SINGLE CELL PROTEIN Aim- The purpose of this topic is to discuss the function, production and properties of

single cell protein. Single cell protein is a very good source of protein and is used as a source of protein from ancient time.
HISTORY Since 2500 BC yeasts have been used in bread and beverage production. In 1781 processes for preparing highly concentrated forms of yeast were established. In 1919 Endomyces vernalis yielded fats from sulphite liquor (from paper manufacture), and similarly in 1941 employing Geotrichum. The term SCP was coined in 1966 by Carol L. Wilson. 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 protein-rich foods, in human and animal feeds.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. The term single cell protein (SCP) refers to dead, dry cells of microorganisms such as yeast, bacteria, fungi and algae which grow on different carbon 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. Producing and harvesting microbial proteins is not without costs, unfortunately. In nearly all instances where a high rate of production would be achieved, the single cell protein will be found in rather dilute solutions, usually less than 5 % solids. Methods available for concentrating include, filtration, precipitation, coagulation, centrifugation, and the use of semi-permeable membranes. These de-watering methods require equipment that is quite expensive and would not be suitable for most small-scale operations. Removal of the amount of water necessary to stabilize the material for storage, in most instances, is not currently economical. Single cell protein must be dried to about 10 % moisture, or condensed and acidified to prevent spoilage from occurring, or fed shortly after being produced. Single cell protein can be produced on a number of different substrates, often this is done to reduce the Biological Oxidation Demand of the effluent streams leaving various type of agricultural processing plants.

EXAMPLES OF SCP Microbes employed include yeasts (Saccharomyces cerevisiae, Candida utilis=Torulopsis and Geotrichum candidum (=Oidium lactis)), other fungi (Aspergillus oryzae, Sclerotium rolfsii,Polyporus and Trichoderma), bacteria (Rhodopseudomonas capsulata), and algae (Chorella and Spirulina ). Typical yields of 43 to 56%, with protein contents of 44 to 60%. The fungus Scytalidium acidophilum grows at below pH 1, offering advantages of (i) low-cost aseptic conditions, (ii) avoiding over 100-fold dilution of the acidic hydrolysates to pH values needed for other microbes, and (iii) after the biomass is harvested, the acids can be reused. Commercial production of SCP (Spirulina) includes Cyanotech in Hawaii and Earthrise in California. SINGLE CELL PROTEIN PRODUCTION 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). The problem with extracting single cell proteins from the wastes is the dilution and cost. They are 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. The single cell protein needs to be dehydrated to approximately 10% moisture content and/or acidified to aid in storage and prevent spoilage. The methods to increase the concentrations to adequate levels, and de-watering process require equipment that is expensive and not always suitable for small-scale operations. It is economically prudent to feed the product locally and shortly after it is produced.

MATERIALS AND METHODS Strain: Penicillium javanicum was isolated and purified from Lawsonia inermis leaves samples. The stock culture was maintained on agar slants, containing (gL-1) dextrose 20.0; peptone 10.0; agar 20.0 and distilled water. The ingredients were thoroughly mixed and kept in culture tubes sterilized at 1.5 kg/cm2 for 20 minutes. The sterilized slants were inoculated with Penicillium javanicum and incubated at 27°C to obtain luxuriant growth. Inoculum: A spore suspension was prepared by adding sterile water to stock culture to get 80x106 spores/ml. Preparation of substrate from rice husk: Rice husk was used as a substrate for the growth of microorganism and production of single cell protein. Rice husk (lignocellulosic material) was degradated to simple components (fermentable sugars) by chemical and enzymatic treatment, the details of the procedures are as under. Chemical treatment: 10.0 G of rice husk was mixed with various concentrations (0.15, 0.30, 0.45, and 0.60 N) of acids (sulphuric acid and perchloric acid) and

