A
PROJECT REPORT
ON
“PRODUCTION OF SINGLE CELL PROTEIN FROM PINEAPPLE
WASTE AND ORANGE PEEL USING YEASTS AND Aspergillus niger”
A PROJECT REPORT
Submitted in partial fulfillment of the
Requirements for the award of the degree of
Master of Science
In
Department Of Biotechnology
By
NIRAV J GHANTALA
BHAGWAN MAHAVIR COLLEGE OF M.Sc BIOTECHNOLOGY
(AFFILIATED TO VEER NARMAD SOUTH GUJARAT UNIVERSITY UNIVERSITY)
SURAT
2012
1
CERTIFICATE
2
ACKNOWLEDGEMENT
I thank the almighty whose blessings have enabled me to accomplish my dissertation work
successfully.
It is my pride and privilege to express my sincere thanks and deep sense of gratitude to Priya
Bande, Department of Biotechnology and Environmental Sciences, MITCON, pune for her
valuable advice, splendid supervision and constant patience through which this work was
able to take the shape in which it has been presented. It was her valuable discussions and
endless endeavors through which I have gained a lot. Her constant encouragement and
confidence-imbibing attitude has always been a moral support for me.
My sincere thanks to Dr.Chandrashekhar kulkarni, Head Department of Biotechnology and
Environmental Sciences, for his immense concern throughout the project work.
I also wish to thank all my friends, for providing the mandatory scholastic inputs during my
course venture.
Finally, I wish to extend a warm thanks to everybody involved directly or indirectly with my
work.
The whole credit of my achievements goes to my parents and my brother who were always
there for me in my difficulties. It was their unshakable faith in me that has always helped me
to proceed further.
3
DECLARATION
I hereby declared that the work presented in the Project entitled ””has been carried out by
GHANTALA NIRAV J. under the guidance of Priya Bande, Project Guide, at MITCON, Pune.
The entitled Work is original and no part of this work is either published or submitted in any
university for the award of any degree or diploma.
Date:
Place: Pune
4
S.NO. CONTENTS
PAGE.NO
1. Introduction
7
2. Materials & methods
22
3.Observation Tables
29
4. Results
36
5. conclusion
41
6.References
41
7.Appendix
43
5
Abstract
“PRODUCTION OF SINGLE CELL PROTEIN FROM DIFFERENT
AGRICULTURE WASTE USING YEASTS”
People in third world and developing countries are suffering from menace of protein
deficiency in their diets resulting in serious protein-energy malnutrition problems.The
worldwide food protein deficiency is becoming alarming day to day and with the fast
growing population of world,Pressure is exerted on the feed industry to produce enough
animal feed to meet the region’s nutritional requirements.Single-Cell Protein (SCP)
represents microbial cells (primary) grown in mass culture and harvested for use as protein
sources in foods or animal feeds. In the present study, pineapple waste was used as sole
carbon source in five concentrations for preparation of fermentation media on which two
strains of yeasts, Saccharomyces cerevisiae,Candida tropicalis,Pichia stipites and Aspergillus
niger and were grown. The increased concentration of pineapple hydrolysate and orange peel
enhanced the biomass yield and the protein formation within the yeast cells. Lower carbon
utilization by the two yeast strains occurred in the wastecontaining media, as compared to
control, increasing the economic value of the waste obtained after 7-day fermentation.The
present finding helps in SCP production from cheap, inexpensive agro waste material.
Key words:
Yeast, SCP, Pineapple waste,Orange peel, Saccharomyces cerevisiae, Candida
tropicalis,Pichia stipites, Aspergillus niger.
6
1.INTRODUCTION
1.1General Introduction
Production of SCP by mass culture of microorganisms is yet to take momentum at industrial
scale and deserve much attention to solve the problem of starvation in the coming decades.
The criteria for microorganisms to be used as food or feed include 1.the organism must be
genetically stable, non toxic and grow rapidly on a simple non specific medium.2. it should
have high nutritional or vitamin content and should be edible to human and other animals.
The organisms should also utilize the energy source without producing any side effects and
any undesirable effects. 3. it should be easy to separate the cells from the medium and the
product must have good quality and composition. Among the various groups of
microorganisms used to produce SCP, yeasts are perhaps the most important groups because
yeasts produce many bioactive substances such as proteins, amino acids, vitamins,
polysaccharides, fatty acids, phospholipids, polyamines, astaxanthins, β-carotenoids,
trehalose, glutathione, superoxide dismutase, chitinase, amylase, phytase, protease, killer
toxin etc. which have been receiving much attention for many decades. The main nutritional
contribution in either human food or animal feed is its high protein content. Because of high
protein and fat content, the contribution of carbohydrates to the nutritional value of SCP is
not of prime importance. But a major constraint for yeast as SCP is the thick cell wall which
is difficult to digest leading to poor protein bioavailability.
Agricultural activities and food industry generate considerable quantities of wastes which are
rich in organic matter and could constitute new materials for value added products. A
growing concern for the acute food shortages for the world’s expanding population has led to
the exploitation of non-conventional food sources as potential alternatives. Among these, the
7
single cell organisms probably present the best chances for the development of unique
independence of agricultural crop based food supply.
Single cell protein is a biomass based on protein extract derived from microorganism.Single
cell protein offers numerous advantages for the productions of proteins because of its high
productivity,low cost media,less effort and product with added
nutritional market
value(pandey et al.,1992). The single cell protein is a dehydrated cell consisting of mixture of
proteins, lipids,carbohydrates, nucleic acids, inorganic compounds and a variety of other non protein
nitrogenous compounds such as vitamins. Agricultural wastes are useful substrate for production of
microbial protein, but must meet the following criteria; it should be non toxic, abundant, totally
regenerable,non-exotic, cheap and able to support rapid growth and multiplication of the organisms
resulting in high quality biomass.
The continued population growth especially in developing and third-world countries is
resulting in increased food demand in parallel and is posing serious threats to food security
due to yawning gap in demand and supply (Anupama and Ravindera, 2000). Chronic
malnutrition and hunger are typically most prevalent in developing countries. Malnutrition is
a consequence of not taking appropriate amount or quality of nutrients comprising diet. The
gap b/w demand and supply is expected to grow unless planned actions are taken to improve
the situation. Therefore it is essential to search for un-conventional or novel proteins to
supplement the available sources. Because of population explosion and the limited land resources,
the world will be soon unable to feed its population. At the present time the food problem is limited
mainly to developing countries of Asia, Africa and South America.However, Predictions show that
more advanced nations will eventually face the same problems. The developing of novel food
production process independent of agricultural land use is thus becoming imperative.
