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Pure

Appi. Chem. Vol.51, pp.2537—2560.
Pergamon Press Ltd. 1979. Printed in Great Britain.
© IUPAC

0033—4545/79/1201—2537 $02.00/0

INTERNATIONAL UNION OF PURE
AND APPLIED CHEMISTRY
APPLIED CHEMISTRY DIVISION
COMMISSION ON FERMENTATION*

and
COMMISSION ON FOOD CONTAMINANTSt

GUIDELINES FOR TESTING OF SINGLE CELL
PROTEIN DESTINED AS. PROTEIN SOURCE
FOR ANIMAL FEED—II
Prepared for publication by
J. C. HOOGERHEIDE and K. YAMADA
Commission on Fermentation

J. D. LITTLEHAILES and K. OHNO
Commission on Food Contaminants

*Chairman: A. E. HUMPHREY (USA); Vice-Chairman: H. DELLWEG (FRG); Secretary: R. C.
RIGHELATO (UK); Titular Members: C. L. COONEY (USA); T. K. GHOSE (India); J. HOLLO
(Hungary); K. YAMADA (Japan); Associate Members: H. T. BLACHERE (France); V. K. EROSHIN
(USSR); A. FIECHTER (Switzerland); J. C. HOOGERHEIDE (Netherlands); F. PARISI (Italy); S. J.
PIRT (UK); J. TAKAHASHI (Japan); J. ZAJIC (Canada); National Representatives: B. P. RALPH
(Australia); R. J. ERTOLA (Argentina); H. WUTZEL (Austria); W. K. BRONN (FRG); M. LINKO
(Finland); 0. ILNICKA-OLEJNICZAK (Poland); A. F. LANGLYKKE (USA); M. RINGPFEIL
(GDR).

tChafrman: J. KOJIMA (Japan); Secretary: L. E. COLES (UK); Titular Members: A. D. CAMPBELL

(USA); M. JEMMALI (France); P. KROGH (Denmark); M. V. TRACEY (Australia); Associate
Members: 0. BILLEK (FRG); F. BRO-RASMUSSEN (Denmark); H. GUTHENBERG (Sweden); W.
KRONERT (FRG); J. D. LITTLEHAILES (UK); K. OHNO (Japan); P. L. SCHULLER (Netherlands);
P. 5. STEYN (S. Africa); National Representatives: H. LANGE (ERG); T. JUSZKIEWICZ (Poland).

GUIDELINES FOR TESTING OF SINGLE CELL PROTEIN
DESTINED AS PROTEIN SOURCE FOR ANIMAL FEED (II)

Definition - By single Cell Protein (SCP) is meant isolated
mass of non—viable, dried cells of micro—organisms (yeasts,
bacteria, fungi or algae) , generally produced by cultivating
such micro-organisms on substrates such as alkanes, lower
hydrocarbons (e.g. methane) , lower alcohols (e.g. methanol
or ethanol) or industrial and agricultural waste products
(e.g. sulfite waste liquor). under standardized and well—
controlled conditions in regard to organism, substrate and
processing, and destined to be used as protein source in
animal feed.
I.

INTRODUCTION

On the premise that there will be a serious world shortage of proteins for
both human and animal consumption in the forthcoming decades, a great deal
of technological effort has been directed to the realization of a novel
source of protein of microbial origin, which is referred to as single cell
protein. The first generation technology of SCP, based on petroleum-derived
hydrocarbon substrates including n-paraffin has already been firmly
established from both engineering and product safety points of view.

IUPAC, in August 1974, published Technical Report No.12 entitled: "Proposed
Guidelines for Testing of Single Cell Protein Destined as Major Protein
Source for Animal Feed" in which "General Standards of Identity for SCP to
be used for Animal Feed" are given, and in addition "Specific Standards of
Identity of SCP prepared from Hydrocarbons" are suggested. Although these
specifications on the assessment. of SCP in general and those specific for
SCP based on hydrocarbon still stand, it is now considered that further
information is required to specify the product. In addition specific
consideration must be given to SCP based on methanol as substrate.
After a wide variety of carbon/energy sources have been explored for SCP
production, methanol and methane have recently been of increasing interest
as alternative feed stocks for SCP production. Methanol has advantages over
liquid or gaseous alkanes in respect to several important points. Normal
alkanes are hardly soluble in water, and the rate of microbial assimilation
of these substrates is limited by their mass transfer rate into the culture
fluid. Methanol, on the other hand, is completely miscible with water. In
addition to this technical advantage, methanol is commercially available as
a highly pure chemical of relatively low cost, which makes methanol highly
promising as alternative feedstock for the second generation of industrially
produced single cell protein.
It is the purpose of the present report to provide additional information on
safety testing, to suggest specific standards for methanol based SCP and in
addition provide a brief description of the new methanol based technology.
This is in order to avoid some mistaken views that have previously arisen
and may arise otherwise because of the unconventional nature of this
feedstock and elaborated technology.
II.
1)

TOXICOLOGICAL AND NUTRITIONAL EVALUATION

General Requirements for Testing

"No novel source of protein should be admitted as animal feed ingredient
unless it has been evaluated thoroughly with respect to safety and absence
of toxicity. This evaluation must be conducted on samples derived from a
fully stabilised process, yielding products of fully reproducible quality.

