SINGLE CELL PROTEIN AND ITS PRODUCTION

Abstract: A process for the production of prokaryotic, particularly bacterial, single cell protein which includes a step in which whole and/or ruptured micro-organism cells are treated with an oxidising agent or are subjected to other oxidising means. The oxidising agent can be added during cultivation and/or during subsequent treatment and/or it can be added to the product obtained. The treatment is preferably carried out at a pH between 7.0 - 9.0. The preferred oxidising agent is hyd­ rogen peroxide. The product of the process has a number of advantages over known prokaryotic single cell protein, most important being that its use alleviates difficulties of reduced feed intake, reduced live weight gain, poor feed conversion ratios and liver damage experienced when single cell protein is fed to domestic fowls in high dietary inclusions.

Description: Improved process for the Production of single cell protein This invention relates to an improved process for the production of single cell protein (SCP). Prot einac eous materials are important dietary components for many animals. A wide variety of such materials are in use including for example fish meal, soya bean meal and materials derived from rape seeds and sunflower seeds. In the past twenty years considerable research effort has been devoted to the product ion of yeast and bacterial proteins for inclusion in animal diets. Such proteins derived from micro-organisms are commonly referred to as single cell protein or SCP. The inclusion of high proportions of some widely used proteinaceous materials in their diets can have adverse effects upon certain species. This is frequently ascribed to "toxic" constituents. The effect of these constituents has been recently reviewed (Toxic Constituents of Plant Foodstuffs, Ed. I.E. Liener, Academic Press, New York). Liener (1969, pages 4 - 6) refers to toxic constituents as substances that produce deleterious effects when fed to men or animals. Re distinguishes between chemical constituents that are lethal and those that produce an adverse physiological response. Processing, or "other means of preparation have proved to be effective in destroying many toxic constituents". Reference is also made to Altshul A.M., (1967, Science, 158, 221) that if it "were not for the fact that such oilseeds as soyabeans and cotton seed can be processed so as to inactivate their toxic constituents, these rich sources of plant protein would not occupy such a position of importance as they now cozzard in the feeding of farm animals". It is known that the feeding of single cell protein derived from prokaryotic micro-organisms, which include bacteria and blue-green algae to certain animal species, particularly domestic fowls, in high dietary inclusions cases reduced feed intake (tri), reduced live weight gain (LWG) and poor feed conversion ratios (FCR). In extreme instances this produces liver damage with organ enlargement, focal necrosistwith phogocytic and inflammstorr cell infiltration being observable on histopathological examination. This effect has been produced with prokaryotic single cell protein derived from different micro-organisms, grown on different nutrient media and in different processes and has been reported by (i) D E Owen and S M A 1)bay (Fd. Cosmet. Toxicol, 16, 633, 1978); (ii) L Farstad (Acta Agric. Scand., 27, 129, 1977) and (iii) C T Whittemore, D W GilcErist - Shirlaw, D 5 Nclonald and A G Taylor (British Poultry Science, 19, 283, 1978). Prom experiments in which single cell protein produced by the process described in our UK Specification No. 1 370 892 was included as a proteinaceous material in normal commercial diets fed to domestic fowls, we have found progressive decreases in LWG and progressive decreases in food intake. Correlated with this is an increase in the incidence of observable liver damage as the percentage inclusion of prokaryotic single cell protein in the diets is increased. The experiments were carried out with diets containing 0%. 2.5%. 