Production of Single Cell Protein From Pineapple Waste and Orange Peel Using Yeasts and...
Transcript of Production of Single Cell Protein From Pineapple Waste and Orange Peel Using Yeasts and...
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A
PROJECT REPORT
ON
PRODUCTION OF SINGLE CELL PROTEIN FROM PINEAPPLE
WASTE AND ORANGE PEEL USING YEASTS ANDAspergillus niger
A PROJECT REPORT
Submitted in partial fulllment of theRequirements for the award of the degree of
Master of Science
In
Department Of BiotechnologyBy
NIRAV J GHANTALA
BH!"# MH$IR %O&&'!' O( M)Sc BIO*'%H#O&O!+
,((I&I*'D *O $''R #RMD SO-*H !-.R* -#I$'RSI*+ -#I$'RSI*+/S-R*
0120
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CERTIFICATE
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ACKNOWLEDGEMENT
I thank the almighty whose blessings have enabled me to accomplish my dissertation work
successfully.
It is my pride and privilege to express my sincere thanks and deep sense of gratitude to Priya
Bande, Department of Biotechnology and Environmental ciences, !I"#$%, 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. &er constant encouragement and
confidence'imbibing attitude has always been a moral support for me.
!y sincere thanks to Dr.#handrashekhar kulkarni, &ead Department of Biotechnology andEnvironmental ciences, for his immense concern throughout the pro(ect work.
I also wish to thank all my friends, for providing the mandatory scholastic inputs during my
course venture.
)inally, I wish to extend a warm thanks to everybody involved directly or indirectly with my
work.
"he 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.
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DECLARATION
I hereby declared that the work presented in the Pro(ect entitled has been carried out by
*&+%"++ %I-+ /. under the guidance of Priya Bande, Pro(ect *uide, at !I"#$%, Pune.
"he entitled 0ork is original and no part of this work is either published or submitted in any
university for the award of any degree or diploma.
Date1
Place1 Pune
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S.NO. CONTENTS PAGE.NO
2) Introduction 7
0) Materials 5 methods 223)Obser6ation *ables07
4) Results 38
9) conclusion
42
8)References 41
:)Appendix 43
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Abstract
PRODUCTION OF SINGLE CELL PROTEIN FROM DIFFERENT
AGRICULTURE WASTE USING YEASTSPeople in third world and developing countries are suffering from menace of protein
deficiency in their diets resulting in serious protein-energy malnutrition problems!he
worldwide food protein deficiency is becoming alarming day to day and with the fast
growing population of world"Pressure is exerted on the feed industry to produce enough
animal feed to meet the region#s nutritional re$uirements%ingle-&ell Protein '%&P(
represents microbial cells 'primary( grown in mass culture and harvested for use as protein
sources in foods or animal feeds )n 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" %accharomyces cerevisiae"&andida tropicalis"Pichia stipites and Aspergillus
niger and were grown !he increased concentration of pineapple hydrolysate and orange peel
enhanced the biomass yield and the protein formation within the yeast cells *ower carbon
utili+ation 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!he
present finding helps in %&P production from cheap" inexpensive agro waste material
Key !r"s, east" %&P" Pineapple waste".range peel" accharomyces cerevisiae, #andida
tropicalis,Pichia stipites, +spergillus niger.
