Source: http://www.patentgenius.com/patent/4466988.html
Timestamp: 2019-01-23 11:17:00
Document Index: 249415015

Matched Legal Cases: ['application No. 8977', 'application No. 8978', 'application No. 30584', 'application No. 8977', 'application No. 23452', 'application No. 23452', 'Application No. 23452', 'Application No. 30584']

Edible protein containing substances - Patent # 4466988 - PatentGenius
Edible protein containing substances
4466988 Edible protein containing substances
Inventor: Towersey, et al.
Application: 05/809,018
Inventors: Cockram; Geoffrey N. (Exeter, GB2)
Longton; John (Berkhamsted, GB2)
Towersey; Peter J. (Wycombe, GB2)
Assignee: Ranks Hovis McDougall Limited (London, GB2)
U.S. Class: 426/656; 530/371; 530/821
Field Of Search: 195/1; 195/2; 195/28R; 195/28N; 195/98; 195/104; 260/112R; 426/62; 426/656
U.S Patent Documents: 3139385; 3243354; 3634194; 3686144; 3775393; 3809776; 4061781
Abstract: A fermentation product comprising a nonviable edible proteinaceous mass derived from a non-toxic fungal mycelium of a non-toxic strain of Fusarium preferably selected from the group consisting of Fusarium graminearum, Fusarium solani and Fusarium oxysporum possessing a reduced level of RNA of below 4%.
1. An edible protein-containing substance comprising a non-viable edible non-toxic fungal mycelium of a non-toxic strain of Fusarium possessing a reduced level of RNA of below 2%, andbeing further characterized by improved ease of processing to a form suitable for food use and an essentially white color such to make the protein-containing substance compatible with food use and a filamentous structure.
2. An edible protein-containing substance comprising a non-viable edible non-toxic fungal mycelium of a non-toxic strain of Fusarium selected from the group consiting of Fusarium graminearum, Fusarium solani, and Fusarium oxysporum possessing areduced level of RNA of below 1.5%, and being further characterized by improved ease of processing to a form suitable for food use and an essentially white color such to make the protein-containing substance compatible with food use and a filamentousstructure.
3. An edible protein-containing substance comprising a non-viable edible non-toxic fungal mycelium of a non-toxic strain of Fusarium graminearum Schwabe deposited with the Commonwealth Mycological Institute and assigned the number IMI 145425(A.T.C.C. No. 20334) possessing a reduced level of RNA of below 1.29%, and being further characterized by improved ease of processing to a form suitable for food use and an essentially white color such to make the protein-containing substance compatiblewith food use and a filamentous structure.
4. An edible protein-containing substance comprising a non-viable edible non-toxic fungal mycelium of a non-toxic strain of Fusarium, said product having a filamentous structure and possessing a reduced level of RNA of between approximately 0.8%and approximately 0.43%, and being further characterized by improved ease of processing to a form suitable for food use and an essentially white color such to make the protein-containing substance compatible with food use.
5. An edible protein-containing substance in the form of a cohesive sheet comprising a non-viable edible non-toxic fungal mycelium of a non-toxic strain of Fusarium, said product having a high net protein utilization value of the order of 41 orabove based on total nitrogen and possessing a reduced level of RNA of between approximately 0.67% and approximately 0.43%, and being further characterized by improved ease of processing to a form suitable for food use and an essentially white color suchto make the protein-containing substance compatible with food use and a filamentous structure.
6. An edible protein-containing substance comprising a non-viable edible non-toxic fungal mycelium of a non-toxic strain of Fusarium graminearum Schwabe I.M.I. 145425 (A.T.C.C. No. 20334), said product having a high net protein utilizationvalue of the order of 52 or above based on total nitrogen and possessing a reduced level of RNA of approximately 0.43%, and being further characterized by a white color such to make the protein-containing substance compatible with food use and afilamentous structure, and being capable of processing by vacuum filtration in the form of a washed moist product suitable for food use.
Description: This invention is for improvements in or relating to theproduction of edible protein containing substances.
It has particular reference to a process for reducing the nucleic acid content of microfungi.
Our Specification No. 1,210,356 describes and claims a process for the production of an edible protein-containing substance which comprises incubating and proliferating, under aerobic conditions, an organism which is a non-toxic strain of amicrofungus of the class Fungi Imperfecti, in a culture medium containing essential growth-promoting nutrient substances, of which carbon in the form of assimilable carbohydrate constitutes the limiting substrate in proliferation, and separating from theassimilable carbohydrate exhausted medium the proliferated organism which constitutes the edible protein-containing substance.
Our application No. 8977/70 (Ser. No. 1,331,471) describes and claims a process for the production of an edible protein-containing substance which comprises incubating and proliferating, under aerobic conditions, a non-toxic strain ofPenicillium notatum or Penicillium chyrsogenum or a variant or mutant thereof, in a culture medium containing essential growth-promoting nutrient substances, of which carbon in the form of assimilable carbohydrate constitutes the limiting substrate inproliferation, and separating from the assimilable carbohydrate exhausted medium the proliferated organism which constitutes the edible protein-containing substance.
Our application No. 8978/70 (Ser. No. 1,331,472) describes and claims our specific novel strain of Penicillium notatum-chrysogenum IMI 138291 and variants and mutants thereof.
Our application No. 30584/70 and cognate No. 10466/71 (Ser. No. 1,346,062) describes and claims a process for the production of an edible protein-containing substance which comprises incubating and proliferating, under aerobic conditions, anon-toxic strain of the genus Fusarium or a variant or mutant thereof, in a culture medium containing essential growth-promoting nutrient substances, of which carbon in the form of assimilable carbohydrate constitutes the limiting substrate inproliferation, and separating the proliferated organism comprising the edible protein-containing substance. The corresponding U.S. Patent is U.S. Pat. No. 3,937,654.
Our British specification contains the following disclosure:
The present invention relates to a process for the production of edible protein-containing substances and has particular reference to the production of fungal protein by microbial action.
Our Specification No. 1210356 relates to a process for the production of an edible protein-containing substance which comprises incubating and proliferating, under aerobic conditions, an organism which is a non-toxic strain of a microfungus ofthe class Fungi Imperfecti, in a culture medium containing essential growth-promoting nutrient substances, of which carbon in the form of assimilable carbohydrate constitutes the limiting substrate in proliferation, and separating from an assimilablecarbohydrate exhausted medium the proliferated organism which constitutes the edible protein-containing substance.
Our copending application No. 8977/70, Ser. No. 1,331,471, claims an edible protein-containing substance comprising fungal mycelium possessing a high net protein utilisation value on rat assays of at least 70 based on the .alpha.-amino nitrogen.
It is also an object of the present invention to provide an edible protein-containing substance comprising non-toxic fungal mycelium possessing a high net protein utilisation value on rat assays of at least 65 preferably at least 70 based on the.alpha.-amino nitrogen and containing a non-toxic strain of the genus Fusarium or a variant or mutant thereof. The non-toxic mycelium possessing a high net protein utilisation value of at least 70 based on the .alpha.-amino nitrogen may contain anon-toxic strain of the species Fusarium graminearum.
