SINGLE CELL PROTEIN (SCP) PRODUCTION BY BACILLUS SUBTILIS FROM PINEAPPLE FRUIT EXTRACT

INTRODUCTION

Single cell protein (SCP) typically refers to sources of mixed protein extracted from pure or mixed cultures of algae, yeasts, fungi or bacteria (grown on agricultural wastes) used as a substitute for proteinrich foods, in human and animal feeds. Since 2500 BC yeasts have been used in bread and beverage production. In 1781 processes for preparing highly concentrated forms of yeast were established. Commonly referred to as SCP, these products are dried cells of microorganisms such as algae, actinomycetes, bacteria, yeasts, molds and higher fungi which are grown in large fermentors. Single cell protein has the potential to be developed into a very large source of supplemental protein that could be used in livestock feeding. In some regions single cell protein could become the principal protein source that is used for domestic livestock, depending upon the population growth and the availability of plant feed protein sources. This could develop because microbes can be used to ferment some of the

vast amounts of waste materials, such as straws; wood and wood processing wastes; food, cannery and food processing wastes; and residues from alcohol production or from human and animal excreta. The term single cell protein (SCP) refers to dead, dry cells of microorganisms such as bacteria, Yeast, Fungi and algae which grown on different carbon sources. The term single cell protein was used for the first time, fifty years ago by the M.I.T professor Carol Wilson to give a better image than Microbial protein (Ware, 1977).

About 70 years ago (1934-1938) the less developed areas of the world, Asia, Africa and South America, were the main exporters of grain to the developed world. Since 1948 flow has reversed, from the developed world to the less developed, mainly due to the rate of growth of the worlds population which was much higher in the less developed countries (Brown, 1968). Based on present trends United Nations population experts project that there will be 8 billion people living on this planet by 2015 and 10.5 billion by the year 2110. This means that during the 35-years period (1980-2015) we must produce as much food as we have since the dawn of agriculture about 12000 years ago (Miller, 1985).

In the 1950s many people predicted a future protein mortgage in the world, due in 30-40 years. We know see that these predictions 2

were rather pessimistic for the developed countries. However death from starvation malnutrition and related diseases is a reality in many countries today. The world Health Organization (WHO) estimates that 12,000,000 people die of hunger and starvation related diseases every year. Half are children under the age of 5 (Miller, 1985).

Due to such a great population growth man could not have depended only on agriculture husbandry and fisheries for food. An energy crisis has an immediate impact on agriculture and hence man should have searched for other types of food for his survival.

During the sixties the idea that the single cell protein could help the less developed countries in future food shortages was gaining research interest among scientists in universities and industry

,particularly in oil. The result was the development of SCP technology either for livestock or for human consumption.

Although man has been familiar with the food and feed usage of microorganisms for centuries, SCP technology for food was developed over the last 100 years while large scale production has developed in the 20th century and particularly after the First World War. This was in Berlin with the growth of Sacharomyees cerevisiae on a production level to replace as much as 60% of the food stuffs Germany had been

importing after the war (Rose, 1981).Also yeast storing of Candida arborea and C. utilis made an important contribution to the diet in Germany in World War II. After World War II, growth of fungi in submerged culture for production of antibiotics led to an investigation of the potential micro fungi as flavour additives to replace mushrooms.

In the 1950s some oil industries became interested in the growth of micro organism on alkanes. The microorganisms which were used developed into a potential diet for feed and food. Many companies producing SCP including BP (UK), Kanegafuichi (Japan) and Liquichimica (Italy) appeared on the scene. In the USSR 12 out of 86 plants making SCP were said to rely on hydrocarbon as the source of carbons and energy for the microorganisms. Other potential substrates for SCP include bagasse, citrus wastes, sulphite waste liquor, molasses, animal manure, whey, rice brawn, starch, sewage, etc. In general the more reduced the substrate, the greater the cell yield (YX/S) and the more oxygen required for the oxidation of the substrate (Mateles, 1979). In this case a serious problem is the great heat which is produced, i.e. the cost which is required for cooling off the fermentors. Finally the cost of the substrate in connection with feasibility of the process.

Nutritional values of SCP

For the assessment of the nutritional value of SCP, factors such as nutrient compostion, amino acid profile, vitamin and nucleic acid content as well as palatability, allergies and gastrointestinal effects should be taken into consideration (Lichtfield, 1968).