8
There have been studies as well as efforts to improve the protein quantity and quality of the
finished food products by augmenting protein-rich cheaper ingredients in food formulations
(Nasir and Butt, 2011;Hussain et al., 2007). Although animal proteins are considered to be
best quality proteins (Saima et al., 2008), however microbial protein also known as single
cell protein grown on agricultural wastes is one of the important optional proteins because of
higher protein content and very short growth cycle of microorganisms,thereby, leading to
rapid biomass production (Bekatorou et al., 2006) Moreover, microbes are also able to grow
on cheap nutrient sources resulting in economical,potentially supplemental protein biomass
for balanced nutrition.
The UN work for the servey of food consumption of worldwide.It recently reported that over
1 billion people currently do not have access to adequate amounts of food and that number
could climb even higher in the near future if current drought conditions persist. Based on UN
research, it was estimated a decrease in agricultural production of 20-40% if drought
conditions continue and concluded that 2009 could be the beginning of a record-breaking
humanitarian crisis throughout much of the world.The demand for protein as food is
expanded for malnourished human populations. Consequently,the cost of protein secondary
use as food for livestock has also risen. Increasingly there was always a stimulus needed to
introduce an additional and or complementary source of animal feed microorganisms that are
considered for food or feed use including algae, bacteria, yeasts, molds, and higher fungi. The
dried cells of these organisms are collectively referred to as single cell protein. Different
types of microorganisms have been recommended for human consumption, including yeast,
molds and algae. But as of now only yeast have been used as food to any extent and then
under unusual conditions. During Second World War when there were shortages in proteins
and vitamins in the diet, the Germans produced yeast and mold Geotrichum candidum in
9
some quantity for food. Research on single cell protein has been stimulated by food crisis or food
shortages that will occur if the world’s populations are not controlled. Scientists believe that use of
microbial fermentations and the development of an industry to produce and supply single cell proteins
from agricultural waste are insufficient.
The present study was focused on yeast single cell protein rather than bacterial, fungal and algal
single cell protein. Algal single cell protein have limitations such as the need for warm temperatures
and plenty of sun light in addition to carbon dioxide, and also that the algal cell wall is indigestible.
Bacteria are capable of growth on a wide variety of substrates, have a short generation time and are
high protein content. Their use is somewhat limited by poor public acceptance of bacteria as food,
small size and difficulty of harvesting and high content of nucleic acid on a dried weight basis. Yeasts
are probably the most widely accepted and used microorganisms for single cell protein. These include
strains of Candida utilis, C. arborea, C. pulcherrima and Saccharomyces cerevisiae.Pichia stipitis is
also used for SCP production in the present study. In recent years increasing attention has been
given to the conversion of food processing wastes into valuable by-products such as the
production of yeast protein from potato (Skogman 1976) and confectionery effluents (Forage
1978). The recovery of such by-products can significantly reduce the costs of waste disposal.
Pineapple is an important fruit crop, belonging to the family Bromeliaceae.Pineapple
(Ananas sativus Schult. f.) is widely grown in the north eastern region of India.In India nearly
100 different cultivars are known, of which ‘kew’ and ‘Queen’ are most useful and cultivated
widely. In India estimates of the area under pineapple vary from 24,350 ha to 56,000 ha. The
world shortage of protein has stimulated the interest of scientists for the production of
unconventional protein – rich food and feed-stuffs. The production of single cell protein
(SCP) by fermentation processes has been mentioned by many investigators and the
impressive advantages of microorganisms for single cell protein (SCP) production compared
10
with conventional sources of protein (soybeans or meat) are well known.A number of
agricultural and agro industrial waste products have been used for the production of SCP and
other metabolites, including orange waste, mango waste, cotton salks, kinnowmandarinwaste, barley straw, corn cops, rice straw, cornstraw, onion juice and sugar cane
bagasse (Nigam et al., 2000), cassava starch (Tipparat et al., 1995), wheat straw (Abou
Hamed, 1993), banana waste (Saquido et al., 1981), capsicum powder (Zhao et al., 2010) and
coconut water (Smith and Bull, 1976). The usage of such wastes as a sole carbon and
nitrogen source for the production of SCP by microorganisms could be simply attributed to
their presence in nature on large scale and their cheap cost.
1.2 Nutritional / Biochemical composition
The commercial value of single cell protein depends on their nutritional performance. The
main nutritional contribution of SCP is its high protein content which varies depending upon
the kind of microorganisms and substrates used for production. The mean crude protein
content in dry matter of yeasts account for about 50 %. The amino acid composition of a
protein primarily determines its potential nutritional value. The protein efficiency ratio and
biological value of yeast protein are known to be relatively high (Munro, 1964). Composition
of growth medium governs the protein and lipid contents of microorganisms. Yeasts, moulds
and higher fungi have higher cellular lipid content and lower nitrogen and protein
contents,when grown in media having high amount of available carbon as energy source and
low nitrogen (Litchfield, 1979). The lipid composition of microorganisms is very responsive
to changes in the chemical and physical properties of the environment. Among the
environmental factors that have been reported to affect the lipid composition of
microorganisms are growth rate, composition of the medium, growth temperature, and
dissolved O2 tension in the culture. Since, with most organisms, the bulk of the cell lipids are
11
in membranes, it is likely that these environmentally induced changes in lipid composition
are of major physiological significance. Microorganisms contain a diverse range of fatty acid
composition which can be useful as a chemotaxonomic tool for classifying species and
strains. Fatty acids typically comprise 70-90 % of the lipids in yeast with oleic acid (18: 1 n9) being the commonest found.
