effect of various factors (carbon sources, nitrogen

www.wjpps.com Vol 7, Issue 6, 2018.

1127

Venkatesh et al. World Journal of Pharmacy and Pharmaceutical Sciences

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


1128


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


1131


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


1132


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.


1138


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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effect of various factors (carbon sources, nitrogen

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