According to the present invention there is provided a process for the production of an edible protein-containing substance which comprises incubating and proliferating, under aerobic conditions, a non-toxic strain of the genus Fusarium or avariant or mutant thereof, in a culture medium containing essential growth-promoting nutrient substances, of which carbon in the form of assimilable carbohydrate constitutes the limiting substrate in proliferation, and separating the proliferatedorganism comprising the edible protein-containing substance.
The separated proliferated organism comprising the edible protein-containing substance may be incorporated into a foodstuff for human or animal consumption.
The substrate employed in the incubation stage may be of vegetable origin, for example starch, starch containing materials or products of their hydrolysis, sucrose, sucrose containing materials or hydrolysed sucrose i.e. invert sugar or mixturesthereof. Thus the substrate may comprise hydrolysed potato, molasses, glucose, maltose, hydrolysed bean starch or cassava. Alternatively substrate of animal origin comprising whey may be employed.
The non-toxic strain of Fusarium may be a strain of Fusarium graminearum.
The preferred non-toxic strain is our strain of Fusarium graminearum Schwabe, which is described and claimed together with variants and mutants thereof in copending United Kingdom application No. 23452/70 (Ser. No. 1346061), has been depositedat the Commonwealth Mycological Institute, Kew, and assigned the number IMI 145425. It is non-pathogenic to wheat.
Our copending United Kingdom application No. 23452/70 (Ser. No. 1346061) also describes and claims specifically five variants of our strain of Fusarium graminearum Schwabe IMI 145425 namely I-7, I-8, I-9, I-15 and I-16 deposited with theCommonwealth Mycological Institute and assigned the numbers IMI 154209, IMI 154211, IMI 154212, IMI 154213 and IMI 154210 respectively.
The temperature of incubation is in general between 25.degree. and 34.degree. C., preferably around 30.degree. C.
Inoculation resulting in commencement of the process is carried out by a pregerminated seed stage comprising for example from 2% to 10% of inoculum, usually in the range 5% to 10% of inoculum based on final fermented operating volume.
The pH of the substrate medium during incubation is preferably kept within a suitable range supporting maximum growth, for example, between 3.5 to 7.
The period of growth in batch culture under the above-mentioned conditions is usually found to range from 20 to 48 hours. In both batch and continuous processes aeration and agitation should be carried out to provide a sufficient level ofdissolved oxygen to overcome deficiency which can be a limiting growth factor.
As will be well understood by those skilled in the art sufficient quantities of essential growth nutrients such as nitrogen, sulphur, phosphorus and other trace elements are maintained in the substrate medium so that growth of the substrate islimited only by the carbohydrate available to the fungus.
In addition to the nutrients stated above the presence of one or more vitamins such for example as biotin may be desirable to maintain maximum growth rate.
It is also desirable to add a non-toxic anti-foaming agent to the substrate medium to control foaming during the fermentation.
The substance produced according to the present invention may be isolated in any suitable manner known in the art. Thus the resulting mycelium may be recovered by separation, washing, filtration and drying. In this connection, however, it hasbeen found that if the moisture content of the substance is reduced below a critical level of about 50% (w/w) by filtration under pressure the subsequent drying methods employed are not subjected to such stringent temperature limitations which is animportant factor in the economic processing of these materials. The method of drying must not cause damage to the nutritional value of the mycelium and may be drying in a current of air at 75.degree. C. or freeze drying.
The fungal mycelium produced by the process of the present invention shows very good water binding capacity and may be useful as a thickening and gelling agent. Not being an isolate, it retains its vitamins as well as other nutritionallyavailable materials such as lipids and some carbohydrates. Fungal mycelium has satisfactory baking characteristics which are of value in protein enriched breads, breakfast foods and food snacks. The fungal mycelium, because of its filamentousstructure, can be baked, fried or puffed and presented to many communities as a food comparable in appearance and acceptability with conventional foods which they are accustomed to eating.
Following is a description by way of example of methods of carrying the invention into effect.
Culture medium or medium percentages given are as weight per unit volume (w/v) or volume per unit volume (v/v) for solids and liquids respectively
NPU=net protein utilisation
NPUop=net protein utilisation: operative
.mu.: a specific growth rate which is the rate of increase/unit of organism concentration ##EQU1## .mu..sub.max. is the growth rate constant (the maximum value of .mu. at saturation levels of substrate).
Yield Factor: weight of organism formed/weight of substrate used.
Examples 1-4 are of batch culture.
10 Liters of the following culture medium were prepared and sterilised as described in a stirred fermenter vessel.
______________________________________ Cane molasses to provide 6% w/v sugar Ammonium sulphate 1.2% NaH.sub.2 PO.sub.4 0.25% Sterilised 30 minutes 15 psig CaCO.sub.3 0.5% w/v Sterilised 3 hours 15 psig ______________________________________
The medium components were added aseptically and attemperated to 30.degree. C. An inoculum equivalent to 5-10% by volume of the culture medium and grown either on a glucose/corn steep liquor medium or other suitable materials in shake flasks wasinoculated with a spore suspension of the organism comprising our strain of Fusarium graminearum Schwabe IMI 145425, for 18-24 hours at 30.degree. C. on a rotary shaker, and added aseptically to the fermenter.
The fermenter incubated at 30.degree. C. was then stirred at 800 rpm with a 6 bladed disc turbine (0.5D) in a full baffled vessel and 1 VVM of sterile air passed through. After 35 hours, the grown mycelium was removed from the fermenter,centrifuged, washed with water and dried in a warm air band drier, air temperature 75.degree. C.
______________________________________ Total Nitrogen 8.0% Ash 5.3% Lipid 2.7% NPUop. 52 based on Total Nitrogen ______________________________________
10 Liters of the following culture medium were prepared and sterilised as described, in a 14 liter New Brunswick, Microferm, fermenter.
______________________________________ Final % ______________________________________ Solution 1 Glucose pH 3.0 3.0 Solution 2 Ammonium sulphate 0.7 Solution 3 Potassium di-hydrogen pH 5.0 1.0 phosphate Solution 4 FeSO.sub.4 7H.sub.2 O pH 2.5 0.001 MnSO.sub.4 4H.sub.2 O 0.0005 CuSO.sub.4 5H.sub.2 O 0.0001 MgSO.sub.4 7H.sub.2 O 0.025 Solution 5 Na.sub.2 MoO.sub.4 2H.sub.2 O 0.0001 CoCl.sub.2 6H.sub.2 O 0.0001 CaCl.sub.2 2H.sub.2 O 0.0015 Solution 6 NaOH 0.1 ______________________________________
All the above solutions were sterilised by heat for 15 minutes at 15 psig. Solution 7 Vitamins and/or amino acids as described below sterilised by filtration.
The solutions were added aseptically to the vessel.
An inoculum was grown and added as in Example 1 except that the final concentration in the fermenter was adjusted so as to provide 0.5 gm/l dry wt. of mycelium.
The conditions of growth were temperature 30.degree. C.; aeration 1 VVM, stirrer speed was adjusted to maintain a level of dissolved oxygen above 25% of the saturation value in the culture medium, measured by a New Brunswick Inc. DO probe. Sterile anti-foam, polypropylene glycol 2000, was added as required to suppress foam and pH was maintained between 6.0-6.3 by the addition of sterile potassium hydroxide solution.
