital pH meter which was pre-calibrated. Titrable

Acidity As given by AOAC, 1984, titratable acidity was estimated by titrating required volume of sample against 0.1N alkali (NaOH)

using phenolphthalein indicator till pink color. Amount of alkali required was noted as V and its normality as N.

2.3.4 Sugars (by Lane and Eyon volumetric method, AOAC , 1984)Sugar content was estimated by titrating Fehling’s solution against the

given sample using methylene blue indicator till color changes from blue to red.

a) Standardization of Fehling’s solution: Done by titrating the Fehling’s solution against standard invert sugar solution using methylene

blue indicator.

Factor for Fehling’s solution = titre x 2.5

(grams of invert sugar) 1000

b) % reducing sugar = mg. of invert sugar x dilution x 1000

Titre value x weight or volume of sample

c) %Total sugar = mg. of invert sugar x dilution x 1000

(As invert sugar) Titre value x weight or volume of sample x 100

Where, Titre value = amount of sample and inverted sample required for reducing Fehling’s solution.

2.4 Pure culture isolation: Part of juice was left at 27oC for 2 weeks for fermentation by indigenous yeasts. Spreading of juice using 0.1

ml, was done on potato dextrose agar plates (PDA) plates and incubated at 32-37oC for 2-3 days. Colonies observed, were individually

selected, and streaked in PDA plates, and kept at 32-37oC for 2-3 days. Isolated culture was stained using lactophenol cotton blue (1%) and

observed under microscope. If fungal hyphae were observed, then culture was streaked in PDA slants and preserved after initial incubation

at 37oC for 3 days. One loop full of pure culture was preserved in glycerol stock eppendorf tubes. The stock was prepared, by adding 1ml

glycerol to 2 ml water. The tubes were kept under refrigeration (4oC), which maintained the cultures in intact form for as much as one year.

2.5 Analysis of cider samples

2.5.1 Microbiological analysis Microbiological analysis was carried out according to the procedure given in APHA (1992). 11ml. of

sample was taken and suspended in 99 ml. of sterilized distilled water as dilution blank. Serial dilutions were also prepared. (Maheshwari

et al., 1998)

2.5.1.1 Total plate count Approximate dilution of sample were transferred aseptically to sterile petriplate in duplicate and mixed with 10-

15 ml. of pre-sterilized melted Plate Count Agar at 45C.

After solidification petriplate were incubated at 37C for 24-48 hours. Average count multiplied by the dilution factor was expressed as

CFU/ml.

Colony forming unit (CFU) = No. of colonies

Dilution factor x ml of sample 2.5.1.2 Yeast and moldApproximate dilution of sample were transferred aseptically to sterile petriplate in duplicate and mixed with 10-15

ml. of pre-sterilized melted Potato dextrose agar at 45°C. After solidification petriplate were incubated at 25°C for 48-72 hours. Average

count multiplied by the dilution factor was expressed as CFU/ml. Above mentioned tests are performed fortnightly to observe the change

in character of the cider.

3.6 Antimicrobial effect: Action of cider in inhibition of pathogenic microorganisms was observed using different isolated pure cultures

of Escherichia coli. Nutrient agar petriplates were prepared from standard Hi-media media. Petriplates were spreaded with E.coli &

incubated at 25-27 °C in incubator for 24 hours. Wells were plucked using a cork borer, and different amount of cider was poured to

observe the change in the culture growth the next day. Measurement of zone of inhibition If any antimicrobial activity was observed the

zone of inhibition around the wells were measured, and the most effective sample quantity giving optimum result was identified.

3.7 Post fermentation procedures & tests

2.7.1 Ethanol content estimation (Caputi et al., 1968) Percentage of ethanol in samples was estimated by the method of Caputi et al.,

(1968). Ethanol standards were made by using ethanol-water solution containing 0 – 20% ethanol (v/v). Potassium dichromate solution

was prepared by adding 325 ml sulphuric acid (H2SO4) to 400 ml distilled water in 1 liter volumetric flask. After mixing & cooling, 33.768

gm of potassium dichromate (K2Cr2O7) was added and then final volume of 1ltr was made with water. Standard curve was prepared by

taking 1 ml of each concentration of the standard solution (1 – 20%) in a 50 ml volumetric flask containing 25 ml of potassium dichromate

solution. The samples were heated at 60 oC for 20 mins in a water bath, cooled & diluted to 50 ml with distilled water. Absorbance was

measured at a wavelength of 600 nm. 1ml of each alcoholic sample was added to the distillation flask, diluted to 30ml with water and then

distilled. Distillation was started at 70 +/-2 oC and 20ml of the distillate was collected in a 50ml volumetric flask containing 25 ml

potassium dichromate solution. The contents of the flask were heated at 60 oC in water bath for 20 mins and the final volume was made up

with distilled water. After the mixing & cooling the contents of the flask, the absorbance was recorded at 600 nm wavelength. The amount

of ethanol (%) present in each sample was determined by using the standard curve of ethanol.