Bacterial protection is similar to fish protein, yeasts protein resembles Soya and the fungi protein is some what lower than the yeasts. Of course microbiological proteins are deficient in the sulphur amino acids cysteine and methionin and require supplementation, while they exhibit better levels of lysine with microbial cells it is important to note that digestibility is low especially with algae cells because of indigestible cell walls. Although the protein efficiency ratio of SCP compares favorably with the protein deficiency ratio of conventional protein sources, acceptability and palatability results on nutritional trials were not always encouraging(Ware,1977).

The problem of nucleic acid is about 70-80% of the total cell nitrogen is represented by amino acids while the rest occurs in nucleic acids. This concentration of nucleic acids is higher than other conventional proteins and is characteristic of all fast growing organisms. The problem which occurs from the consumption of proteins with high

concentration of nucleic acid (78-25g/100g protein dry weight) is the high level of uric acid in the blood, sometimes resulting in the disease gout (While et al, 1964).Uric acid is a product of purine metabolism. Most mammals reptiles and molluses possess senyme uricase, and the end product of purine metabolism is allantonin.

REVIEW OF LITERATURE
The protein from microorganisms, popularly known as single cell protein, offers the best hope for being a new source of major proteins, independent of agriculture. However, high cost of production limits the feasibility of SCP. To bring down the cost, the domestic and industrial wastes, which are ready available, should be used to produce SCP. India is primarily an agricultural country with the largest livestock population of more than 300 million. Biogas plants, where animal during and other agricultural wastes are anaerobically digested to generate methane, used in turn as fuel gas, are quit popular and are fast becoming an internal part of rural economy. Bacteria are frequently among the predominant populations of microorganisms in ponds, ditches, and other water sources, polluted by sewage or other types of organic matter (Sudhanshu Vrati, 1984). Use of microbes as a food source may appear to be unacceptable to some people but the idea of consumption of microbes as food for man and animals is certainly innovative to solve the global food problem. Algae, fungi, yeast and bacteria are the chief of microbial protein that can be utilized as a protein supplement (Faust, 1987 and Anupama, 2000). Different means by which to guarantee the global

supply and distribution of available sources of proteins were discussed in the 1960s (Brown, 1968). At that time there were doubts of the ability of agriculture and the fishing industry to supply the increasing need for protein feed supplements. An exploitation of microorganisms was considered as one possibility to cover the protein shortage (Abbort, 1973).Many microorganisms have high protein content and may be produced in large quantites by industrial fermentation from simple raw materials with a low or no nutritional value. A new technology was developed that exploited fungi, yeasts, and bacteria to produce socalled single cell protein (SCP) with a high nutritional value from inorganic nutrients and simple organic chemicals, such as hydrocarbons, alcohols, or carbohydrates (Humphrey, 1974; Hamer and Harrison, 1980).

Most of the developing countries of the world have been facing malnutrition problem. The deficiency of protein in human food and animal feed is well recognized due to the rapid growth of population. It has been reported that in India the protein measures are adopted to handle the situation. It is there fore, important to increase protein production by utilizing all the available ways and means. The increasing world demand for food and feed protein spurred the search for nonconventional protein source. A great deal of interest has been focused on the potential of agricultural wastes to microbial protein or single cell

protein. The impetus behind single cell protein production lies partly in the need for more protein and partly in the commercial increase in the economic advantages gained by substitution of microbial protein for the conventional protein supplements used in live stock feeding.

Many microbial species are used as protein rich food. They provide the B-complex group of vitamins and they also show a low level of nucleic acid content (Frazier and Westhoff, 1990). The amino acid composition of Aspergillus niger according to FAO standards is well balanced (Kuzmanova et al.1989). Yeast contains thiamine, riboflavin, biotin, choline, niacin, pantothenic acid, pyridoxine, streptogenin, glutathione folicacid p-benzoic acid (Frazier and Westhoff, 1990). Single cell protein from mixed cultures of Trichoderma reesei and

kluyveromyces marxianus are reported to contain essential amino acids which compares favorably with FAO guidelines and soybean oil meal (Ghanem, 1992).

