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EFFECT OF VARIOUS FACTORS (CARBON SOURCES, NITROGEN
SOURCES, TEMPERATURE, AND PH) ON GROWTH OF YEAST
ISOLATED FROM CURD AND CHEESE
G. Venkatesh1*, G. Vijaya Geetha
2 and N. Mounika Reddy
3
1Contractual Teacher, College of Horticulture, Anantharajupeta, Railway Kodur, A.P.
2G.Vijaya Geetha, M.Sc., Dept. of Microbiology, S.V.University, Tirupati, A.P.
3N.Mounika Reddy, M.Sc., Dept. of Microbiology, S.V.University, Tirupati, A.P.
ABSTRACT
Yeast is single-celled fungi. Some yeast is beneficial, being used to
produce bread or allow the fermentation of sugars to ethanol that
occurs during beer and wine production (e.g., Saccharomyces
cerevisiae). Other species of yeasts are detrimental to human health.
An example is Candida albicans, the cause of vaginal infections,
diaper rash in infants and thrush in the mouth and throat. Among the
samples screened for of yeast, 6 isolates were selected i.e., four from
curd and two from cheese sample. Isolation and Screening of the
thermotolerant yeasts was performed by culturing each isolate of the
yeast on YPD culture medium. The isolated cultures were tested for
their antimicrobial property, antibiotic sensitivity and the effect of different carbon sources,
nitrogen sources, pH levels, and temperatures were also tested in vitro. Results indicated that
the Enteropathogens like E.coli, Enterococcus and Klebsiella are sensitive to yeast where as
proteus vulgarius and s.aureus resistant to saccharomyces spp and it was resistance to
Gentamycin, Flucanazole, Ceftazidime and sensitive to Ampicillin, tetracycline, streptomycin
antibiotics. The maximum growth of saccharomyces spp. was found to be in glucose (1.51),
beef extract (1.43), pH 6.5 (1.64) and temperature 350c compared with reference strain
baker’s yeast. Further, the strain was found to vary morphologically with the baker’s yeast
under the study.
KEYWORDS: Yeast, saccharomyces spp, baker’s yeast, antimicrobial property, antibiotic
sensitivity test, temperature, pH.
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
SJIF Impact Factor 7.421
Volume 7, Issue 6, 1127-1139 Research Article ISSN 2278 – 4357
*Corresponding Author
G. Venkatesh
Contractual Teacher, College
of Horticulture,
Anantharajupeta, Railway
Kodur, A.P.
Article Received on
05 April 2018,
Revised on 26 April 2018,
Accepted on 16 May 2018,
DOI: 10.20959/wjpps20186-11761
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INTRODUCTION
Yeast is ascomycetous or basidiomycetous fungi that reproduce vegetatively by budding or
fission, and that for sexual states which are not enclosed in a fruiting body (Boekhout and
Kurtzman, 1996). The yeast species are all characterized by a similar set of features, both
morphological and physiological. This type of description, in which physiological characters
are important, distinguishes yeast taxonomy from other fungal taxonomy (Kreger-van Rij,
1984). Yeasts, which form one of the important subclasses of fungi, are rather more complex
and usually larger than bacteria. They are distinguished from most fungi by their usual
existence as single ovoid cells about 8μm long and 5μm in diameter, doubling every 1-3
hours in favourable media (Way man and Parekh, 1990). Yeast is unicellular fungus
containing 40% protein, 15% nucleic acid, 25% polysaccharides and 15% fat and the
remaining is accounted for water soluble compounds such as nucleotides, amino acids,
vitamins and minerals. The essential amino acid lysine content in yeast (7.8g/16g protein) is
comparable to Fish meal (7.4g/16g protein). Out of the 60 genera of yeast listed by kreger-
vanris (1984, 1987), the genus Saccharomyces has the greatest interest in the industrial world.
Although this genus consists of 41 species, only S.cerevisiae and related species S.boulardi
are utilized to a large extent in the industry. Yeast requires basic carbon and energy source,
minerals, vitamins, salts, source of nitrogen and phosphate for growth. These can be achieved
by manipulating various parameters including the pattern of substrate and nutrient addition.
Yeasts occur widely in nature and have been recovered from widely differing terrestrial as
well as marine sources. Certain yeasts are more or less ubiquitous while others appear to be
restricted to very specific habitats. Yeasts seldom occur in the absence of either molds or
bacteria (Kreger-van Rij, 1984). Yeasts are used in many industrial processes, such as the
production of alcoholic beverages, biomass and various metabolic products. The last category
includes enzymes, vitamins, capsular polysaccharides, carotenoids, polyhydric alcohols,
lipids, glycolipids, citric acid, ethanol, carbon dioxide and compounds synthesized by the
introduction of recombinants DNA into yeasts. Some of these products are produced
commercially while others are potentially valuable in biotechnology (Kurtzman and Fell,
1997). Some yeast species have potential to be uses in food, beverage and fermentation
industries. Some potential uses of yeasts in the food, beverage and fermentation industries
(Jacobson and Jolly, 1989).