Fatty acids, the simplest of lipids are required for membrane structure, function, transport of
cholesterol, formation of lipoproteins etc. Composition of growth medium governs the lipid
content of microorganisms. Microbial fatty acid profiles are unique from one species to
another. The fatty acids occur as esters in triacylglycerol, phospholipids, glycolipids or
sterols in membranes and other cytoplasmic organelles, such as the mitochondria,
plasmalemma, endoplasmic reticulum, nuclei, vacuoles, spores and lipid particles. The 14: 0
fatty acids are only seen as trace fatty acyl residues. The microbial identification system
based on fatty acid methyl ester (FAME) analysis has been used in laboratories for the
identification of clinical yeast strains (Peltroche-Liacsahuanga et al., 2000). The system
analyses long-chain fatty acids containing 9–20 C atoms, identifying and quantifying the
FAMEs
of
microorganisms.
The
database
library
searches
for
fatty
acid
composition,compares the FAME profile of the isolate with those of well characterized
strains and defines the most likely species of the isolate. Fatty acid composition of cold
adapted carotenogenic basidiomycetous yeasts was studied by Libkind et al. (2008). Total
fatty acids of six yeast species isolated from the temperate aquatic environments in Patagonia
ranged from 2 to 15 % of dry biomass.Linoleic, oleic, palmitic and α-linolenic acids were the
major fatty acid constituents,which accounted for as much as 40%, 34%, 13% and 9% of total
fatty acids, respectively. The proportion of each varied markedly depending on the taxonomic
12
affiliation of the yeast species and on the culture media used. The high percentage of
polyunsaturated fatty acids (PUFAs) found in Patagonian yeasts, in comparison to other
yeasts, is indicative of their cold-adapted metabolism.
Yeast proteins are easily digestible compared to those from bacteria. Chemical analysis of
microorganisms tested for SCP reveal that they are comparable in amino acid content to the
plant and animal sources with the exception of sulphur amino acid methionine which is low
in some SCP sources, especially yeasts. However, this can be alleviated by culturing yeasts
on molasses (Bhalla et al., 1999). The only species of yeast fully acceptable as food for
humans is S. cerevisiae (baker’s and brewer’s yeast) (Bekatorou et al., 2006).
The majority of the SCP are either deficient in one or more amino acids or they suffer from
an amino acid imbalance (Tacon and Jackson, 1985; Kiessling and Askbrandt, 1993). The
supplementation of yeast-based diets with the deficient amino acids was shown to have
beneficial effects on fish growth (Nose, 1974; Spinelli et al., 1979; Murray andMarchant
1986).
Another concern with SCP is their high concentration in nucleic acids, ranging from 5 % to
12 % in yeast and 8 % to 16 % in bacteria (Schulz and Oslage, 1976). In rapidly proliferating
microbial cells, RNA forms the bulk of nucleic acids. The RNA content of yeast cells is
known to be dependent on the culture conditions and C/N ratios. The marine yeasts with high
levels of nucleic acids could be used as a feed to marine animals because some of them can
produce uricase which convert uric acid, the toxic intermediate of nucleic acid catabolism
into the non-toxic allantoin. The nutritional value of the yeast depends also on the
concentrations of other micronutrients such as sterols, vitamins and minerals. Yeasts have
usually high concentrations of sterols (typically 1-10 % of total lipids) which are required for
growth and survival of molluscs (Brown et al., 1996).
13
1.3 Factors Affecting Yeast Growth
Yeast growth is affected by a number of factors. These include composition of medium
commonly sugar source, aeration (oxygen), agitation of the medium, pH, temperature and period
of propagation. Some of the factors affecting yeast growth are discussed below.
14
1.3.1 Sugar feed (media composition)
The main carbon and energy source for most yeast is glucose which is converted via the
glycolytic pathway to pyruvate and by the Krebs cycle to anabolytes and energy in the form of
ATP.Yeasts are further classified according to their modes of further energy production from
pyruvate: respiration and fermentation. These processes are regulated by environmental factors,
mainly glucose and oxygen concentrations.
In respiration, pyruvate is decarboxylated in the mitochondrion to acetyl- CoA which is
completely oxidized in the citric acid cycle to CO , energy and intermediates to promote yeast
2
growth.
15
In anaerobic conditions, glucose is slowly utilized to produce the energy required just to keep the
yeast cells alive. This process is called fermentation, in which the sugars are not completely
oxidized yielding CO and ethanol (Scragg, 1991; Bekatorou et al., 2006) as final product. When
2
the yeast cell is exposed to high glucose concentration, catabolite repression occurs, during which
gene expression and synthesis of respiratory enzymes are repressed, and fermentation prevails
over respiration(Rincon et al.,2001: Bekatorou et al.,2006).
In industrial practice, catabolite repression (repression of gluconeogenesis, the glyoxylate cycle,
respiration and the uptake of less preferred carbohydrates) by glucose and sucrose, also known as
Crabtree effect, may lead to several problems, such as incomplete fermentation, development of
off flavors and undesirable by products as well as decreased biomass and yeast vitality
(Verstrepen et al., 2004; Bekatorou et al., 2006).
Industrial production of Saccharomyces cerevisiae is therefore, performed in aerobic, sugar
limited fed-batch cultures (Van Hoek et al., 1998; Mickiewicz and Borowiak, 2005).
16
When the yeast cell is exposed to high glucose concentration, catabolite repression occurs, during
which gene expression and synthesis of respiratory enzymes are repressed, and fermentation
prevails over respiration(Rincon et al.,2001: Bekatorou et al.,2006).
In industrial practice, catabolite repression (repression of gluconeogenesis, the glyoxylate cycle,
respiration and the uptake of less preferred carbohydrates) by glucose and sucrose, also known as
Crabtree effect, may lead to several problems, such as incomplete fermentation, development of
off flavors and undesirable by products as well as decreased biomass and yeast vitality
(Verstrepen et al., 2004; Bekatorou et al., 2006).
Industrial production of Saccharomyces cerevisiae is therefore, performed in aerobic, sugar
limited fed-batch cultures (Van Hoek et al., 1998; Mickiewicz and Borowiak, 2005).
17
1.3.2 Aeration
Highly aerobic culture conditions are used in the production of yeast specifically
baker’s yeast to maximize cell growth (Campelo and Belo, 2004). The modern
technique of baker’s yeast production is based on applying the principle of the Pasteur
reaction at the limit value of its aeration. Pasteur defined fermentation as life without
air. Its biochemistry involves the breakdown of carbohydrates only to the stage of
ethanol.
Under aerobic conditions, however, maximum growth occurs and the efficiency of
utilization of carbohydrate increases as respiration and the breakdown of the
carbohydrate to carbon dioxide and water becomes complete (Cook, 1958).
