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3. MATERIALS AND METHODS
The present study was undertaken in the Department of Microbiology, College of
Basic Sciences, Himachal Pradesh Agricultural University, Palampur which is located in
the mid-hills and sub-humid agro-climatic zone of Himachal Pradesh, India. This town is
located at 32º6’N, 76º18’E and 1220 m msl.
The chemicals and media components used were of analytical grade (AR)
obtained from Merck limited - India, Sigma-Aldrich Inc. USA, and Hi Media
Laboratories, Mumbai, India. The materials used and the methods employed are
presented below under the following sub-headings:
3.1 Yeast isolates and culture maintenance
3.2 Morphological and biochemical studies on S. cerevisiae strains
3.3 ITS region sequencing
3.4 PCR-RFLP of ITS region
3.4.1 DNA extraction, amplification and visualization
3.4.2 Digestion of PCR products
3.5 Baking traits
3.5.1 Specific growth rate
3.5.2 Acid tolerance
3.5.3 Maltose adaptation
i. Anthrone colorimetric method
ii. Glucose oxidase method
3.5.4 Invertase activity
i. DNS method
3.5.5 Latent time
3.6 Brewing traits
3.6.1 Alcohol production
i. Estimation of ethanol content
3.6.2 Alcohol tolerance
3.6.3 Molasses concentration
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3.6.4 Attenuation and fermentation efficiency
3.6.5 Flocculation
3.7 Killer activity
3.7.1 Assay for mycocinogenic activity
3.7.2 Determination of killer activity by the well test method
3.8 Allele Mining of ADH1 and ATF1 genes
3.9 Organoleptic studies
3.9.1 Selection of fruits
3.9.2 Physico-chemical analysis of apple pulp
3.9.3 Preparation of inoculum
3.9.4 Alcoholic fermentation of apple juice
3.9.5 Analytical techniques
i. Estimation of pH
ii. Estimation of total soluble solids
iii. Estimation of titrable acidity
iv. Estimation of brix-acid ratio
v. Estimation of ethanol content
vi. Estimation of ascorbic acid content
vii. Estimation of reducing sugars
viii. Estimation of total sugars
3.9.6 Sensory evaluation
3.10 Screening of bio-emulsifier producing strains
3.10.1 Growth medium
3.10.2 Extraction of emulsifier
3.10.3 Measurement of emulsification activity
3.11 Statistical analysis of data
3.1 Yeast isolates and culture maintenance
Out of twenty-three S. cerevisiae strains, eighteen strains (Table 3.1) were used in
the present study, because five strains were lost as these could not be revived. S.
cerevisiae strains available with the Department of Microbiology of Himachal Pradesh
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Agricultural University, Palampur were maintained on potato dextrose agar at 4 °C, and
were also preserved in 50 % glycerol at -80 °C.
The composition of potato dextrose agar was:
Potato infusion 300.0 g
Dextrose 20.0 g
Agar 15.0 g
Distilled water 1000 mL
pH 5.6±0.2
Table 3.1 S. cerevisiae strains used in the study along with their source, place of
collection and GenBank accession numbers
S.
No.
Strain
code
Source Place of
collection
GenBank
accession number
aNBAIM
Accession No.
1. Sc01 Chhang Lahaul&Spiti KC515355 Awaited
2. Sc02 Lugari Lahaul&Spiti KC515356 NAIMCC-F-02236
3. Sc03 Dhaeli Lahaul&Spiti KC515357 NAIMCC-F-02237
4. Sc04 Aara Lahaul&Spiti KC515358 NAIMCC-F-02238
5. Sc05 Chiang Lahaul&Spiti KC515359 NAIMCC-F-02239
6. Sc06 Chilra Lahaul&Spiti KC515360 NAIMCC-F-02240
7. Sc07 Bhaturu Lahaul&Spiti KC515361 Awaited
8. Sc08 Babru Lahaul&Spiti KC515362 NAIMCC-F-02241
9. Sc09 Khameer Lahaul&Spiti KC515363 NAIMCC-F-02242
10. Sc10 Faasur Sangla KC515364 Awaited
11. Sc11 Chuli Sangla KC515365 Awaited
12. Sc12 Apple
wine
Sangla KC515366 Awaited
13. Sc15 Beverage Bharmour KC515369 Awaited
14. Sc16 Beverage Pangi KC515370 Awaited
15. Sc19 Wine Sangla KC515373 Awaited
16. Sc20 Wine Sangla KC515374 Awaited
17. Sc21 Wine Sangla KC515375 Awaited
18. Sc24 Fermented
product
Palampur KC515376 Awaited
aNational Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan, Uttar Pradesh, India
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3.2 Morphological and biochemical studies on S. cerevisiae strains
The indigenous yeast strains were streaked on potato dextrose agar. Their
morphological studies with respect to colony characteristics, characteristics in broth
culture, and cell shape were carried out.