#.INTRODUCTION
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#.#Ge$era% I$tr!"&ct'!$
Production of %&P by mass culture of microorganisms is yet to ta/e momentum at industrial
scale and deserve much attention to solve the problem of starvation in the coming decades
!he criteria for microorganisms to be used as food or feed include 1the organism must be
genetically stable" non toxic and grow rapidly on a simple non specific medium2 it should
have high nutritional or vitamin content and should be edible to human and other animals
!he organisms should also utili+e 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 $uality and composition Among the various groups of
microorganisms used to produce %&P" 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" 0-carotenoids"
trehalose" glutathione" superoxide dismutase" chitinase" amylase" phytase" protease" /iller
toxin etc which have been receiving much attention for many decades !he main nutritional
contribution in either human food or animal feed is its high protein content ecause of high
protein and fat content" the contribution of carbohydrates to the nutritional value of %&P is
not of prime importance ut a maor constraint for yeast as %&P is the thic/ cell wall which
is difficult to digest leading to poor protein bioavailability
Agricultural activities and food industry generate considerable $uantities of wastes which are
rich in organic matter and could constitute new materials for value added products A
growing concern for the acute food shortages for the world#s expanding population has led to
the exploitation of non-conventional food sources as potential alternatives Among these" the
:
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single cell organisms probably present the best chances for the development of uni$ue
independence of agricultural crop based food supply
%ingle cell protein is a biomass based on protein extract derived from microorganism%ingle
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 mar/et
value'pandey et al"12(!he single cell protein is a dehydrated cell consisting of mixture of
proteins" lipids"carbohydrates" nucleic acids" inorganic compoundsand a variety of other non protein
nitrogenous compounds such as vitaminsAgricultural 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 $uality biomass
!he continued population growth especially in developing and third-world countries is
resulting in increased food demand in parallel and is posing serious threats to food security
due to yawning gap in demand and supply 'Anupama and 5avindera" 2666( &hronic
malnutrition and hunger are typically most prevalent in developing countries alnutrition is
a conse$uence of not ta/ing appropriate amount or $uality of nutrients comprising diet !he
gap b8w demand and supply is expected to grow unless planned actions are ta/en to improve
the situation !herefore it is essential to search for un-conventional or novel proteins to
supplement the available sourcesecause 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 %outh America9owever" Predictions show that
more advanced nations will eventually face the same problems !he developing of novel food
production process independent of agricultural land use is thus becoming imperative
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!here have been studies as well as efforts to improve the protein $uantity and $uality of the
finished food products by augmenting protein-rich cheaper ingredients in food formulations
':asir and utt" 26119ussain et al., 2667( Although animal proteins are considered to be
best $uality proteins '%aima et al" 266;(" however microbial protein also /nown 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 'e/atorou et al" 266
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some $uantity for food5esearch on single cell protein has been stimulated by food crisis or food
shortages that will occur if the world#s populations are not controlled %cientists believe that use of
microbial fermentations and the development of an industry to produce and supply single cell proteins
from agricultural waste are insufficient
!he present study was focused on yeast single cell protein rather than bacterial" fungal and algal
single cell protein Algal single cell protein have limitations such as the need for warm temperatures
and plenty of sun light in addition to carbon dioxide" and also that the algal cell wall is indigestible
acteria are capable of growth on a wide variety of substrates" have a short generation time and are
high protein content !heir use is somewhat limited by poor public acceptance of bacteria as food"
small si+e and difficulty of harvesting and high content of nucleic acid on a dried weight basis easts
are probably the most widely accepted and used microorganisms for single cell protein !hese include
strains of #andida utilis, #. arborea, #. pulcherrima andaccharomyces cerevisiaePichia stipitis is
also used for %&P production in the present study)n recent years increasing attention has been
given to the conversion of food processing wastes into valuable by-products such as the
production of yeast protein from potato '%/ogman 17
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with conventional sources of protein 'soybeans or meat( are well /nownA number of
agricultural and agro industrial waste products have been used for the production of %&P and
other metabolites" including orange waste" mango waste" cotton sal/s" /innow-
mandarinwaste" barley straw" corn cops" rice straw" cornstraw" onion uice and sugar cane
bagasse ':igam et al." 2666(" cassava starch '!ipparat et al." 1E(" wheat straw 'Abou
9amed" 13("banana waste '%a$uido et al." 1;1(" capsicum powder 'Ghao etal., 2616( and
coconut water '%mith and ull" 17 !he amino acid composition of a
protein primarily determines its potential nutritional value !he protein efficiency ratio and
biological value of yeast protein are /nown to be relatively high 'unro" 1
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in membranes" it is li/ely that these environmentally induced changes in lipid composition
are of maor physiological significance icroorganisms contain a diverse range of fatty acid