Several studies have involved various microbial species in producing single cell protein from waste celluloses and fungal mycelial biomass is an acceptable source of edible protein (Mary and James, 1969; Reade et al, 1972; Soloman, 1973; Yakoub Khan et al, 1992; Nigan, 1998; Anupama and Ravindra, 2001; Uysal, 2002; Schultz et al., 2005).

The aim of the present work was to investigate the potential and effectiveness of using pineapple extract as a substrate for the production of single cell protein by Bacillus subtilis.

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MATERIALS AND METHODS

Glassware and chemicals
In all the experiments acid washed (0.4N HCl) Pyrex glassware rinsed with double distilled water was used. For the preparation of culture media and other chemical reagents analytical grade chemicals (E Merk GR/BDH, Analar R) were used.

Sterilization
All the glassware used in the experiments were sterilized in hot air oven at 160C for 4 hours. The media used in the experiments were sterilized in an autoclave at 15 lbs per square inch for 15 minutes.

Composition of media employed

For isolation and the study of bacteria, nutrient agar and nutrient broth medium used in the present work. Nutrient agar medium Nutrient agar medium was performed as the medium for the culture of the bacteria. It was prepared with the following composition. Beef extract Peptone NaCl Agar Distilled water 5g 5g 20g 1 lit. 3g

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After preparation, it was mixed well until the contents are dissolved and then autoclaved at 121c for 15 minutes. Nutrient broth Nutrient broth does not contain agar. Nutrient broth (NB) is used for enrichment of specified bacteria. Beef extract Peptone NaCl Distilled Water 5g 5g 1000 ml 3g

Collection of water sample

Water samples were collected from marine water in Mypad beach, Nellore District, A.P, India. The samples were collected in sterilized bottles for isolation of Bacillus subtilis (single cell protein- produce bacteria).

Isolation of bacteria
The bacteria strain was isolated by serial dilution plate method (Aneja, 2001) described as follows. 1 ml of marine water samples were taken into 9 ml of sterilized water blanks to get 10-1 dilutions. From these dilutions, 1ml was transferred to another 9ml sterile water and thus six dilutions (10-1 -10-6) were maintained. 1 ml of aliquots was transferred from six dilutions to the corresponding labeled Petri plates. Triplicates were maintained for

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each dilution. The molten and cooled nutrient agar medium was poured into the Petri plates and rotated gently for proper distribution of the inoculum suspension with the medium. After solidification, the plates were incubated at 27 1C for 48 hours. After incubation, the colonies were separated, sub cultures were maintained.

Maintenance of pure cultures

The identified colonies were sub cultured on nutrient agar slants and preserved at 4C. Sub-culturing was performed at one month interval.

Identification of bacterial strain

For identification, bacterial cultures were prepared for

morphological and biochemical characters were performed observed their morphological characters and identified with the help of the standard keys. The macro/microphotographs of selected bacteria were also taken wherever it was found necessary.

Morphological characterization
Grams Staining 1. Smear and heat fix a clean microscope slide with your bacterial culture.

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2. Put 10 to 15 drops of Crystal Violet stain on your bacteria smear and leave on for one minute. 3. Rinse the Crystal Violet stain off with water (from the washing bottle). 4. Put the Grams' stain on the smear and leave it on for one minute, then rinse it off with water from the washing bottle. 5. Add 10 to 15 drops of 95% ethanol and leave on for 10 to 15 seconds. Rinse off with water for the washing bottle. 6. Place the safranin on the slide and leave on for 45 seconds. Rinse off with water from the washing bottle. Add a cover slip. 7. Look under the microscope. Determine whether or not your bacterial culture stained positive or negative. Endospore Staining 1. Prepare smears of organisms to be tested for endospores. 2. Heat fixes the smear. 3. Cover the smears with a piece of absorbent paper cut to fit the slide and place the slide on wire gauze on a ring stand 4. Saturate the paper with malachite green and holding the Bunsen burner in the hand heat the slide until steam can be seen rising from the

