SINGLE-CELL PROTEIN

SINGLE-CELL PROTIEN

What is Protein ??? Any of a group of complex organic macromolecules that contain carbon, hydrogen, oxygen, nitrogen, and usually sulphur and are composed of one or more chains of amino acids. Proteins are fundamental components of all living cells and include many substances, such as enzymes, hormones, and antibodies, that are necessary for the proper functioning of an organism. They are essential in the diet of animals for the growth and repair of tissue and can be obtained from foods such as meat, fish, eggs, milk, and legumes.

Protein requirement: In inactive lifestyle, the recommended dietary allowance (RDA) for a sedentary individual is about 0.8 grams per kilogram of body . So, if weight of body is 90 kg then protein requirement 90 x 0.8 = 72g per day protein is required. If lifestyle is active or hard working then protein required is 1.4g/kg to 1.8 g/kg of body weight daily.

Essentials amino acids Daily requirements (g) of essential aminoacids for the human adult Data retrieved from FAO (http://www.fao.org) Essentials amino acids FAO recommendation Minimum Phenylalanine 2.2 1.1 Methionine Leucine Valine 1.6 0.8 Lysine Isoleucine 1.4 0.7 Threonine 1.0 0.5 Tryptophan 0.25 Total 12.7 6.35

WHY DO WE NEED ALTERNATIVE SOURCES OF FOOD? About 50 years ago (1934-1938) the less developed areas of the world were the main exporters of grain to the developed world. Since 1948 the food flow has reversed, from the developed world to the less developed, mainly due to the rate of growth of the world's population which was much higher in the less developed countries. This means that during the 35-year period (1980-2015) we must produce as much food as we have since the dawn of agriculture about 12000 years ago.

REALITY Death from starvation, malnutrition and related diseases is a reality in many countries today. The World Health Organisation (WHO) estimates that 12,000,000 people die of hunger and starvation related diseases every year. Half are children under the age of 5.

The increasing world deficiency of protein is becoming a main problem of humankind. Since the early fifties, intense efforts have been made to explore new, alternate and unconventional protein. For this reason, in 1996, new sources mainly yeast, fungi, bacteria and algae named Single Cell Protein (SCP) as coined to describe the protein production from biomass, originating from different microbial sources. Microbial biomass has been considered an alternative to conventional sources of food or feed.

Large-scale processes for SCP production show interesting features, including: The wide variety of methodologies, raw materials and microorganisms that can be used for this purpose High efficiency in substrate conversion High productivity, derived from the fast growth rate of microorganisms Independence of seasonal factors

History A survey of the history of the use of microorganisms for human consumption indicates three major trends: microbes as a source of enzymes in the food industry (baking, brewing, distilling, wine making, cheese production) has at present a new aspect-the use of immobilized enzymes of microbial origin, (b) microbes as producers of nutritive substances (amino acids, nucleotides, vitamins, organic acids, sugars, aromatizers) and (c) direct utilization of microbial biomass as foodstuff.

HISTORY Used to named as Microbial Proteins. 1966: M.I.T Professor Carroll L.Wilson renamed it as “Single Cell Proteins” Transportation of food was common in the past but with the increase in population, energy crisis has encounter the world. In 60s’, idea that the dried cells of micro-organisms can become an ultimate part to solve this problem. Thus gained research interest among the scientists & industries (specifically oil industry).

In 20th Century, the SCP technology for the production of protein-rich contents from the microbes on the large scale was established. 1950s: Food-from-oil. 1960s: British Petroleum Industry developed technique named as “Protein- from-oil Process” using yeast fed on waxy paraffin, a product of oil refineries.

ADVANTAGES OF USING MICROORGANISMS MO grow at very fast rate under optimal conditions Quality and quantity is better than higher plants and animals Wide range of raw materials can be used Culture and fermentation conditions are simple MO can be genetically manipulated

Advantages of SCP over conventional protein sources are: It has high protein and low fat content. It is good source of vitamins particularly B-complex. e.g. Yeasts It can be produced through-out the year. Waste materials are used as substrate for the production of these proteins. It reduces the environmental pollution and helps in recycling of materials. SCP organisms grow faster and produce large quantities of SCP from relatively small area of land and time. These have proteins with required amino acids that can be easily selected by genetic engineering. During the production of SCP biomass, some organisms produce useful by products such as organic acids and fats. It can be genetically controlled. It causes less pollution. Algal culture can be done in space which is normally unused.