The strains were identified on the basis of biochemical characteristics as
described by Lodder (1970) and Barnett et al. (1983). These biochemical characteristics
were growth at different temperatures, growth in presence of 10% ethanol and
fermentation pattern of different sugars.
3.3 ITS region sequencing
Thermo Scientific Phire Plant Direct PCR Kit (F-130) was used to perform
polymerase chain reaction (PCR) directly from the yeast cells with some modifications.
Based on optimization trials, the standardized PCR protocol for 20 µl reaction mixture
included 10 µl of 2X phire plant PCR buffer, 0.4 µl of 10 µM of primer set containing
forward and reverse primers, 0.4 µl of phire hot start II DNA polymerase, 0.5 µl of yeast
cells grown in potato dextrose broth (PDB) and sterilized milli-Q water to make up the
final reaction volume.
Universal primers viz., ITS-1 (5′TCCGTAGGTGAACCTGCGG3′) and ITS-4
(5′TCCTCCGCTTATTGATATGC3′) targeting ribosomal gene region [Internal
Transcribed Spacer region (ITS1 and ITS2), 5.8S and partial regions for 18S -28S] were
used to amplify the genomic DNA of the yeast strains (White et al. 1990).
Amplification was carried out in a thermal cycler (B-96G, TC-PRO, BOECO,
Germany) with an initial denaturation at 94 °C for 5 min, followed by 30 cycles of 30 s at
94 °C, 30 s at 55 °C, 1 min at 72 °C and a final elongation step at 72 °C for 2 min. The
PCR products were resolved in 1.2 % agarose gel in 1x tris acetate EDTA buffer
containing ethidium bromide (0.5 μg/ mL) at 75 V for 90 minutes (Plate 3.1).
The PCR products were freeze dried (CHRIST ALPHA I-2LD) and custom
sequenced (ABI 3730xl automated sequencer) by using same upstream and downstream
primers, by a commercial sequencing facility (Xcelris Labs Ltd., Ahmedabad, India). The
sequences of different isolates of yeasts were edited manually and analyzed using NCBI
BLASTN program (http://www.ncbi.nih.gov/blast; Altschul et al. 1997). The
phylogenetic analyses were carried out in MEGA 5.1 software program.
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The sequences were submitted to GenBank, National Center for Biotechnology
Information (NCBI), USA (Table 3.1). The yeast cultures were deposited with the
National Bureau of Agriculturally Important Microorganisms (NBAIM), Maunath
Bhanjan, Uttar Pradesh, India.
Plate 3.1 PCR amplification of ITS region of S. cerevisiae, Rhodotorula mucilaginosa
and Torulaspora delbrueckii using ITS-1 and ITS-4 primers
3.4 PCR-RFLPof ITS region
3.4.1 DNA extraction, amplification and visualization
Thermo Scientific Phire Plant Direct PCR Kit, F-130 was used to perform
polymerase chain reaction (PCR) directly from the yeast cells. Based on optimization
trials, the standardized PCR protocol for 50 µl reaction mixture; included, 25 µl of 2X
phire plant PCR buffer, 0.6 µl of 10 µM of primer set containing forward and reverse
primers, 1 µl of phire hot start II DNA polymerase, 0.5 µl of yeast cells grown in potato
dextrose broth (PDB) and sterilized milli-Q water to make up the reaction volume. PCR
amplification and visualization of PCR products were done as described in section 3.3.
3.4.2 Digestion of PCR products
The PCR products were digested without further purification with four restriction
endonucleases i.e. AluI, HaeIII, HinfI and TaqI by the following protocol:
PCR product: 10 µl
Buffer: 2 µl
Restriction enzyme: 0.5 µl
Distilled water: 7.5 µl
ITS region of Rhodotorula mucilaginosa
(~800 bp)
ITS region of Torulaspora delbrueckii
(~850 bp)
Ladder
ITS region of
S. cerevisiae (~880 bp)
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The mixture was incubated at 37 ºC for two hours except for TaqI, where
incubation temperature was 60 ºC, the restriction fragments were separated on 1.2 %
agarose gel in 1X TAE buffer. The gels were stained with ethidium bromide, visualized
under UV light and photographed by UVITEC, Cambridge, GeNeiTM
. Sizes of different
bands were estimated by comparison with a DNA length standard. The PCR-RFLP
analyses were carried out using Treeconw software program.