5%, 7.5%, 10%, 15%, 20%, 25%, and 30% of prokaryotic single cell protein produced by the process of UK Specification No. 1 370 892. With low inclusions of SCP (eg 2.5%), the incidence of liver damage did not differ significantly from the incidence with diets containing no SCP. However, with high inclusions of SCP significant differences in the incidence of liver damage between SCP containing diets and non-SCP diets were observed. For instance with broiler chick diets containing nonstressful levels of crude protein, say 25, and also non-stressful levels of vitamins and minerals, the incidence of liver damage was between 10 and 40duo usually 10%. With laying hens, fed stressful levels (30%) of crude protein, contained in diets having a 30% SOP content the incidence of liver damage was between 30 and 70 /0, usually 50 /0. We have also found that the effect upon growth performance of domestic fowls resulting from the inclusion of SCP in their diets is not affected significantly by varying the microbial culture conditions or by varying the method whereby the microorganisms are dried to produce a dietary ingredient, i.e. freeze-, flash- and spray-dried products give similar effects. The effect of using prokaryotic SCP as a dietary ingredient upon certain species restricts the proportions in which the SCP can be included in the diets of these species and, in consequence, reduces the utility of prokaryotic SCP in relation to these species. According to the present invention we provide a process for the production of single cell protein which comprises cultivating prokaryotic micro-organisms and subsequently treating the cultivated micro-organisms to produce a product comprising whole non-viable cells and/or ruptured cells wherein the micro-organisms are treated with an oxidising agent or are subjected to other oxidation means, the oxidising agent being added or the other means being applied during cultivation and/or during the subsequent treatment and/or to the product after the subsequent treatment, the oxidising agent being added in an amount between 3 and 300 m moles per litre of a solids suspension containing the micro-organisms when it is added during cultivation or during subsequent treatment, or, when added to the product, being added in an amount between 3 and 300 m moles per litre of the product moisture content. The prokaryotic micro-organisms generally used to date in processes for the production of single cell protein are bacteria. Thus the process of the present invention is mainly of interest insofar as it relates to bacteria and will hereafter be described as a process for the production of bacterial SOP. The present invention is applicable to any process for the production of bacterial SOP. Such a process is that described in our UK Specification No. 1 370 892 in which the bacteria employed are strains of the species Pseudomonas metkylotropha (now known as MethYloPhilus methylotrophus), Pseudomonas rosea, Ryphomicrobium variabile and Microcyclus polymorphum. A number of strains of these species have been deposited at the following culture collections: : (a) National Collection of Industrial Bacteria (NC7B), Torrey Research Station, Aberdeen, Scotland, UK; (b) US Department of Agriculture (NRRL), Peoria, Illinois, USA; (c) Fermentation Research Institute (FRI), Japan; and have been assigned the following acoession numbers: NCIB Nos. 10508 - 10515; 10592 - 10596; 10516; 10517; 10597 - 10612. KRRL Nos. 35352 - 35359; 35360 - 35364; B 5381; 35382; 3 5365 - 35380. FRI Nos. 1215 - 1222; 1223 - 1227; 1228; 1229; 1230 1245. The characteristics of the above four species and the particular strains listed are given in our BE Specification No. 1 370 892. Of the species specifically mentioned above Pseudomonas metkylotropha (etvlophilus methslotrophus) is especially preferred. Among the specific strains listed above this species is represented by strains NOIB Nos. 10508 - 10515 and 10592 - 10596. The process of the invention may be a batch or a continuous process preferably it is a continuous process. In a continuous process a culture of micro-organisms is grown in a fermenter and an aqueous suspension containing microbial cells is continuously withdrawn and subjected to a series of treatment steps to concentrate the suspension before it passes to a drier. In a typical process sequence the suspension leaving the fermenter is subjected to the following steps before