Factors that impairs the usefulness of Unicellular biomass All single-celled microorganisms of interest from the industrial point of view have a nondigestible envelope, which makes protein assimilation difficult. The content of nucleic acids in the unicellular biomass is higher than the permissible level and may cause disorders of purine metabolism in the human body. The biomasses of some unicellular microorganisms have an unpleasant color (algae), taste, and smell, which make them unsuitable even for animal consumption. Food grade production of SCP is more expensive than other sources of proteins, as it depends on the raw materials.SCP for human consumption is 10-12 times more expensive than SCP for animal feed. Digestion of microbial cells is rather slow, and is frequently associated with indigestion and allergy reactions.

SINGLE CELL PROTEINS

SINGLE CELL PROTEINS The term “Single Cell Protein” refers to the total protein extracted from the pure cultures of microorganisms (e.g. yeast, algae, filamentous fungi, bacteria) and can be used as a protein-rich food supplements by humans and animals. Also known as ”Microbial Protein”

Single-cell proteins develop when microbes ferment waste materials (including wood, straw, cannery, and food-processing wastes, residues from alcohol production, hydrocarbons, or human and animal excreta). 60-80% dry cell weight; contains nucleic acids, fats, CHO, vitamins and minerals Rich in essential amino acids (Lys-Met) The problem with extracting single-cell proteins from the wastes is the dilution and cost. Found in very low concentrations, usually less than 5% . Engineers have developed ways to increase the concentrations including centrifugation, flotation, precipitation, coagulation, and filtration, or the use of semi-permeable membranes.

Microorganisms Bacteria Methylophilus methylotrophus Pseudomonas sp. Brevibacterium sp. Yeasts Lactobacillus bulgaricus Candida lipolytica Bakers yeast Kluyveromyces fragilis Fungi Trichoderma viridae Aspergillus niger Actinomycetes Nocardia sp Thermomonospora fusca Algae Chlorella and Spirulina Mushrooms Agaricus Morchella Vovariella

“Possible Substrates for SCP” They can be subdivided into three categories: High energy sources (natural gas, n-alkanes, gas-oil, methanol, ethanol, acetic acid); Different wastes (molasses, sulfite waste liquor, milk, whey, fruit wastes); and Renewable plant resources (sugar, starch, cellulose).

A wide range of substrates can be used to grow microbial proteins whey, orange peel residue, sweet orange residue, sugarcane bagasse, paper mill waste, rice husks, wheat straw residue, cassava waste, sugar beet pulp, coconut waste, yam waste, banana pulp, mango waste, grape waste, sweet potato

Fungi Algae Yeasts Bacteria Average composition of the main groups of micro-organisms (% dry weight) Table : 1 Fungi Algae Yeasts Bacteria Protein 30-45 40-60 45-55 50-65 Fat 2-8 7-20 2-6 1.5-3.0 Ash 9-14 8-10 5-9.5 3-7 Nucleic Acids 7-10 3-8 6-12 8-12

Yeasts and Fungi Filamentous Fungi used for SCP production are Chaetomium celluloliticum, Fusarium graminearum, Paecilomyces varioti which grows on cellulose waste, starch, and sulphite waste liquor respectively and content about 30 – 55 % protein. SCP is produced from yeasts viz. Candida utilis, Candida lipolytica, Saccharomyces cervicea. Torula yeast (which grows on Ethanol) as a food is obtained through fermentation using molasses as substrate and it has high protein–carbohydrate ratio than forages. It is rich in lysine but poor in methionine and cysteine. Saccharomyces consists of high protein with good balance of amino acids and rich in B–complex vitamins. It is more suitable as poultry feed. Yeast are higher in lysine content. Strict aseptic conditions are required when using Yeast as a SCP production. Disadvantages: High nucleic acid content. Slow growth is observed in Fungi vis-à-vis than yeast & bacteria. Contamination risk. Mycotoxins are also produced.

2. BACTERIA They have more than 60% protein but are poor in sulphur containing amino acids. Brevibacterium uses hydrocarbons while Methylophilus methylitropous uses methanol as a substrate. Disadvantages: It has high nucleic acid content. Recovering the cells is a bit problematic. Endotoxin production should be carefully tested.

3. ALGAE: Chlorella, Scenedesmus acutus and Spirulina maxima are grown for SCP. These have about 60% protein with good amino acid composition but less in sulphur containing amino acids. Chlorella and Spirulina are used for commercial scale production in Taiwan, Thailand, Japan, Israel, Mexico and USA. It is spray dried and sold as pills and powders. Disadvantages: As they are rich in Chlorophyll, it is not advised for human consumption (except Spirulina). It has low density. There is lot of risk of contamination during growth.