3.5 Baking Traits
Eighteen S. cerevisiae strains were screened for following five baking traits as per
the method of Clement and Hennette (1982):
3.5.1 Specific growth rate
This test included determination of the mean or average multiplication coefficient
(µ) of a given strain by following the variation in optical density of a standard medium
(potato dextrose broth, Hi-Media) seeded with the suspension of yeast cells, at 600nm,
which could be calculated by using formula,
µ= 1/x X dx/dt
where, x represents the cellular population at time t.
3.5.2 Acid tolerance
The log CFU/mL of the yeast strains were measured after incubation of 48 hours
at 30 °C in the presence of an inhibitor i.e. glacial acetic acid at a concentration of 0.466
% (v/v) added in potato dextrose broth where, pH corresponded to 3.45.
3.5.3 Maltose adaptation
Maltose adaptation of the strains was measured in presence of glucose by
determination of the amount of maltose subsisting in a standard medium after a known
amount of glucose added to the medium had been completely consumed. The test was
carried out in 0.01M phosphate buffer medium, pH 6.5, the reaction mixture comprising
of: yeast-20 to 25 mg of dry matter/mL, glucose-4 mg/mL and maltose-2 mg/mL. The
reaction was carried out at 30 °C for one hour and was arrested by sudden cooling and
cold centrifugation. Disappearance of the total sugars in the supernatant was determined
by anthrone colorimetric method (Hodge and Hofreiter 1962) and that of glucose by
glucose oxidase method (Meites 1965). The amount of maltose was determined by the
difference in readings and percentage of maltose consumed was worked out.
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i. Anthrone colorimetric method
a) Standard stock solution of anthrone
Standard stock solution was prepared by dissolving 100 mg of glucose in 100 mL
of distilled water and working standard was prepared by diluting 10 mL of stock solution
to 100 mL with distilled water.
b) Preparation of anthrone reagent
Anthrone reagent was prepared by dissolving 200 mg of anthrone in 100 mL of
ice cold 95 per cent H2SO4.
c) Preparation of standard curve
Standard curve was prepared by taking 0, 0.2, 0.4, 0.6, 0.8 and 1 mL of the
working standard and the volume was made up to 1 mL by adding distilled water. 4 mL
of anthrone reagent was added and the mixture was heated for eight minutes in a boiling
water bath. After rapid cooling, the absorbance was recorded at a wavelength of 630 nm
using SPECTRONIC® GENESYS
TM5 Spectrophotometer. The standard curve so formed
is shown as below (Fig 3.1):
Fig 3.1 Standard curve of anthrone colorimetric method
d) Determination of total sugar from samples
One mL of sample was added directly in the tube. 4 mL of anthrone reagent was
added and the mixture was heated for eight minutes in a boiling water bath. After rapid
cooling, the absorbance was recorded at 630 nm. The amount of total sugars in each
sample was determined by using the standard curve of anthrone.
y = 0.1371x + 0.0065 R² = 0.9988
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.02 0.04 0.06 0.08 0.1
Glucose conc. (mg/mL)
Abso
rban
ce a
t 630 n
m
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ii. Glucose oxidase method
a) Standard stock solution of glucose
Standard stock solution was prepared by dissolving 100 mg of glucose in 100 mL
of distilled water and working standard was prepared by diluting 10 mL of stock solution
to 100 mL with distilled water.
b) Preparation of glucose oxidase peroxidase reagent
Twenty five mg of O-dianisidine was dissolved completely in 1 mL of methanol
and 49 mL of 0.1M phosphate buffer (pH 6.5) was added to it. Then 5 mg of peroxidase
and 5 mg of glucose oxidase were added to the above prepared O-dianisidine solution.
c) Preparation of standard curve
Standard curve was prepared by taking 0, 0.2, 0.4, 0.6, 0.8 and 1 mL of the
working standard glucose solution and the final volume was made up to 1 mL by adding
distilled water. 1 mL of glucose oxidase peroxidase reagent was added and the mixture
was incubated at 35 ºC for 40 minutes. The reaction was terminated by the addition of 2
mL of 6N-HCl. The absorbance was recorded at a wavelength of 540 nm using
SPECTRONIC® GENESYS
TM5 Spectrophotometer. The standard curve so formed is
shown as below (Fig 3.2):
Fig 3.2 Standard curve of glucose oxidase method
y = 0.3782x + 0.0504 R² = 0.9919
0
0.5
1
1.5
2
2.5
0.02 0.04 0.06 0.08 1
Abso
rban
ce a
t 540 n
m
Glucose conc. (mg/mL)
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44
d) Determination of glucose concentration from samples
One mL of sample was added directly in the tube. 1mL of glucose oxidase
peroxidase reagent was added and the mixture was incubated at 35 ºC for 40 minutes.