composition which can be useful as a chemotaxonomic tool for classifying species and
strains Batty acids typically comprise 76-6 > of the lipids in yeast with oleic acid '1;, 1 n-
( being the commonest found
Batty acids" the simplest of lipids are re$uired for membrane structure" function" transport of
cholesterol" formation of lipoproteins etc &omposition of growth medium governs the lipid
content of microorganisms icrobial fatty acid profiles are uni$ue from one species to
another !he 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 !he 14, 6 fatty acids are
only seen as trace fatty acyl residues !he microbial identification system based on fatty acid
methyl ester 'BAH( analysis has been used in laboratories for the identification of clinical
yeast strains 'Peltroche-*iacsahuanga et al" 2666( !he system analyses long-chain fatty
acids containing F26 & atoms" identifying and $uantifying the BAHs of microorganisms
!he database library searches for fatty acid composition"compares the BAH profile of the
isolate with those of well characteri+ed strains and defines the most li/ely species of the
isolate Batty acid composition of cold adapted carotenogenic basidiomycetous yeasts was
studied by *ib/ind et al '266;( !otal fatty acids of six yeast species isolated from the
temperate a$uatic environments in Patagonia ranged from 2 to 1E > of dry biomass*inoleic"
oleic" palmitic and " 13> and > of total fatty acids" respectively !he proportion of
each varied mar/edly depending on the taxonomic
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affiliation of the yeast species and on the culture media used !he high percentage of
polyunsaturated fatty acids 'P=BAs( found in Patagonian yeasts" in comparison to other
yeasts" is indicative of their cold-adapted metabolism
east proteins are easily digestible compared to those from bacteria &hemical analysis of
microorganisms tested for %&P reveal that they are comparablein amino acid content to the
plant and animal sources with the exception of sulphuramino acid methionine which is low
in some %&P sources" especially yeasts9owever" this can be alleviated by culturing yeasts
on molasses 'halla et al" 1( !he only species of yeast fully acceptable as food for
humans is . cerevisiae'ba/er#s and brewer#s yeast( 'e/atorou et al" 266
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13 Bactors Affecting east rowth
east growth is affected by a number of factors !hese include composition of medium
commonly sugar source" aeration 'oxygen(" agitation of the medium" p9" temperature and period
of propagation %ome of the factors affecting yeast growth are discussed below
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131 %ugar feed 'media composition(
!he main carbon and energy source for most yeast is glucose which is converted via the
glycolytic pathway to pyruvate and by the Jrebs cycle to anabolytes and energy in the form of
A!Peasts are further classified according to their modes of further energy production from
pyruvate, respiration and fermentation !hese processes are regulated by environmental factors"
mainly glucose and oxygen concentrations
)n respiration" pyruvate is decarboxylated in the mitochondrion to acetyl- &oA which is
completely oxidi+ed in the citric acid cycle to &.2" energy and intermediates to promote yeast
growth
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)n anaerobic conditions" glucose is slowly utili+ed to produce the energy re$uired ust to /eep the
yeast cells alive !his process is called fermentation" in which the sugars are not completely
oxidi+ed yielding &.
2
and ethanol '%cragg, 11 e/atorou et al., 266
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@hen the yeast cell is exposed to high glucose concentration" catabolite repression occurs" during
which gene expression and synthesis of respiratory en+ymes are repressed" and fermentation
prevails over respiration'5incon et al."2661, e/atorou et al."266
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132 Aeration
9ighly aerobic culture conditions are used in the production of yeast specifically
ba/er#s yeast to maximi+e cell growth '&elo and elo" 2664( !he modern
techni$ue of ba/er#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 )ts biochemistry involves the brea/down of carbohydrates only to the stage of
ethanol
=nder aerobic conditions" however" maximum growth occurs and the efficiency of
utili+ation of carbohydrate increases as respiration and the brea/down of the
carbohydrate to carbon dioxide and water becomes complete '&oo/" 1E;(
enerally oxygen has the following basic functions 'Prescott and ?unn" 1E(
1 )nhibits fermentation
2 )ncreases respiration
3 Agitation of the medium
4 5emoval of toxic end products
E %timulation of vegetative growth
2;
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.xygen is used in the synthesis of unsaturated fatty acids and sterols which form the cell
membrane !hese molecules are important for both growth and fermentation and serve as a means
for storing oxygen within the cell !hey are also necessary for increasing cell mass 'growth(
involving the over all upta/e of nutrients and determining alcohol tolerance .xygen stimulates
the synthesis of molecules necessary for yeast to metaboli+e and ta/e up maltose and other sugars
133 !emperature
!he temperature most favorable to the growth of ba/er#s yeast varies from strain to strain
.ptimum temperature is usually between 2E6
&-3E6& !he maximum survival temperature is
376
& '&oo/" 1E;( Propagation at low temperature" the rate of growth is slower and gives a
decreased yield of yeast east grown in low temperature is less stable when stored and
transported as a pressed ca/e and the dry matter of a yeast ca/e of standard consistency becomes
progressively less at low temperature" ie" it affects the relationship between intracellular and
extra cellular water content
134 p9
easts grow well at acidic p9 'acidophilic organisms( Bor industrial propagation low p9 is
helpful in restricting the development of many bacterial contaminations however" the color of the
yeast may be affected at low p9 !he p9 of the media is commonly adusted by the addition of
92
%.4"
:93
" :a2&.