Generally oxygen has the following basic functions (Prescott and Dunn, 1959).
1. Inhibits fermentation
2. Increases respiration
3. Agitation of the medium
4. Removal of toxic end products
5. Stimulation of vegetative growth
18
Oxygen is used in the synthesis of unsaturated fatty acids and sterols which form the cell
membrane. These molecules are important for both growth and fermentation and serve as a means
for storing oxygen within the cell. They are also necessary for increasing cell mass (growth)
involving the over all uptake of nutrients and determining alcohol tolerance. Oxygen stimulates
the synthesis of molecules necessary for yeast to metabolize and take up maltose and other sugars
1.3.3. Temperature
The temperature most favorable to the growth of baker’s yeast varies from strain to strain.
0
0
0
Optimum temperature is usually between 25 C-35 C. The maximum survival temperature is 37 C
(Cook, 1958). Propagation at low temperature, the rate of growth is slower and gives a decreased
yield of yeast. Yeast grown in low temperature is less stable when stored and transported as a
pressed cake and the dry matter of a yeast cake of standard consistency becomes progressively
less at low temperature, i.e., it affects the relationship between intracellular and extra cellular
water content.
1.3.4 pH
Yeasts grow well at acidic pH (acidophilic organisms). For industrial propagation low pH is
helpful in restricting the development of many bacterial contaminations; however, the color of the
yeast may be affected at low pH. The pH of the media is commonly adjusted by the addition of
H SO NH , Na CO or NaHCO (Prescott and Dunn, 1959) to the substrate.
2
4,
3
2
3
3
1.3.5 Yeast Dry Matter
The main elements present in baker’s yeast are carbon, hydrogen, oxygen and nitrogen which
normally account for as much as 94% of the dry matter (White, 1954). Table 3 shows elementary
analysis of yeast dry matter.
19
TABLE:1 Elementary analysis of yeast dry matter (White, 1954).
Constituent
Percent of yeast dry
matter
carbon (C)
45.0-49.0
Hydrogen ( H)
5.0- 7.0
Oxygen ( O)
30.0 -35.0
Nitrogen (N)
7.1 -10.8
Total ash
4.7- 10.5
Phosphate ( P2O5)
1.9- 5.5
Potash ( K2O)
1.4- 4.3
Calcium (CaO)
0.005 – 0.2
Magnesium ( MgO )
0.1 – 0.7
Aluminum ( as Al2O3)
0.002 – 0.02
Sulphate ( as SO4)
0.01- 0.05
These four elements are present on the yeast in the form of carbohydrates (glycogen,
cellulose, and yeast mannan), protein and lipids (true fats, lecithin, and sterols). Yeasts also
contain high amount of vitamin B complex, nucleic acids and organic bases (pyramidine and
purine bases). The inorganic substances may constitute up 6 to 8 % of the yeast dry matter
(White,1954;Roman,1957) Table 4 gives the quantities of some substances present in yeast
dry matter (White, 1954).
20
Table 4. Composition of yeast dry matter (White, 1954).
Constituent
Percent of yeast dry matter
Ash
Normally 6 -8
Glycogen
1 -30
Fat soluble fraction(true fats, sterols. lipids
1 -2. 2
Yeast gum (mannan )
Up to 4
Cellulose ( yeast )
Up to 5
Proteins and organic nitrogenous bases
44 – 47
REVIEW OF LITERATURE
The work carried out on Comparative assessment of various agro-industrial wastes for
Saccharomyces cerevisiae biomass production and its quality evaluation as Single cell
protein(U. Bacha, M. Nasir, A. Khalique, A. A. Anjum* and M. A. Jabbar in 2011)
This study was planned to assess the feasibility of using agro-industrial wastes for
Saccharomyces cerevisiae production and to evaluate protein quality of produced single cell
protein (SCP) biomass. Potato peels contained significantly highest dry matter and
carbohydrate content as compared to other wastes. Significantly higher SCP biomass was
produced using potato peels followed by carrot peels. On the basis of higher SCP biomass
production, potato peels were selected for further biomass production. The SCP biomass
contained 49.29±1.126% crude protein which was non-significant (P=0.1710) compared to
commercially available Saccharomyces cerevisiae. The parameters for in-vivo protein quality
assay in Sprague Dawley rats were; 93.68% true digestibility, 67.02% net protein utilization,
21
70.56% biological value, 4.55net protein ration, and 2.75 protein efficiency ratio, which are
higher in comparison to most of cereal proteins. The present exploration depicted that
Saccharomyces cerevisiae can be efficiently produced utilizing wastes and the produced
biomass can potentially be used as protein source in various food formulations.
The work carried out on Production of Single cell protein from Pineapple waste using
yeast(Dharumadurai
DHANASEKARAN1*,
Subramaniyan
LAWANYA,
Subhasish
SAHA1, Nooruddin THAJUDDIN 1, Annamalai PANNEERSELVAM2)
In this work pineapple waste was used as sole carbon source in five concentrations for
preparation of fermentation media on which two strains of yeasts, Saccharomyces cerevisiae
and Candida tropicalis were grown. The increased concentration of pineapple hydrolysate
enhanced the biomass yield and the protein formation within the yeast cells. Lower carbon
utilization by the two yeast strains occurred in the wastecontaining media, as compared to
control, increasing the economic value of the waste obtained after 7-day fermentation.
2. MATERIALS AND METHODS
(a) GLASSWARE
• Petri dishes,
• Glass slides,
• Glass beakers,
• Cover slips,
• Media bottles,
• Conical flasks,
• Pipette,
• Test tubes,
22
(b )REAGENTS REQUIRED
• Alcohol,
• Distilled water,
• Ethanol
• Concentric sulfuric acid
(c)EQUIPMENTS REQUIRED
• Ph meter
• Inoculating loops,
• Weighing machine,
• Incubator,
• Autoclave(Meta instrument,Mumbai),
• Refrigerator,
• Laminar air flow,(Microfilt)
• Microscope,
• Measuring cylinder

Spectrophotometer

Vortex machine

Centrifuge(Remi)

Waterbath
2.1COLLECTION OF PINEAPPLE WASTE AND ORANGE PEELS
The ripe, yellowish and firm pineapple fruits ,Orange peels collected from local market.Pineapple
waste and Orange peels were cut into slices in order to hydrolysis.Put this slices in oven at
135 C for 24 hours For the moisture content to know.
23
2.2MICROORGANISM
The yeast culture such as Saccharomyces cerevisiae ,Candida tropicalis and pichia stipitis were
obtained by using isolation. The cultures were maintained on slant of yeastpeptone dextrose
medium(Yeast extract 10g, Dextrose 20g, Peptone 20g, Agar 20g, pH = 6.0,Distilled water 1000ml)
and stored at 4oC.Aspergillus niger is also used in this study.