The reaction was terminated by the addition of 2 mL of 6N-HCl and the absorbance was
recorded at 540 nm. The amount of glucose in each sample was determined by using the
standard curve of glucose oxidase.
3.5.4 Invertase activity
Each invertase unit is defined as production of a micromole of reducing sugars in
five minutes per mg of yeast dry matter at 30 °C and at pH 4.7, without plasmolysis of
the yeast. 0.2 mg of dry yeast matter was placed in the presence of saccharose at a final
concentration of 0.1 M in an acetate buffer medium, pH 4.7, in a test tube and was placed
in a water bath at 30°C. At the end of five minutes, inversion reaction of saccharose was
blocked by the addition of a reactant sodium dinitrosalycilate which also helped in
determining the reducing sugars formed, by colorimetric method (Miller 1950).
i. DNS method
a) Standard stock solution of glucose
Standard stock solution was prepared by dissolving 100 mg of glucose in 100 mL
of distilled water and working standard was prepared by diluting 10 mL of stock solution
to 100 mL with distilled water.
b) Preparation of dinitrosalicylic acid (DNS) reagent
One gram of dinitrosalicylic acid, 200 mg of crystalline phenol, and 50 mg of
sodium sulphite were simultaneously dissolved in 100 mL of 1 per cent NaOH solution
by stirring.
Forty per cent of Rochelle salt solution (sodium-potassium tartrate solution) was
prepared.
c) Preparation of standard curve
Standard curve was prepared by taking 0, 0.2, 0.4, 0.6, 0.8 and 1 mL of the
working standard glucose solution and the volume was made up to 3 mL by adding
distilled water. 3 mL of DNS reagent was added and the mixture was heated for five
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minutes in a boiling water bath. After the development of the color, 1 mL of 40 per cent
Rochelle salt solution was added (in warm contents) and mixed thoroughly. After cooling
the tubes, the absorbance was recorded at a wavelength of 540 nm using SPECTRONIC®
GENESYS TM
5 Spectrophotometer. The standard curve so formed is shown as below
(Fig 3.3):
Fig 3.3 Standard curve of DNS colorimetric method
d) Determination of invertase activity in samples
One mL of sample was added directly in the tube and the volume was made up to
3 mL by adding distilled water. 3 mL of DNS reagent was added and the mixture was
heated for five minutes in a boiling water bath. The reaction was terminated by the
addition of 1 mL of Rochelle salt solution. After cooling, the absorbance was recorded at
540 nm. The invertase units in each sample were determined by using the following
formula:
Units of invertase activity in each assay tube = ABS/5.0 min * Final volume
a * 1.0 cm
Where, ΔABS = absorbance change
a = milli-molar absorptivity constant
Total units = Number of units / Final volume*Final volume/mL of dilute enzyme added*total
mL of dilute enzyme / mL of stock added*mL of stock fraction / 1
y = 0.2318x - 0.04 R² = 0.9983
0
0.2
0.4
0.6
0.8
1
1.2
0 1 2 3 4 5 6
Abso
rban
ce a
t 540 n
m
Glucose conc. (mg/mL)
46
46
3.5.5 Latent time
The latent time was measured by following the variation in the optical density
(600 nm) of yeast suspension applied in the specific growth rate test after having
conferred on this suspension a sugar concentration of 30 %.
3.6 Brewing Traits
S. cerevisiae isolates were screened for different brewing traits using molasses as
a substrate because it is being used as a carbon source in most of the industries for large
scale production (Keo 1967).
3.6.1 Alcohol production
For the estimation of maximum alcohol content produced by yeasts, molasses was
diluted to 15 °Brix. The solution was then poured into seven different flasks. To each
individual flask different doses of sucrose were added to achieve the final sugar
concentration varying from 22 to 28 °Brix with a difference of 1 °Brix in two consecutive
flasks (Table 3.2).
The molasses was then inoculated with 1 % inoculum and was incubated at 30 °C
for three days.
Table 3.2: Different doses of sucrose added to achieve the final sugar concentration
in the range of 22-28 °Brix
Flask Initial °Brix Sucrose added (%) Final °Brix
1 15 7 22
2 15 8 23
3 15 9 24
4 15 10 25
5 15 11 26
6 15 12 27
7 15 13 28
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i. Estimation of ethanol content
Per cent of ethanol in alcoholic samples was estimated by the method of Caputi et
al. (1968).
a) Standard stock solution of ethanol
Ethanol standards were made by using ethanol-water solution in the range of 0 –
20 % ethanol (v/v).
b) Preparation of potassium dichromate solution
Potassium dichromate solution was prepared by adding 325 mL conc. H2SO4 to
400 mL distilled water in 1 liter volumetric flask. After mixing and cooling (80-90 0C),
33.768 g K2Cr2O7 was added and then final volume of 1 liter was made with distilled
water at 20 0C.
