Production of Single Cell Protein from Pineapple Waste and Orange Peel Using Yeasts And

A

PROJECT REPORT

ON

“PRODUCTION OF SINGLE CELL PROTEIN FROM PINEAPPLE WASTE AND ORANGE PEEL USING YEASTS AND Aspergillusniger”

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

CERTIFICATE

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 PriyaBande, 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.Chandrashekharkulkarni, 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 mandatoryscholastic 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.

DECLARATION

I hereby declared that the work presented in the Project entitled””has been carried out by GHANTALA NIRAV J. under the guidance of PriyaBande, Project Guide, at MITCON, Pune. The entitled Work is original and no partof this work is either published or submitted in any university for the award ofany degree or diploma.

Date:

Place: Pune

S.NO. CONTENTS PAGE.NO

1. Introduction 7

2. Materials & methods 22

3.Observation Tables 29

4. Results 36

5. conclusion 41

6.References41

7.Appendix 43

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 proteinsources in foods or animal feeds. In the present study, pineapple waste was used as sole carbon source in fiveconcentrations for preparation of fermentation media on which two strains of yeasts, Saccharomyces cerevisiae,Candidatropicalis,Pichia stipites and Aspergillusniger and were grown. The increased concentration of pineapple hydrolysateand 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,Aspergillusniger.

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 contentand 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 ofwastes 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, thesingle cell organisms probably present the best chances forthe development of unique independence ofagricultural 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 ofproteins 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 compoundsand 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.

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;Hussainet al., 2007). Although animal proteins are considered to be best quality proteins (Saimaet 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 (Bekatorouet al., 2006) Moreover, microbes are also able to grow on cheap nutrient sources resulting in economical,potentially supplemental protein biomass for balancednutrition.

The UN work for the servey of food consumption of worldwide.Itrecently 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 Geotrichumcandidumin 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 andSaccharomyces cerevisiae.Pichiastipitis 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 (AnanassativusSchult. f.) is widely grown inthe north eastern region of India.In India nearly 100different 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 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, kinnow-mandarinwaste, barley straw, corn cops, rice straw, cornstraw, onion juice and sugar cane bagasse (Nigam et al., 2000), cassava starch (Tipparatet al., 1995), wheat straw (AbouHamed, 1993), banana waste (Saquidoet al., 1981), capsicum powder (Zhao etal., 2010) and coconut water (Smith and Bull, 1976). The usage of such wastes as a sole carbonand 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.

1.2Nutritional / 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 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 n-9) 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 acidcomposition of cold adapted carotenogenicbasidiomycetous yeasts was studied byLibkind et al. (2008). Total fatty acids of six yeast species isolated from thetemperate 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

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 comparablein amino acid content to the plant and animal sources with the exception of sulphuramino 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 orthey 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 andMarchant1986).

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 forgrowth and survival of molluscs (Brown et al., 1996).