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surface. Remove the heat and reheat the slide as needed to keep the slide steaming for about three minutes. As the paper begins to dry add a drop or two of malachite green to keep it moist, but don't add so much at one time that the temperature is appreciably reduced. overheat. The process is steaming and not baking. 5. Remove the paper with tweezers and rinse the slide thoroughly with tap water. 6. Drain the slide and counter stain 45 seconds with 0.5% safranin. 7. Wash, blot, and examine. 8. The vegetative cells will appear red and the spores will appear green Capsule Staining Procedure 1. Place one loopful of India ink at one end of the microscope slide. 2. Mix 1 loopful of sterile saline with the Ink. 3. Aseptically transfer and mix small amount of bacteria in the loopful India ink. 4. Take a second slide and hold at a 45 degree angle touch the end of the slide to the other slide and pull the slide to meet the drop. 5. Without raising the slide push the top slide back to spread the stain. Do not

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6. Dispose of the second slide. 7. Do not Heat Fix! Allow slide to thoroughly air dry. 8. Flood smear with Ethylene blue for 3 minutes. 9. Remove Ethylene blue and gently rise the slide with water. 10. Do not Heat Fix! Allow slide to thoroughly air dry. 11. Add immersion oil to smear and observe. Motility Test (Hanging Drop Method) 1. Place a small drop of liquid bacterial culture in the center of a cover slip 2. Place a small drop of water at each corner of the cover slip 3. Invert a slide with a central depression over the cover slip 4. The cover slip will stick to the slide and when the slide is inverted the drop of bacterial culture will suspended in the well 5. Examine microscopically (X400) for motile organisms.

Biochemical characterization
Catalase Test A bacterial colony was picked up with a toothpick or a platinum loop and mixed with a drop of hydrogen peroxide (10% v/v in water) taken on a

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glass slide. The effervescence indicates the presence of the catalase enzyme. Oxidase Test 1. Pick a well-isolated colony from the culture plate or slant culture and place it on a piece of filter paper. 2. Add one drop of the reagent (if it is dark blue, it is old and should not be used). 3. Or you can drop the reagent directly onto the slant or plate, but that might damage your culture. TIME the reaction: a positive reaction will occur within 20 seconds. DO NOT READ the reaction after 30 seconds. Preparation of Culture Media and Reagents for Biochemical characterization The cultivation of microorganisms in the laboratory requires that the needed nutrients and suitable environmental conditions be provided. Nutrients are those raw materials that are used to sustain living organisms, promote growth, replace cellular constituents, and provide energy for metabolic reactions and cell movement. In the laboratory, nutrients are supplied to microorganisms in culture media. A culture medium generally contains water, an energy source, and nutritionally suitable sources of carbon, nitrogen, sulfur, phosphorus, oxygen, 17

hydrogen and other ingredients such as metal ions, vitamins, etc.The culture medium may be in a liquid or in a solid gel form: Liquid culture medium for bacteria is often prepared from basal media such as nutrient broth. Enriched medium is any basal medium, which has been supplemented (enriched) with serum, blood or extracts of plant or animal tissues etc. in order to be able to support or enhance the growth of particular microorganisms. Procedure 1) LB broth Prepare 100ml LB medium by dissolving the appropriate amount of yeast extract, tryptone and NaCl in distilled water. Adjust pH to 7.2. Sterilize in autoclave for 20 minutes. (Do not forget to cover the top of the flask with aluminum foil and put autoclave tape) 2) LB-agar medium (1000 ml) Prepare 100ml LB-agar medium by dissolving the appropriate amount of yeast extract, tryptone, NaCl and agar in distilled water. Adjust pH to 7.2. Sterilize in autoclave for 20 minutes. 3) Citrate Agar Medium 1. Dissolve ingredients, as mentioned in the materials then autoclaved at 121C for 15 minutes. 2. The media was poured into test tubes and allowed to solidify the slants.

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3. 24 hr old culture was taken by means of inoculation loop and streaked on the agarified slants. 4. Observed for colour change. 4) Tryptone Broth 1. Dissolve the ingredients in the water. Cooled to room temperature. 2. PH was adjusted to 7.5 0.1 and filter if necessary. Dispense and autoclave at 121C for 15 minutes. 3. Broth was poured into test tubes and then inoculate with 24 hr old culture. 5) Indole Test 1. Inoculate the tryptone (or peptone) broth that was prepared as mentioned in the materials with the test organism and incubate at 37C for 24-48h 2. Add 0.5mL of Kovacs reagent and gently agitate 3. Examine the upper layer of liquid. Positive result Red colour (occurring within a few seconds) Negative result Yellow colour

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MR TEST 1. Using sterile technique, inoculate each experimental organism into its appropriately labeled tubes of medium by means of an inoculation loop. The last tube will serve as a control. 2. Incubate all cultures for 24 to 48 hrs, at 37C 3. after 24 hrs. of incubation add 2 to 3 drops of MR reagent.