passing to a drier: (i) a flocculation step, eg involving heating and acid treatment as described in our BE Specification No. 1 381 306; (ii) a separation step or steps to separate the bulk of the liquid from the microbial cells, separated liquid may be returned to the fermenter while a concentrated suspension often termed "drier feed cream" passes to the drier. The cells may be treated with the oxidising agent at any stage in the process. However in choosing the stage at which to perform treatment the following factors are relevant: 1. The oxidising agent employed may be toxic to living micro-organisms and thus have deleterious effects upon a growing culture; 2. Many oxidising agents are degraded and wholly or partially de-activated by enzymes such as peroxidases and catalase which are present in most bacteria; 3. The amount of oxidising agent required depends upon the volume of liquid associated with the cells and thus the invention is more economically applied by adding the oxidising agents to concentrated suspensions or to the product; 4. Treatment with oxidising agent is preferably carried out at temperatures between 100 and 1400C, especially between 500 and 800C, a temperature of 600C being particularly preferred; 5. The pH at which treatment with oxidising agent is carried out is preferably in the range pH 7.0 9.0, particularly in the range 7.5 - 8.5 with pH values of 8.0 to 8.5 being most suitable. Lower pH values are acceptable but the oxidation con ditions are altered. Equally higher pH values are also acceptable but in this instance decomposition of some oxidising agents tends to occur to an en hanced extent making treatment less effective. Add itionally lysed and flocculated cells react in high pH conditions to produce a highly viscous fluid which is difficult to mix. Taking account of the above considerations addition of tn oxidising agent to a growing culture is not preferred because of problems caused by the action of the oxidising agent on a grow Ir cell and by its degradation by catalase peroxidase. After cell lysis and suspension concentration catalase degradtion is minimal and the solids concentration is high. Thus it is preferred to treat the cell suspension with the oxidising agent shortly beore the suspension passes to the drier, ie to treat the drier feed cream. Alternatively the product from the drier may be treated with the oxidising agent. Although this product tends to be referred to loosely as "dried" cells, there is in fact a measurable proportion of water associated with the "dried" cells typically Thus (w/w). Thus if the oxidising agent is added in an amount of 3 to 300 m moles per litre of the product moisture content, this can be calculated as follows: Typical "dry" product contains 10% moisture = 100 mls water/kg. Therefore add 3 - 300 m moles oxidising agent per 10 kg product. When the product is treated with certain oxidising agents further process steps may be required in order to remove traces of the oxidising agents from the final product. Suitably the oxidising agent is one which is pure, leaves no toxic residues and does zero or minimal damage to the nutritional quality of the product. The preferred oxidising agent is hydrogen peroxide. Other suitable oxidising agents in dude organic peroxides, inorganic peroxides, ozone and permanganate. Other suitable oxidising means may include electrolytic processes. The preferred amount of oxidising agent to be added varies to some extent depending upon the pH at which addition takes place. However a preferred range of amounts effective at a variety of pH values is from 30 to 175 m moles, particularly 70 to 150 m moles, per litre of a solids suspension or per litre of the product moisture content. In the studies we have made we have found the greatest effect with minimal oxidation when adding the oxidising agent in an amount of 86 m moles per litre. The duration of the treatment with the oxidising agent is suitably within the range 1 to 300 minutes, 10 to 40 minutes and particularly 30 minutes being preferred. Using the process of the invention a product is obtained which has a number of advantages over previously known prokaryotic SCP. Most importantly use of this product alleviates the