Microorganism Substrate Used as Used commercially Algae   Chlorella sp. CO2 + sunlight Feed Yes (Japan and Taiwan) Scenedesmus acutus - Spirulina maxima Yes (Mexico) Yeasts Candida utilis (Torula Yeast) 1. Confectionery Yes (U.K.), Symba process 2. Ethanol Yes (USA) 3. Sulphite liquor Yes (Europe, USA, Russia) C. intermedia Whey Yes; Vienna process C. krusei (+ Lactobacillus bulgarius) Yes; Kiel process C. lipolytica n-alkanes (C10 - C23) + ammonia Yes (Russia) Kluyveromyces fragilis Food Yes (France); Le Bel process Saccharomyces cerevisiae Molasses (Food)* Yes Fungi Chaetomium cellulolyticum Cellulosic wastes Promising Fusarium graminearum Starch hydrolysate Yes (U.K) Paecilomyces varioti Sulphite liquor Yes (Finland); Pekilo process Bacteria Brevibacterium sp. C1 - C4 hydrocarbons Process developed Methylophilus methylotrophus Methanol Yes (U.K.),

Properties of SCP

Organism Mass Doubling One of the main advantages of SCP compared to other types of protein is the small doubling time of cells (td) as shown in Table Table : 2 Organism Mass Doubling Bacteria and yeast 10-120 min Mold and Algae 2-6h Grass and some plants 1-2wk Chickens 2-4 wk Pigs 4-6 wk Cattle 1-2 mo People 0.2-0.5 yrs

Organism (1,000 kg) Amount of Protein Due to this property, the productivity of protein from micro-organisms is greater than that of traditional proteins Efficiency of protein production of several protein sources in 24 hours (16)   Table : 3 Organism (1,000 kg) Amount of Protein Beef Cattle 1.0 kg Soybeans 10.0 kg Yeast 100.0 tn Bacteria 100x10,000,000 tn

Fungi Algae Yeasts Bacteria Average composition of the main groups of micro-organisms (% dry weight) Table : 4 Fungi Algae Yeasts Bacteria Protein 30-45 40-60 45-55 50-65 Fat 2-8 7-20 2-6 1.5-3.0 Ash 9-14 8-10 5-9.5 3-7 Nucleic Acids 7-10 3-8 6-12 8-12

Bacterial protein is similar to fish protein, yeast's protein resembles soya and the fungi protein is somewhat lower than the yeast's. Of course microbiological proteins are deficient in the sulphur amino acids cysteine and methionine and require supplementation, while they exhibit better levels of lysine.

(grams of amino acid per 100 grams of protein) Saccharomyces cerevicia Essential amino acid content of the cell protein in comparison with several reference proteins (grams of amino acid per 100 grams of protein) Table : 4 Amino Acids Cellulomonas Saccharomyces cerevicia Spirulina maxima PeniciIllium notatum Wheat Egg Cow milk Lysine 7.6 7.7 4.6 3.9 2.8 6.3 7.8 Threonine 5.4 4.8 - 2.9 5.0 Methionine 2.0 1.7 1.4 1 1.5 3.2 2.4 Cysteine 0.4 2.5 Tryptophan 1.0 1.25 1.1 1.6 Isoleucine 5.3 6.0 3.3 6.8 6.4 Valine 6.5 4.4 7.4 6.9 Phenylalanine 4.1 Histidine 2.7 4.9 Arginine

The vitamins of micro-organisms are primarily of the B type, B12 occurs mostly in bacteria, while vitamin A is usually found in algae. Table shows the vitamin content of various food MO; Vitamin content of various food micro-organisms (mg/100 g dry weight)

Other nutritional parameters which evaluate the quality of a given SCP are: - the digestibility (D) - the biological value (BV) - the protein efficiency ratio (PER) - the net protein utilisation (NPU)

The quality of SCP is an important factor for commercial production. First parameter which reflects the quality of a protein, is the is the percentage of the total nitrogen Digestibility Coefficient (DC) consumed which is absorbed from the digestive tract. Estimation of the Biological Value (BV) is a measure of nitrogen retained for growth or maintenance. An accurate method to evaluate the quality of proteins is the determination of the Protein Efficiency Ratio (PER), expressed in terms of weight gain per unit of protein consumed by the test animal in short-term feeding trials. Finally, the Net Protein Utilization (NPU) -equivalent to the calculation BVxDC-is a measure of the digestibility of the protein and the biological value of the amino acids absorbed from the food.