3or :a9&.
3'Prescott and ?unn" 1E( to the substrate
13E east ?ry atter
!he main elements present in ba/er#s yeast are carbon" hydrogen" oxygen and nitrogen which
normally account for as much as 4> of the dry matter '@hite" 1E4( !able 3 shows elementary
analysis of yeast dry matter
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!A*H,1 Hlementary analysis of yeast dry matter '@hite" 1E4(
C!$st't&e$t Perce$t ! yeast "ry
,attercarbon '&( 4E6-46
9ydrogen ' 9( E6- 76
.xygen ' .( 366 -3E6
:itrogen ':( 71 -16;
!otal ash 47- 16E
Phosphate ' P2.E( 1- EE
Potash ' J2.( 14- 43
&alcium '&a.( 666E F 62agnesium ' g. ( 61 F 67
Aluminum ' as Al2.3( 6662 F 662
%ulphate ' as %.4( 661- 66E
!hese 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( easts also
contain high amount of vitamin complex" nucleic acids and organic bases 'pyramidine and
purine bases( !he inorganic substances may constitute up < to ; > of the yeast dry matter
'@hite"1E45oman"1E7( !able 4 gives the $uantities of some substances present in yeast
dry matter '@hite" 1E4(
Tab%e /. &omposition of yeast dry matter '@hite" 1E4(
C!$st't&e$t Perce$t ! yeast "ry ,atterAsh :ormally < -;
01
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lycogen 1 -36
Bat soluble fraction'true fats" sterols lipids 1 -2 2east gum 'mannan ( =p to 4
&ellulose ' yeast ( =p to E
Proteins and organic nitrogenous bases 44 F 47
RE0IEW OF LITERATURE
!he wor/ carried out on &omparative assessment of various agro-industrial wastes for
accharomyces cerevisiaebiomass production and its $uality evaluation as %ingle cell
protein1= acha" :asir" A Jhali$ue" A A AnumL and A Iabbar in 2611(
!his study was planned to assess the feasibility of using agro-industrial wastes for
accharomyces cerevisiaeproduction and to evaluate protein $uality of produced single cell
protein '%&P( biomass Potato peels contained significantly highest dry matter and
carbohydrate content as compared to other wastes %ignificantly higher %&P biomass was
produced using potato peels followed by carrot peels .n the basis of higher %&P biomass
production" potato peels were selected for further biomass production !he %&P biomass
contained 42M112 crude protein which was non-significant 'P23.4543( compared to
commercially available accharomycescerevisiae. !he parameters for in'vivoprotein $uality
assay in %prague ?awley rats were 3 true digestibility" net protein utili+ation"
76E biological value" 4EEnet protein ration" and 27E protein efficiency ratio" which are
higher in comparison to most of cereal proteins !he present exploration depicted that
accharomyces cerevisiae can be efficiently produced utili+ing wastes and the produced
biomass can potentially be used as protein source in various food formulations
!he wor/ carried out on Production of %ingle cell protein from Pineapple waste using
yeast'?harumadurai ?9A:A%HJA5A:1L" %ubramaniyan *A@A:A" %ubhasish %A9A1"
:ooruddin !9AI=??): 1" Annamalai PA::HH5%H*KA2(
02
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)n this wor/ pineapple waste was used as sole carbon source in five concentrations for
preparation of fermentation media on which two strains of yeasts" accharomyces cerevisiae
and #andida tropicalis were grown !he increased concentration of pineapple hydrolysate
enhanced the biomass yield and the protein formation within the yeast cells *ower carbon
utili+ation 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
(. MATERIALS AND MET2ODS
1a3 GLASSWARE
= >etri dishes?