2.7.2 Sensory analysis: Sensory score of different verities of hard honey cider were determined using test panel consisting of eight panel

members drawn from the students and the staff of Department of Microbial and Food Technology .Panelists were asked to evaluate

samples for different quality attributes namely color, body, flavour and overall acceptability on the following nine point hedonic scale.

Qulaity scores were given as follows:

Like extremely-9, Like very much-8,Like moderately-7, Like slightly-6, Neither like nor dislike -5, Dislike slightly-4, Dislike moderately -

3,Dislike very much-2 & Dislike extremely-1.

RESULTS AND DISCUSSION

4.1. Selection of apples

Among the three varities of apple available, selection was done on basis of maximum pulp less juice production. According to table 4.1, it

was observed that, shimla apples gave highest percentage of pulp less juice (around 44%).

Table 4.1: Selection of apple according to juice content

S.no Type Size Juice content(per 10 ml)

Observation 1 Observation 2 Observation 3

1. Kashmir apples large 2.4 ml 2.3 ml 2.4 ml

2. Kinnaur apples large 3.2 ml 3.3 ml 3.5 ml

3. Shimla apples small 4.2 ml 4.4 ml 4.5 ml

Figure 1, demonstrates the higher efficency of juice production by apples of shimla region as compared to apples of Kashmir and kinnaur

region.

4.2 Physico-chemical tests of apple juice

Total soluble solids (TSS) of apple juice was found to be 22 o Brix using Baume’s refractometer. Titrable acidity of the apple juice was

found to be 0.799% as malic acid. pH of the apple juice was found to be 3.8, using standardized pH meter. Reducing sugar percentage for

the apple juice was found to be 5.7142%. total sugar percentage was found to be 10.5263%.

4.3 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. This was concluded by an experiment, using pasteurized apple from which isolation of microflora was done.

The microorganisms isolated earlier were not found as colonies of similar character in streak/spread plate, and the pasteurized juice

showed no further fermentation. Once this juice was re-inoculated by the isolated microorganisms, fermentation was observed within a

period of 5 days, giving characteristic aroma.Two principal types of colonies with similar texture, microscopic morphology but different

colour were observed, and specifically isolated.

Table 4.3: Isolated colony characteristics

Sl. No. Colony colour Microscopic morphology Texture

1. White Oval, in chains gummy

2 Fluroscent orange Oval –spherical, in chains gummy

4.4 Analysis of cider samples

Sample code(Abbreviations):

SC- Sacharomyces cerevsiae (pure culture), SU- Sacharomyces uvarum (pure culture) & A- Sacharomyces cerevsiae + Sacharomyces

uvarum (mixed culture; 1:1).

Sample A had least value for pH which denoted that, maximum acids were produced in the mentioned sample. Due to fermentation,

sugars get converted to alcohol, which are acidic in nature .thus higher the alcohol, lower the pH. However the acidity for sample A falls in

the middle between Sample SU & SC, due to some unverified contributing factors, not a part of this project. From the above discussion we

also understand the reason for Sample A to have least total sugar percent. Also, TSS (total soluble solids) gives an approximate quantity of

dissolved sugars and other solids other than the solvent present in the sample. So the lowest TSS of Sample A confirms the sugar percent

in the cider. SC comes 2nd as per physico-chemical values are concerned, since it is the most popular yeast used in liquor/wine industry, the

potential of ethanol production, sugar utilization (total sugar %), TSS & pH are higher than Sample SU (rarely used as a pure culture in

industry), but lesser than Sample A (mixed xulture).

The microbiological analysis of the cider samples is also related to the production of ethanol & lactic acid, which is microbicidal in nature.