The yeasts culture was used in the experiments because of its ability to ferment sugar. The
cultures of yeasts were isolated from soil by pour plate method. Dry powdered yeast (Instant
Yeast, Levure Instantane) was also used. The samples were streaked on YPD-agar medium
composition yeast extract (3.0g/L); peptone (10g/L); dextrose (20g/L); agar-agar (15g/L)
(YPG-agar) and incubated at 30ºC for 24h.. The best culture was selected for further studies.
Selected cultures were stored at 4ºC
2.3 METHODS
2.3.1 ISOLATION OF Aspergillus niger
1. Soil samples were collected from agriculture college campus,pune.
2. 1 g of each soil sample was individually suspended in 1 mL of sterile distilled water
3. Serially diluted to 10–3 fold
4. And plated on potato dextrose agar (PDA) plates containing kanamycin to a final
concentration of 10–6.
5. The plates were incubated at 30 °C for 3 days in an environmental chamber under
controlled conditions.
6.Single colony of fungus was isolated and transferred repeatedly to a new PDA plate
until pure cultures were obtained. These were grown on PDA slants as above and
stored at 4°C.
24
2.3.2 PREPARATION OF INOCULUM
Inoculum was prepared by adding 80% twin 80 in the slant and scrapping the slant with nicrome
wire and then make 2.0 ml volume.
2.3.3 IDENTIFICATION TESTS
The identification of the fungi is based upon a combination of morphological and biochemical
criteria. Morphology is primarily used to establish the genera, whereas biochemical assimilations
are used to differentiate the various species. The morphological identifications of saccharomyces
cervicies were the colonies, which are light yellow in colour; round in shape, smooth, slightly
convex i.e. bulged in the Petri plates.
2.3.4Biochemical analysis of pineapple waste
The pineapple fruits skin waste was weighed and peeled. The moisture, protein,reducing and
total sugars were determined by Bradford,anthrone and DNSA method.
2.4 FERMENTATION
2.4.1 PROCEDURE
1.Five media, other than control, were prepared.
2.Control consisting of the basal media (D-glucose-10.0g; (NH2)2SO4 – 5.0g ; KH2PO4 –
1.0g ; MgSo4, 7H2O – 0.5g ; NaCl – 0.1g ; CaCl2 – 0.1g ; Distilled water 1000 ml ; pH –
5.5)with glucose
3.Other media were not free from glucoses but supplied with 1 to 5% pineapple hydrolysate.
4.The medium were distributed in Erlenmeyer flasks and sterilized at 121oC for 15 minutes.
5.The Yeast strains were inoculated in the media and incubated at 28oC for 7 days.
6, The yeast cells were separated by washing from the fermented broth and analyzed
7.Other synthetic media also used for the Single cell protein production..
25
2.4.2 MOISTURE CONTENT
PROCEDURE
1 Weigh moisture sample immediately and record as “wet weight of sample”
2 Dry the wet sample to a constant weight, at a temperature not exceeding 239º F (115º C)
using the suitable drying equipment.
3 Allow the sample to cool.
4 Weigh the cooled sample again, and record as the “dry weight of sample”
5. CALCULATION
5.1 The moisture content of the sample is calculated using the following equation:
A=A-B/B x 100
Where:
%W = Percentage of moisture in the sample,
A = Weight of wet sample (grams), and
B = Weight of dry sample (grams)
5.2 Report the moisture content to the nearest tenth of one percent.
2.4.3REDUSING SUGAR ESTIMATION BY DINITROSALISYLIC
METHOD
PROCEDURE
1. Weigh 100 mg of the sample and extract the sugars with hot 80% ethanol twice (5 mL
each time).
2. Collect the supernatant and evaporate it by keeping it on a water bath at 80°C.
3. Add 10 mL water and dissolve the sugars.
4. Pipette out 0.5 to 3 mL of the extract in test tubes and equalize the volume to 3 mL
with water in all the tubes.
5. Add 3 mL of DNS reagent.
6. Heat the contents in a boiling water bath for 5 min.
7. When the contents of the tubes are still warm, add 1 mL of 40% Rochelle salt solution.
8. Cool and read the intensity of dark red colour at 510 nm.
9. Run a series of standards using glucose (0–500 μg) and plot a graph.
26
CALCULATION
Calculate the amount of reducing sugars present in the sample using the standard graph.
2.4.4 TOTAL SUGAR ESTIMATION BY ANTHRONE METHOD
PROCEDURE
1. Weigh 100 mg of the sample into a boiling tube.
2. Hydrolyse by keeping it in a boiling water bath for three hours with 5 mL of 2.5 N HCl and
cool to room temperature.
3. Neutralise it with solid sodium carbonate until the effervescence ceases.
4. Make up the volume to 100 mL and centrifuge.
5. Collect the supernatant and take 0.5 and 1 mL aliquots for analysis.
6. Prepare the standards by taking 0, 0.2, 0.4, 0.6, 0.8 and 1 mL of the working standard.
‘0’ serves as blank.
7. Make up the volume to 1 mL in all the tubes including the sample tubes by adding
distilled water.
8. Then add 4 mL of anthrone reagent.
9. Heat for eight minutes in a boiling water bath.
10. Cool rapidly and read the green to dark green colour at 630 nm.
11. Draw a standard graph by plotting concentration of the standard on the X-axis versus
absorbance on the Y-axis.
12. From the graph calculate the amount of carbohydrate present in the sample tube.
CALCULATION
Amount of carbohydrate present in 100 mg of the sample= mg of glucose
× 100
Volume of test sample

NOTE:
Cool the contents of all the tubes on ice before adding ice-cold anthrone reagent.