= !lass slides?
= !lass bea@ers?
= %o6er slips?
= Media bottles?
= %onical Aas@s?
= >ipette?
= *est tubes?
1b 3REAGENTS RE4UIRED
= lcohol?
= Distilled water?
= 'thanol
= %oncentric sulfuric acid
1c3E4UIPMENTS RE4UIRED
00
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= >h meter
= Inoculating loops?
= "eighing machine?
= Incubator?
= utocla6e,Meta instrument?Mumbai/?
= Refrigerator?
= &aminar air Aow?,Microlt/
= Microscope?
= Measuring cylinder
Spectrophotometer
$orte machine
%entrifuge,Remi/
"aterbath
(.#COLLECTION OF PINEAPPLE WASTE AND ORANGE PEELS!he ripe" yellowish and firm pineapple fruits ".range peels collected from local
mar/etPineapple waste and .range peels were cut into slices in order to hydrolysisPut this
slices in oven at 13E & for 24 hours Bor the moisture content to /now
2.2MICROORGANISM!he yeast culture such as accharomyces cerevisiae "#andida tropicalis and pichia stipitis were
obtained by using isolation !he cultures were maintained on slant of yeastpeptone dextrose
medium'east extract 16g" ?extrose 26g" Peptone 26g" Agar 26g" p9 N
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composition yeast extract '36g8*( peptone '16g8*( dextrose '26g8*( agar-agar '1Eg8*(
'P-agar( and incubated at 36O& for 24h !he best culture was selected for further studies
%elected cultures were stored at 4O&
(.5 MET2ODS
(.5.# ISOLATION OF As-er6'%%&s $'6er
1 %oil samples were collected from agriculture college campus"pune
2 1 g of each soil sample was individually suspended in 1 m* of sterile distilled water
3 %erially diluted to 16F3 fold
4 And plated on potato dextrose agar 'P?A( plates containing /anamycin to a final
concentration of 16F
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were the colonies? which are light yellow in colourE round in shape? smooth?
slightly con6e i)e) bulged in the >etri plates)
(.5./*'!c+e,'ca% a$a%ys's ! -'$ea--%e aste
!he pineapple fruits s/in waste was weighed and peeled !he moisture" protein"reducing and
total sugars were determined by radford"anthrone and ?:%A method
(./ FERMENTATION
(./.# PROCEDURE
1Bive media" other than control" were prepared
2&ontrol consisting of the basal media '?-glucose-166g ':92(2%.4 F E6g J92P.4 F
16g g%o4" 792. F 6Eg :a&l F 61g &a&l2 F 61g ?istilled water 1666 ml p9 F
EE(with glucose
3.ther media were not free from glucoses but supplied with 1 to E> pineapple hydrolysate
4!he medium were distributed in Hrlenmeyer flas/s and sterili+ed at 121o& for 1E minutes
E!he east strains were inoculated in the media and incubated at 2;o& for 7 days
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E1 !he moisture content of the sample is calculated using the following e$uation,
ANA-8 x 166
@here,
>@ N Percentage of moisture in the sample"
A N @eight of wet sample 'grams(" and N @eight of dry sample 'grams(
E2 5eport the moisture content to the nearest tenth of one percent
(./.5REDUSING SUGAR ESTIMATION *Y DINITROSALISYLIC
MET2OD
PROCEDURE
1 @eigh 166 mg of the sample and extract the sugars with hot ;6> ethanol twice 'E m*
each time(