VP Test 1. Using sterile technique, inoculate each experimental organism into its appropriately labeled tubes of medium by means of an inoculation loop. The last tube will serve as a control. 2. Incubate all cultures for 24 to 48 hrs, at 37 c 3. After 24 hrs incubation add 1 to 2 drops of Barretts A reagent and Barretts reagent and observe for change in colour.

Triple Sugar Iron Agar 1. Using sterile technique, inoculate each experimental organisms into its appropriately labeled tube by means of a stab-and streak inoculation. The last tube will serve as control. 2. Incubate for 18 to 24 hrs at 37 c. Carbohydrate Fermentation Rapid Biochemical Assay: The API-20E employs a plastic strip composed of 13 individual micro tubes, each containing a dehydrated medium in the bottom and an

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upper cupule. The media become hydrated during inoculation of a suspension of the test organism, and the strip is then incubated in a plastic covered tray to prevent evaporation. In this manner 13 carbohydrates tests are performed. Following incubation, identification of the organism is made by using differential charts supplied by the manufacture or by means of a computer-assigned system called PRS. The traditional method of noting the characteristic color changes and interpreting them according to manufacturers instructions Effect of PH on cell growth 1. The effect of pH on the cell growth was observed by adjusting the pH of the production media from pH 4.0 - 8.0 by the addition of either HCl or 2N NaOH with the help of a sensitive pH meter. 2. Then the inoculation of the production media was carried out and the cultivation was carried out for 24 hours on a platform shaker. Controls were prepared for each pH. 3. After 24 hours, the absorbance was read at 600 nm. Effect of Temperature on cell growth 1. The pH of the production media was adjusted to the optimum pH at which maximum growth was observed. 2. Four flasks containing the media were inoculated and maintained at 20C; 30C; 37C and 50C for 24 hours. Control was set up in each case. 3. After 24 hrs, the absorbance was read at 600 nm.

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Preparation of pineapple fruit extract

Pineapple fruit extract was used as a substrate for the growth of microorganism and production of single cell protein. A 500g ripe pineapple fruits were obtained from the local fruit market, TIRUPATI. The fruits were washed with several changes of sterile water to remove dust particles and peeled. Fruits cut in to sliced pulp, cleaned initially with 2% solution of H2SO4 sliced into cubes, rinsed in sterile water, and pulverized into slurry using a sterile blender. The fruit extract was obtained from slurry filtered with the use of cheese cloth. The extract was placed into a sterile container and the crude protein was determined.

Preparation of inoculum
Bacterial cells were harvested from a 48 hour old Bacillus subtilis in 5ml of sterile distilled water to get 8010-6 cells/ml.

Fermentation medium
From the extract, 100ml was measured into sterilized 250ml conical flasks. To each was added glucose 2%, Fructose 2%, sucrose 2%, maltose 2%, ribose 2% and sodium chloride, sodium nitrate, potassium nitrate, casein which served as nitrogen source supplement. The conical flasks were plugged loosely with sterile cotton wool and aluminum foil. Sterilization was achieved by autoclaving at 121C for 15 min. On cooling, the medium in the flasks was inoculated with 1ml of bacterial

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suspensions (1010-6 cells/ml). Aeration was achieved by shaking in an orbital shaker at 200 rpm for 10 days at 27C. The contents of each flask were harvested weighed separately and homogenized in a homogenizer. The homogenized contents of sample from each flask were centrifuged at 600 rpm for 45 min. The supernatant volume was measured and used for further analysis.