2539

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To achieve a reasonably high level of the test SCP within the diet it is advisable to operate at two dietary protein levels (say 15 and 30%) .5 1/12—p . Within the intensive farming industry growth rate and productivity is sensitive to small changes in dietary nutrients. Mortality beyond that normally experienced in farm livestock must be carefully evaluated. and Mg are known to induce nephrocalcinosis in rats. Changing dietary raw materials by the substitution of SCP will change the dietary nutrient profile. Note a. exercised in the interpretation of data. but due regard should be paid to the normal practices followed in animal production. Care should be. simple changes in dietary levels of Ca. Long term life—span studies equivalent to those carried out with the rodent are not necessary. but rather a change in nutrient balance. such as 90 days rat studies. Primary screening methods such as the in vitro Ames Test conducted on the soluble part of SCP and the more sophisticated in vivo Dominant' Lethal Test may give some preliminary indication of. and the subsequent effects that this has on the nutrient balance of the test diet. The toxicological evidence gained from the laboratory rodent species will form the principle basis for evaluation 'of hazard. and biochemical parameters should be measured ahd evaluated. This in turn can induce changes in the parameters used for evaluation relative to the control. These experiments with farm livestock should follow the same experimental concepts as those employed for the laboratory animal. The experimental designs used in these studies should simulate those employed for the laboratory animal. Poor productivity in the absence of any other toxicological symptoms should not be interpreted as a toxicological abnormality. which if not recognised. P. adequate controls are needed using conventional protein sources of similar crude protein content to the test product. It is possible to induce a 'toxicological change' due to simple alterations of dietary minerals or energy.C. When evaluating the toxicological data it is necessary to bear in mind the changes that may be induced by such nutritional changes. are valuable during the As evaluation using longer development stages of the process (see note a) . The specific time length will depend upon the strain of the rat and the husbandry conditions under which the animals are kept. thus experimental design must reflect conventional animal production systems used by the farming industry. possible cellular mutagenicity. and terminal pathology. The normal econOmic life span of afarm animal. Nevertheless it is still necessary to test the SCP in the diets of farm animals.Testing of single cell proteins 2541 Short term tests. All conventional pathological. again using the rodent. Considerable care must be exercised in equating the nutrient balance of the diet. Reference should be made to Guidelines Numbers 6 and 15 issued by the Protein-Calorie Advisory Group of the United Nations System. The objective of these tests is to evaluate potential carcinogenicity and they should therefore be of sufficient duration to induce tumour formation. could be ascribed to the use of the single cell protein as such.A. this includes biochemistry. The tests should include bone marrow cytology to detect any host chromosome abberations. forms a sensible basis for the evaluation of risk in the actual target species. Specific teratological investigations should also be made in at least one species' apart from the rodent. . Multigeneration experiments made with both poultry and pigs need to have a sound statistical basis. P. term rodent studies should use material derived from the pilot plant.A. haematological. The main problem associated with these types of investigation is the inclusion level of the test material. Individual laboratory experience will define this time period. Experimental evidence should also be obtained from specific multigeneration and teratological experiments. haematology. These changes could be wrongly ascribed to the SC? and hence extreme care must be exercised in the formulation of the test diets. The reproductive potential and the viability of the off-spring are important both scientifically and commercially. Such features are common place with naturally occurring raw materials.

. It is a testing programme that should cover any adverse eventuality as reflected by current knowledge within the subject. Extensive tissue analysis of eggs. . and there is no need for the regulatory authorities to be unduly involved in the overall utilization of the SC?. The hazards. Experiments of this nature are best regarded as applied animal nutrition and are of commercial significance only. Nevertheless the tissues resulting from the toxicological and nutritional experiments utilising large amounts of SCP should be evaluated for their potential effects upon the human food chain. further attention needs to be paid to the components that the cells manufacture themselves. digestibility of amino acids. fat and edible internal organs needsto be undertaken. digestible energy. Nutritional Testing of the Product Conventional nutritional tests such as PER. When this has been achieved SCP irrespective of source. This will indicate the potential limitations on the use of the product. These nutritional studies will require large animal populations housed under practical conditions which will define with statistical precision the correct mode of use for the product. In the future it is possible. The current testing programme as outlined above is long and costly.depending upon the animal and usual mode of evaluation which. if any. For the future it needs to be understood that the testing programme described above may not be necessary. To help the nutritionists in their role of dietary formulation the following information on nutrient value will be of assistance: Available energy (defined as metabolizable energy. digestible crude protein. Abnormal profiles for minerals or amino acids within the SC? may give spurious results which need to be recognised at the time of dietary formulation and corrected. they will include for instance fatty acids and trace elements. At this stage in the development of our knowledge it is both prudent and necessary to accept that totally novel materials. Considerable attention needs to be taken over dietary nutrient balance. with a potential understanding of this topic then the toxicological testing programme outlined above may become redundant. are to be derived from the substrate. Organoleptic evaluation on the meat is also desirable. then work on other animals should be aimed at raw material acceptability and dietary formulation. varies from country to country).2542 COMMISSIONS ON FERMENTATION AND ON FOOD CONTAMINANTS It should notbe necessary to repeat the toxicological tests on every potential target species. meat. 3. in particular the known toxic metals that may accumulate within the body system. The nature of the analysis will be dependent upon the basic analysis of the scp product. total digestible nutrients . or from cellular metabolites. The toxicity of the current substrates used for SCP production is well understood. the fowl. can be regarded on an exactly similar basis to soya bean meal and fish meal. net energy. the extent of this data will not be sufficient to demonstrate its nutritional potential as a novel material within the animal feedingstuff industry. It is unlikely that single cell protein will provide all the supplementary protein within an animals diet: mixtures of proteins optimise animal productivity. availability of amino acids. From a practical animal nutrition standpoint there is no substitute for the incorporation of the SCP with locally available raw materials and its evaluation under local practical animal husbandry' conditions. that these products will not be considered novel. If the livestock do not thrive in the toxicological tests it is unlikely that th product will be of any great value as a nutrient source. given a satisfactory rate of industrial development. and the pig. This is considered to be a seller-customer relationship. and products should be evaluated. The toxicological evaluation of the SCP within the target species will have provided a considerable amount of evidence on the nutritional value and efficacy. However. NPU are of little practical value in animal nutrition although they may have some application during the early stages of product evaluation to monitor the effects of process changes at the pre-pilot plant stage. If there are no abnormalities arising within the profile of studies undertaken on the rat.