alkali (sodium hydroxide and ammonium hydroxide). These mixture were frequently agitated on flame for one hour, maintaining the level of solution constant. After cooling at room temperature, the slurry was autoclaved at 1.5 kg/cm2 for 30 minutes. The autoclaved slurry was cooled at room temperature and unsolubilized rice husk was removed by filtration through suction pump. The filtrate of solubilized rice huck was incorporated into the culture medium as a carbon and energy source. The loss in weight of rice husk sample was determined after drying at 110°C to constant weight. SCP from N. Alkanes In the late 1950's, British Petroleum (BP) became interested in the growth of a micro-organism in C12-C20 alkanes. This constitutes the wax fraction of gas oils for treating. Some crude oils contain up to 15% in wax, and these waxes must be removed since they make oil more viscous, precipitate out at low temperatures, block tubes etc. BP uses two yeasts, Candidor lipolytica and C. tropicals and built a 16,000 tons/year plant in Cap Lavera, France, and a 4,000 tons/year plant in England. The product produced was called "TOPRINA". In the UK the product "TOPRINA G" was a purer product while the one in France was not separated from alkanes. Both processes employed NH3 as N-source and Mg ions to increase yields. No other carbon source was used. For 12 years TOPRINA was tested for toxicity and carcinogenecity and was marketed as a replacement for fish meal in high protein feeds and as a replacement for skimmed milk powder in milk replacers. There were no signs at all for toxicity or carcinogenicity. In spite of this, people were concerned that aromatic hydrocarbons may be carried over to SCP. The main opposition came from Japan, where environmental groups and university professors condemned SCP as dangerous, and the matter became political. In 1972 a specialised committee decided that SCP was only for animal feeding but later, Japan was the first country to ban petrochemical protein. Meanwhile BP and an Italian company constructed a 100,000 tn/year plant in Sardinia. Following the Japanese attack on SCP, there has been great concern about and opposition to the use of SCP from environmental groups in government. The Italian government ordered further studies which showed that there was no hazard or carcinogenesis due to SCP. Pigs fed on 30% TOPRINA in their diets showed less n-paraffins in their fat tissue than those fed on pasture. Based on this evidence the Italian government agreed to the use of TOPRINA in limited amounts and only for export. In 1977 Italy stopped the SCP production for alkanes altogether due to the increase in oil prices. The price of soya was more competitive. Now there is no factory which produces any petrochemical protein.

SCP from Methane Methane is cheap, abundant and without the toxicity problems of alkanes. It is a constituent of North Sea Gas and is also produced during anaerobic digestion. Methane contains the most highly reduced form of carbon and consequently gives high cell yields relative to the amount of gas consumed. The general Methylomonas and Methylococcus have been recognised as utilising methane as a carbon source. The species which has been extensively studied is Methylomonas methanica. Nitrates or ammonium salts can serve as N-source. Perhaps the most important work in this field was carried out by Shell in England. The process involves methane oxidation by stable mixed cultures. These were 1. a methane utilising G(-) rod; 2. a Hyphomicrobium; 3. two g(-) rods; Acinetobacter and Flavobacterium This mixed culture was one of the best examples of symbiosis. The process began in 1970 in a 300 e pilot plant at Sittingbourne, UK. In 1974 Shell announced plans for a construction of a larger pilot-plant in the same area and a development program in Amsterdam with a goal of producing 100,000 tn/year. In spring 1976, Shell stopped commercialisation and its development plans were indefinitely postponed. This decision was based on 3 factors: 1. the low price of soybeans & maize; 2. the potential of many countries for expanding existing protein sources; 3. the difficulty in applying Shell's sophisticated process in underdeveloped countries.

SCP from Methanol The technology of SCP from methanol has been well studied and the most advanced process belongs to ICI. The fermentation was carried out in a big airlift fermentor with the bacterium. Methylophilus methylotropha. This organism was selected among other methanol utilises after screening tests for pathogenicity and toxicity. As a nitrogen source ammonia was used. The product was named "PRUTEEN". Pruteen contained 72% crude protein and was marketed for feed as a source of energy, vitamins and minerals as well as a highly balanced protein source. The methionine and lysine content of Pruteen compared very favourably with white fish meal. ICI has commissioned a 60,000 tn/year plant utilising the single largest fermentor in the world (2 x 10,000,000 l). Unfortunately Pruteen now cannot compete with soya and fish meal. ICI hopes to be able to sell their technology, because they have given up the idea of making money out of Pruteen. So today Pruteen although a major engineering success is not economical to run.