Protein estimation
Protein in the culture supernatant was measured by Folin method of Lowry et al (1951) using bovine serum albumin as standard. Preparation of Reagents A = 2% sodium bicarbonate in 0.1N NAOH B = 0.5% copper sulphate in 1% sodium tartarate C = Mixture of 50ml reagent A with 1ml of reagent B D = Folin phenol reagent. All the above reagents were freshly prepared for the protein estimation. 1ml of culture supernatant was taken for protein extraction using 20ml of 20% TCA. The contents were centrifuged and the supernatant was discarded. The protein precipitate was washed twice with cold TCA, centrifuged again and the protein precipitate was dissolved in 0.1N sodium hydroxide and made up to 2ml, out this 0.1 ml was taken in a test tube, 5ml of reagent c was added, mixed thoroughly and the solution allowed to stand for 10 min at room temperature. The 0.5 ml of

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reagent D was added with instantaneous and vigorous shaking. After 30 min the color intensity was read at 660 nm on Shimadzu SpectroPhotometer. A blank was prepared by using the same volume of glass distilled water in place of protein extract. A reference curve was prepared with the known concentration of bovine serum albumin.

RESULTS

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The bacterium was obtained in pure culture from marine water sample. The bacterium was related mainly to the generic nomenclature Bacillus known as Bacillus subtilis.

Bacillus subtilis
The bacterial strain was identified based on their morphological characters and biochemical characters and was pure cultures were streaked on agar plates (Fig. 1 a & b) and also maintained subculture on agar slants at 40C prior to use (Fig. 2 a & b).

Morphological characteristics
Grams staining, capsule and spore staining, motility and colony morphology of the isolates were studied for identification. The results of these observations are listed in Table 1. Table 1: Morphological characterizations of bacterial isolate property Morphology of the cells in nutrient broth Name of test 1.Gram stain 2. Shape 2.Motility 3.Spores 4.growth Isolate Gram-positive Rod shaped bacilli Motile Sporulate Turbid, with sediment

Fig. 1 A & B

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(A)

(B)

Fig. 2 A & B 26

(A)

(B)

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Biochemical Tests
The isolates were further characterized by biochemical studies. The results are given in table 2.
Table 2: Biochemical characterization of selected bacterial isolates
Biochemical tests Catalase ONPG Lysine decarboxylas e Ornithine Urease Phenyl alanine deamination Nitrate reduction H2S production Citrate utilization Voges proskaeurs Methyl red Indole Malonate Blue Positive Reaction Bubbles were produced Purple Purple Purple Orangish Yellow Colourless Isolate Positive Negative Positive Negative Negative Negative Positive Positive

Colourless Orangish yellow

Colourless Methyl red Colourless Light green

Negative Positive Negative Positive

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Pineapple extract characterization

The physical and chemical characteristic of ultra filtration permeate pineapple extract are given in Table 3. The initial PH value of the pineapple extract used in this study was 7.2; however the optimum PH for the growth and survival of Bacillus subtilis is between 5.0 and 6.0.

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It has also been recognized that keeping the PH at about 5.5 eliminates possible contamination by fungi and bacteria that grow at PH above 6.0. Thus, in this study, PH of the medium was adjusted to 5.5 by the addition of N HCl solution.

Since the protein content of pineapple was low, no difficulties related to precipitation of protein during preparation were encountered and therefore pineapple extract is a more suitable substrate for SCP production than others substrate.

Table 3: The measured values of major composition of pineapple __________________________________________________________________

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Ingredients

Percents

__________________________________________________________________ Water Carbohydrates Reducing sugars Proteins Nitrogen Phosphate Ash PH 69.5 18.0 11.6 1.5 0.9 1.3 0.7 6.2

__________________________________________________________________ * Average values of triplicate samples.

Single cell protein production

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For protein yield, various media combination with different carbon and nitrogen souses were supplemented with pineapple substrate was studied. The biomass in terms of the final protein content of the moldy groundnut cake harvested on the seven day is shown in the Table 4.

Pineapple extract when supplemented with carbon and nitrogen sources individually or together improved the biomass yield (expressed in terms of total protein yield). Among all these combination, the supplementation of Pineapple extract with sodium nitrate gave the highest protein yield. Supplementation of Pineapple extract with carbon individually also improved the biomass yield. But the biomass yield was not as high as that with the sodium nitrate supplementation. Individual nitrogen sources in combination with carbon source supported the biomass yield to a lesser extent as compared to sodium nitrate or carbon source (Table 4).

The yield of protein content from pineapple extract supplemented with different carbon and nitrogen sources ranged from 8.40 to 52.50 mg. Total protein content, the supplementation of Pineapple extract

with sodium nitrate gave the highest protein yield (Table 4).