Higher the conversion of sugars to lactic acid (followed by conversion of lactic acid to malic acid during malo-lactic fermentation), lesser

the microbial population, thus Sample A gave the lowest values for TPC and Yeast & mold count. However, Sample SU came close 2 nd,

which may be due to the higher acidity found earlier.

Table 3.4: Comparative analysis of different cider samples

Sl.No. CHARACTERESTICS SU SC A

1. TSS (o Brix) 14 16 13

2. TOTAL SUGAR (%) 12.34 12.8 11.62

3. pH 3.3 3.4 3.2

4. ACIDITY (%) 0.1316  0.1038 0.188 

5. T.P.C(10-2 dilution)

(10-3 dilution)

1 X 103c.f.u./ml 

0c.f.u/ml

8 X 103c.f.u./ml

6 X 104c.f.u./ml

2 X 103c.f.u/ml 

5 X 104c.f.u./ml 

6. YEAST & MOULD(10-2 dilution)

(10-3 dilution)

3 X 103c.f.u./ml 

1X104c.f.u./ml 

9 X 103c.f.u./ml

3 X 104c.f.u./ml

15X103c.f.u./ml 

7 X 104c.f.u./ml 

7. ETHANOL CONTENT % 2.90  3.44 5.45

8. ANTIMICROBIAL EFFECT (diameter, zone of inhibition-E.coli, cm)

0.73 1 2.67

Figure 2 discusses the comparative physico-chemical analysis results for different cider samples. Although the acidity and pH values are

fairly constant, the values of total sugar and TSS, reduces from SC<SU<A, showing more utilization of sugar to produce alcohol by the

later sample.

3.5 Sensory evaluation:

Based on the sensory evaluation scores on colour, body, flavour and overall acceptability, calculations were done to get the respective

average scores of the respective marked fields, to find the best or most acceptable sample among the three verities. Based on the

calculations of the sensory scores of the different samples of cider (SC, SU & A), sample A, was selected as the most acceptable sample,

with highest average overall acceptability score of 8.33.

Figure 3 discusses the comparative analysis of different cider samples, based on the sensory score provided by the selected 9 panelists, and

taking average of them. The average scores plotted on the graph clearly show that flavour and overall acceptability scores are highest for

cider A and lowest for SC, where as colour and body scores are highest for cider SC. Cider SU scores fall in between SC and A.

3.7 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. The graph was plotted using MS-EXCEL chart wizard. 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. Values are shown in Table 4.4.

Figure 4 - The standard curve for different ethanol concentrations, which give corresponding optical density values at 600nm. Figure 5

discusses, the ethanol conc graph plotted as per the standard OD curve plotted earlier for known ethanol concentrations, which is used to

obtain the ethanol concentrations(%) for cider samples(SC,SU&A) by drawing perpendicular on X-axis.

3.7 Antimicrobial effect of cider on E.coli:

The zone of inhibition of the E.coli lawn on the nutrient agar petriplates was measured, usuing a centimeter scale.Values obtained (shown

in table 4.6), 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 (0.73cm), followed by SC (1cm) and finally the mixed culture cider (A), has the highest diameter of

2.67cm. This is because, the combined effect of pure cultures (Sacharomyces cerevsiae & Sacharomyces uvarum), produced higher

ethanol content, already determined by alcohol estimation test (4.5), than pure single cultures. Moreover, Sacharomyces cerevsiae is

commonly used for alcohol production for commercial purpose, so it had larger diameter, than less frequently used Sacharomyces uvarum.

Figure 6 - The comparative results for antimicrobial activity by different cider samples, which clearly indicates that cider A, gives a larger

zone of inhibition’s diameter than cider SC, which comes next, followed by cider SU.

Table 3.8 Antimicrobial effect of cider

Sl.No. Sample

name/code

Diameter of Zone of

inhibition (cm)

Average(cm)

1st 2nd 3rd

1. SC 1.0 0.9 1.1 1

2. SU 0.7 0.7 0.8 0.73

3. A 2.6 2.7 2.7 2.67

SUMMARY & CONCLUSSION

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). It was also found that

mixed culture cider had a higher antibacterial effect on E.coli 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. As a part of the last objective of this project, indigenous microflora responsible fermentation in fresh apple juice

was isolated. Two types of yeast like colonies were isolated having yeast like morphology (gummy colonies, oval shaped, in chains) .The

colonies had different colours i.e. white & fluorescent orange. A table is shown below, to demonstrate the comparative evaluation of the

different cider varieties prepared.

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