difficulties of reduced feed intake, reduced live weight gain and poor feed conversion ratics experienced with domestic fowls and reduces the incidence of liver damage when the product is fed at high dietary inclusions. In addition the product has improved colour and improved dispersion properties which are important when it is used for feeding young animals, eg veal calves. These advantages increase the utility of prokaryotic SCP generally. The invention is illustrated by the following Examples: MAMPI;E 1 In a series of large-scale trials male and female chicks and laying hens were fed diets containing Methylophilus methYlotroPhus (Pseudomonas methylotropha) single cell protein in some instances untreated and in others treated with hydrogen peroxide by the process of the present invention. During production of the treated single cell protein used, cell lysate (ie drier feed cream) having a solids content of 1 % (w/v) was treated with quantities of concentrated hydrogen peroxide solution (ltfo w/v) such that the initial concentration of hydrogen peroxide in the cream was between zero and 175 mM. After mixing the hydrogen peroxide with the cream, the treated cream was held at a temperature of 600C and at pH 7.0 for 30 minutes prior to drying by flash drier. Typical results, for birds fed diets containing treated and untreated single cell protein and for birds fed diets in which single cell protein was replaced by alternative proteinaceous materials, are given in Table 1. Figure 1 is a graph of the occurs ence of gross observable liver damage (per cent of the test population against the concentration m)M* of hydrogen peroxide in ne drier feed cream for laying hens (plot A), female chicks (plot B) and and male chicks (plot C). Plots A, B anc C are linear regression analyses of all chick and laying hen data obtained in this series of trials. The incidence of lesions was highest with Laying nan and least with male chicks. The graph indicates that the minimum concentration of hydrogen peroxide required to reduce @@@er damage in in chicks and laying hens effectively to zero levels 15 114 mm. . In our tests on birds fed diets containing alternative proteinaceous materials a number of birds exhibited liver damage. TA3LE 1 EMI8.1 <tb> liver <SEP> damage <SEP> in <SEP> liver <SEP> damage <SEP> in <SEP> liver <SEP> damage <SEP> in <tb> laying <SEP> hens <SEP> male <SEP> chicks <SEP> female <SEP> chicks <tb> single <SEP> incidence <SEP> total <SEP> incidence <SEP> total <SEP> incidence <SEP> total <tb> tcell <SEP> mM <SEP> H202* <SEP> ie <SEP> % <SEP> of <SEP> na <SEP> of <SEP> ie,' <SEP> of <SEP> no <SEP> or <SEP> ie <SEP> % <SEP> of <SEP> no. <SEP> of <tb> protein <SEP> in <SEP> drier <SEP> birds <SEP> birds <SEP> birds <SEP> birds <SEP> birds <SEP> birds <tb> tin <SEP> diet <SEP> feed <SEP> developing <SEP> in <SEP> developing <SEP> in <SEP> developing <SEP> in <tb> (% <SEP> W/w) <SEP> cream <SEP> damage <SEP> trial <SEP> damage <SEP> trial <SEP> damage <SEP> trial <tb> <SEP> 0.0 <SEP> 0 <SEP> 1.3 <SEP> 78 <SEP> 0.7 <SEP> 1067 <SEP> - <SEP> <tb> <SEP> 7.5 <SEP> 0 <SEP> i <SEP> - <SEP> 5.2 <SEP> 408 <SEP> <SEP> - <SEP> - <SEP> <tb> <SEP> 10.0 <SEP> 0 <SEP> 8.3 <SEP> 60 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> <tb> <SEP> 25.0 <SEP> 0 <SEP> - <SEP> - <SEP> 15.3 <SEP> <SEP> 144 <SEP> 21.0 <SEP> 276 <tb> <SEP> 30.0 <SEP> 0 <SEP> 47.1 <SEP> 102 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> <tb> <SEP> 25.0 <SEP> 36.o <SEP> - <SEP> - <SEP> 5.9 <SEP> 187 <SEP> 16.5 <SEP> 284 <tb> <SEP> 25.0 <SEP> 175.0 <SEP> - <SEP> - <SEP> 2.0 <SEP> 100 <SEP> 0.0 <SEP> 93 <tb> <SEP> 30.0 <SEP> 70.0 <SEP> 19 <SEP> 42 <SEP> j <SEP> - <SEP> - <SEP> <tb> <SEP> 30.0 <SEP> 87.5 <SEP> 7.1 <SEP> 42 <SEP> - <SEP> - <SEP> - <SEP> <tb> * In the Table and in Figure 1 the concentration of hydrogen peroxide in m moles per litre was calculated assuming the drier feed cream to have a density of 1.0 g/cm . EXAMPLE 2 In a further series of large-scale trials domestic fowls were fed diets containing Methylophilus methylotrophus (Pseudomonas methylotropha) SCP, in some instances untreated and in others treated with hydrogen peroxide according to the process of the present invention. The SCP was produced