c) Preparation of standard curve
Standard curve was prepared by taking 1 mL of each concentration of the
standard solution [0-20% (v/v)] in a 100 mL volumetric flask containing 25 mL of
potassium dichromate solution. The samples were heated at 60 0C for 20 minutes in a
water bath and then cooled and diluted to 50 mL with distilled water. Absorbance was
recorded at a wavelength of 600 nm using SPECTRONIC® GENESYS
TM5
Spectrophotometer. The standard curve so formed is shown in Fig 3.4.
d) Estimation of alcohol in the samples
One mL of alcoholic sample was added directly to the distillation flask, diluted to
30 mL with distilled water and then distilled. Distillation was carried out at 70+ 2 0C and
20 mL of distillate was collected in a 50 mL volumetric flask containing 25 mL of
potassium dichromate solution. The contents in the volumetric flask were heated at 60 0C
in a water bath for 20 minutes and final volume was made to 50 mL with distilled water.
After mixing and cooling the contents of the flask, the absorbance was recorded at 600
nm. The amount of ethanol in each sample was determined by using the standard curve of
ethanol.
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Fig 3.4 Standard curve for estimation of ethanol content
3.6.2 Alcohol tolerance
To determine the maximum alcohol content tolerated by yeasts, molasses was
diluted to 22 °Brix concentration and poured into five flasks. To each flask, various doses
of alcohol were added to achieve an initial concentration of 2 % in flask 1, 3 % in flask 2
and so on. The flasks were then inoculated and incubated at 30 °C for three days and then
per cent alcohol was estimated by Caputi et al. (1968) method.
3.6.3 Molasses concentration
For the determination of maximum molasses concentration at which the yeasts
remain functional, the molasses was diluted to 22 to 40 °Brix. Then the substrate was
inoculated with 1 % inoculum and incubated at 30 °C for three days. After incubation,
per cent alcohol was estimated in the samples by Caputi et al. (1968) method.
3.6.4 Attenuation and fermentation efficiency
Attenuation and fermentation efficiency were calculated by using the following
formulae:
Attenuation refers to the percentage of sugars converted to alcohol and carbon dioxide, as
measured by specific gravity,
Attenuation= [(OG-FG) / (OG-1)] X 100
y = 0.1136x + 0.0063 R² = 0.9998
0
0.2
0.4
0.6
0.8
1
1.2
0 2 4 6 8 10 12
Ethanol conc. (v/v)
Abso
rban
ce a
t 600 n
m
49
49
Where, OG is original gravity (specific gravity before pitching), FG is final
gravity (specific gravity at the end of the fermentation).
Fermentation efficiency is an expression of how much alcohol was actually
produced in relation to the theoretical production,
Fermentation efficiency (%) = (Actual ethanol recovery (v/v) / Theoretical
ethanol recovery (v/v)) X 100
3.6.5 Flocculation
For measuring the flocculation ability of the yeast strains, modified helm
sedimentation test (Soares and Mota 1997) was performed. All pre-cultures were
prepared by inoculating a loopful of yeast cells in 100 mL of potato dextrose broth. The
cells were incubated at 30 °C on an orbital shaker at 150 rpm, for 48 hours. Flocculent
cells were harvested by centrifugation (4500 X g, 5 min, 4 °C), washed twice in 250 mM
EDTA solution, followed by washing the cells with NaCl solution (250 mM) at pH 2.0
and with NaCl solution (250 mM) at pH 4.5. Cells were finally suspended in NaCl
solution (250 mM) at pH 4.5 at a final concentration of 1 x 108cells/mL. Cell suspension
(24 mL) in NaCl solution (250 mM), at pH 4.5, was placed in a 25 mL cylinder. The
suspension was adjusted to 4 mM Ca2+
with the addition of CaCl2 solution (1.0 mL from
a stock solution of 100 mM, at pH 4.5), and then agitated to promote flocculation.
Eighteen inversions of the cylinder were used to promote flocculation. At defined periods
of time, samples were taken (200-1000 µl) from a fixed position of the cylinder (the level
corresponding to 20 mL), and dispersed in NaCl solution (250 mM) at pH 2.0. Cell
concentration was determined by measuring the absorbance of the suspension at 620 nm.
3.7 Killer activity
The killer yeasts were detected on Yeast Extract Peptone Dextrose (YEPD) agar.
The composition of YEPD agar buffered at pH 4.2 (0.1M citrate-phosphate buffer) was
as follows:
Yeast extract 1 g/L
Peptone 2 g/L
Glucose 2 g/L
Methylene blue 0.05 g/L
50
50
3.7.1 Assay for mycocinogenic activity
Determinations were performed according to the method of Salek et al. (1990).