______________________________________ Growth rates (hr..sup.-1) ______________________________________ (i) Omitting solution 7 (Minimal medium) very slow (ii) Solution 7 such that the final 0.2 concentration of Biotin in the culturemedium was 50 .mu.g/l (iii) Solution 7 such that the final 0.25 concentration of Biotin in the culture medium was 50 .mu.g/l; Choline chloride 30 mg/l and Methionic 300 mg/l ______________________________________
Medium and conditions were as in Example 2, but the glucose was replaced with maltose.
______________________________________ (i) Solution 7 as Example 2 (ii) 0.18 (ii) Solution 7 as Example 2 (iii) 0.21 ______________________________________
100 Liters of the following culture medium were prepared and sterilised as described in a 130 l stainless steel fermenter.
______________________________________ % final concentration ______________________________________ Glucose 4.0 Corn steep liquor (50% Total 0.8 Solids) Ammonium sulphate 0.2 Potassium di-hydrogen phosphate 0.2 MgSO.sub. 4 7H.sub.2 O 0.025 ZnSO.sub. 4 7H.sub.2 O 0.0005 FeSO.sub. 4 7H.sub.2 O 0.0005 MnSO.sub. 4 4H.sub.2 O 0.0001 ______________________________________
The medium was sterilised at pH 3.0 at 15 psig for 30 minutes and on cooling to 30.degree. C. adjusted to pH 5.0 by the sterile addition of ammonia.
Biotin sterilised by filtration to give 40 .mu.g/l final concentration, was added aseptically.
The vessel was inoculated with 10 liters of culture grown in a sparged vessel, for 18 hours, at 30.degree. C., on a medium containing: Glucose 2%; tryptone ("Oxoid") 0.4%; yeast extract ("Oxoid") 0.1%; ammonium sulphate 0.15%; potassiumdi-hydrogen phosphate 1%; sodium hydroxide 0.1%; magnesium sulphate 0.025%; ferrous sulphate 0.001%; zinc sulphate 0.001%; manganese sulphate 0.0005%; copper sulphate 0.001%; anti-foam, polypropylene glycol 2000 0.05% and sterilised for 45 minutes at 15psig, inoculated with a spore suspension of our organism Fusarium graminearum Schwabe IMI 145425. The word "Oxoid" is a Registered Trade Mark.
The conditions for growth were temperature 30.degree. C., aeration adjusted to provide dissolved oxygen concentrations above 10% of the saturation value for the culture broth. Sterile anti-foam, polypropylene glycol 2000, was added to suppressfoaming, and the pH was maintained at 5.0 by means of sterile ammonia additions. Samples of the mycelium were taken at intervals over the period of batch culture, filtered, washed with water and dried. On dry weight basis analysis gave Total nitrogen8.0-8.6%; .alpha.-Amino nitrogen 6.4-6.6%. The initial growth rate in this complex medium derived from both the batched culture medium and inoculum was approximately 0.3 hr..sup.-1.
The following examples 5 and 6 are of continuous culture.
Culture medium of the following composition was prepared:
______________________________________ Final % ______________________________________ Solution 1 Glucose 3.0 Ammonium sulphate 0.25 Potassium di-hydrogen phosphate 0.3 Magnesium sulphate 0.025 Anti-foam, polypropylene 0.01 glycol 2000 Sterilised at pH 3.0 for 30 minutes at 15 psig Solution 2 MnSO.sub.4 4H.sub.2 O 0.0005 FeSO.sub.4 7H.sub.2 O 0.0005 ZnSO.sub.4 7H.sub.2 O 0.0005 CoCl.sub.2 6H.sub.2 O 0.0001 CuSO.sub.4 5H.sub.2 O 0.0001 NaMoO.sub.4 2H.sub.2 O 0.0001 Sterilised15 minutes at 15 psig Solution 3 Vitamins and or amino acid as described below sterilised by filtration. ______________________________________
All solutions were added as necessary, aseptically. In 8.5 liter chemostat the conditions of growth were as follows:
Temperature 30.degree. C.; aeration 1 VVM; agitation 800 rpm single 6 bladed disc turbine 0.5 D in fully baffled vessel. Organism, our strain of Fusarium graminearum Schwabe IMI 145425. The pH maintained at 5.0 by automatic addition of sterileammonia.
Samples were taken, filtered, washed with water and dried.
__________________________________________________________________________ .mu. Max. Yield Mycelium NPU based NPU based hr..sup.-1 factor TN % AN % on TN on AN __________________________________________________________________________ (i) Solution 3 such that the final 0.17-0.19 0.5 7.2 to 7.9 6.3 to 6.8 54 65 concentrate of Biotin in the culture medium was 20 .mu.g/l (ii) Solution 3, such that the 0.20-0.21 0.5 7.7 to 8.6 6.1 to 6.5 59 78 final concentration of Biotin inthe culture medium was 20 .mu.g/l and of methionine was 600 mg/l __________________________________________________________________________
______________________________________ % ______________________________________ Bean starch (.alpha.-amylase treated) 3.0 carbohydrate Corn steep liquor 1.33 Ammonium sulphate 0.25 Potassium di-hydrogen phosphate 0.15 Magnesium sulphate0.025 Antifoam polypropylene 0.025 glycol 2000 (v/w) Sterilised pH 4.0 for 30 minutes at 15 p.s.i.g. ______________________________________
The medium was fed to the 8.5 liter chemostat under the same conditions as in Example 5 except that the pH was varied between 3.5 and 6.0 and growth rate throughout 0.1 hr.sup.-1. Samples were taken, filtered, washed with water and dried. Thefollowing result was obtained:
______________________________________ TN AN NPU based NPU based % % on TN on AN ______________________________________ Product grown at pH 4.0 7.8 6.6 54 67 Product grown at pH 5.0 8.6 7.1 57 71 Product grown at pH 6.0 7.7 5.9 61 80 ______________________________________
The culture medium and conditions were as in Example 6 except that the pH was held at 5.0 throughout the run and the temperature was varied between 26.degree. and 34.degree. C. The optimum temperature was found to be 30.degree.-32.degree. C.
Examples 7 to 11 describe the fermentation of five variants or isolates of Fusarium graminearum Schwabe IMI 145425.
Duplicate shake flasks of 1-liter capacity were prepared containing 200 mls. of the following medium:
______________________________________ Final con- centration % ______________________________________ Solution 1 Glucose (sterilised 3.0 separately pH 3.0, 10 p.s.i./10 min.) Solution 2 Ammonium sulphate 0.565 Potassium Dihydrogen 1.0 Phosphate MgSO.sub.4 :7H.sub.2 O 0.025 FeSO.sub.4 :(NH.sub.2).sub.2 SO.sub.4 :6H.sub.2 O 0.0005 MnSO.sub.4 :4H.sub.2 O 0.0005 CuSO.sub.4 :5H.sub.2 O 0.0001 CoCl.sub.2 :6H.sub.2 O 0.0001 CaCl.sub.2 :2H.sub.2 O 0.0015 Na.sub.2 MoO.sub.42H.sub.2 O 0.00001 NaOH 0.2 Salts sterilised at 15 p.s.i.g./15 min. Final pH 6.0 Solution 3 Vitamins as described below were sterilised by filtration ______________________________________
The solutions were added aseptically to give a final volume of 200 ml. then the flasks were inoculated with washed spores of our strain of Fusarium graminearum I-7 IMI 154209 to give a concentration of 8.times.10.sup.3 /ml.