in a large pilot plant using the process of our UK Specification No. 1370892. In the process bacterial cells were cultivated in a methanol-containing medium in a fermenter of our UK Specification No. 1 353 008. The cell suspension leaving the fermenter was treated using the method of our Et Specification No. 1 381 306 and was concentrated to produce a drier feed cream (DFC) which was subsequently fed to a drier. In these trials samples of DFC at pH 4 and having a 12 - 1596 solids content were taken from the plant and were subjected to the following treatment steps in a laboratory to produce hydrogen-peroxide-treated material (steps 2 to 4 being omitted to produce a non-hydrogen peroxide treated control material):1. add KOHaOH (1.68 : 1.00 molar ratio) tq give desired pH; 2. add H202 - see note below; 3. maintain at 600C for 30 minutes; re-neutralize to pH 7 with H2SO,; 5. spray dry; and 6. granulate feed sample. * In trial No. APC021J this step was found to be unneoeseasy. In step 3 an amount of 12% (40 vol) hydrogen peroxide was added to the DFC. The number of millilitres of hydrogen peroxide added to 100 g of DFC is hereinafter designated as the "percentage addition" of hydrogen peroxide, i.e. 2.5 mls added to 100 g gFC gives a "2.5%" hydrogen peroxide treatment - corresponding to the addition of 86 m moles per litre of hydrogen peroxide. Samples of p obtained as described above were included in the diets of the following fowis: (a) Broiler chicks (Ross I) fed diets containing 250/0 of the test sample from approximately 8 to 22 days of age. (b) Laying hens (Light White Hybrids over 40 weeks of age) fed diets containing 30%o of the test sample for 6 days. In each of a series of 6 trials SCP was treated with "2.5%" hydrogen peroxide at various pH values and was included in the diets of the above fowls (4 trials with chicks and 2 with laying hens). In each case a control diet containing untreated SCP was also used. The biological data generated from these trials are summarized in the graph shown in Figure 2. In this the ordinate is the percentage incidence of liver damage, relative to that occurring with a control diet including untreated SOP in the same trial (taken as 1009), using diets containing SCP treated with "2.5%n hydrogen peroxide. The abscissa is the pH of hydrogen peroxide treatment for the treated SOP. The different symbols on the graph represent the different trials from which the values plotted were obtained. The percentage of fowls exhibiting liver damage amongst those fed a control diet containing untreated SCP in each trial is given in Table 2. TABLE 2 EMI10.1 <tb> <SEP> Trial <SEP> No. <SEP> and <SEP> % <SEP> incidence <SEP> of <tb> symbol <SEP> on <SEP> Fig. <SEP> 2. <SEP> liver <SEP> damage <SEP> in <SEP> control <SEP> Type <SEP> of <SEP> fowl <tb> BC <SEP> 117 <SEP> 22.0% <SEP> chicks <tb> APC003J <SEP> 32.7% <SEP> chicks <tb> O <SEP> APC016J <SEP> 27.0% <SEP> 1 <SEP> chicks <tb> n <SEP> APC021J <SEP> 14.096 <SEP> chicks <tb> CL <SEP> L <SEP> 32 <SEP> 30.0% <SEP> laying <SEP> hens <tb> IPL4 <SEP> XXV <SEP> 27.*6 <SEP> laying <SEP> hens <tb> The percentages given in Table 2 were taken as the 100% values for the graph in Figure 2 in respect of the individual trials. These data clearly indicate a pH optimum for such hydrogen peroxide treatment in the region of pH 8 - 8.5. The average percentage incidences of liver damage represented by the broken line in the graph in Figure 2 are: - Untreated Control - 25.6% DFC/2.5% H202 . pH 7 - 11.7% pH 7.5 - 3.6% pH 8 - l.4 pH 8.5 - 2.3% pH 9 - 10.2% In a further trial APC024J carried out in the manner described above the level of hydrogen peroxide addition and the pH of treatment with hydrogen peroxide were both varied. The liver damage data resulting from this trial are summarised in the graph shown in Figure 3. In this graph the ordinate is the percentage liver damage incidence, relative to that occurring when an untreated control was used (taken as 100%), using feeds containing SCP treated with varying levels of hydrogen peroxide at varying pH values. The abscissa is the pH of hydrogen peroxide treatment. These data imply that an optimum treatment lies in the range "2%" H202 at pH 8 and "2.5%" H202 at pH 8.5.