5% SCP derived from methanol: Residual methanol < 2oppm Total lipids Fatty acid profile Glycerols. Recommended additional specifications: be be be be be be be be declared declared declared declared declared declared declared declared (as indicator for presence of Benzo(a)pyrene polycyclic aromatic hydrocarbons) < 5ppb Acety lacetone reagent-positive substance (as formaldehyde) < 2oppm SCP derived from alkanes: * Total hydrocarbons Total aromatic hydrocarbons < < 0.Mo) 2.2543 Testing of single cell proteins III GENERAL AND SPECIFIC STANDARDS OF IDENTITY FOR SC' USED FOR ANIMAL FEED 1. aureus Clostridia total Cl. a) b) to to to to to to to to Specific Standards of Identity for SC? * For 0. perfringens Lancefield Group D streptococci Moisture content (to be declared) Ash content Lead Arsenic Mercury Total nitrogen Amino acid nitrogen and profile Nucleic acid nitrogen Ammonia and urea nitrogen Nitrate and nitrite nitrogen Available lysine as % of total lysine * to < < 100 000 100 10 < lper50g 1 < 1000 < 10 000* < 12% 100 to be declared to to to to to to < < < 5ppm 2ppm 0.12.Cu. 12 .Se.Zn. The product should conform to the following standards: Per gram Viable bacteria count (total aerobic bacteria) Viable moulds Enterobacteriaceae Salmonella Staph.Co.1 ppm be be be be be be declared declared declared declared declared declared be aimed at 1000 per gram For chemical and biological assay methods see IUPAC Technical Report No.05% analytical methods see IUPAC Technical Report No. phospholipids non-saponifiables Fibre Proximate carbohydrate profile Vitamins Nutritional trace elements (Mn. General Standards of Identity for SC? The final product must consist of the dead cells of the defined microbial species used for its production and contamination should be no more than that anticipated by good and standard food processing practices.

The water is recovered by centrifugation and is then analysed for methanol content by the standard analytical procedure for aqueous methanol. 10-20 ml of water is added to the tube by means of a pipette. d) Chromatographic Method for Determination of Aqueous Methanol Outline of Method (i) Gas chromatographic separations are used in the analysis. Quantitative results are achieved by the injection of reproducible volumes of sample. 2jl of the supernatant is injected into the gas chromatograph. METHOD a) Summary The method relies upon extraction of methanol from the cells by added water. plus 4% NaOH. and connect this end via a catch pot to a vacuUm pump. Remove all water by rotating in a heated rotary evaporator. Add this to the dried Chromosorb and remove solvent by placing in the rotary evaporator. b) Column: 5 meters x¼OD s/s tubing. Determination of Methanol in Single Cell Protein SCOPE The method is suitable for the determination of methanol at the 8 ppm to 1000 ppm (w/w) level in single cell proteins. c) Column Packing: 20% UCON. Shut down the pump and remove funnel and close the column end with glass wool. . Leave the pump running for a further ten minutes then make up any fall in packing level. Pour the prepared packing into the funnel vibrating continuously until the column is full. d) Diluent for Standards: Water. b) Preparation of Column Bend a 5 meter length of ¼" OD tubing into convenient coils to suit the chromatograph oven. and then centrifuged at about 15.tl with 3" needle. HB 5100 on Chromosorb W. Weigh 10 grams of UCON and dissolve in 125 mls of dlichloromethane. b) Apparatus Centrifuge tube (i) (ii) Centrifuge Gas chromatograph. Connect a small funnel to the other end of the column and switch on the pump. (ii) Apparatus and Materials a) Gas Chromatograph: Any flame ionisation chromatôgraph capable of providing similar sensitivity to that in the specimen may be used. and the component peak heights obtained are converted to concentration by direct reference to peak heights from synthetic mixtures. e) Syringe: 5j. Close one end of the column with a glass wool plug. set up as in (d) (iii) c) Procedure About 5 g of sample is weighed accurately into the centrifuge tube. f) Recorder: 1 mV 0.000 rpm for 20 minutes.25 cms/min. The tube is allowed to stand for 2 hours.COMMISSIONS ON FERNENTATION AND ON FOOD CONTAMINANTS 2544 3. (iii) Preparation of Apparatus a) Preparation of PackIng Weigh 50 grams of Chromosorb "W" and add 200mls of 4% NaOH to form a slurry. A note should be taken of the weight of packing used.

is treated in the same way. After cooling. analytical grade. 55. J. APPARATUS Steam Distillation Apparatus a) b) Spectrophotometer Any instrument capable of operating at the required sensitivity at the wavelength of 420 mix.tg HCHO). c) 20 ml of the Stock Solution is diluted with water to 100 ml (Standard Solution A: 1 ml = 4 g HCHO). Nash. PRINCIPLE The sample is suspended in a diluted phosphoric acid solution and the formaldehyde is steam-distilled. The distillateis made up to 200 ml with distilled water to make a sample solution. T. 5 ml of this solution is diluted with water to 100 ml to make the Stock Solution. To the distillate is added a neutral solution of acetylacetone and ammonium salt. The solution is steam-distilled. 416 (1953). A or B. . SCOPE This method is applicable for not less than 5 ppm of formaldehyde. which should be colourless) 150 g ammonium acetate 3 ml acetic acid to 1000 ml 2 ml acetylacetone distilled water J Formaldehyde Stock Solution b) Hexamethylenetetramine (311. 2545 Determination of Formaldehyde in Single Cell Protein.223 (1973). Formaldehyde Standard Solution. Biochem. At the same time. into which the leg of the cooler is to be dipped. Stop the distillatiOn when nearly 200 ml of distillate is obtained. optical absorption of each solution is measured as A and CALCULATION Formaldehyde content (ppm) = K x A x 200 x 100 As sample weight (g) K: content of HCHO (tg) in 1 ml of the Standard Solution REFERENCES 1) 2) Standard Methods of Analysis for Hygienic Chemists with Commentary. Determination of Formaldehyde b) The sample solution (5m1) is mixed with 5 ml of acetylacetone solution and heated for 10 minutes in a boiling water bath. redistilled acetylacetone. 5 ml of the Stock Solution is diluted with water to 100 ml (Standard SolutionlB: 1 ml = 1 j. 5 ml of the Standard Solution. Pharmaceutical Society of Japan. Prior to the distillation 10 ml of water is added to the receiver. is made up to 1000 ml in water. REAGENTS a) Acetylacetone Solution (Analytical grade reagents. The colour is measured at the wavelength of 420 m. PROCEDURE a) Preparation of Sample Solution About 10 g of the SCP is weighed accurately and added to 10 ml of water and 1 ml of 20% H3PO. whereby a yellow colour develops due to the synthesis of diacetyldihydrolutidine.2 g). content of single cell proteins. p.Testing of single cell proteins 4.