SCP from Ethanol Ethanol although expensive as a substrate has been used for SCP. The process comes from the Amoco Company in the US utilising a food grade yeast: "Torula". The product is sold by the name "TORUTEIN" and government clearances have been obtained to market Torutein in Canada and Sweden. The yeast is about 52% protein and due to its relatively low Methionine level has a PER of about 1.7. The PER of wheat from 1.1 to 2.0. Torutein is being marketed as a flavour enhancer of high nutritional value, and a replacement for meat, milk and egg protein. However it is not very successful in the United States since soya which is plentiful and cheap can serve as an alternative or substitute to meat and egg diets.

Mycoprotein This is a development of Ranks Hovis McDougall and is the only mycoprotein (except edible mushrooms) that has been cleared for human consumption. It uses a Fusarium graminearum growing in molasse, or glucose. The medium contains NH3 for nitrogen source and pH control. The product is heat treated for RNA reduction. The mycelium is separated by vacuum filtration, and can be technologically treated to match food texture. In the UK it is marketed as pies and is considered a success since having less fat than meat, it can be sold at a premium price. SCP from Lignocellulose The lignocellulosic wastes, mainly from agriculture, constitute the most abundant substrate for SCP which is also renewable. The world annual production of straw for example reaches 600 million tons every year. In Greece the straw from wheat and rye, the two most important cereals, is an estimated 1.5 million tons per year. For the utilisation of lignocellulose, a pre-treatment is usually necessary. Many pre-treatment methods have been reported which vary from alkali or acid treatment, steam explotion or even x-ray radiation. To the present time the only economical utilisation of lignocellulosic wastes is in mushroom production. Besides our well know cultivated mushroom Agaricus bisporus there are many important ones which contain lignocellulolytic enzymes and are cultivated for food mainly in Asia and Africa. Some are of great economic significance and are cultivated on an industrial scale. Examples of important ones include the following species: Volvariella sp., Lentinus edodes and Pleurotus sp. SCP's Evaluation and Future Prospects The development of SCP was really the beginning of biotechnology. Prior to this the industrial

fermentation was mainly focused on antibiotics and other products which did not have to compete. This was not the case with SCP which had to compete with similar products in the market. The development was brought up by the oil companies rather than the food companies, because they could take the risk of a highly costly product out with no real expected profit. They also had all the high technology required. The efforts tried so far by adding dry SCP as a supplement to diets in order to solve the problems of the hungry in the Third World Countries, certainly have not given the expected results. Every new food which appears in the market should have not only high nutritive quality, but also satisfactory organoleptic supplementary element. Today in most countries where market forces operate. SCP cannot compete with soya, alfalfa or fish meal. Mushroom production from lignocellulosics seems to be one economical and promising use for SCP. 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. Nutritional Value of SCP For the assessment of the nutritional value of SCP, factors such as nutrient composition, amino acid profile, vitamin and nucleic acid content as well as palatability, allergies and gastrointestinal effects should be taken into consideration. Also long term feeding trials should be undertaken for toxicological effects and carcinogenesis. Table 1 shows the average cell composition of the major groups of micro-organisms. Table1 Average composition of the main groups of micro-organisms (% dry weight)

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 (Table 2). Table 2 Essential amino acid content of the cell protein in comparison with several reference proteins (grams of amino acid per 100 grams of protein)

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 3 shows the vitamin content of various food micro-organisms. Table 3 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) With microbial cells it is important to note that digestibility is low especially with algae cells because of indigestible cell walls. Although the PER of SCP compares favourably with the PER of conventional protein sources, acceptability and palatability results on nutritional trials were not always encouraging. Furthermore we can make no assumptions with regard to human acceptability and tolerance based on results with animal feeding trials.