27
2.4.5PROTIEN ESTIMATION BY BRADFORD METHOD
Procedure (Standard Assay, 20-150 µg protein; 200-1500 µg/ml)
1. Prepare a series of protein standards using BSA diluted with 0.15 M NaCl to final
concentrations of 0 (blank = NaCl only), 250, 500, 750 and 1500 µg BSA/mL. Also
prepare serial dilutions of the unknown sample to be measured.
2. Add 100 µL of each of the above to a separate test tube (or spectrophotometer tube if
using a Spec 20).
3. Add 5.0 mL of Coomassie Blue to each tube and mix by vortex, or inversion.
4. Adjust the spectrophotometer to a wavelength of 595 nm, and blank using the tube
which contains 0 BSA.
5. Wait 5 minutes and read each of the standards and each of the samples at 595 nm
wavelength.
6. Plot the absorbance of the standards vs. their concentration. Compute the extinction
coefficient and calculate the concentrations of the unknown samples.
28
3.OBSERVATION TABLE
1.BRADFORD METHOD
STANDARD
SR NO.
STD
DIST.WATER
COOMASIAE O.D
SOLUTION(ML)
BRILLIANT
BLUE(ml)
1
BLANK
5.0
1.0
0.00
2
0.2
4.8
1.0
0.16
3
0.4
4.6
1.0
0.32
4
0.6
4.4
1.0
0.50
5
0.8
4.2
1.0
0.63
6
1.0
4.0
1.0
0.76
FOR SAMPLE(S.cerevisiae)
PINEAPPLE WASTE
1%
2%
3%
4%
5%
0.5
0.5
0.5
0.5
0.5
4.5
4.5
4.5
4.5
4.5
1
1
1
1
1
0.15
0.27
0.32
0.58
0.73
4.5
4.5
4.5
4.5
4.5
1
1
1
1
1
0.10
0.23
0.39
0.55
0.66
Candida tropicalis
1%
2%
3%
4%
5%
0.5
0.5
0.5
0.5
0.5
Pichia stipitis4.5
29
1%
2%
3%
4%
5%
0.5
0.5
0.5
0.5
0.5
4.5
4.5
4.5
4.5
4.5
1
1
1
1
1
0.08
0.12
0.33
0.49
0.53
0.5
0.5
0.5
0.5
0.5
4.5
4.5
4.5
4.5
4.5
1
1
1
1
1
0.10
0.12
0.32
0.48
0.54
4.5
4.5
4.5
4.5
4.5
1
1
1
1
1
0.07
0.12
0.28
0.41
0.53
0.5
0.5
0.5
0.5
0.5
4.5
4.5
4.5
4.5
4.5
1
1
1
1
1
0.09
0.13
0.20
0.38
0.55
0.5
0.5
0.5
0.5
0.5
4.5
4.5
4.5
4.5
4.5
1
1
1
1
1
0.06
0.15
0.22
0.36
0.44
0.5
0.5
0.5
0.5
4.5
4.5
4.5
4.5
1
1
1
1
0.11
0.17
0.30
0,43
Aspergillus niger
1%
2%
3%
4%
5%
FOR ORANGE PEELS
Saccheromyces cerevisiae
1%
2%
3%
4%
5%
0.5
0.5
0.5
0.5
0.5
Candida tropicalis
1%
2%
3%
4%
5%
Pichia stipitis
1%
2%
3%
4%
5%
Aspergillus niger
1%
2%
3%
4%
30
5%
0.5
4.5
1
0,56
0.5
0.5
0.5
0.5
4.5
4.5
4.5
4.5
1
1
1
1
0.39
0.32
0.31
0.30
0.5
0.5
0.5
0.5
4.5
4.5
4.5
4.5
1
1
1
1
0.38
0.34
0.27
0.29
OTER SYNTHETIC MEDIUM
PINEAPPLE WASTE
S.c
C.t
P.s
A.n
4%
4%
4%
4%
ORANGE PEEL
S.c
C.t
P.s
A.n
4%
4%
4%
4%
2.DNSA METHOD (STANDARD)
SR NO.
ALIGUOTES
DISTILLED
DNSA
(ml)
WATER(ml)
REAGENT
40%
O.D
Rochelle
salt
solution(ml)
1.
BLANK
2.0
3.0
1
0.00
2.
0.2
1.8
3.0
1
0.09
3.
0.4
1.6
3.0
1
0.25
4.
0.6
1.4
3.0
1
0.38
5.
0.8
1.2
3.0
1
0.50
6.
1.0
1.0
3.0
1
0.62
1
0.13
PINEAPPLE WASTE
1%
1
1
3
31
2%
3%
4%
5%
1
1
1
1
1
1
1
1
3
3
3
3
1
1
1
1
0.24
0.31
0.47
0.56
1
1
1
1
1
3
3
3
3
3
1
1
1
1
1
0.09
0.17
0.27
0.36
0.44
1
1
1
1
1
3
3
3
3
3
1
1
1
1
1
0.10
0.19
0.28
0.39
0,.46
1
1
1
1
1
3
3
3
3
3
1
1
1
1
1
0.08
0.16
0.22
0.32
0.40
1
1
1
1
1
3
3
3
3
3
1
1
1
1
1
0.08
0.17
0.30
0.42
0.51
1
3
1
0.08
Candida tropicalis
1%
2%
3%
4%
5%
1
1
1
1
1
Pichia stipitis
1%
2%
3%
4%
5%
1
1
1
1
1
Aspergillus niger
1%
2%
3%
4%
5%
1
1
1
1
1
1
FOR ORANGE PEELS
Saccheromyces cerevisiae
1%
2%
3%
4%
5%
1
1
1
1
1
Can1dida tropicalis
1%
1
32
2%
3%
4%
5%
1
1
1
1
1
1
1
1
3
3
3
3
1
1
1
1
0.13
0.27
0.36
0.45
1
1
1
1
1
3
3
3
3
3
1
1
1
1
1
0.12
0.22
0.35
0.42
0.48
1
1
1
1
1
3
3
3
3
3
1
1
1
1
1
0.13
0.20
0.31
0.40
0.47
1
1
1
1
3
3
3
3
1
1
1
1
0.23
0.29
0.36
0.44
1
1
1
1
3
3
3
3
1
1
1
1
0.25
0.27
0.35
0.42
Pichia stipitis
1%
2%
3%
4%
5%
1
1
1
1
1
1
Aspergillus niger
1%
2%
3%
4%
5%
1
1
1
1
1
OTER SYNTHETIC MEDIUM
PINEAPPLE WASTE
S.c
C.t
P.s
A.n
4%
4%
4%
4%
1
1
1
1
ORANGE PEEL
S.c
C.t
P.s
A.n
4%
4%
4%
4%
1
1
1
1
33
3.ANTRONE METHOD
SR NO.