Operating Conditions (iv) 108°C Nitrogen 8. connect the column to the detector and adjust the conditions to those specified. It is recommended that standards in the lower range i. methanol in single cell protein wt SCP in supernatant (ppm w/v) f) Limit of Detection The limit of detection is fixed by The sensitivity of the chromatographic method and (i) the amount of water used in the extraction.COMMISSIONS ON FERMENTATION AND ON FOOD CONTAMINANTS 2546 c) Preparation of Chromatogp Install the column in the chromatograph leaving the detector end disconnected and the carrier gas flowing.5 lbs 17. A sample was therefore made up containing 780 ppm w/w methanol in solid.000 ppm. After conditioning.— 7 —' — — 51 so —t————— 0 SO . —- —— —-———- ---- i—----Fig 1. Concentration of methanol = ml water Conc. (v) Typical traces of aqueous standards are shown in Figure 1. 100 ppm to 1. . e) Calculation of Results The concentration of methanol in the supernatant as found from the gas chromatogram is expressed in mg/l (ppm w/v).16 ppm (w/w).- 90 in . No methanol could be found in manufactured SCP.25 cms/min Preparation of Calibration Standards Calibration is carried out by the preparation of standard mixtures of pure methanol and distilled water covering the range anticipated in the sample..e. (ii) For the method described above it is 8 . This was extracted into 10 ml water. Sensitivity setting was 1/2 max. are always freshly prepared. 1-----.5 lbs 29 lbs Column temperature Carrier gas Carrier gas pressure Hydrogen pressure Air pressure 2 jl sample injected Recorder Chart speed (3" needle) 0-lmV 0. Raise the oven temperature to 190° C and leave overnight. This conditioning period is necessary to allow all traces of solvent and possible impurities in the UCON to elute without contamination of the detector. 1 l of water extract was injected.

yielding practically pure methanol. Presence of such minor impurities in the substrate are not considered to give any adverse effect on the safety of SCP prOducts produced from methanol. LPG. naphtha to heavier hydrocarbons. On the basis of available information it must be concluded that commercial refined methanol contains only traces of impurities.S. ranging from natural gas. Lurgi Apparate Téchnik of FDR and Mitsubishi Gas Chemical Company of Japan. Sulphur containing impurities are carefully removed since they are catalyst poisons. After cooling this results in a crude methanol. containing water and small amounts of by-products. TABLE 2 gives a typical analysis of crude and refined methanol. all of relatively small molecular weight. light hydrocarbons are used. Even if "crude" methanol were to be used for SCP production its composition does not indicate that an accumulation of impurities in the cell would occur as a consequence of substrate impurities.Testing of single cell proteins 2547 APPENDIX 1 Methanol Production Process and Methanol Quality Industrial methanol production has reached levels of 8-10 million tons per year. mainly lower alcohols and their derivatives. The three major process owners of methanol are Imperial Chemical Industries of Britain. Federal Standards Specification Grade A or Grade AA (see TABLE I). As a commercial industrial product it normally meets the standards represented by U. Crude methanol is normally refined by distillation. A brief description of the present technical process of methanol production might be in order since it might indicate possible (toxic) impurities present in crude or refined methanol that could be expected in methanol derived SCP. CO2 and H2. . These gases are passed over a Zn-Cr or a Cu catalyst at a temperature of 200-400°C and at a pressure of 50-400 kg/cm2. and hence this does not warrant establishing special limits for the use of crude methanol. The synthetic reactions of methanol syntheses are: CO +2H2=CH3OH CO2 +3H2= CH3OH As feedstock for production of the CO.

Percent methanol by weight.003 0.7928 at 20°/20°C 99.S. 150 2—Butanol (ppm) 1-Butanol (ppm) 15 n.10°C at 760 mm No cloudiness or opalescence 0. mm Nonvolatile content 0. as NH3 Appearance 0.85 0.003 Specific gravity. non-residual Permanganate test No discharge o colour in 30 minutes No discharge of colour in 30 minute Water.d.d. non—residual Characteristic.0010 Odour Characteristic.6°C ± . 0. Alkalinity.2548 CONMISS IONS ON FERMENTATION AND ON FOOD CONTAMINANTS TABLE 1 U. No discolouration 0. percent max.d.012 n.d.' of ASTM platinumcobalt scale Not more than 1°C and shall include 64. 0. 0. 30 30 .10°C at 760 mm No cloudiness or opalescence Hydrocarbons - TABLE 2 - Typical Analysis of crude and refined methanol Composition Methanol (%) Water Refined methanol Crude methanol 99.003 Acetone. per cent max.003 0.96 0. 20 trace trace 84 16 130 200 trace - (%) Dimethyl ether (ppm) Methyl formate (ppm) Acetone Methyl acetate - trace Ethanol (ppm) 30 - 2-Propanol (ppm) Methyl ethyl ketone 1-Propanol (ppm) 35 n.0010 0. per cent max.10 Carbonizable substances Colour Not darker than colour standard No.6°C ± .d.003 Clear and free from suspended matters or sediment. n.7928 at 20D /20°-C No discolouration Not darker than colour standard No.001 Acidity (as Acetic acid) per cent max. max.15 0.003 0.5 of ASTM platinumcobalt scale Distillation range Not more than 1°C and shall include 64. 150 80 n. per cent max. per cent max. Federal Standards Grade A Grade AA - Characteristics Requirement Acetone and aldehydes. Requirement 0.001 Ethanol.85 99.