Nitrogen and Protein contents of microbial cells compared with selected foods of animal and plant origin Table : 5 Source Nitrogen (%) Crude Protein Filamentous fungi 5-8 31-50 Algae 7.5-10 47-63 Yeast 7.5-8.5 47-53 Bacteria 11.5-12.5 72-78 Milk 3.5-4.0 22-25 Beef 13-14.4 81-90 Egg 5.6 35 Rice 1.2-1.4 7.5-9.0 Wheat Flour 1.6-2.2 9.8-13.5 Corn meal 1.1-1.5 7.0-9.4

Problem of Nucleic Acids 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 acids (78 g/100 g protein dry weight) is the high level of uric acid in the blood, sometimes resulting in the disease gout. Uric acid is a product of purine metabolism.

Problem of Nucleic Acids Most mammals, reptiles and molluscs possess the enzyme uricase, and the end product of purine metabolism is allantoin. Man, birds and some reptiles lack the enzyme uricase and the end product of purine degradation is uric acid. The removal or reduction of nucleic acid content of various SCP's is achieved with one of the following treatments: chemical treatment with NaOH; treatment of cells with 10% NaCl; thermal shock. These methods aim to reduce the RNA content from about 7% to 1% which is considered within acceptable levels.

Production Of SCP: Production of SCP involves following steps: Selection of Strain of microbe and Substrate Fermentation Harvesting Post harvest treatment Processing of SCP

Basic Steps of SCP production:

Selection of Strain of Microbe & Substrate: Very Crucial step. Microbe selected shouldn’t produce toxicity in its biomass. It should not be harmful for a consumer to consume. Selected microbe should produce a large quantity of protein. Substrate should be cheap, effective, allow favorable growth and ease of isolation.

Fermentation: Is done in a large chamber either of glass or stainless steel called “Fermentor”. Fermentation should be done under sterilized conditions. Controlled conditions as necessary e.g. Temperature, Pressure, pH, Humidity etc. Usually fed-batch cultures are used for the fermentation of microbes.

General process for production of SCP

Harvesting For the producing and harvesting of microbial proteins cost is a major problem. There are many methods available for concentrating the solutions like filtration, precipitation, centrifugation and the use of semi-permeable membranes. The equipment used for these methods of de-watering is expensive and so would not be suitable for small scale productions and operations. Single cell proteins need to be dried to 10% moisture or they can be condensed and denatured to prevent spoilage.

Biomass Harvesting The microbial biomass can be harvested by a variety of methods. Single cell organisms like yeast and bacteria are normally recovered by centrifugation, flocculation and floatation. Filamentous organisms are recovered by filtration. It is important to recover as much water as possible prior to final drying. The whole operation is to be done under clean and hygenic conditions to keep the product and the broth that leaves the plant free of bacterial contamination. In some cases, an after-treatment of the biomass is desirable to reduce the unwanted compounds in the product or to isolate the protein. One of the important tasks is to reduce nucleic acid content, which is high in microorganisms (4-6% in algae, 10-16% in bacteria, 6-10% in yeasts and 2.5-6% in fungi) and can be hazardous to health.

Post-harvesting Fermentation: Isolated microbial colonies are subjected to various differential techniques. E.g. Centrifugation, Washing, Drying etc. Produced protein contain impurities in it e.g. carbohydrates, nucleic acids, lipid contents, salts etc Pure protein isolation can be done by disrupting the cell wall through crushing, crumbling, cycles of freezing & thawing, grinding & thermal shocks. Nucleic acid can be remove by: By treatment with Nacl 10% By Chemicals e.g. NaoH Thermal shocks Enzymes Treatment e.g. ribonucleases

Kluyveromyces fragilis 5000 tons/year Food yeast fermentation nutrient Raw material Organism Scale Product Organization Cheese whey Kluyveromyces fragilis 5000 tons/year Food yeast fermentation nutrient Universal Foods Corporation, Juneau, Wisconsin Ethanol Candida utilis 10,000 tons/year Torula yeast, food ingredient Pure culture products, Hutchinson, Minnesota N-paraffins Candida guillienmondis 20,000 to 40,000 tons/year Food yeast All Union Research Institute of Protein Biosynthesis, USSR Sulfite waste liquor 15 tons/year Torula yeast Rhinelender paper corporation Rhinelender Wisconsin