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MATERIALS AND METHODS
Yeast strains were isolated from curd and cheese based on their growth at 30-370c.The
isolated cultures were tested for their antimicrobial property, and antibiotic sensitivity.
Optimization of cultural conditions and ethanol production, were performed for the selected
yeast isolates. Baker’s yeast was used as reference culture for comparison throughout the
study.
Samples
Samples (curd, cheese) were collected from market. Curd of room temperature and
refrigerated temperature and cheese of room and, refrigerated temperatures were collected.
Bakery yeast was collected from bakery units. Enteropathogens like E.coli, Enterococcus,
S.aureus, Proteus vulgarius and Klebsiella spp was collected from SVIMS hospital, Tirupati.
METHODS
Screening and isolation of yeast
The yeast was isolated from the above samples by using YPD culture medium.
Ypd Medium
Yeast extract - 2%
Peptone - 2 %
Glucose - 1%
Agar Agar - 2.5g
Distilled water - 100ml
One gram of the sample (cheese and curd) was taken and it was serially diluted up to 10-7
dilutions.0.1ml aliquots of 10-4
, 10-5
and 10-6
dilutions was spread on to the agar medium and
incubated at 370c for 24hrs.
Morphological Characteristics
Shape, size elevation, colour, state, pseudo mycelium, reproduction feature of surface were
studied under colony morphology.
In Vitro screening of Antimicrobial property
For detection of antagonistic activities of isolated strain well diffusion assay method was
used. For the agar well diffusion assay, an overnight cultures of enteropathogens was used to
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inoculate agar growth media. Wells of 5 mm diameter were made in agar plates and 50 µl of
isolated and screened yeast culture broth was added on the well made on the agar plates
culture containing antibacterial activity. The yeast culture broth was allowed to diffuse into
the agar. The plates were then incubated at 370c and examined after 24hr for zone of
inhibition. Previously the actively growing enteropathogens like E.coli, Enterococcus,
S.aures, Proteus vulgarius and Klebsiella culture suspension was spread evenly on the
surface of agar plate. After incubation the growth inhibition zones were observed around the
well on each plate.
Antibiotic Sensitivity Test
For detection of antibiotic sensitivity of the isolated strain disk diffusion method was used.
For the disk diffusion method, culture medium was prepared and poured into the plates and
allowed to solidify. After solidification, the suspension of actively growing isolated yeast was
spread on the agar plates. Different antibiotic discs Gentamycin (G) Streptomycin (S),
Tetracycline (T), Ampicillin (A), flucanazole and Ceffazidine (C) was placed on culture
inoculated plates with the help of sterile forceps and gently press to make proper contact with
agar and kept for incubation at 370c for 24- 48hrs.After incubation the zone of inhibition were
observed around each disc.
Optimization of Cultural Conditions
The yeast isolated yeast was carried out to optimize the parameters namely carbon sources,
nitrogen sources, pH, and incubation temperatures.
Effect of carbon sources on the growth of yeast
Various carbon sources such as Glucose, Sucrose, Fructose, Maltose and Lactose were
supplemented at 1 %( w/v) to study their effect on yeast production. The media with different
carbon sources were sterilized, cooled and poured in the sterilized tubes and then inoculated
with the isolated spp. After 24hrs incubation, the turbidity was measured in colorimeter at
600nm.
Effect of nitrogen sources on the growth of yeast
The effect of various nitrogen sources were studied by growing the yeast in the medium
containing various nitrogen sources like tryptone, peptone ammonium nitrate, yeast extract,
beef extract. The media with different nitrogen sources were sterilized, cooled and poured in
the sterilized tubes and then inoculated with the isolated spp. After 24hrs incubation, the
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turbidity was measured in colorimeter at 600nm.
Effect of pH on the growth of yeast
The effect of pH on the growth of the selected yeast was studied. Different pH levels viz., 3.5,
4.5, 5.5, 6.5 and 7.5 were used. The pH levels of the medium were adjusted in a digital pH
meter using 0.1 N Hydrochloric acid and 0.1 N Sodium hydroxide. The media with different
pH levels were sterilized, cooled and poured in the sterilized tubes and it is inoculated with
selected yeast. The tubes were incubated at 350c ± 2°C for 24hrs. The growth of yeast was
measured at 600 nm on the next day after inoculation.