2 &ollect the supernatant and evaporate it by /eeping it on a water bath at ;6&
3 Add 16 m* water and dissolve the sugars
4 Pipette out 6E to 3 m* of the extract in test tubes and e$uali+e the volume to 3 m*
with water in all the tubes
E Add 3 m* of ?:% reagent
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2 9ydrolyse by /eeping it in a boiling water bath for three hours with E m* of 2E : 9&l and
cool to room temperature
3 :eutralise it with solid sodium carbonate until the effervescence ceases
4 a/e up the volume to 166 m* and centrifuge
E &ollect the supernatant and ta/e 6E and 1 m* ali$uots for analysis
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2 Add 166 U* of each of the above to a separate test tube 'or spectrophotometer tube if
using a %pec 26(
3 Add E6 m* of &oomassie lue to each tube and mix by vortex" or inversion
4 Adust the spectrophotometer to a wavelength of EE nm" and blan/ using the tube
which contains 6 %A
E @ait E minutes and read each of the standards and each of the samples at EE nm
wavelength
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STANDARD
SR NO. STD
SOLUTION1ML
3
DIST.WATER COOMASIAE
*RILLIANT
*LUE1,%3
O.D
1 *A:J E6 16 666
2 62 4; 16 61 6E 4E 1 627
3 > 6E 4E 1 632
4 > 6E 4E 1 6E;
E > 6E 4E 1 673
Candida tropicalis
1 > 6E 4E 1 616
2 > 6E 4E 1 623
3 > 6E 4E 1 63
4 > 6E 4E 1 6EE
E > 6E 4E 1 6
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Picia stipitis!"#
1 > 6E 4E 1 66;
2 > 6E 4E 1 612
3 > 6E 4E 1 633
4 > 6E 4E 1 64
E > 6E 4E 1 6E3
Aspergillus niger
1 > 6E 4E 1 616
2 > 6E 4E 1 612
3 > 6E 4E 1 632
4 > 6E 4E 1 64;
E > 6E 4E 1 6E4
FOR ORANGE PEELS
Sacc+er!,yces cere?'s'ae
1 > 6E 4E 1 667
2 > 6E 4E 1 612
3 > 6E 4E 1 62;
4 > 6E 4E 1 641
E > 6E 4E 1 6E3
Ca$"'"a tr!-'ca%'s
1 > 6E 4E 1 66
2 > 6E 4E 1 613
31
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3 > 6E 4E 1 626
4 > 6E 4E 1 63;
E > 6E 4E 1 6EE
P'c+'a st'-'t's
1 > 6E 4E 1 66 6E 4E 1 61E
3 > 6E 4E 1 622
4 > 6E 4E 1 63 6E 4E 1 644
As-er6'%%&s $'6er
1 > 6E 4E 1 611
2 > 6E 4E 1 617
3 > 6E 4E 1 636
4 > 6E 4E 1 6"43
E > 6E 4E 1 6"E 6E 4E 1 63
&t 4 > 6E 4E 1 632
Ps 4 > 6E 4E 1 631
An 4 > 6E 4E 1 636
ORANGE PEEL
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%c 4 > 6E 4E 1 63;
&t 4 > 6E 4E 1 634
Ps 4 > 6E 4E 1 627
An 4 > 6E 4E 1 62
(.DNSA MET2OD 1STANDARD3
SR #O)
&I!-O*'S
,ml/
DIS*I&&'D
"*'R,ml/
D#S
R'!'#*
41C
Rochelle
salt
solution,
ml/
O)D
2) BF 0)1 3)1 2 1)110) 1)0 2); 3)1 2 1)173) 1)4 2)8 3)1 2 1)09
4) 1)8 2)4 3)1 2 1)3;9) 1); 2)0 3)1 2 1)918) 2)1 2)1 3)1 2 1)80
PINEAPPLE WASTE
1 > 1 1 3 1 613
2 > 1 1 3 1 624
3 > 1 1 3 1 631
4 > 1 1 3 1 647
E > 1 1 3 1 6E 1 1 3 1 66
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2 > 1 1 3 1 617
3 > 1 1 3 1 627
4 > 1 1 3 1 63 1 1 3 1 644
Picia stipitis
1 > 1 1 3 1 616
2 > 1 1 3 1 61
3 > 1 1 3 1 62;
4 > 1 1 3 1 63
E > 1 1 3 1 6"4 1 1 3 1 66;
2 > 1 1 3 1 61 1 1 3 1 622
4 > 1 1 3 1 632
E > 1 1 3 1 646
1
FOR ORANGE PEELS
Sacc+er!,yces cere?'s'ae
1 > 1 1 3 1 66;
2 > 1 1 3 1 617
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3 > 1 1 3 1 636
4 > 1 1 3 1 642
E > 1 1 3 1 6E1
Ca$#"'"a tr!-'ca%'s
