Production of Single Cell Protein From Pineapple Waste and Orange Peel Using Yeasts and...

7/26/2019 Production of Single Cell Protein From Pineapple Waste and Orange Peel Using Yeasts and Aspergillusniger

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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

2


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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

9


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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 '&ampelo 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 < -;

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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(

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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

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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


31/46

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

32


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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

30


33/46

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


1 > 1 1 3 1 66;

2 > 1 1 3 1 617

33


34/46

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


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(

Production of Single Cell Protein From Pineapple Waste and Orange Peel Using Yeasts and...

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