The conditions of growth were temperature 30.degree. C., 140 r.p.m. on orbital shaker with 2" throw.
At hourly intervals the growth was measured by measuring the Optical Density of a sample at 600 m.mu.. From the results obtained the following growth rates were established.
______________________________________ Growth rate h.sup.-1 ______________________________________ (i) Solution 3 omitted (minimal very slow medium) (ii) Solution 3 such that the final 0.22 concentration of Biotin in the culture medium was50.mu.g/l (iii) Solution 3 such that the final 0.27 concentration of Biotin in the culture medium was 50.mu.g/l and Choline chloride 50 mg/l. ______________________________________
The procedure of Example 7 was repeated but the strain I-7 was replaced by our strain of Fusarium graminearum Schwabe I-8. The following growth rates were established
______________________________________ Growth rate h.sup.-1 ______________________________________ (i) As 7 (i) very slow (ii) As 7 (ii) 0.22 (iii) As 7 (iii) 0.27 ______________________________________
The procedure of Example 7 was repeated but the strain I-7 was replaced by our strain of Fusarium graminearum Schwabe I-9. The following growth rates were established:
______________________________________ Growth rate h.sup.-1 ______________________________________ (i) As 7 (i) very slow (ii) As 7 (ii) 0.21 (iii) As 7 (iii) 0.27 ______________________________________
The procedure of Example 7 was repeated but the strain I-7 was replaced by our strain of Fusarium graminearum Schwabe I-15. The following growth rates were established:
The procedure of Example 7 was repeated but the strain I-7 was replaced by our strain of Fusarium graminearum Schwabe I-16. The following growth rates were established:
The procedure of Example 7 was repeated but the strain I-7 was replaced by the parent strain Fusarium graminearum Schwabe IMI 145425. The following growth rates were established:
Examples 13 and 14 describe fermentation using strains of Fusaria other than Fusarium graminearum.
A spore suspension of Fusarium solani strain A7-16 (IMI 154217) was inoculated into a seed fermenter of 80 liter volume containing a glucose, corn steep liquor medium. After growing up the seed fermenter to 10-20 gms per liter, it was split intwo, 40 liters to each 400 liter vessel. The seed was inoculated into a medium of the following composition:
______________________________________ % ______________________________________ Starch 6.0 KH.sub.2 PO.sub.4 0.20 (NH.sub.4).sub.2 SO.sub.4 0.25 Corn Steep Liquor 0.50 (50% Total Solids) ______________________________________
pH was 5.5 maintained by addition of sterile ammonia; Temperature 30.degree. C. Pressure 30 p.s.i.g. Air rate 1.0 v.v.m. The revolutions of the stirrer were increased steadily from 92 to 184 r.p.m. to maintain dissolved oxygen in the vessel. The agitator consisted of two turbines 0.4D. When the carbohydrate had been utilised the grown mycelium was removed from the fermenter, filtered, washed with water, centrifuged, and dried in a warm air band drier at 75.degree. C. The dried product hadthe following composition:
______________________________________ Total nitrogen 9.1% Ash 8.3% ______________________________________
When fed to rats this material gave an NPUop of 41 based on Total Nitrogen.
Fusarium oxysporum strain A9-23 (IMI 154214) was grown exactly as in the previous example except the starch was replaced by cane molasses at a concentration that produced 6.0% sugars.
______________________________________ Total Nitrogen 9.9% Ash 10.8% NPUop 47.0 based on Total Nitrogen ______________________________________
Methods of analysis for Total Nitrogen (TN) Automatic Kjeldahl digestor (Technicon). A Ferrari, Ann. N.Y. Sci. 87, 792 (1960).
Amino nitrogen (AN) 2:4:6-Tri-nitro benzene sulphuric acid (modified). M. A. Pinnegar, Technicon Symposium 1965, p. 80.
Our Application No. 23452/70 (Ser. No. 1,346,061) describes and claims our specific novel strain of Fusarium graminearum Schwabe IMI 145425 and variants and mutants thereof.
The separated proliferated organism comprising the edible protein-containing substance obtained by the fermentation processes of our Applications Nos. 8977/70 (Ser. No. 1,331,471) and 30584/70 and Cognate No. 10466/71 (Ser. No. 1,346,062) maybe incorporated into a foodstuff for human or animal consumption.
The processes of our Applications Nos. 8977/70 (Ser. No. 1,331,471) and 30584/70 and Cognate No. 10466/71 (Ser. No. 1,346,062) are capable of producing an edible protein-containing substance comprising fungal mycelium which possesses a highnet protein utilisation value on rat assays of at least 70 based on the .alpha.-amino nitrogen.
If single-cell protein is to be used as a primary protein source for human consumption the Protein Advisory Group of the Food and Agricultural Organization (FAO) World Health Organisation (who) has advised that the nucleic acid content should bereduced to a level which would allow a maximum intake in the range of 2 grams of nucleic acid per day.
For a processing method to be acceptable, it must not only decrease the nucleic acid level to the required degree, but it also must be inexpensive and must not contaminate the food product with undesirable chemicals.
It is an object of the present invention to provide a process for the reduction of levels of nucleic acid in particular ribonucleic acid (RNA) in proliferated microorganisms combined with the minimum loss of protein to render them more acceptableas human food.
We have developed a process for treating cells of grown non-toxic microfungus of the class Fungi Imperfecti which can meet the above requirements of the World Health Organisation.
The invention provides fungal mycelium possessing a reduced level of RNA of below 4%.
Thus the invention provides fungal mycelium containing Fusarium graminearum Schwabe IMI 145425 possessing a reduced level of RNA of below 3% by weight, preferably below 2% by weight.
The invention also provides fungal mycelium containing Penicillium notatum-chrysogenum IMI 138291 possessing a reduced level of RNA of below 4%.
According to the present invention there is provided a process for reducing the nucleic acid content in the production of an edible protein-containing substance comprising contacting a grown non-toxic microfungus of the class Fungi Imperfectiwith a solvent comprising between 40% and 100% (by volume) of a lower alkanol containing up to three carbon atoms and thereafter incubating at a pH between 5 and 9.5 and at a temperature between 30.degree. C. and 80.degree. C. for a time of at least 90seconds.
The process may be applied to a grown non-toxic strain of Fusarium, Penicillium notatum or Penicillium chrysogenum, Penicillium funiculosum or Aspergillus niger.
The strain of Fusarium may be a strain of Fusarium graminearum Schwabe in particular IMI 145425, Fusarium oxysporum or Fusarium solani as described and claimed in our Applications Nos. 23452/70 (Ser. No. 1,346,061) and 30584/70 and Cognate No.10466/71 (Ser. No. 1,346,062).