SINGLE CELL PROTEIN IN FUTURE According to many theories, the bluegreen algae were among the first forms of plant life to appear on the earth some billions of years ago... and they're about the simplest to produce, most nutritious food source we could ever hope to find. Among the many varieties of such primitive plants, Spirulina scores incredibly high on a nutritional scale: It's rich in vitamins A and E, and the B-complex (in fact, this species is the highest nonanimal source of B-12, a vitamin that's often difficult to obtain in a vegetarian diet), and has an impressive array of trace minerals. Spirulina is also chock-full of protein ... containing a whopping 65-70% by dry weight (as compared to the approximately 35% protein content of soybeans and 45% of brewer's yeast). Furthermore, the algae providecomplete protein, containing all of the eight essential amino acids that the body cannot manufacture. Unlike other single-celled algae,Spirulina contains no cellulose in its cell walls, so the substance is much easier to digest than are the more fibrous forms, such as Chlorella. But perhaps the best news is that one needs to ingest only a small portion in order to receive the benefits of this nutritional powerhouse: Just two tablespoons—or 20 grams—of Spirulina powder (an amount which, by the way, contains only 78 calories) will provide 13 grams of complete protein ... almost one-third of the minimum daily amount most people require. Exactly where do these protein-rich algae come from, anyway? Well, worldwide production of Spirulina is currently between 500 and 800 tons a year ... most of which is grown on modern algae farms in Mexico. Other producers—of much smaller amounts—include Thailand, Japan (where several algal strains have been commonly eaten for generations), and Israel. In addition to the overseas operations, however, a few experimental farms have been established in the United States ... to investigate the possibility of quantity cultivation in this country. Several years ago, two enterprising researchers at the University of Texas—Dr. Tom Newsom and his colleague, Dr. Michael Leesley—built algae aquariums, troughs, and pools to test the productivity of Spirulina. The Texas engineers estimated—from the results of their tests—that a ten-square-mile farm, growing only the marine plant, could feed two and a half million people a year ... and (on a smaller scale) that a group of 80 men and women could sustain themselves by keeping a mere 10 or 12 acres under Spirulina cultivation! Before the team was forced to abandon the promising project late last year (because of a shortage of research funds), Newsom and Leesley had worked out an equipment setup that could be duplicated for at-home production of the nutritious food. Their simple plan involved a windmill-powered paddle wheel to stir the water gently and move the algae over a set of baffles ... assuring constant exposure of the growing organisms to necessary sunlight. Meanwhile, in 1975, an enthusiastic young entrepreneur named Larry Switzer formed the Proteus Corporation, to conduct further research into the mysteries of Spirulina platensis. His venture soon became successful enough to support the opening of a five-acre experimental

farm in California's Imperial Valley, where a closed pond system is now processing several tons of the product each year.

LATEST RESEARCHES PAPERS Production of Single-Cell Protein from Ram Horn Hydrolysate Esab› Baflaran KURBANO⁄LU Atatürk University, Science and Letters Faculty, Department of Biology, 25240 Erzurum TURKEY 22.02.2000 Abstract : Candida utilis NRRL Y-900 was grown on horn hydrolysate for single-cell protein production. First, ram horns obtained from slaughterhouse in Erzurum were hydrolyzed by physical and chemical methods and crude horn hydrolysate (CHH) was obtained. The contents of protein, nitrogen, ash, some minerals, total sugars and amino acids of CHH were determined and it was seen that it has sufficient organic and inorganic materials to allow its use as a substrate source in the production of single–cell protein. The CHH was enriched by addition of yeast extract, glucose and KH2PO4. The effects of different CHH concentrations (1, 2, 3, 4, 5, 6, 7, 8, 9 and 10%) on the growth of C. utilis were investigated and 4% of CHH (Horn Broth=HB) was found to be optimal. The biomass yield of C. utilis and its protein content were found to be 6.8 g l-1 and 49.8% respectively. On the other hand, biomass contained 5.4% lipids, 5.94% RNA, 1.53% DNA and 9.7% ash. The biomass contained all of the essential amino acids and when compaired with FAO reference protein it

showed a good profile. The results demonstrated that ram horns can be used as a substrate source in the production of single-cell protein.