Fusarium graminearium 50-100 tons/year Mycoprotein (human food) Raw material Organism Scale Product Organization Glucose (Food Grade) Fusarium graminearium 50-100 tons/year Mycoprotein (human food) Ranks Hovis Mc Dougall, High Wycombe,UK Cheese whey Penicillium cyclopium 300 tons/year Animal feed Heurty , S.A.,France Coffee waste Trichoderma harzianum 40,000 lit ICAITI,Guatemala and El Salvador Sulfite waste liquor Paecilomyces varioti 10,000 tons/year Animal feed(Pekilo protein) Tampela and Finnish Pulp and paper Research Institute,Jamsankoski, Finland Pulp mill wastes Chaetomium cellulyticum 1 tons/day Animal feed(Waterloo process) Envirocon Ltd,Vancouver, BC, Canada;University of Waterloo,Ontario

Taiwan Chlorella Manufacture Co.Ltd, Taipei Organism Raw material Production Producer or developer Chlorella sp. CO2 2 metric tons/day Taiwan Chlorella Manufacture Co.Ltd, Taipei Scenedesmus acutus CO2,Urea 20 grams/ square meter/day Central Food Technological Research Institute,Mysore,India Spirulina maxima CO2, or NaHCO3, Na2CO3 320 metric tons/year Sosa Texococo,SA Mexico City

Economic parameters : The Scope of SCP production Products obtained via microbiological synthesis must be competitive with traditional food sources. When estimating costs involved in SCP production, such major factors as the biomass yield, cooling, and oxygen requirements should be taken into consideration. They depend not only on the choice of substrate, but on the choice of the microorganism as well. All this determines the cost of production and economic feasibility. It is obvious that one of the major factors limiting the use of hydrocarbon yeast is the residual hydrocarbon content. Demands of the country or its separate regions for protein of a particular type Expenditures for the delivery of finished products to the places of their consumption Disposal of by products.

ACCEPTABILITY OF SCP AS A HUMAN FOOD AND ITS TOXICOLOGY CONCERN Raw materials used in production of SCP are the main safety hazard. The acceptability of SCP when presented as a human food does not depend only on its safety and nutritional value but also the mind frame of people to consume material derived from microbes which is concerned to social and ethical issues like psychological, sociological and religious implications. A more intensive clinical and toxicology testing including short-term acute toxicity testing (animal species) and followed by extensive and detailed long term studies. And in return incurs a huge scientific and financial investment.

Biolipsticks and herbal face creams are produced in Japan from the phycocyanin pigment of Spirulina.  Formulation of SCP

APPLICATIONS 1. As protein supplemented food- Also source of vitamins, amino acids, minerals, crude fibers, etc. Supplemented food for undernourished children. 2. As health food- Controls obesity Provides instant energy . Example- Spirulina- part of diet of US Olympic team.

Reduce body weight, cholesterol, stress. 3. In therapeutic and natural medicines- Reduce body weight, cholesterol, stress. Lowers blood sugar level in diabetic(due to presence of B - linolenic acid) Prevents accumulation of cholesterol in body. Healthy eyes and skin (beta carotene) Beta carotene ( anti cancer substance- UN National Cancer Research Institute) Increase lactation.

MEDICINAL USES OF SPIRULINA Strengthen and improve immune system Phycocyanins build blood cells Increase antiviral activity Exhibits anti cancer activity The US Olympic teams take spirulina tablet as a source of instant energy. Studies showed that spirulina consumption of 4 weeks reduced serum cholestrol level in human beings by 4.5% and significantly reduced body weight by 1.4±0.4 kg after 4 weeks. There is no changes in clinical parameters (Blood Pressure) or in biochemical variables (haemoglobin, blood cells, sedimentation rate) and absence of adverse effects.

4. In cosmetics- Important role in maintaining healthy hair (vitamin A and B). Many herbal beauty products. Biolipstics and herbal face cream(Phycocyanin). Capable of replacing coal tar dye based cosmetics. 5. Poultry and cattle feed- Excellent, convenient source of protein and other nutrients. Used to feed cattle, fishes etc. 6. In the technical field as: Paper processing, leather processing and as foam stabilizers.

CONCLUSION At present SCP production is in its infancy. One of the ways to enhance productivity and quality is genetic improvements of micro-organisms. Using microbial biomass as a food source deserves serious consideration because of insufficient world food supply and high protein content of most micro- organisms.

For future success of SCP First, food technology problems have to be solved in order to make it similar to familiar foods and Second, the production should compare favourably with other protein sources.

Sample of the single cell protein biosolids after the drum dryer.

THANK YOU -PHARMA STREET