The strain of Penicillium notatum or Pencillium chrysogenum may be a strain of Penicillium notatumchrysogenum, for example IMI 138291, as described and claimed in our Applications Nos. 8977/70 (Ser. No. 1,331,471) and 8978/70 (Ser. No.1,331,472).
The lower alkanol containing up to three carbon atoms may be methyl alcohol, ethyl alcohol, propyl alcohol or isopropyl alcohol. Ethyl alcohol and isopropyl alcohol are solvents permitted by the Solvents in Food Regulations, 1967. The preferredsolvent in the process of the present invention is isopropyl alcohol (IPA). Instead of pure isopropyl alcohol aqueous solutions containing between 40 or 50% by volume and up to 100% I.P.A. may be employed.
The incubation may conveniently be carried out at a temperature between 45.degree. C. and 60.degree. C. for a time of between 1.5 minutes and 40 minutes.
The incubation step may conveniently be carried out in the presence of a buffer solution for example NH.sub.4 Cl/NH.sub.4 OH or NH.sub.4 Cl/HCl.
The post fermentation process of the present invention for reducing the nucleic acid content of microorganisms is essentially a two stage process.
The grown microbial protein or fungal mycelium obtained for example by the fermentation process described and claimed in our Applications Nos. 8977/70 (Ser. No. 1,331,471) and 30584/70 and Cognate No. 10466/71 (Ser. No. 1,346,062) may beharvested, filtered to remove growth medium and washed, if desired. It may then be suspended in the alkanol solvent for example for 1 minute at 20.degree. C. or contacted with an alkanol solvent water mixture. The majority or all of the alkanolsolvent may be removed by such methods as vacuum filtration, filter pressing or centrifugation. The duration of contact with the alkanol solvent may be varied but is generally in the range between 15 seconds and 15 minutes. The temperature may varybetween 0.degree. C. and 60.degree. C.
The cells may then be brought into intimate contact with aqueous buffer solutions in the pH range 5 to 9.5. Thus the solvent treated cells may then be resuspended and incubated in aqueous buffer solution at pH 8.6 and temperature 45.degree. C.An example of a suitable buffer solution is 0.1M ammonium chloride solution with ammonium hydroxide added to adjust the pH to 8.6.
The resulting treated cells may then be harvested again for example by filtration and washing with water and thereafter formulated into foods or dried by various methods.
When the process is carried out in the pilotplant the pH is adjusted to 5 after RNA removal. The purpose of this acidification is twofold (a) the material becomes "whiter" and (b) the texture changes and this enables harvesting by vacuumfiltration to be carried out easier.
The resulting solvent treated microbial protein or fungal mycelium may have a RNA content of 1-4% compared to 7 to 10% of the untreated proliferated organism.
The cells may be analysed to determine their chemical composition and to evaluate the efficiency of the nucleic acid reduction process.
References to "Biomass Loss" denote weight lost during processing.
Ribonucleic acid (RNA) content was determined by a modification of the method of Schmidt G. and Thannhauser, S. J., J. Biol.Chem., 1945, 161, 83.
Method of analysis for Total Nitrogen (TN) Automatic Kjeldahl digestor (Technicon). A Ferrari, Ann. N.Y. Sci. 87, 792 (1960).
Amino nitrogen (AN) TNBS (modified). M. A. Pinnegar, Technicon Symposium 1965, p.80.
Reduction of the Nucleic Acid Levels in Various Micro-Organisms
Fusarium graminearum IMI 145425 was cultivated by the following procedure:
Medium in distilled water:
______________________________________ K.sub.2 HPO.sub.4 15.05 gL.sup.-1 (NH.sub.4).sub.2 HPO.sub.4 6.64 gL.sup.-1 tri Sodium Citrate 15.7 gL.sup.-1 Citric Acid 5.48 gL.sup.-1 K.sub.2 SO.sub.4 1.0 gL.sup.-1 Choline chloride 50 mgL.sup.-1 Biotin 50 .mu.gL.sup.-1 Glucose 30 gL.sup.-1 ______________________________________
______________________________________ MgCl.sub.2.6H.sub.2 O 0.2 gL.sup.-1 ZnSO.sub.4 0.003 gL.sup.-1 MnCl.sub.2 4H.sub.2 O 0.005 gL.sup.-1 FeCl.sub.3.6H.sub.2 O 0.01 gL.sup.-1 CuCl.sub.2.6H.sub.2 O 0.001 gL.sup.-1 NaMoO.sub.4.2H.sub.2 O 0.001 gL.sup.-1 CoCl.sub.2.6H.sub.2 O 0.001 gL.sup.-1 CaCl.sub.2.2H.sub.2 O 0.015 gL.sup.-1 ______________________________________
All components with the exception of glucose are sterilised together, and the amounts of these materials required for 1 liter of medium are dissolved, made up to 850 ml. and distributed into 5 1 liter conical flasks, each containing 170 ml. A30% w/v solution of glucose is prepared and sterilised in 20 ml. portions in universal bottles. Sterilisation is effected in an autoclave at 15 p.s.i. for 15 minutes.
Before inoculation with 10 ml. of a growing culture, the contents of one bottle of sterile glucose solution is added to each flask. Culture of A3/5 then proceeds on an Orbital Shaker, with 2 inch throw, at 160 r.p.m. and a temperature of30.degree. C. The culture is harvested after 18 hours.
Cells were collected and washed on a Buchner filtration system and treated as follows:
(i) Suspended in 66% v/v isopropyl alcohol for 1 minute at 20.degree. C.
(ii) Isopropyl alcohol was removed by filtration.
(iii) The treated cells were incubated in 0.1M NH.sub.4 Cl/NH.sub.4 OH buffer at pH 8.6 and 45.degree. C. for various times. The incubations were carried out at a slurry concentration of approximately 10 g/l with stirring.
______________________________________ Time of Incuba- Micro- tion % RNA % Amino % Total fungi Treatment Minutes Content Nitrogen Nitrogen ______________________________________ F. None None 10.86 7.57 9.80 gramine- arum Nucleic Zero9.86 8.23 10.98 acid Reduction Nucleic 20 2.29 8.84 10.45 acid Reduction Nucleic 40 1.88 8.68 9.91 acid Reduction Nucleic 60 1.69 8.73 10.56 acid Reduction ______________________________________
The level of nucleic acid was effectively reduced by the treatment described.
Penicillium notatum chrysogenum IMI 138291 was cultivated by the following procedure:
______________________________________ 2% Soluble starch 0.2% Spray dried corn steep liquor 0.2% Mycological peptone 0.4% (NH.sub.4).sub.2 SO.sub.4 0.2% KH.sub.2 PO.sub.4 1% Sucrose ______________________________________
The medium is made up with hot tap water, and dispensed in 200 ml. aliquots into conical shake flasks.
0.1 ml. of liquid amylase was added to each shake flask and incubated at 70.degree. C. for 15 minutes so that the starch was broken down and the viscosity reduced.
The flasks were sterilised in an autoclave at 15 p.s.i. for 20 minutes.