1 > 1 1 3 1 66;
2 > 1 1 3 1 613
3 > 1 1 3 1 627
4 > 1 1 3 1 63 1 1 3 1 64E
P'c+'a st'-'t's
1 > 1 1 3 1 612
2 > 1 1 3 1 622
3 > 1 1 3 1 63E
4 > 1 1 3 1 642
E > 1 1 3 1 64;
#
As-er6'%%&s $'6er
1 > 1 1 3 1 613
2 > 1 1 3 1 626
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3 > 1 1 3 1 631
4 > 1 1 3 1 646
E > 1 1 3 1 647
OTER SYNT2ETIC MEDIUM
PINEAPPLE WASTE
%c 4> 1 1 3 1 623
&t 4 > 1 1 3 1 62
Ps 4 > 1 1 3 1 63 1 1 3 1 644
ORANGE PEEL
%c 4
>
1 1 3 1 62E
&t 4 > 1 1 3 1 627
Ps 4 > 1 1 3 1 63E
An 4 > 1 1 3 1 642
3.ANTRONE METHOD
SR #O)
&I!-O*'S
,ml/
DIS*I&&'D
"*'R,ml/
#*HRO#'
R'!'#* O)D2) BF 0)1 4)1 1)110) 1)0 O); 4)1 1)233) 1)4 1)8 4)1 1)0:4) 1)8 1)4 4)1 1)409) 1); 1)0 4)1 1)948) 2)1 1)1 4)1 1)88
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PINEAPPLE WASTE
Sacc+ar!,yces cere?'s'ae
1 > 6E 1E 4 611
2 > 6E 1E 4 624
3 > 6E 1E 4 632
4 > 6E 1E 4 64E
E > 6E 1E 4 6E3
Candida tropicalis
1 > 6E 1E 4 66
2 > 6E 1E 4 626
3 > 6E 1E 4 62
4 > 6E 1E 4 642
E > 6E 1E 4 64;
Picia stipitis
1 > 6E 1E 4 66
2 > 6E 1E 4 622
3 > 6E 1E 4 62
4 > 6E 1E 4 643
E > 6E 1E 4 6E2
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Aspergillus niger
1 > 6E 1E 4 612
2 > 6E 1E 4 62 6E 1E 4 632
4 > 6E 1E 4 643
E > 6E 1E 4 6E4
FOR ORANGE PEELS
Sacc+er!,yces cere?'s'ae
1 > 6E 1E 4 667
2 > 6E 1E 4 61 6E 1E 4 636
4 > 6E 1E 4 642
E > 6E 1E 4 6E1
Ca$"'"a tr!-'ca%'s
1 > 6E 1E 4 66 6E 1E 4 61;
3 > 6E 1E 4 62 6E 1E 4 646
E > 6E 1E 4 6E2
P'c+'a st'-'t's
1 > 6E 1E 4 66
3:
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2 > 6E 1E 4 614
3 > 6E 1E 4 62 6E 1E 4 63
E > 6E 1E 4 6E6
As-er6'%%&s $'6er
1 > 6E 1E 4 616
2 > 6E 1E 4 614
3 > 6E 1E 4 627
4 > 6E 1E 4 642
E > 6E 1E 4 6E6
OTER SYNT2ETIC MEDIUM
PINEAPPLE WASTE
%c 4 > 6E 1E 4 62
&t 4 > 6E 1E 4 631
Ps 4 > 6E 1E 4 633
An 4 > 6E 1E 4 642
ORANGE PEEL
%c 4>
6E 1E 4 62 6E 1E 4 62;
Ps 4 > 6E 1E 4 633
An 4 > 6E 1E 4 641
4.RESULT
4.1IDENTIFICATION OF FUNGI
*he fungus isolated from soil was identied as spergillus niger by theire
morphological characteristics? cultural characterstics"&onidiospores were found
upright"simple"terminating in a globose or swelling bearing phialides at the apex and or
radiating from the entire surface"conidia was one celled"globose"variously colored in the
mass"catenulate"produced basipetally
Big 1Aspergillus niger on potato dextrose agar plate
/.(EFFECT OF PINEAPPLE WASTE 2YDROLYSATE AND ORANGE PEELS ON
PROTEIN CONTENT OF YEAST ISOLATES ANDA"niger
PINEAPPLE WASTE
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!he . cerevisiae recorded high protein content '1;
concentration of pineapple waste followed by #.tropicalis '1
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PINEAPPLE ASTE
)n general the reducing sugar content increased with the increase in the concentration of
carbon source in the yeast basal medium !he content of reducing sugar in the yeast was
reduced from 116mg8166 ml to 43< mg8166ml on 7th day of observation on fermentation
process Among the four isolates used in the study . cerevisiae recorded highest reducing sugar