Sensitive lawns were made by mixing 200 μl of fresh culture of the sensitive strain
MTCC-473, with 20 mL of YEPD and poured in Petriplates. The yeast isolates were
seeded on the sensitive lawns and the plates were incubated at 22, 30 or 37 ºC for 3 to 7
days. Positive killer activity was observed by a clear zone, surrounded by a blue
precipitated halo, indicative of cellular death.
3.7.2 Determination of killer activity by the well test method
The cultures were grown in 200 mL potato dextrose broth in a 500 mL
Erlenmeyer flask for 24 hrs at room temperature on a rotary shaker (120 rpm). After
centrifugation (10,000 X g, for 10 min. at 5 ºC), the supernatant was filtered through 0.45
µm pore size membrane filter (Millipore). The resulting filtrate was incubated for 4 hrs at
4 ºC and the precipitate was collected by centrifugation (10,000 X g, for 20 min.) and
resuspended in equal volume of 0.1 M citrate-phosphate buffer (pH 4.2). This toxin
supernatant was stored at 4 ºC until use (Sawant et al. 1988).
A volume of 100 μl of toxin supernatant was inoculated into wells (10-mm
diameter) cut into sensitive cell lawns and the diameters of the death zones were
measured after incubation for 3 to 7 days at 22 or 30 ºC.
3.8 Allele Mining of ADH1 and ATF1 genes
Ten yeast strains viz., Sc01, Sc03, Sc04, Sc05, Sc11, Sc12, Sc15, Sc19, Sc21 and
Sc24; based on their variation in baking and brewing traits were selected for allele
mining. For DNA isolation, Yeast DNA isolation Kit was used (Xcelgen). The DNA
stock samples were quantified using Nanodrop spectrophotometer at 260 and 280 nm.
Quality and purity of DNA were checked by agarose gel electrophoresis.
The length of ADH1 gene is 1047 bp, hence, two separate primer pairs amplifying
two parts of the gene with overlapping flanking sequences were used (Table 3.3). The
second gene consisted of 2088 bases with 1578 bp long protein coding region of ATF1
gene. ATF1 gene sequence (1578 bp), preceding promoter and TATA box sequences (293
bases) and proceeding 217 bases were amplified and sequenced. For amplification and
sequencing, this 2088 bp region was divided into three overlapping sequences
51
51
(Appendix-1). Three separate primer pairs were used to amplify these three overlapping
sequences (Table 3.3). PCR was carried out in a final reaction volume of 25 µl: DNase-
RNase free water 7.50 μl, 2x PCR master mix (MBI Fermentas) 12.50 μl, forward primer
(10 pmole/μl) 1.00 μl, reverse primer (10 pmole/μl) 1.00 μl and diluted DNA (30 ng/μl)
3.0 μl. Amplification was carried out in the thermal cycler (B-96G, TC-PRO, BOECO,
Germany) with an initial denaturation at 95 °C for 2 min, followed by 30 cycles of 30 s at
94 °C, 30 s at 51 °C, 90 s at 72 °C and a final elongation step at 72 °C for 10 min. To
confirm the targeted PCR amplification, electrophoresis was done (Pate 3.2 and Plate 3.3)
and the amplified PCR products were purified using Qiagen Mini elute Gel extraction kit
according to manufacturer’s protocol.
The purified PCR products were freeze dried (CHRIST ALPHA I-2LD) and
custom sequenced (ABI 3730xl automated sequencer) by using same upstream and
downstream primers, by a commercial sequencing facility (Xcelris Labs Ltd.,
Ahmedabad, India). The complete gene sequences were then recovered by aligning the
overlapping regions of the obtained contigs. The homology search for these sequences
was carried out using an on-line NCBI BLASTN program http://www.ncbi.nih.gov/blast
(Altschul et al. 1997). All the phylogenetic analyses were conducted in MEGA 5.1
software program. The ADH1 and ATF1 gene sequences were submitted at GenBank,
National Center for Biotechnology Information (NCBI), USA (Table 3.4).