Effect of temperature on the growth of yeast
The effect of temperature on growth of the selected yeast was studied. Different temperatures
maintained for the growth of selected yeast in broth media were 33°C, 35°C, 37°C, 39°C,
41°C and 43°C. Inoculated tubes were kept in incubator and temperature was adjusted to
required level. The growth of yeast was measured at 600 nm on the next day after inoculation.
Production of ethanol (Brady July, 1994)
The selected yeast was used for fermentative production. The ethanol production efficiency of
the yeast strains was tested in 250-mL conical flasks containing 100 mL of YM broth and 200
g/L of glucose. The YM medium was inoculated with selected yeast strains at an initial yeast
cell concentration of approximately 1 × 107 cells/mL. Then the cultured flask was tightly
plugged; centrifuged at 13,000 rpm for 10 min and incubated at 37 °C or 40 °C for 3-4 days.
Viable yeast cell numbers were determined by the direct counting method using a
haemocytometer and the methylene blue staining technique. The isolated yeast spp. was
grown as thick mat in the medium during incubation period. After incubation, the medium
was filtered and this filtrate was used for ethanol estimation.
Estimation of ethanol
Required volume of stock solution was prepared by using absolute ethanol. Different gradient
was prepared from the stock solution and used for preparation of standard curve by using
potassium dichromate method.
RESULTS AND DISCUSSION
Isolation
Among the samples screened for of yeast, 6 isolates were selected i.e., four from curd and
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two from cheese sample was made as described in material and methods. The pure culture
obtained was again sub cultured and kept in the refrigerator at 5°C for further studies.
Growth and colony characteristics
The morphological features of yeast colonies grown on culture agar for 48hrs are shown in
Table 1. On the culture agar plate colonies observed were white, powdery, rough, showing
budding stage. The morphologically tested isolates was grown on agar plates 350c ± 2°C for
screening of yeast. Among the six isolates screened only one strain shows maximum growth
at 370c. The selected yeast was tested for antimicrobial, antibiotic properties, optimization
and ethanol production of yeast (Table-2).
Table-2: Yeast cells morphological features.
S.No. Morphological features Isolated yeast
1. Colour White
2. State Powdery
3. Shape of rising Convex
4. Feature of surface Rough
5. Reproduction Budding
Microscopic characteristics
The yeast was observed under microscope. Yeast cells were simple filaments to elaborate pseudo
hyphae and budding cells was also noticed.
Antimicrobial property
The antimicrobial property of yeast was studied. The results revealed that, enteropathogens
like E.coli, Enterococcus and Klebsiella are sensitive to yeast where as proteus vulgarius and
s.aureus resistant to isolated yeast (Table-3).
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Table-3: Antimicrobial property of yeast.
Enteropathogens Zone of inhibition
E.coli +
Enterococcus +
Klebsiella +
S.aureus -
Proteus vulgarius -
Note: (+) indicates clear zone around disc (resistant to Enterococcus, E.coli and klebsiella (-)
no zone formation (sensitive to S.aureus and Proteus vulgarius)
Antibiotic sensitivity test
The antibiotic sensitivity of yeast was studied. The results revealed that the yeast was
resistance to Gentamycin, Flucanazole, Ceftazidime and sensitive to Ampicillin, tetracycline,
streptomycin (Table-4).
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Note: A = Ampicillin, T = Tetracycline
S = Steptomycin G = Gentamycin
Fu = Flucanazole Cac = Caftazidime
Table-4: Antibiotic sensitivity test of yeast.
Antibiotic discs Zone of inhibition
Gentamycin -ve
Flucanazole -ve
Ceftazidime -ve
Ampicillin +ve
Tetracycline +ve
Streptomycin +ve
Note: (+ve) indicates clear zone around disc (resistance) (-ve) no zone formation (sensitive)
Effect of carbon sources on the growth of yeast
Effect of different carbon sources on the growth of yeast isolated was studied. The results
revealed that a highest growth of (1.51) was observed at 37°C in Glucose which was followed
by Sucrose (1.39); Maltose (1.35); Fructose (1.25) and the growth was found to be the lowest
in Lactose as it recorded 1.06 and these are compared with reference strain baker’s yeast
(Table-5).
Table 5: Effect of carbon sources on the growth of yeast.
S.No. Carbon source Isolated yeast Baker’s yeast
1. Sucrose 1.39 1.45
2. Glucose 1.51 1.13
3. Fructose 1.25 1.03
4. Maltose 1.35 0.98
5. Lactose 1.06 1.00
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Effect of nitrogen sources on the growth of yeast
Effect of different nitrogen sources on the growth of yeast isolated was studied. The results
revealed that a highest growth of (1.43) was observed at 37°C in Beef extract which was
followed by Peptone (1.37); Yeast extract (1.26); Tryptone (1.08) and the growth was found
to be the lowest in Ammonium nitrate as it recorded 0.11 and these are compared with
reference strain baker’s yeast (Table-6).