Single-cell protein induction dynamics reveals a period of vulnerability to antibiotics in persister bacteria Orit Gefen, Chana Gabay, Michael Mumcuoglu, Giora Engel, and Nathalie Q. Balaban Racah Institute for Physics and Center for NanoScience and Nanotechnology, Hebrew University, Jerusalem 91904, Israel Edited by Susan Gottesman, National Institutes of Health, Bethesda, MD, and approved March 5, 2008 (December 12, 2007) Phenotypic variability in populations of cells has been linked to evolutionary robustness to stressful conditions. A remarkable example of the importance of cell-to-cell variability is found in bacterial persistence, where subpopulations of dormant bacteria, termed persisters, were shown to be responsible for the persistence of the population to antibiotic treatments. Here, we use microfluidic devices to monitor the induction of fluorescent proteins under synthetic promoters and characterize the dormant state of single persister bacteria. Surprisingly, we observe that protein production does take place in supposedly dormant bacteria, over a narrow time window after the exit from stationary phase. Only thereafter does protein production stop, suggesting that differentiation into persisters fully develops over this time window and not during starvation, as previously believed. In effect, we observe that exposure of bacteria to antibiotics during this time window significantly reduces persistence. Our results point to new strategies to fight persistent bacterial infections. The quantitative measurement of single-cell induction presented in this study should shed light on the processes leading to the dormancy of subpopulations in different systems, such as in subpopulations of viable but nonculturable bacteria, or those of quiescent cancer cells. Single-Cell Protein Production by the Acid-Tolerant Fungus Scytalidium acidophilum from Acid Hydrolysates of Waste Paper K. C. Ivarson and H. Morita Chemistry and Biology Research Institute, Agriculture Canada, Ottawa, Ontario K1A 0C6, Canada Contribution 1260 from the Chemistry and Biology Research Institute The bioconversion of waste paper to single-cell protein at pH <1 by Scytalidium acidophilum is described. Waste paper pretreated with 72% H2SO4 at 4°C was diluted with water to a pH of <0.1 and hydrolyzed. This yielded an adequate sugar-containing substrate for the growth of the fungus. A total of 97% of the sugars (glucose, galactose, mannose, xylose, arabinose) in the

hydrolysates were converted to cell biomass. Microbial contamination was not observed. Based on the sugars consumed, S. acidophilum produced higher yields in shake cultures than many other Fungi Imperfecti. In aerated cultures, productivity increased, and yields of 43 to 46% containing 44 to 47% crude protein were obtained. This compares favorably with Candida utilis, a yeast used commercially to produce single-cell protein. The chemical constituents and the essential amino acids of the fungal cells were similar to those of other fungi. The nucleic acid content was characteristic of microbes containing low levels of nucleic acid. The advantages of using S. acidophilum for single-cell protein production are discussed.

Single cell protein as food and feed
Doc. Dr. A. Giec, Prof. Dr. J. Skupin Academy of Agriculture, Faculty of Food Technology, Institute of Microbiology, Biochemistry and Food Analysis, 60-623 Poznan, Mazowiecka 48, Poland This review pertains the current knowledge concerned with the application of SCP in human and animal nutrition. General factors limiting the utilization of microbial proteins in human nutrition, such as toxicological barriers, nutritive value and functional properties, are discussed. Special attention is paid to several modern procedures of protein extraction from microbial cells, reduction of nucleic acids level as well as preparation of protein isolates. According to the data presented the latter can be considered as valuable protein substitutes. Significantly less nutritive problems are being concerned with the application of SCP in animal nutrition. Successful feeding experiments with chicken and pigs are discussed. Under these experimental conditions 10-20% of the protein in the feedstuff can be replaced by SCP. Moreover, several in Poland obtained protein-vitamin preparations are described. These originated from selected yeast and propionic acid bacteria grown in whey and its ultrafiltrates and can be considered as valuable food and feed supplements. Dietary single cell protein reduces fatty liver in obese Zucker rats. There is growing evidence that dietary proteins may interfere with lipid metabolism. We therefore examined the effects of feeding obese Zucker rats a single cell protein (SCP) with low ratios of methionine:glycine and lysine:arginine for 6 weeks. SCP feeding reduced the hepatic steatosis and lowered the plasma transaminase levels when compared with casein-fed rats (controls). The fatty acid oxidation was increased in liver mitochondria and peroxisomes, whereas the activities of enzymes involved in lipogenesis and TAG biosynthesis were unaffected. SCP feeding affected the fatty acid composition of liver lipids and plasma, and reduced the mRNA levels of the fatty acid desaturases. The decreased gene expression of