Table 3.3: ADH1 and ATF1 gene primer sequences
Primer Sequence
ADH1FL CAGGAAAGAGTTACTCAAGAATAAGAA
ADH1FR TGGGTAACGAATCCAACTGTC
ADH1SL ACGGTGATACCAGCACACAA
ADH1SR CTCGTTCCCTTTCTTCCTTG
ATF1FL TGCACTCGATGGTCTTCTCA
ATF1FR GACAAATTAGCCGCCAACTC
ATF1SL TGCAATGTTCTGCACGTTATT
ATF1SR TAGTTGTGAGCGGCAATCTG
ATF1TL GAACTTCGAATGGCTTACGG
ATF1TR TGCAATGTTCTGCACGTTATT
52
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Table 3.4: Genbank accession numbers of ADH1 and ATF1 genes along with their
strain codes
S.No. Strain Code ADH1 GenBank
Accession Number
ATF1 GenBank
Accession Number
1 Sc04 KF429720 KF429730
2 Sc24 KF429721 KF429731
3 Sc01 KF429722 KF429732
4 Sc03 KF429723 KF429733
5 Sc05 KF429724 KF429734
6 Sc19 KF429725 KF429735
7 Sc15 KF429726 KF429736
8 Sc11 KF429727 KF429737
9 Sc21 KF429728 KF429738
10 Sc12 KF429729 KF429739
3.9 Organoleptic studies
3.9.1 Selection of fruits
Healthy fruits were selected, washed in hot water, mixed with 0.1 % of potassium
metabisulphite and then used for the extraction of juice. Juice was extracted aseptically
under hygienic conditions.
3.9.2 Physico-chemical analysis of apple juice
The physico-chemical analysis of apple juice was done which included estimation
of TSS, pH, titrable acidity, brix acid ratio, total sugars, reducing sugars and ascorbic
acid.
3.9.3 Preparation of inoculum
The inoculum of six randomly selected S. cerevisiae strains viz. Sc01, Sc02,
Sc05, Sc12, Sc21 and Sc24 was prepared in 250 mL Erlenmeyer flasks containing 100
mL potato dextrose broth inoculated with a loopful of culture and incubated at 28 ± 2 ºC
for 24 hrs with 100 rpm shaking. From this seed inoculum, starter culture was prepared
by inoculating 2 % of seed inoculum to pasteurized apple juice and incubated at 28 ºC for
24 hrs under shaking conditions.
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Plate 3.2 PCR amplification products of ADH1 gene contigs
Plate 3.3 PCR amplification products of ATF1 gene contigs
First part of ADH1 (850 bp)
Second part of ADH1 (500 bp)
First part of ATF1 (850 bp)
Second part of ATF1 (850 bp)
Third part of ATF1 (850 bp)
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3.9.4 Alcoholic fermentation of apple juice
The pasteurized apple juice (1500 mL) was taken in 3000 mL Erlenmeyer flasks
which was adjusted to sugar level of 18 ºBrix using granulated sucrose procured from
local market. Juice was inoculated using 1 per cent inoculum supplemented with
diammonium hydrogen orthophosphate (DAHP) (300 mg w/v) and incubated at room
temperature. The periodic samples were taken, spun at 6000 rpm for 5 minutes and
analyzed for TSS, pH and ethanol content till no further decrease in ºBrix was noticed.
3.9.5 Analytical techniques
i. Estimation of pH
The pH of the periodic samples was determined by using Digital pH meter
(Eutech Instruments, Germany).
ii. Estimation of total soluble solids
Total soluble solids (TSS) in juice were determined by using Erma hand
refractometer of 0-32 ºBrix. A drop of distilled water (at 20 ºC) was placed on clean and
dry prism and calibration was done at zero line on the scale. Then the samples of juice
and cider were analyzed for their TSS value by reading the line of demarcation on the
scale.
iii. Estimation of titrable acidity
It was expressed as per cent acidity and analyzed using the method of Amerine et
al. (1967). Titrable acidity was determined by titrating known quantity of apple juice or
cider sample (10 mL) against standardized 0.2 N NaOH using a few drops of 1 per cent
phenolphthalein solution as indicator to achieve pink colourend point which should
persist for 15 seconds.
Acidity (%) = volume of 0.2 N NaOH used X 0.2 X 6 X 100
10 (volume of sample taken)
iv. Estimation of brix-acid ratio
It was calculated by dividing TSS value with titrable acidity of the juice and cider.
v. Estimation of ethanol content
The estimation was done by chemical oxidation method of Caputi et al. (1968) as
described in section 3.6.1 (i).
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vi. Estimation of ascorbic acid content
Ascorbic acid content was estimated by the method of Ranganna (1976). A dye
solution was prepared in which 42 mg of sodium bicarbonate and 52 mg of
dichlorophenol indophenol were mixed in 200 mL of distilled water. In stock standard
solution, 100 mg ascorbic acid was dissolved in 100 mL of 4 % oxalic acid solution in a
flask (1 mg/mL). For the preparation of working solution, 10 mL of the stock solution
was diluted to 100 mL with 4 % oxalic acid and the concentration of this working
solution was taken 100 µg/mL.
In next step, 5 mL of the working solution was pipetted out into 100 mL conical
flask. 10 mL of 4 % oxalic acid was then added to it and titrated against the dye (V1 mL).