Table 6: Effect of nitrogen sources on the growth of yeast.
S.No. Nitrogen source Isolated yeast Baker’s yeast
1. Yeast extract 1.26 1.02
2. Beef extract 1.43 1.18
3. Peptone 1.37 1.04
4. Tryptone 1.08 0.88
5. Ammonium nitrate 0.11 0.05
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Effect of pH on the growth of yeast
Effect of pH on the growth of yeast isolated was studied. The results revealed that maximum
growth of (1.64) was observed at pH6.5 followed by pH7.5 (1.52); pH5.5 (1.47); pH4.5
(1.41) and the lowest growth were recorded at pH-3.5 (1.18) and these are compared with
reference strain baker’s yeast (Table-7).
Table 7: Effect of pH on the growth of yeast.
S.No. pH Isolated yeast Baker’s yeast
1. 3.5 1.18 0.99
2. 4.5 1.41 1.00
3. 5.5 1.47 1.11
4. 6.5 1.64 1.38
5. 7.5 1.52 1.33
Effect of temperature on the growth of yeast
Effect of temperature on the growth of yeast isolated was studied. The results revealed that
maximum growth of (0.61) was observed at 370C followed by 390C (0.63); 350C & 410C
(0.60); 330C (0.55); and the lowest growth were recorded at 430C (0.52) and these are
compared with reference strain baker’s yeast (Table-8).
Table 8: Effect of temperature on the growth of yeast.
S.No. Temperature Isolated yeast Baker’s yeast
1. 330C 0.55 0.42
2. 350C 0.60 0.43
3. 370C 0.61 0.45
4. 390C 0.63 0.48
5. 410C 0.60 0.49
6. 430C 0.52 0.51
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ETHANOL PRODUCTION
The amount of ethanol estimated in isolated yeast species was 0.18ml/100ml
DISCUSSION
Yeast are used in many industrial processes, such as the production of alcoholic beverages,
biomass and various metabolic products. The last category includes enzymes, vitamins,
capsular polysaccharides, carotenoids, polyhydric alcohols, lipids, glycolipds, citric acid,
ethanol, carbon dioxide, and compounds synthesized by the introduction of recombinants
DNA in to yeasts. Some of these products are produced commercially while others are
potentially valuable in biotechnology. Some yeast species have potential to be uses in food,
beverages and fermentation industries. The yeast grows rapidly and can be cultured easily.
Saccharomyces species are emerging as potential organisms.
In our present study yeast species were isolated from curd and cheese samples for yeast
production. Six yeast isolates were isolated and screened on YPD agar plates by incubating at
370c. The yeast species were identified using morphological and physiological characteristics.
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The selected yeast species was subjected for detection antimicrobial and antibiotic sensitivity
properties. Baker’s yeast was used as reference culture for comparison. Among the isolates
screened only one strain was selected for optimization of cultural conditions was optimized
for enhanced production.
Previously it was proved that glucose; fructose and maltose were good carbon sources. The
highest growth was observed with supplementation of sucrose then in glucose, maltose,
fructose and lactose. Different nitrogen sources were used for maximum growth of yeast
species. The highest growth was observed in beef extract then in yeast extract, peptone,
ammonium nitrate and tryptone. In different pH like 3.5, 4.5, 5.5, 6.5 and 7.5 the highest
growth was observed in 6.5. In different temperatures like 330C; 35
0C; 37
0C; 39
0C; 41
0C and
430C, the highest growth was observed at 37
0C. The selected yeast specie was subjected to
antimicrobial property against enteropathogens (E.coli, Enterococcus, S.aureus, Proteus
vulgarius, klebsiella). Saccharomyces species was resistant to E.coli, Enterococcus, and
klebsiella. The yeast specie was sensitive to antibiotics like ampicillin, tetracycline and
streptomycin and resistant to gentamycin, flucanazole, ceftazidime.
CONCLUSION
In our present study yeast species were isolated from curd and cheese samples for yeast
production. Six yeast isolates were isolated and screened on YPD agar plates by incubating at
370c. The selected yeast species was subjected for detection of antimicrobial and antibiotic
sensitivity properties. Baker’s yeast was used as reference culture for compression. The yeast
species have maximum growth when compared with baker’s yeast. The optimal conditions
were studied for the isolation of yeast species. The isolated yeast showed maximum growth
with Beef extract (1.43); Glucose (1.51); temperature (370C); pH6.5 (1.64); when compared
to Baker’s yeast. Hence yeast species were successfully isolated and optimized.
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