A spore inoculum was added to each flask and the culture grown at 30.degree. C. on an orbital shaker with a 2 inch throw at 160 r.p.m. After growth for 24 hours, 10 ml. of the growing culture was used as growing inoculum which was added tomore flasks containing the starch medium. Cells produced after a further 24 hours growth were harvested, washed and used as follows:
(i) Suspended in 66% (v/v) isopropyl alcohol for one minute at 20.degree. C.
(iii) The treated cells were incubated in 0.1M NH.sub.4 Cl/NH.sub.4 OH buffer at pH 8.6 and 40.degree. C. for various times. The incubations were carried out at a slurry concentration of approximately 10 gm/l with stirring.
__________________________________________________________________________ Time of Micro- Incubation % RNA % Amino % Total % Biomass fungi Treatment Minutes content Nitrogen Nitrogen Loss __________________________________________________________________________ P. notatum- None None 7.19 5.78 7.58 0 chrysogenum P. notatum- Nucleic 15 3.60 6.64 8.47 30 chrysogenum Acid Reduction P. notatum- Nucleic 40 3.25 6.47 8.52 32 chrysogenum Acid Reduction P. notatum- Nucleic 60 3.32 6.34 8.04 32 chrysogenum Acid Reduction __________________________________________________________________________
The level of nucleic acid was reduced by the treatment described.
Penicillium funiculosum IMI 79195 was cultivated by the following procedure:
______________________________________ KH.sub.2 PO.sub.4 15 g/l NaOH 1 g/l Dextran 1 g/l Caster Oil 10 g/l Solution A.sup.+ 5 ml/l Solution B.sup.+ 5 ml/l Solution C.sup.+ 5 ml/l Yeast extract 10 g/l ______________________________________
______________________________________ A.sup.+ B.sup.+ C.sup.+ ______________________________________ MgSO.sub.4 50 g/l CaCl.sub.2 3 g/l FeSO.sub.4 1 g/l ZnSO.sub.4 1 g/l MnSO.sub.4 1 g/l CoCl.sub.2 0.2 g/l CuSO.sub.4 0.2 g/l ______________________________________
All in distilled water.
Adjust pH of medium to 5.5 before sterilisation. Autoclave all components together. (50 minutes 15 p.s.i.)
______________________________________ (Batch culture) Volume 101. (Fermenter) Temperature 28.degree. C. Stirrer 400 r.p.m. Air flow 101/minutes Harvest time 80 hours Inoculum size 5% by volume (shake flask culture) ______________________________________
(i) Suspended in 80% isopropyl alcohol for 1 minute at 20.degree. C.
(iii) The treated cells were incubated in 0.1M NH.sub.4 Cl/NH.sub.4 OH buffer at pH 8.6 and 37.degree. C. for 60 minutes. The incubation was carried out at a slurry concentration of approximately 10 g/l with stirring.
______________________________________ Micro- % RNA % Amine organism Treatment content N % Total N ______________________________________ P. None 4.23 3.74 5.81 funiculosum P. Nucleic 2.80 4.46 7.34 funiculosum Acid Reduction ______________________________________
Aspergillus niger NRRL 330 was cultivated by the following procedure:
The medium and sterilisation procedure were identical to that described for P. notatum-chrysogenum.
Growth conditions were also identical except that cells grown directly from spores were used instead of cells cultivated from growing inoculum.
(i) Suspended in 66% (v/v) isopropyl alcohol for 1 minute at 20.degree. C.
(iii) The treated cells were incubated in 0.1M NH.sub.4 Cl/NH.sub.4 OH buffer at pH 8.6 and 40.degree. C. for various times. The incubations were carried out at a slurry concentration of approximately 10 g/l with stirring.
__________________________________________________________________________ Time of Micro- Incubation % RNA % Amino % Total % Biomass fungi Treatment Minutes Content Nitrogen Nitrogen loss __________________________________________________________________________ A. niger None None 6.36 4.03 5.70 none A. niger Nucleic zero acid Reduction A. niger Nucleic 15 1.88 4.40 6.30 27 acid Reduction A. niger Nucleic 30 1.86 4.35 5.7728 acid Reduction A. niger Nucleic 60 1.82 4.25 5.62 32 acid Reduction __________________________________________________________________________
Effect of the % Iso-propyl Alcohol on the Efficiency of the Nucleic Acid Reduction Process
F. graminearum IMI 145425, cultivated as described in Example A, was contacted with various isopropyl alcohol/water mixtures at 20.degree. C. for 2 minutes. The treated cells were then incubated in 0.1M NH.sub.4 Cl/NH.sub.4 OH buffer pH 8.5 at37.degree. C. for 20 minutes. The incubations were carried out at a slurry concentration of approximately 10 g/l with stirring.
______________________________________ % IPA % Biomass (by volume) Treatment loss % RNA remaining ______________________________________ 0 None 0.0 9.33 0 Nucleic 1.8 9.33 acid reduction 10 Nucleic 1.1 10.68 acid reduction 20 Nucleic11.3 9.74 acid reduction 30 Nucleic 19.4 8.87 acid reduction 40 Nucleic 25.6 4.58 acid reduction 50 Nucleic 26.5 3.03 acid reduction 60 Nucleic 28.0 3.25 acid reduction 70 Nucleic 27.6 2.86 acid reduction 80 Nucleic 26.3 3.35 acid reduction 90 Nucleic 23.8 3.69 acid reduction 100 Nucleic 25.1 4.58 acid reduction ______________________________________
The nucleic acid removal process is most effective in the range of 40-100% isopropyl alcohol.
In the case of the treatment with 10 & 20% IPA the final RNA content is greater than the starting material; this is because RNA is removed to a lesser extent than biomass lost.
Effect of Contact With IPA at Various Temperatures on the Subsequent Nucleic Acid Reduction Process
F. graminearum IMI 145425, cultivated as described in Example A, was contacted with 100% IPA at 0.degree., 20.degree., 40.degree., and 60.degree. C. for 2 minutes, then incubated with 0.1M NH.sub.4 Cl/NH.sub.4 OH buffer pH 8.5 for 20 minutes at39.degree. C. The incubations were carried out at a slurry concentration of approximately 10 g/l with stirring.
______________________________________ Temperature of IPA treatment % RNA remaining ______________________________________ No treatment 9.04 0.degree. C. 3.42 20.degree. C. 3.47 40.degree. C. 2.52 60.degree. C. 2.33 ______________________________________
The nucleic acid reduction process is effective over the temperature range studied.
Effectiveness of Various Alcohols on the Nucleic Acid Reduction Process
F. graminearum IMI 145425, cultivated as described in Example A, was contacted with 100% iso-propyl alcohol, 70% iso-propyl alcohol, 70% propyl alcohol, 100% ethyl alcohol or 100% methyl alcohol at 20.degree. C. for two minutes, then incubatedwith 0.1M NH.sub.4 Cl/NH.sub.4 OH buffer pH 8.5 at 37.degree. C. or 40.degree. C. for various time periods. The incubations were carried out at a slurry concentration of approximately 10 g/l with stirring.