End point was determined by the appearance of pink color which persisted for a few
minutes. 10 mL of sample was taken and 100 mL of volume was made with 4 % oxalic
acid solution. 5 mL of this supernatant was added to 10 mL of 4 % oxalic acid and
titrated against the dye (V2 mL). The results thus, obtained were expressed in terms of mg
ascorbic acid/100 mL of juice or cider. The ascorbic acid content was calculated by using
the following formula:
Ascorbic acid (mg/100 g sample) =
Where, V1 (mL) is the volume of dye used for the end point of standard
V2 (mL) is the volume of dye used for the end point of sample
vii) Estimation of reducing sugars
Reducing sugars were estimated by the method of Miller (1950). Test tubes
containing 3 mL sample and 3 mL DNS reagent were heated for 15 min. in a boiling
water bath. One mL Rochelle salt solution was added to each tube and the tubes were
allowed to cool to room temperature and O.D. was measured at 575 nm using
SPECTRONIC® GENESYS
TM5 Spectrophotometer. The concentration of reducing
sugars (as glucose) was calculated from the standard curve (Fig 3.5).
viii) Estimation of total sugars
Total sugars were estimated by phenol-sulphuric acid method of Dubois et al.
(1956) using glucose as standard. For estimation purposes, different aliquots of 0.2 mL
0.5 mg×V2×100 mL×100
V1×15 mL×Wt. of the sample
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cider were taken in test tubes and distilled water was added to make the volume 1 mL. It
was followed by addition of 1 mL of 5 per cent phenol and 5 mL conc. sulphuric acid.
The acid was poured directly in the center of the test tube to ensure that temperature rises
Fig 3.5 Standard curve of DNS colorimetric method
to 70 ºC for optimal color development. The test tubes were kept for 10 min at room
temperature and then cooled under tap water. A stable yellow orange color developed
after about 20 min. Absorbance was recorded at 490 nm using SPECTRONIC®
GENESYS TM
5 Spectrophotometer against a reagent blank. The concentration of total
sugars was calculated from the standard curve by using glucose as standard (Fig 3.6).
Fig 3.6 Standard curve of phenol-sulphuric acid method
y = 0.0071x - 0.0046 R² = 0.9992
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0 2 4 6 8 10 12
y = 0.0066x + 0.022 R² = 0.9963
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 20 40 60 80 100 120
Abso
rban
ce a
t 5
75
nm
Glucose conc. (µg/mL)
Glucose conc. (µg/mL)
Ab
sorb
ance
at
49
0 n
m
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3.9.6 Sensory evaluation
The organoleptic evaluation of cider was done on the basis of appearance, color,
flavor, mouthfeel and overall acceptability by a panel of judges. Consumer acceptance for
the products was evaluated on a nine point “Hedonic scale” (Amerine et al. 1965) with
following scale
Table 3.5 Nine point Hedonic scale for sensory evaluation of apple cider
Scale Sensory Score
Liked extremely 9
Liked very much 8
Liked moderately 7
Liked slightly 6
Neither liked nor disliked 5
Disliked slightly 4
Disliked moderately 3
Disliked very much 2
Disliked extremely 1
3.10 Screening of bio-emulsifier producing strains
3.10.1 Growth medium
For screening of emulsifier production by intact cells, yeast strains were grown in
Yeast Casamino Glucose (YCG) medium; 0.5% (w/v) yeast extract, 1% casamino acids
and 1% glucose, for 48 hours on a rotary shaker (150 rpm) at 28 ºC. Cells were harvested
by centrifugation at 3000 rpm for 10 min.
3.10.2 Extraction of emulsifier
Emulsifier was extracted from the cells of S. cerevisiae strains by the method of
Cameron et al. (1988). Yeast cells were suspended in 100 mL of 0.1 M potassium citrate
and 0.02 M potassium metabisulphite buffer (pH 7.0) and autoclaved (121ºC) for 3 hours.
The resulting suspensions were centrifuged at 5000 X g for 10 min at 4 ºC. The
supernatant was retained for the assay of emulsification activity
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3.10.3 Measurement of emulsification activity
Emulsification activity was measured using the method described by Cooper and
Goldenberg (1986). Kerosene (6 mL) was added to 4 mL of the bio-emulsifier and
vortexed at high speed for 2 min. A vernier caliper was used to take measurements after
24 hours. The emulsification index (E24) was obtained by dividing the height of emulsion
layer by the total height and multiplied by 100.
3.11 Statistical analysis of data
All the experiments were performed in triplicates and results were statistically
analyzed by one-way and two-way ANOVA and are presented as mean values with the
standard error calculated at 95 % confidence level.