ALIGUOTES
DISTILLED
ANTHRONE
(ml)
WATER(ml)
REAGENT
1.
BLANK
2.0
4.0
0.00
2.
0.2
O.8
4.0
0.13
3.
0.4
0.6
4.0
0.27
4.
0.6
0.4
4.0
0.42
5.
0.8
0.2
4.0
0.54
6.
1.0
0.0
4.0
0.66
PINEAPPLE WASTE
Saccharomyces cerevisiae
1%
2%
3%
4%
5%
0.5
0.5
0.5
0.5
0.5
1.5
1.5
1.5
1.5
1.5
4
4
4
4
4
0.11
0.24
0.32
0.45
0.53
4
4
4
4
4
0.09
0.20
0.29
0.42
0.48
Candida tropicalis
1%
2%
3%
4%
5%
O.D
0.5
0.5
0.5
0.5
0.5
1.5
1.5
1.5
1.5
1.5
Pichia stipitis
34
1%
2%
3%
4%
5%
0.5
0.5
0.5
0.5
0.5
1.5
1.5
1.5
1.5
1.5
4
4
4
4
4
0.09
0.22
0.29
0.43
0.52
1.5
1.5
1.5
1.5
1.5
4
4
4
4
4
0.12
0.26
0.32
0.43
0.54
1.5
1.5
1.5
1.5
1.5
4
4
4
4
4
0.07
0.16
0.30
0.42
0.51
1.5
1.5
1.5
1.5
1.5
4
4
4
4
4
0.06
0.18
0.26
0.40
0.52
1.5
1.5
1.5
1.5
1.5
4
4
4
4
4
0.09
0.14
0.26
0.39
0.50
Aspergillus niger
1%
2%
3%
4%
5%
0.5
0.5
0.5
0.5
0.5
FOR ORANGE PEELS
Saccheromyces cerevisiae
1%
2%
3%
4%
5%
0.5
0.5
0.5
0.5
0.5
Candida tropicalis
1%
2%
3%
4%
5%
0.5
0.5
0.5
0.5
0.5
Pichia stipitis
1%
2%
3%
4%
5%
0.5
0.5
0.5
0.5
0.5
Aspergillus niger
35
1%
2%
3%
4%
5%
0.5
0.5
0.5
0.5
0.5
1.5
1.5
1.5
1.5
1.5
4
4
4
4
4
0.10
0.14
0.27
0.42
0.50
1.5
1.5
1.5
1.5
4
4
4
4
0.29
0.31
0.33
0.42
1.5
1.5
1.5
1.5
4
4
4
4
0.26
0.28
0.33
0.41
OTER SYNTHETIC MEDIUM
PINEAPPLE WASTE
S.c
C.t
P.s
A.n
4%
4%
4%
4%
0.5
0.5
0.5
0.5
ORANGE PEEL
S.c
C.t
P.s
A.n
4%
4%
4%
4%
0.5
0.5
0.5
0.5
4.RESULT
4.1IDENTIFICATION OF FUNGI
The fungus isolated from soil was identified as Aspergillus niger by theire morphological
characteristics, cultural characterstics,Conidiospores were found upright,simple,terminating
in a globose or swelling bearing phialides at the apex and or radiating from the entire
surface,conidia was one celled,globose,variously colored in the mass,catenulate,produced
basipetally.
36
Fig 1.Aspergillus niger on potato dextrose agar plate.
4.2EFFECT OF PINEAPPLE WASTE HYDROLYSATE AND ORANGE PEELS ON
PROTEIN CONTENT OF YEAST ISOLATES AND A.niger
PINEAPPLE WASTE
The S. cerevisiae recorded high protein content (186mg/100ml). The protein content
increased with increase in concentration of carbon source inthe medium. The maximum
protein content of S.cerevisiae was recorded on 7th day of the fermentation at 5%
concentration of pineapple waste followed by C.tropicalis (168mg/100ml),pichia
stipites(134mg/ml) and Aspergillus niger ( mg/100ml)
ORANGE PEELS
The C.tropicalis recorded high protein content (140 mg/100ml). The protein content
increased with increase in concentration of carbon source inthe medium. The maximum
protein content of C.tropicalis was recorded on 7th day of the fermentation at 5%
concentration of pineapple waste followed by S.cerevisiae (134 mg/100ml),pichia
stipites(112 mg/ml) and Aspergillus niger ( 140 mg/100ml)
37
Fig 2; protein estimation by b Bradford method
4.3EFFECT OF PINEAPPLE WASTE HYDROLYSATE and ORANGE PEELS ON
REDUCING SUGAR OF YEAST ISOLATES
PINEAPPLE WASTE
In general the reducing sugar content increased with the increase in the concentration of
carbon source in the yeast basal medium. The content of reducing sugar in the yeast was
reduced from 11.0mg/100 ml to 4.36 mg/100ml on 7th day of observation on fermentation
process. Among the four isolates used in the study S. cerevisiae recorded highest reducing sugar
(6.64mg/100ml) followed by C. tropicalis (5.91 mg/100ml).
ORANGE PEELS
The content of reducing sugar in the yeast was reduced from 9.45 mg/100 ml to 3.75
mg/100ml on 7th day of observation on fermentation process. Among the four isolates used in
38
the study S. cerevisiae recorded highest reducing sugar (5.70 mg/100ml) followed by C. tropicalis
(4.92 mg/100ml).
fig 3 :Redusung sugar estimation by DNSA method
4.4EFFECT OF TOTAL SUGAR PINEAPPLE WASTE HYDROLYSATE and
ORANGE PEELS ON TOTAL SUGAR OF YEAST ISOLATES
Pineapple waste
The total sugar estimate is highest in S.cerevisiae(20.3 mg/100 ml )and C.tropicalis (19.3
mg/100ml) and lowest in Aspergillus niger(16.8 mg/100ml).