stearoyl-CoA desaturase suggested that the fatty acids were directed towards oxidation rather than esterification as TAG. The decreased mRNA levels of VLDL-receptor and lipoprotein lipase in the liver after SCP feeding suggested that the uptake of TAG-rich lipoprotein to the liver was decreased. To conclude, the reduced fatty liver by SCP feeding may be caused by the increased capacity for fatty acid beta-oxidation in the liver, combined with changed fatty acid composition and possibly a reduced hepatic clearance of circulating VLDL. An increased awareness of the effect of dietary proteins on lipid metabolism could be of relevance in future dietary treatment of non-alcoholic fatty liver disease.

Single-cell protein production from Jerusalem artichoke extract by a recently isolated marine yeast Cryptococcus aureus G7a and its nutritive analysis. After crude protein of the marine yeast strains maintained in this laboratory was estimated by the method of Kjehldahl, we found that the G7a strain which was identified to be a strain of Cryptococcus aureus according to the routine identification and molecular methods contained high level of protein and could grow on a wide range of carbon sources. The optimal medium for single-cell protein production was seawater containing 6.0 g of wet weight of Jerusalem artichoke extract per 100 ml of medium and 4.0 g of the hydrolysate of soybean meal per 100 ml of medium, while the optimal conditions for single-cell protein production were pH 5.0 and 28.0 degrees C. After fermentation for 56 h, 10.1 g of cell dry weight per liter of medium and 53.0 g of crude protein per 100 g of cell dry weight (5.4 g/l of medium) were achieved, leaving 0.05 g of reducing sugar per 100 ml of medium and 0.072 g of total sugar per 100 ml of medium total sugar in the fermented medium. The yeast strain only contained 2.1 g of nucleic acid per 100 g of cell dry weight, but its cells contained a large amount of C(16:0) (19.0%), C(18:0) (46.3%), and C(18:1) (33.3%) fatty acids and had a large amount of essential amino acids, especially lysine (12.6%) and leucine (9.1%), and vitamin C (2.2 mg per 100 g of cell dry weight). These results show that the new marine yeast strain was suitable for single-cell protein production. Microbial production of single-cell protein from deproteinized whey concentrates. Deproteinized sweet and sour cheese whey concentrates were investigated for their suitability as substrates for the production of single-cell protein with Kluyveromyces marxianus CBS 6556 up to a 100-l scale. An important factor for gaining high cell concentrations was the use of the Crabtree-negative strain K. marxianus CBS 6556. Supplements such as trace elements, ammonium and calcium were required for the complete conversion of sweet whey concentrates

into biomass, whereas sour whey concentrates had to be supplemented with ammonium, trace elements and vitamins. After improvement, biomass dry concentrations of up to 50 g l-1 could be reached with Yx/s values of 0.52 for sweet whey and of up to 65 g l-1 with Yx/s values of 0.48 for sour whey concentrates. The chemical oxygen demand of the whey concentrates were reduced by 80%. The cells were used for the analysis of amino acid and ash composition, showing a distinct increase of eight out of ten essential amino acids compared to sweet and sour whey protein and exceeding the World Health Organisation guidelines for valine, leucine, isoleucine, threonine, phenylalanine and tyrosine.


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