as information on methanol-grown microorganisms is sparse due to the limited capability of organisms to grow on this simple alcohol. Bacteria Extensive taxonomic studies on methylotrophic bacteria have been undertaken (Ref s 1-2). facultative methylotrophs involve species of genera Protaminobacter. This situation requires new approaches of modern systematic bacteriology using chemo-taxonomical information and techniques in order to avoid confusion in strain identification. Gen. 61. Thus in recent years. Pichia and Saccharomyces as well as asporogenic yeasts such as Kloeckera. Jiybi and A. new approaches in taxonomy involving subcellular structures. Microbial Growth on C1-compounds.5 R. Alcaligenes. Achromobacter.205 (1970) J. methanol and other C1-compounds. different microbial strains and species. 2. Methylotrophic bacteria are divided into two domains: obligate methylotrophs that can assimilate exclusively methane. 1975 1/l2— . are being emphasized to clarify interrelationship of. Strains so far identified as new species are based on differing physiological and biochemical properties relative to the established species. P. REFERENCES 1. Difficulties may exist in their classification at species level using conventional taxonomic keys. Methylotrophs are a group of organisms that are difficult to deal with by the conventional methods of classification. K. Methanomonas. J. or strain level. Acinetobacter. At the species. Methylococcus and Methylomonas and. Whittenbury. a large number of microorganisms capable of growing on m?thanol has been isolated from nature. criteria. Ozaki. cell constituents. Both yeasts and bacteria have industrial potential.Rhodotorula have been reported as methanol utilizers. Wilkinson. and this in turn will necessitate new approaches and methods in taxonomy in order to eliminate undesirable confusion. Yeasts Methanol assimilating yeasts include both those isolated from nature and those selected from among strains of stock cultures. Microcyclus. A considerable number of new species has been identified but some of them are likely to be synonymous. specific DNA parameters. Sporogenic yeasts including Hansenula. Osaka.A.F.A. Pseudomonas.C. respiratory mechanism. Microbiol. Candida and. Obligate methylotrophs include species of genera Pseudomonas. The fundamental principles of microbial taxonomy are based on morphology and this is given the heaviest weighting at the genera level. Hyphomicrobium.C. Bacillus and some coryneforms. Torulopsis. and facultative methyiotrophs that can assimilate different carbon compounds besides C1-compounds. Phillips and J. differences in biochemical and physiological characters are more important and are used as classification. No obligate methylotrophic yeasts are known.Testing of single cell proteins 2549 APPENDIX 2 Methanol-Utilizing Microorganisms Since methanol has emerged as a novel feedstock for SCP production.

Some of the pioneering work has reached the stage of commercial plant production both yeasts and bacteria have shown to have industrial potential. and fed continuously to a fermenter. a) Aeration Sterile compressed air is supplied continuously to the culture by means of a compressor after passing an air-filter and thus rendered free from contaminating organisms. . c) Biomass Separation A microbial culture is continuously taken out of the culture vessel at the same rate of feeding. b) Biomass Propagation Inoculum of the microbial culture used for SCP production is propagated batchwise until a certain cell density is obtained. minerals and water (often recycled water from biomass separation process) is sterilized continuously in medium sterilizer. When the steady state of microbial culture is established. In the case of bacterial cultures the biomass aggregates and is sedimented prior to separation and concentration. ammonia. Culture medium is then continuously fed to maintain a culture of microbial biomass under the desirable conditions. Wet biomass is then subjected to a drying process. A typical example of an SCP manufacturing process. d) e) Drying and Pelleting Wet biomass separated is usually spray-dried to a powdery SCP product. methanol is the limiting substrate in the continuous culture and the concentration of substrate methanol remains negligibly small in the microbial culture. applicable to both yeast and bacteria SCP production is schematically given in Fig. Feedstock Preparation and Sterilization Nutrient medium for microbial growth containing methanol as carbon and energy source. Table 3 summarizes the properties of some selected yeast and bacteria strains being considered for commercial development.2550 COMMISSIONS ON FERNENTATION AND ON FOOD CONTAMINANTS APPENDIX 3 SCP Production Process Based on Methanol Single cell protein production based on methanol is an area of applied microbiology where at present a great deal of activity is involved. Culture medium after biomass removal is recycled to the fermenter after readjustment of mineral constituents and pH. 2. When necessary the powdery product is made into a pellet form of appropriate size to meet the requirements for proper feeding of farm animals. Numerous scientific and technical publications as well as patent literature are proof of the great interest in this field.

2. FERMENTER FURNACE SEPARATOR DRYER Methanol-5Cp Pilot Plant of Schematic Flow Diagram PRODUCT PRODUCT GRANULATOR Ui C/) CD 0 CD C.) CD ci) 0 c/Q C/) CD .METHANOL WATER MINERALS AMMONIA AIR FILTER COOLER STERILIZER AIR FUEL CENTRATOR PRIOR CON- Fig.

5 40% 40 Yield 6—7 Mitsubishi Gas Chemical Co. Methylomonas sp. Poland.5 3—5 35 27—30 29 28 Opt.0 4.5—4.1% 5. 3) 36—42 3.9 3.0 8.8 6. 6—7 1) 2) 3) 4) 5) 6) 7) 7) 6) 5) 4) Methylophilus methylotrophus Methylomonas methanolis Aeromonas sp.5% 53.5 78.7 8.2 Crude Protein Typical Analysis 13. pH Opt. Temp.5—7. Hoechst Institute of the Fermentation Industry.0 Moisture 87 80 78 82. Cultural Parameters 5 5. Methylomonas sp.9 45 57. Candida sp.4 6. Unitica ICI Mitsubishi Gas Chemical Co.0% Nucleic Acid Typical Microorganisms used for SCP production on Methanol and Analysis of the SCP Pichia anobii Hansenula sp.37 37 36—42 35—40 45 35 50 45—50 6—7 6. Asahi Chemical Industries Co.8 — 10 10 15. 3 BACTERIA 1) 2) 7) YEASTS Table 0 Ui Ui .7 2—5 5.5 50 31.

whereas foreign brandies contained 1700-3700 ppm methanol. rose wines 70 ppm and dessert wines 150 ppm.000 ppm methanol in brandy. Similarly treated apple juices contained 7 to 15 ppm methanol. Zamorani (5) reported 100. fruit juices and (c) alcoholic products. Rebellein (7) analysed 200 samples of German wines and found the following mean concentrations of methanol: white wines 50 ppm. whisky was found to contain 50-70 ppm. Natural occurrence of methanol Like ethanol. 100-200 ppm in whisky and 200-800 ppm in Finnish berry wine.2553 Testing of single cell proteins APPENDIX 4 A.000 ppm. whiskies were found to contain 150 ppm (4). sweetened blackcurrant Orange 145 45 (2) 181 USA 31 Grapefruit 23 Bananas 17 Markh et al (3) found 0. Radmine (8) found 138-340 ppm in 22 Yugoslavian wines. 300-700 ppm in liqueurs. . a) Occurrence in non-fermented fruit juices The following Table of methanol concentrations in fruit juices is taken from a publication by Jacquin and Travernier (2) (TABLE 4). ppm Apple Tomato France (2) (3) 25 46 64 (1) (2) (3) (4) 180 218 195 204 (1) Blackcurrant 680 Raisin/blackcurrant (1) France 65 Gooseberry Conc. (b) non-fermented Methanol occurs naturally in (a) vegetables and fruits. red wines 14 ppm. Nykanen (6) found 700-800 ppm in Russian brandy. Table 4 Fruit Juices Country of Origin Methanol Conc. It is produced from pectin containing substances in fruits as a result of the action of the enzyme pectin methylesterase and also possibly as a secondary product of alcoholic fermentation (1). in fruits and vegetables belonging to Our daily diet. b) Occurrence in fermented products Brandy appears to contain the highest levels of methanol with concentrations quoted as high as 20. methanol is a natural product that occurs abundantly in the plant kingdom.000 to 150.7 to 12 ppm methanol in Russian grape juices treated with pectolytic enzymes and subsequently pasteurised. Dyer (15) reported 180-670 ppm methanol in USA brandies.