______________________________________ Time and temperature % RNA Alcohol used of second incubation remaining ______________________________________ None None 9.16 100% iso-propyl 30 mins. at 37.degree. C. 2.53 alcohol 100% iso-propyl 120 mins. at 37.degree. C. 0.70 alcohol iso-propyl 20 mins. at 40.degree. C. 1.81 alcohol propyl alcohol 20 mins. at 40.degree. C. 1.93 100% ethyl alcohol 30 mins. at 37.degree. C. 2.17 100% ethyl alcohol 120 mins. at 37.degree. C. 0.64 100% methyl alcohol 30 mins. at 37.degree. C. 5.50 100% methyl alcohol 120 mins. at 37.degree. C. 1.17 ______________________________________
The RNA reduction process is successfully activated by a lower alkanol containing up to three carbon atoms.
Duration of Contact With Iso-propyl Alcohol
F. graminearum IMI 145425, cultivated as described in Example A, was contacted with 66% (v/v) IPA at 20.degree. C. for various times then incubated in 0.1M NH.sub.4 Cl/NH.sub.4 OH buffer pH 8.5 at 37.degree. C. for 60 minutes. The incubationswere carried out at a slurry concentration of approximately 10 g/l with stirring.
______________________________________ Contact time % RNA % Biomass with 66% IPA remaining lost ______________________________________ 0 9.25 0 15 seconds 1.12 36 1 minute 1.14 38 2 minutes 0.98 40 5 minutes 1.23 -- 15 minutes 1.27 41 ______________________________________
Over the contact times studied nucleic acid removal was efficient. In practice the contact time for best RNA reduction is around 2 minutes, at longer contact times the % biomass lost tends to rise to unacceptably high values.
Efficiency of Nucleic Acid Reduction With Buffers Over a pH Range of 4-10
F. graminearum IMI 145425, cultivated as described in Example A, was contacted with 100% iso-propyl alcohol at 20.degree. C. and incubated with the following series of buffers at 30.degree. C. for 3 hours. The incubations were carried out at aslurry concentration of approximately 10 g/l with stirring.
______________________________________ % Buffer in second stage RNA Remaining ______________________________________ 0.1 M NH.sub.4 Cl + HCl to bring to pH 4.0 11.49 0.1 M NH.sub.4 Cl + HCl to bring to pH 4.5 9.07 0.1 M NH.sub.4 Cl + HClto bring to pH 5.0 5.85 0.1 M NH.sub.4 Cl + HCl to bring to pH 5.5 3.52 0.1 M NH.sub.4 + NH.sub.4 OH to bring to pH 6.0 2.63 0.1 M NH.sub.4 + NH.sub.4 OH to bring to pH 6.5 1.60 0.1 M NH.sub.4 + NH.sub.4 OH to bring to pH 7.0 0.96 0.1 MNH.sub.4 + NH.sub.4 OH to bring to pH 7.5 0.97 0.1 M NH.sub.4 + NH.sub.4 OH to bring to pH 8.0 0.59 0.1 M NH.sub.4 + NH.sub.4 OH to bring to pH 8.5 0.91 0.1 M NH.sub.4 + NH.sub.4 OH to bring to pH 9.0 1.69 0.1 M NH.sub.4 + NH.sub.4 OH to bring topH 9.5 3.84 0.1 M NH.sub.4 + NH.sub.4 OH to bring to pH 10.0 7.00 ______________________________________
The nucleic acid removal is effective with this buffer system over the pH range 5-9.5.
Efficiency of Nucleic Acid Reduction Carried Out in Buffers of Varying Ionic Strengths
F. graminearum IMI 145425 cultivated as described in Example A, was contacted with 66% (v/v) IPA at 20.degree. C. for 1 minute, and incubated in buffers or non-buffered solutions of varying ionic strengths at 45.degree. C. The incubations werecarried out at approximately 10 g/l with stirring.
__________________________________________________________________________ Time of incubation Buffer at 45.degree. C. % Amino % Total system Treatment (Minutes) % RNA Nitrogen Nitrogen __________________________________________________________________________ None None None 10.89 7.57 9.80 Distilled Nucleic acid reduced 0 11.21 7.82 10.53 water " 20 7.38 8.07 10.22 pH 5.7 " 40 4.79 7.97 10.12 " 60 2.57 8.40 9.84 Non-buffered "0 11.17 8.35 10.81 ammonia " 20 4.54 8.85 10.07 solution sufficient " 40 3.14 8.85 10.68 to bring to pH 8.5 " 60 2.55 8.79 10.30 0.02M " 0 9.96 8.46 10.89 NH.sub.4 Cl/NH.sub.4 OH " 20 3.21 8.62 10.38 buffer " 40 2.39 8.73 10.36 pH 8.5 " 601.82 8.90 10.12 0.1M " 0 9.86 8.23 10.98 NH.sub.4 Cl/NH.sub.4 OH " 20 2.29 8.84 10.45 buffer " 40 1.88 8.68 9.91 pH 8.5 " 60 1.69 8.73 10.56 0.5M " 0 10.02 8.21 10.58 NH.sub.4 Cl/NH.sub.4 OH " 20 5.85 8.35 10.13 buffer " 40 5.63 8.42 10.01 pH8.5 " 60 5.58 8.54 10.12 1.0M " 0 10.19 7.56 10.89 NH.sub.4 Cl/NH.sub.4 OH " 20 10.45 7.72 10.48 buffer " 40 9.96 7.99 10.81 pH 8.5 " 60 9.89 8.15 10.58 __________________________________________________________________________
Nucleic acid is most effectively reduced at lower ionic strengths. The optimum conditions for rapid reduction being 0.1M buffer.
The Nucleic Acid Reduction Process Studied at Various Temperatures
F. graminearum IMI 145425, cultivated as described in Example A, was contacted with 66% (v/v) IPA at 20.degree. C. for 2 minutes, and incubated in 0.1M NH.sub.4 Cl/NH.sub.4 OH buffer pH 8.5 for various durations at various temperatures. Theincubations were carried out at approximately 10 g/l with stirring.
______________________________________ Temperature Time of incubation of buffer minutes % RNA remaining ______________________________________ Control -- 9.44 30.degree. C. 0 10.83 30.degree. C. 20 8.40 30.degree. C. 40 7.06 30.degree.C. 60 7.45 30.degree. C. 90 4.76 30.degree. C. 120 4.11 37.degree. C. 0 10.12 37.degree. C. 20 5.02 37.degree. C. 40 3.55 37.degree. C. 60 3.55 37.degree. C. 90 1.80 37.degree. C. 120 1.02 45.degree. C. 0 10.09 45.degree. C. 20 2.58 45.degree. C. 40 0.99 45.degree. C. 60 0.69 45.degree. C. 90 0.69 45.degree. C. 120 0.61 55.degree. C. 1.5 2.16 55.degree. C. 3.0 1.10 55.degree. C. 4.5 0.76 60.degree. C. 1.5 1.96 60.degree. C. 3.0 1.78 60.degree. C. 4.5 1.11 70.degree. C. 2.0 4.19 70.degree. C. 3.5 3.30 70.degree. C. 5.0 3.56 80.degree. C. 1.5 5.65 80.degree. C. 3.0 5.40 80.degree. C. 4.5 5.31 ______________________________________
Nucleic acid reduction takes place over the temperature range 30.degree. C.-80.degree. C. The most efficient conditions are at a temperature of 60.degree. C., where satisfactory reduction of RNA was achieved within 90 seconds.