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/./EFFECT OF TOTAL SUGAR PINEAPPLE WASTE 2YDROLYSATE a$"
ORANGE PEELS ON TOTAL SUGAR OF YEAST ISOLATES
P'$ea--%e aste
!he total sugar estimate is highest in %cerevisiae'263 mg8166 ml (and &tropicalis '13
mg8166ml( and lowest in Aspergillus niger'1 sugar" 6 protein and trace
level of calcium" phosphorous" ions and vitamins 9ence the availability of nutrients in
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pineapple has rapidly promoted growth of yeast cells &oncerning protein content" the highest
protein content was recorded on the 3rd day of fermentation at E> concentration and
thereafter decreased !he present findings are in agreement with the findings of Abou 9amed
'13( !he protein content increased as the concentration of wheat straw hydrolysate
increased" at the same time the protein content gradually decreased when the fermentation
period increased
7.CONCLUSION
!he bioconversion effect of pineapple waste into %&P was evaluated using yeast !he
increase in biomass contents were observed when there was increase in pineapple waste
concentration !he highest biomass content of . cerevisiae and #.tropicalis was recorded on
7th day fermentation !he highest protein content of .cerevisiae and # tropicalis was
recorded on the 7thday of fermentation at E > concentration !he highest reducing sugar
content of yeast was recorded on 3 rdday of fermentation at E> concentration !he utili+ation
of reducing sugar was increased with the increase in the concentration of substrates !he
present findings reveals that pineapple waste can be used as effective alternate carbon source
for %&P production Also the orang peels have the same effects on all the para meters
@.REFERENCES
Argyro" " &ostas" P" Athanasios" AJ '266
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!ipparat" 9" Jitti/un" A9 '1E( .ptimi+ation of single cell protein production from
cassava starch using chwanniomyces castellii 0./.!icrobiol. 6 Biotechnol 11"
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MEDIA
1 POTATO DE0TROSE AGAR
P"#(#" I%,&i"% 3g
D$#!"&$ 2g
Ag(! 2g
PH 3.5
D'st'%%e" ater 9 #;;;,%
2 YEAST PEPTONE DE0TROSE AGARYeast etract 9 #;6:
Detr!se 9 (;6:
Pe-t!$e 9 (;6:
A6ar 9 (;6:
-2 9 @.;
D'st'%%e" ater 9 #;;;,%
53 basa% ,e"'a
D
1N2(3(SO/ 9 7.;6
K2(PO/ 9 #.;6
M6S!/: B2(O 9 ;.76
NaC% 9 ;.#6
CaC%( 9 ;.#6
D'st'%%e" ater 9 #;;; ,%
-2 9 7.7
REAGENTS RE4UIRED
12E : 9&l
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2+nthrone reagent1
?issolve 266 mg anthrone in 166 m* of ice-cold E> 92%.4 Prepare fresh before
use
3) tandard glucose1
St!cD'ss!%?e #;; mg in 166 m* water @or/ing standard of stoc/ diluted to 166
m* with distilled water %tore refrigerated after adding drops of toluene
4)Dinitrosalicylic +cid -eagent 'D% -eagent(
?issolve by stirring 1 g dinitrosalicylic acid" 266 mg crystalline phenol and E6 mg sodium
sulphite in 166 m* 1> :a.9 %tore at 4& %ince the reagent deteriorates due to sodium
sulphite" if long storage is re$uired" sodium sulphite may be added at the time of use
9)46> 5ochelle salt solution 'Potassium sodium tartrate(