5 mg/cigarette Reference 16 17 17 18 19 19 19 19. a figure which he considered to be a safe level. ppm Source Apricots (canned) Bananas—ripe —half ripe Pineapples Brussel sprouts (volatiles) " 5-13 1-6 18 1 "very large" (but no conc. he often loses sight (5-10 g) or dies.COMMISSIONS ON FERMENTATION AND ON FOOD CONTAMINANTS 2554 Other levels observed in wines include:- Table 5 Reference Conc. .214-217 (1955). 2. REFERENCES 1. kidneys and lungs (1). Tavernier J. Jaquin P. without any apparent ill-effect. ppm Origin . however. The toxicity greatly varies depending on the species of animal. 6th Symposium "Substances etranges aliments" Madrid 960. Methanol has a coagulating action on albumin and results in irritation of mucous tissue particularly in the digestive system. This is especially true in the appendix. toxicologique et analytique de la question". The lethal dose. and appendix contents of guinea pigs. Summer's studies were made to refute objections to the use of fruit juices treated with pectinase.21 19. Flanzy P. the liver.. (1969) (23) gives TLV = 260 mg/m3 (air concentration to which it is believed that nearly all workers may be repeatedly exposed. The physiological fate of the methanol liberated from pectin was studied by Summer (24) by means of suspensions. large intestine. The mechanism of the toxicity has not yet been clearly demonstrated. 7-150 180—240 220 170—500 100—5000 100-700 50—325 Mendoza Japan France Irpinia Various Crimean Various 9 10 10 11 12 13 14 Fermandez et al (16) found that in Spanish vinegar 400 ppm was rarely exceeded. when administered in a large amount. c) Other sources of methanol Table 6 Methanol Conc. LD5o. "Sur la Teneur des Jus de Fruits en Methanol" in 'L' alimentation et la Vie'. In man. It was shown that methanol was released from pectin by pectinase in these organs.. when using pectin-containing fruit and fruit products must come into contact with small amounts of methanol. therefore. is its attack on the nervous system (particularly the optic nerves). small intestine. p 449—463. quoted) Celery " " Parsnip " " Potato " " Onion Tobacco (Russian & Turkish) 0. "L'alcool methylique dans les vms et les jus de fruits clarifies: aspects biologique. 43.M. of stomach.2-0. Collon Y. The human and animal organism. for rates by ingestion of methanol was 10 g/kg body weight. day after day without adverse effect). The Handbook of Analytical Toxicology.20 22 A large dose of methanol is toxic for animals. The most serious effect.

47. 12(1) . 48—52 (1967). 74 7-8 (1975). . 8. 15.. 735-6 (1971). Vapours are intensely irritating to mucous membranes. Topical application raay produce an irritant dermititis. 1(6). Memo No.. Agr. "Methanol and Fusel Oil in Alcoholic Beverages". Agr..E.F. 11. "Determination of Methyl Alcohol in Wine by Gas Chromatography". (CA 59-2130 g). 35. 12. "Oxygen-containing Volatiles (CA 70—95573w). Academic Press. of Vegetables".. Pisanti A. 20. (1970). (CA 58—10692 a). Obst. Arch . Radmie. Station. (ed). "Toxicology of Drugs & Chemicals". Quim md. Kamenhichikova S. Natural Occurrence of Formaldehyde Formaldehyde and its aqueous solution.Testing of single cell proteins 3. 4(2) .. Vitocolt. Swan T. Bienc. 20. p 640. Australan_J. 24.. "Methyl Alcohol Content in Mendoza Wines". Deut. (CA 63—12278 a) Rundschau. 17. Acree T. 13. Lee C.T. Datimashvili E.. London. "Volatile Reducing Substances as a Criterion of Quality of Canned Apricots". 10.. Stepanov L. 47(4). Geralde H.D. 172-3 (1962). "Determination of Methanol in Wines by Gas Chromatography".. Paulina Ya. "Handbook of Analytical Toxicoloy". 19.. B. "Isolation and Identification of the Lachrymogenic Compound of Onion". 163—9 (1962). Lataeva D.G.W. 9. Dadykin V. "Comparison of GIC and Colorimetric Methods for Determination of Methanol in Alcoholic Beverages". 7. Casey J. 61(8) .. 18. "Catechol and Methanol Content of Wines". vertigo. p 863-4 (1963). Butts R. Vinodel Mold (Russ) 25-6 5.. and thus destroys cell functions and causes death. haematuria. MOrales. Vega.M. Biol. Summer.S. Germueseverwert. Kemian Aikakauslekti. "The Physiological Fate of Methanol Liberated from Pectin". acidosis. (CA 61-2400 e). Blackwell. Expt. Formaldehyde acts as a powerful coagulant denaturing proteins of cell protoplasm. 440—4 (1961) Connel D. \7itocolt. berry. 2555 Markh A. Zamorani A.. formaldehyde is widely used as an aseptic or a disinfectant. "The Low Boiling Volatiles of Cooked Foods".E. Nippon NogeikagakuKaishi.d). 1969. Vinodel.. (CA 6 1—8860 d). p 383—384. Chem. (CA 71—99036 s) Sunshine I. "Methyl Alcohol in Wines of Irpinia". Tyntyun. 14(12) 425—31 (1961) Eck L. Storage and Processing". JAOAC. Procter R. 45-56 (1963) (CA 68—86116 f) Tomotsune T.92—6 (1961). For this reason. "Volatile Flavouring Constituents of the Pineapple". Luh B. 9-11 (1964). 21. Anal. "Determination of Methanol in Alcoholic Beverages by Gas Chromatography". Cuoy (Mendoza) . Cornell Univ. "Methyl alcohol in fruit. and grape juices" Sadovod Vinograd."Determination of Methanol Content of Tobacco and Tobacco Smoke". (CA 82—16 8785 b) 15. 8. Med. iv Farm (Belgrade) . 12 (3) . 23. Riv. 54. Enol (Conegliano) . 14(4). Teknillisen 4. 129—132 (1961) (CA 60—6173 c. Buscarons F.. (CA 59-13316 h). 239—40 (1965) . Nac.. 17(1). Hultin H. "Methanol Content of Grape 6. 14. Self R. (1973) CA 81—62316) Drechraan W. Bulg. haematemesis.O. (Bilbao). LD50 orally in rats is 800 mg/kg. Makhnachev I.B. Riv. 385 (1964). Fac. 165-7 (1961). 130-40 (1964).H. (CA 63-3531 f).25—27 (1973). Food Technologr. "Methanol Levels in Wines". Chemistry and Industry. 129-133 (1963) Rebelein H. formalin.N. Ingestion may cause severe abdominalpain. 22. coma and death. Brandy". Maksimovic "Quantitative Determination of Arsenic. Enol (CA 60—2295 k) (Conegliano). l4(3). Ryzhkova V. Kosm. "Changes in Some Volatile Constituents of the Banana During Ripening. Nykanen V. Dyer R. 298-9 (1961) . Lead and Methanol in Wines" .Y.N. 39—41 (1969) . (CA 61—9771 e).P. Nikolai I. Univ.. Rev. anuria. May 25. is a toxic substance for human and animal. Zykina T. 15(3). Food Technology. md. 16. Wilkens W.. Vinograd. 11(1). Chem. Ofrica. proteinuria.C. SSSR.