Table 4: Final protein yield in various media combinations 32

________________________________________________________________________ Content of the flask Protein yield (mg/g) C/N ratio

________________________________________________________________________ Control with pineapple extract Control with water PE+glucose+sodium chloride PE+sucrose+sodium nitrate PE+fructose+potasium nitrate PE+maltose+sodium chloride PE+sucrose+ammonium nitrate PE+fructose+ammonium sulphate PE+ribose+casein 21.60 8.40 30.20 52.50 31.50 30.96 31.50 33.56 35.40 1.530 1.530 1.530 1.530 1.387 1.387 1.387 1.387 1.387

________________________________________________________________________ *Mean values of triplicate samples PE = Pineapple extract C/N ratio = Carbon and Nitrogen ratio

DISSCUSSION

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Members of the genus Bacillus produce a large variety of extracellular enzymes, of which proteases are particularly significant industrial importance. Bacillus subtilis was isolated from marine water by using enrichment and screening procedure for protein that are thermostable and alkali tolerant. Reade and Gregory (1975) reported that autolysis is likely to be increased with high initial inoculum. This is because there is the existence of disproportionate amount of nutrients, as well as lower conversion of efficiency. According to Chikwendu (1987), at lower inoculum level cells are larger and further indicated that this type of growth arises when competition for available nutrients is not great among the cells present. Data obtained from the amount of biomass yield corresponds with that of protein produced during fermentation in this study. The total amount of protein in 10 days was measured highest which is considerable and represents pineapple extract suitable for protein production under conditions treated. From the above observations it is clear that the availability of the nitrogen is the major controlling factor in the final biomass yield. Supplementation with carbon sources individually, gives higher yield because it provides continuous acidic medium to support the growth. As the organism grows in this condition it produces more of cellulases. These enzymes make available more of glucose monomers from the

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pineapple in the medium as well as help in the release of nitrogen for the growth. The biomass yield was the highest when sodium nitrate supplements the medium alone. But when it was used along with the carbon source its free availability for the growth seems to be hindered. The better yield and growth rates were observed when supplemented with carbon and nitrogen sources. This suggests that continuous culture may be more successful in obtaining higher yields of SCP. For industrial large scale production, the protein cost could be still reduced by using cheaper carbon and nitrogen sources. The level of crude protein of more than 50% obtained from the biomass

recommends the product as a potential food and feed supplement when compared to the lower limits of 8% for cattle and poultry feed (Han and Anderson, 1974). The cost effective SCP process can be performed in an industrial scale and the product can be consumed instead of expensive proteins present in the market (Schultz et al., 2005). The product yield could even be increased further by diluting the substrate, but the costs of harvesting the bacteria would then increased and proved to be uneconomical. Such a fermentation process could be carried out on a commercial scale but the economics depend upon a number of factors, such as availability of the substrate (pineapple extract) throughout the year. On other hand, continuous

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culture appears to be more suitable and would compare favourably with the present bacterial production facilities. The latter is important for efficient utilization of the fermentation equipment. The production of bacterial protein from pineapple extract should receive high priority. Furthermore, the economics would be favourable if the strength of the substrate could be increased.

CONCLUSIONS
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A higher yield of SCP production from Bacillus subtilis was possible by fermentation of pineapple fruit extract. Though, utilization of liquid state fermentation for SCP production is emerging field, encouraging results are obtained and some success is achieved in improving the overall protein yield by supplementation of pineapple extract based liquid state fermentation medium. The C/N ratio was not very effective in controlling biomass yield but the supplementation of pineapple extract with various nitrogen sources or carbon sources in combination improved the Bacillus subtilis growth. The biomass yield was best with pineapple fruit extract based medium with sodium nitrate as the nitrogen source for single cell protein production. With regards to the results obtained from this work, pineapple extract is a proper substrate for single cell protein production under conditions provided in this study, however, for profitable production, interruption of the process in the fermentation, in which more than 50% of total protein would be produced, can be useful. Also, further studies should be done to investigate the nucleic acid content and find some ways to reduce it to permitted levels. Finally, cost effective SCP process can be performed in an industrial scale and the product can be consumed instead of expensive proteins present in the market.

REFERENCES

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