ORAGE PEEL
The total sugar estimate is highest in C.tropicalis (19.8 mg/100 ml )and S.cerevisiae (17.9
mg/100ml) and lowest in Pichia stipitisd(14.6 mg/100ml).
39
Fig 4 : Total sugar estimation by antrone method
The biomass level was increased with the increase in concentration of pineapple waste
concentration, because the pineapple waste itself contains 13% sugar, 0.6% protein and trace
level of calcium, phosphorous, ions and vitamins. Hence the availability of nutrients in
pineapple has rapidly promoted growth of yeast cells. Concerning protein content, the highest
protein content was recorded on the 3rd day of fermentation at 5% concentration and
thereafter decreased. The present findings are in agreement with the findings of Abou Hamed
(1993). The protein content increased as the concentration of wheat straw hydrolysate
increased, at the same time the protein content gradually decreased when the fermentation
period increased.
40
5.CONCLUSION
The bioconversion effect of pineapple waste into SCP was evaluated using yeast. The
increase in biomass contents were observed when there was increase in pineapple waste
concentration. The highest biomass content of S. cerevisiae and C. tropicalis was recorded on
7th day fermentation. The highest protein content of S.cerevisiae and C tropicalis was
recorded on the 7th day of fermentation at 5 % concentration. The highest reducing sugar
content of yeast was recorded on 3rd day of fermentation at 5% concentration. The utilization
of reducing sugar was increased with the increase in the concentration of substrates. The
present findings reveals that pineapple waste can be used as effective alternate carbon source
for SCP production. Also the orang peels have the same effects on all the para meters.
6.REFERENCES
Argyro, B., Costas, P., Athanasios, A.K. (2006). Production of Food Grade Yeasts. Food
Technol. Biotechnol. 44, 407–415.
Nigam, N.M. (2000). Cultivation of Candida langeronii in sugarcane bagasse hemi cellulose
hydrolysate for the production of single cell protein. W.J.Microbiol.and biotechnol. 16, 367372.
Tipparat, H., Kittikun, A.H. (1995). Optimization of single cell protein production from
cassava starch using Schwanniomyces castellii. W.J. Microbiol. & Biotechnol. 11, 607-609.
Abou Hamed, S.A.A. (1993). Bioconversion of wheat straw by yeast into single cell protein.
Egypt. J. Microbiol. 28(1), 1-9.
Saquido, P.M.A., Cayabyab, V.A ., Vyenco, F.R. (1981). Bioconversion of banana waste into
single cell protein. J. Applied Microbiol. &Biotechnol. 5(3), 321-326.
Zhao, G., Zhang, W., Zhang, G. (2010). Production of single cell protein using waste
capsicum powder produced during capsanthin extraction. Lett Appl Microbiol. 50. 187-91.
Smith, M.E., Bull, A.T. (1976). Protein and other compositional analysis of Saccharomyces
fragilis grown on coconut water waste. J. Applied Bacteriol. 41, 97-107.
AOAC. (1975).Official methods of Analysis, 16th edition, Association of official Analytical
chemists, Washington D.C.
41
Ranganna, S. (1978). Handbook of analysis and quality of fruit and vegetable products, Tata
McGraw Hill Publishing Co. Ltd, New Delhi
Sadhu, M.K., Chattopadhy, P.K. (2001). Introduction to fruit crops. Naya Prakash Publication,
Calcutta, 252.
Phaff, H.J., Miller, M.W., Mark, E.M. (1996). The life of yeasts. Harvard university press,
Cambridge, Massachrsetts. 186.
Fawcett, J.K., Scott, J.E. (1960). A rapid and precise method for determination of urea. J. Clin. Path.
13, 156.
Krishnaveni. S., Theymoli, B., Sadasivam, S. (1984). Estimation of reducing sugar by dinitrosalicylic
acid method. Food Chem. 15, 186.
Hedge, J.E., Hofreiter, B.T. (1962). Carbohydrate Chemistry 17th Edition, Whistler RL and Be Miller,
J.N. Academic Press New York.
Karla, K.L., Grewal, H.S., Kahlon, S.S. (1989). Bioconversion of kinnowmandarin waste in to single
cell protein. J. Appl. Microbial Biotechnol. 5(3), 153-157.
Rashad, M.M., Moharib, S.A., Jwanny, E.W. (1990). Yeast conversion of mango waste or methanol
to SCP and other metabolites. Biol. Waste. 32(4), 277.
Prosser, J.I., Tough, A.J. (1991). Growth mechanism and growth kinetics of filamentous
microorganism. Critical Reviews in Biotechnol. 10(4), 253-274.
7.APPENDIX
MEDIA
1) POTATO DEXTROSE AGAR
Potato Infusion
:
300g
Dextrose
:
20g
Agar
:
20g
PH
:
3.5
Distilled water
:
1000ml
42
2) YEAST PEPTONE DEXTROSE AGAR
Yeast extract
:
10g,
Dextrose
:
20g,
Peptone
:
20g,
Agar
:
20g,
pH
:
6.0
Distilled water
:
1000ml
3) basal media
D-glucose
:
10.0g;
(NH2)2SO4
:
5.0g
KH2PO4
:
1.0g
MgSo4, 7H2O
:
0.5g
NaCl
:
0.1g
CaCl2
:
0.1g
Distilled water
:
1000 ml
pH
:
5.5
REAGENTS REQUIRED
1.2.5 N HCl
2. Anthrone reagent:
Dissolve 200 mg anthrone in 100 mL of ice-cold 95% H2SO4. Prepare fresh before
use.
3. Standard glucose:
Stock—Dissolve 100 mg in 100 mL water. Working standard of stock diluted to 100
mL with distilled water. Store refrigerated after adding drops of toluene.
4.Dinitrosalicylic Acid Reagent (DNS Reagent)
Dissolve by stirring 1 g dinitrosalicylic acid, 200 mg crystalline phenol and 50 mg sodium
43
sulphite in 100 mL 1% NaOH. Store at 4°C. Since the reagent deteriorates due to sodium
sulphite, if long storage is required, sodium sulphite may be added at the time of use.
5.40% Rochelle salt solution (Potassium sodium tartrate).
6.Standard bovine serum albumin (BSA)(1 mg/ml)
7.Coomassie Brilliant Blue 1
8 0.15 M NaCl
Dissolve 0.877g sodium chloride in 1000 ml Distilled water
44