6 Caught off Olyutorskii Cape. Soon after this it was demonstrated that formaldehyde is widely contained in cod and Alaska Pollock and the detection of formaldehyde in the previous instance was not the case of illegal addition of the chemical for the purpose of preservation.s of a blue or indigo colour of Rimini's reaction and a purple colour of the modified Herner's reaction (Yanagizawa—Maruyama's reaction).4 Caught off West Kamchatka. (TABLE 7).s and Food Additives. Three out of five fresh mushroom and five out of six dried mushroom samples were found to be positive. treated with dry ice immediately after catching • In 1968. 1962. The State Standards on Food. frozen on board 6. the Public Health Department of Osaka Prefecture reported that some commercial Chinese mushroom samples gave an odour of formaldehyde and were found to be positive in official formaldehyde tests.5 Caught at Essa Strait. In the decree issued on February 12.particularly in some typical edible mushrooms.6 Caught in Bering Sea. Yamada and Bito (1963) on the occurrence of formaldehyde in fish demonstrated that cod and Alaskan pollock contain considerable amounts of formaldehyde in their muscle and other tissues.1 " 6. frozen Caught off Kushiro. consequently.5 3. Source and Status of Fish (mg/bOg) TYM-l Cod TYY-8 " " " HKR-l7 II TYY—l8 II TYT-5 TYS—7 4.4 Fishing ground was not certain. Species No. Extensive studies undertaken by Amano.2 7. cod muscle was isolated and chemically identified. frozen on board Caught in Bering Sea. The National. The maximal level of formaldehyde determined in cod muscle was 15 mg/bOg (150 ppm).1 5. frozen on board 15 Caught off Olyutorskii Cape. In 1961.. frozen on board " 8. frozen on board W-l9 K-l5 2. .COMMISSIONS ON FERNENTATION AND ON FOOD CONTAMINANTS 2556 In the Japanese food sanitation regulations.5 Caught off Olyutorskii Cape. a processed product of cod meat obtained in the Tokyo Fisheries Market was found to be positive with the formaldehyde test. under the Food Sanitation Act state that formaldehyde detection should be conducted on the bas. Hokkaido: iced K—16 5. salted and dried 0. Formaldehyde in. formaldehyde is among the chemicals listed as those prohibited for use in manufacture for preservation of foods destined to commercial sale. The decree enforced disposal of cod with formaldehyde content of above a fixed level. conducted extensive investigations on the occurrence of formaldehyde in various natural foods.1 Purchased at Shiogama. the Health Ministry advised tentative measures including determination of formaldehyde using the chromotropic acid method with the cod samples that were found to be positive for formaldehyde using both Rimini's and modified Herner's reactions. Sample Formaldehyde Content in the Muscle of Cod and Alaskan Polbock. Formaldehyde content . iced Fishing ground was not certain: salted S-42 K-I-20 " Y-I-20 II 10 and dried N-55 Alaska Polbock 2. Institute of Hygienic Sciences. The investigation demonstrated that mushrooms particularly Chinese mushroom (Cortinellus edodos) contain small amounts of formaldehyde as natural constituent (TABLE 8). TABLE 7.

lettuce. when they are specified by the Health Minister as not harmful to human health. Bito. 1970.4 100—406 Formaldehyde was also detected by acetylacetone method in the following vegetables: turnip. chrysanthemum. K. pimient and buttérbur. K.5 13. The Japanese Food Sanitation Counàil advised that this low level of naturally occurring formaldehyde should not have any adverse effect on human health. onion. parsley. Food Sanitation Bureau.2 54. The Japanese Food Sanitation Act allows exceptional admission of chemical compounds (referred to as toxic or harmful) in foods. Scientific Fisheries. in Japan.5 7. Amano. Formaldeyç1e Content in Vegetables Vegetable Tomato Cucumber Kikurage (Mushroom) Hiratake (Mushroom) Nameko (Mushroom) Naratake (Mushroom) Chinese Mushroom (fresh) (dried. egg plant. Japanese Soc. Ministry of Health and Welfare. 20 No 12 (1970) . 695 1963) 2. "Detection and Identification of Formaldehyde in Gadoid Fish". 29. Since this decree there is no legal regulation.2557 Testing of single cell proteins TABLE 8. leek. cucumber. Yamada arid M. Partial Amendment of Standards of Food and Food Additives Food Hygiene Research. however. REFERENCES 1. of naturally occurring The use of formaldehyde as an additive is.3 1. On September 11. Bull. radish. formaldehyde in foods strictly prohibited.. 9 samples) ppm 0 2. the Health Ministry issued a decree setting aside the provision that foods must contain no detectable formaldehyde.7 34.