Claims: 1. A process for the production of single cell protein which comprises cultivating prokaryotic micro-organisms and subsequently treating the cultivated micro-organisms to produce a product comprising whole non-viable cells and/or =rup- tured cells wherein the micro-organisms are treated with an oxidising agent or are subjected to other oxidation means, the oxidising agent being added or the other means being applied during cultivation and/or during the subsequent treatment and/ or to the product after the subsequent treatment, the oxidising agent being added in an amount between 3 and 300 m moles per litre of a solids suspension containing the micro-organisms when it is added during cultivation or during subsequent treatment, or, when added to the product, being added in an amount between 3 and 300 m moles per litre of the product moisture content. 2. A process for the production of single cell protein which comprises cultivating prokaryotic micro-organisms and subsequently treating the cultivated micro-organisms to produce a product comprising whole non-viable cells and/or ruptured cells wherin the micro-organisms are treated with an oxidising agent, the oxidising agent being added during cultivation and/or during the subsequent treatment and/or to the product after the subsequent treatment, the oxidising agent being added in an amount between 3 and 300 m moles per litre of a solids suspension containing the micro-organisms when it is added during cultivation or during subsequent treatment, or, when added to the product, being added in an amount between 3 and 300 m moles per litre of the product moisture content. 3. A process according to claim 1 or claim 2 wherein the prokaryotic micro-organism is a bacterium. 4. A process according to claim 3 wherein the bacterium is a strain of the species Methylophilus methylotronhus. 5. A process according to claim 4 wherein the bacterium is any one of strains NCIB Nos. 10508 to 10515 or 10592 to 10596. 6. A process according to any one of the preceding claims wherein, during production of the single cell protein, a concentrated suspension of micro-organism cells is prepared and is subjected to drying means, the oxidising agent being added to the concentrated suspension before said suspension is subjected to drying means. 7. A process according to any one of claims 1 to 5 wherein the oxidising agent is added during subsequent treatment. 8. A process according to any one of the preceding claims wherein the micro-organisms are treated with the oxidising agent at a temperature in the range 100 to 1400C. 9. A process according to claim 8 wherein treatment takes place at a temperature in the range 500 to 8000. 10. A process according to any one of the preceding claims wherein the micro-organisms are treated with the oxidising agent at a pH in the range 7.0 to 9.0. 11. A process according to claim 10 wherein the pH is in the range 7.5 to 8.5. 12. A process according to any one of the preceding claims wherein the oxidising agent is added in an amount in the range 30 to 175 m moles per litre of the solids suspension or per litre of the product moisture content. 13. A process according to claim 12 wherein the oxidising agent is added in an amount in the range 70 to 150 m moles per litre. 14. A process according to any one of the preceding claims wherein the duration of the treatment with the oxidising agent is in the range 1 to 300 minutes. 15. A process according to claim 14 wherein the duration of the treatment is in the range 10 to 40 minutes. 16. A process according to any one of the preceding claims wherein the oxidising agent is an organic peroxide, an inorganic peroxide, ozone or permanganate. 17. A process according to claim 16 wherein the oxidising agent is hydrogen peroxide. 18. A process for the production of single cell protein wherein micro-organisms are treated with hydrogen peroxide substantially as described herein and as shown in either of the Examples. 19. Single cell protein whenever produced by a process according to any one of the preceding claims. 20. A feedstuff containing single cell protein as claimed in claim 19.

Inventors: SENIOR, PETER JAMES, TALBOT, COLIN JOHN
Applicants: IMPERIAL CHEMICAL INDUSTRIES PLC
Application year: 1980
Main classification: C12N1
IPC codes: C12N1, A23K1, A23J1, C12R1