76 7.53% 1.25 0.10 0.76 2.15% 3.01 0.060 0.86 12.88 2.005 0.002% 0.79 5.5 8.20 1.79 0.01 3.53 6.03 0.82 1.67 0.014 .35 0.35 6.18 2.018 0.66 2.36 2.39 2.32 0.43 0.90% 7.01 1.48 1.37 5.26 1.000 0.20% 2.6 8.04 0.43 2.71 2.8 5% 76 4.71 4.16% 9.49 3.27 1.87 0.31 3.47 5.17 Dodecanoic Acid Tetradecanoic Acid (14:0) Tetradecanoic Acid (14:1) Pentadecanoic Acid (15:0) Hexadecanoic Acid (16:0) Cis—9—Hexadecanoic Acid (16:1) Heptadecanoic Acid (17:0) Octadecanoic Acid (18:0) Octadecanoic Acid (18:1) OctadecadienoicAcid (18:2) Octadecatrienoic Acid (18:3) 9.16 2.26 0.40 2.57 3.66% 8.67 Alanine Arginine Aspartic Acid Glutamic Acid Glycine Histidine Isoleucine Leucine Phenylalanine Proline Serine.19 0.001 0.19 (Gross Proximate Composition) Moisture Protein Fat Ash Fibre 8.01 2.6 2.43% 3.29 2.29 5.52 0.006 8 ppm (Amino Acids) 3.0 4.0 7.3 11.11 0. Threonine Tryptophan Tyrosine Valine Cystine Methionine Lysine Available Lysine 0.084 1.21 0.95 0.24 2.2558 COMMISSIONS ON FERMENTATION AND ON FOOD CONTAMINANTS APPENDIX 5 Typical Analysis of Pilot Plant Samples of SCP Produced from Methanol Bacteria-SCP (2) Yeast—SCP (3) Granular Powder Granular 10% 73 7.3 0.44 4.25 0.5% 1.12 2.01 0.96 4.24 2.68 3.07 3. phospholipids Non-saponifiables 7.61 (1) (Fats & Fatty Acids) 0.45 1.87 4.015 0.02 0.000 Glycerols.000 0.3 0.97 4.9 6.07 3.15 0.3 3.6 not detectable (Nitrogen Components) Total N Amino Acid N Nucleic Acid N Ammonia and Urea N Ethanolamine N Glucosamine N Nitrate and Nitrite N 0.31 3.37 2.66 3.5% 55.11 5.48 0.5% 1.80 1.95 0.62 0.97 0.01% 0.01% 0.2.70 4.54 1.005 0.3 0.61 2.14 6.79 2.15 0.26 0.14 0.10-Methylene hexadecanoic Acid 2-Hydroxyhexadecanoic Acid 3—Hydroxyhexadecanoic Acid 0.0 7.3 1.63 4.08 2.41 7.54 3.18 8.75 2.

003 0.31 0.033 0. Dry Matter Basis) 1. Japan.04 less than 0.093 0. (2) Data supplied by Imperial Chemical Industries Ltd.01 (1) Data are for guidance only.5 Choline Ergosterol (Nutritional Minerals.5 31.1 6.03 0. 14 0.00003 .2559 Testing of single cell protein (Vitamins) Bacteria-SCP Granular and Powder Thiamine (B1) Riboflavin (B2) Pyridoxine (B6) Cobalamine (B12) Folic Acid Ca d-pantothenate Nicotinic Acid Biotin Vitamin E Yeast-SCP Granular 137 mg/kg 3.1 not detectable not detectable less than 1 ppb 0.016 21 ppm 26 21 0.74 ppm less than 0.01 0.2 10 500 Zn Cu Se Co Cl 4 less than less than 0.6 2.87% 0.25 P Mg K Na Ca Fe Mn 1.32 70 718 0.12. they should not be interpreted as specific production standards or recommendations. UK.05 ppm 0..1 2000 (Contaminants) Methanol Formaldehyde Benzo (a) pyrene Lead Arsenic Mercury Cadmium not detectable less than 1 ppb 0.3 39. (3) Data supplied by Mitsubishi Gas Chemicals mc.034 30 ppm 317 2.51 33 8260 2600 4000 0.1 ppm 0.01 0.19 0.4 1.6 mg/kg 219 33.22 2.05 0.6 16 0.5 Inos itol 5.80% 0. .

Ohno. A Kanbayashi. K Yamada (Chairman). A Kitai and Dr. Dr. . K Kameoka. Dr. K Ohno. At the Joint Meeting of the Coordinating Committee on Food Chemistry and Commission on Fermentation in Madrid 1975. I Nagai. J Takahashi. Mr.was recognised and an ad hoc Working Group was organized. 12 entitled 'PROPOSED GUIDELINES FOR TESTING OF SINGLE CELL PROTEIN DESTINED AS MAJOR PROTEIN SOURCE FOR ANIMAL FEED' in August 1974. Dr. Dr. Dr. The final version was approved by the Food Contaminants Commission in Budapest (August 1978) and the Fermentation Commission in Munich (September 1978). Commission Commission Commission Commission on on on on Fermentation Fermentation Food Contaminants Food Contaminants. D Littlehailes. Yamáda. Dr. J Kato. Dr. K Aibara. Dr. Dr. K Kato Japanese Drafting Committee: (Secretary). M Kuraishi. Y Shirasu. Dr. K Komagata and Dr. Dr. The Working Group acknowledges the cooperation extended by the following members within and outside of IUPAC: Consultants: Dr. for the methanol based SCP's. At the Joint Meeting of the Commissions in Warsaw 1977. the first draft was discussed and revised. D A Stringer. S J Kubacki. the need for a supplimentary volume of the Guideline.CONMISSIONS ON FERMENTATION AND ON FOOD CONTAMINANTS 2560 IUPAC published the Technical Reports No. Membership of the Working Group on Single Cell Protein responsible for the preparation of this report is as follows: Dr Dr Dr Dr J K J K C Hoogerheide.