According to the present invention there is provided a process for reducing the nucleic acid content in the production of an edible protein-containing substance which comprises maintaining a grown non-toxic microfungus of the class FungiImperfecti in a suspension at a pH between 4.7 and 7.0 and at a temperature between 55.degree. and 72.degree. C. for a time of at least 60 seconds.
The process may be applied to a grown non-toxic strain of Fusarium.
The strain of Fusarium may be a strain of Fusarium graminearum Schwabe in particular IMI 145425, Fusarium oxysporum or Fusarium solani as described and claimed in our Applications Nos. 23452/70 (Ser. No. 1,346,061) and 30584/70 and cognate10466/71 (Ser. No. 1,346,062).
The grown non-toxic microfungus of the class Fungi Imperfecti may conveniently be maintained in a suspension at a pH between 4.7 and 7.0 and at a temperature between 55.degree. and 68.degree. C. for a time of at least 200 seconds.
The post fermentation process of the present invention for reducing the nucleic acid content of micro-organisms is essentially a single-stage process.
The grown microbial protein or fungal mycelium obtained for example by the fermentation process described and claimed in our Application No. 30584/70 and cognate No. 10466/71 (Ser. No. 1,346,062), may be harvested, filtered to remove growthmedium and washed, if desired. The cells may then be brought into intimate contact with aqueous buffer solutions in the pH range 4.7 to 7.0. Thus the cells may then be resuspended and incubated in tap water at pH 6.3 and temperature 63.degree. C. fora period of 20 minutes.
In order to confine the loss of protein to a minimum it is desirable to raise the temperature of the cell suspension to a given temperature within the range of 55.degree. and 72.degree. C. as rapidly as possible; substantially the sametemperature may subsequently be maintained for a period of 5 to 60 minutes.
An optional prior step designed to inhibit or destroy the proteolytic activity comprises maintaining a grown non-toxic microfungus of the class Fungi Imperfecti at the selected isothermal temperature of between 55.degree. and 72.degree. C. at apH where there is no proteolytic activity for a time sufficient to destroy the protease but not the ribonuclease.
Thus with a view to improving the protein economy of the present isothermal process the cells may be held at a pH of 8.5 at the selected isothermal temperature, preferably 65.degree. C., for a duration of between 1/2 minute and 5 minutes,preferably 1 minute before the isothermal process is commmenced (i.e. with an adjustment of the pH to between 4.7 and 7).
The resulting fungal mycelium may have an RNA content of 1 to 4% compared to 7 to 12% of the untreated proliferated organism. In certain instances the RNA content may be less than 1%.
Following is a description of methods of determining the chemical composition.
References to "Biomass Loss" denote weight loss during processing.
Ribonucleic acid (RNA) content is determined by a modification of the method of Schneider, W. C. Analyst, 1945, 161, 293.
Method of analysis for Total Nitrogen (TN) Automatic Kjeldahl digester (Technicon), A. Ferrari, Ann. N.Y. Sci 87, 792.
Amino nitrogen (AN) TNBS (modified). M. A. Pinnegar, Technicon Symposium 1965, p. 80.
Fusarium graminearum IMI 145425 was cultivated continuously by the following procedure:
______________________________________ Medium in tap water. Potato Starch 60 g/l (treated with .alpha.-amylase and glucamylase) MgSO.sub.4 7H.sub.2 O 0.75 g/l ZnSO.sub.4 7H.sub.2 O 10.0 mg/l CuSO.sub.4 5H.sub.2 O 2.0 mg/l NaMoO.sub.42H.sub.2 O 2.0 mg/l CoCl 6H.sub.2 O 2.0 mg/l MnSO.sub.4 4H.sub.2 O 10.0 mg/l FeSO.sub.4 7H.sub.2 O 20.0 mg/l NH.sub.4 H.sub.2 PO.sub.4 3.0 g/l K.sub.2 SO.sub.4 2.4 g/l NaCl 0.125 g/l Boric acid 0.5 mg/l Biotin 0.005 mg/l PPG 2000 0.04 mg/l Fermenter operation conditions Temperature 30.degree. C. pH 6.0 Pressure 15.5 psig Stirrer speed 230 rpm Air flow 800 1/minute Dissolved oxygen 6.5 (arbitary units where air saturation is 80) Sterilisation Temperature 135.degree. C. bycontinuous sterilisation (90 secs holding time) Dilution rate 0.14 hr.sup.-1 Fermenter Volume 1300 1 Inoculum: Prior to continuous growth the fermenter was operated in a batch fashion from a 20 1 inoculum of a growing culture. When batch growth wascomplete the fermenter was put on stream under the above conditions. ______________________________________ Note In some of the examples which follow the % RNA content is not corrected for "Biomass Loss". ##STR1## The % RNA content not correctedfor Biomass Loss is 2% (ie 2 g from the original 100 g of cells ##STR2## 100 = 2.86% (ie 2 g in a final 70 g of product) The Isothermal Process like other RNA reduction processes generally results in approximately 30% Biomass loss. However becausethe biomass loss is not generally determined on each sample it becomes necessary to quote RNA content of the product as a function of the starting material. Results EXAMPLE A The Nucleic Acid Reduction Process Studied at Various
F. graminearum IMI 145425, cultivated as described earlier was harvested and washed on a Buchner filtration system. The cells were suspended at a slurry concentration of approximately 10 g/l in tap water at various temperatures for variousdurations.
______________________________________ % RNA Content Duration (Not % of the Temperature of corrected original of Incubation Incubation for Biomass RNA Treatment .degree.C. minutes Loss) remaining ______________________________________Control 55 -- 8.09 100 Nucleic Acid 55 5 8.09 100 reduction Nucleic Acid 55 10 6.58 81 reduction Nucleic Acid 55 20 3.40 42 reduction Nucleic Acid 55 40 1.16 14 reduction Nucleic Acid 55 60 0.80 10 reduction Control 58 -- 7.56 100 Nucleic Acid 58 5 6.26 83 reduction Nucleic Acid 58 10 3.55 47 reduction Nucleic Acid 58 20 1.79 24 reduction Nucleic Acid 58 30 1.21 16 reduction Nucleic Acid 58 45 0.88 12 reduction Control 60 -- 10.84 100 Nucleic Acid 60 5 8.15 75 reduction Nucleic Acid 60 10 4.92 45 reduction Nucleic Acid 60 20 2.39 22 reduction Nucleic Acid 60 30 1.68 15 reduction Nucleic Acid 60 45 1.29 12 reduction Control 62 -- 9.85 100 Nucleic Acid 62 1 11.25 -- Reduction Nucleic Acid 62 56.37 65 Reduction Nucleic Acid 62 10 4.19 43 Reduction Nucleic Acid 62 20 2.30 23 Reduction Nucleic Acid 62 30 1.83 19 Reduction Control 64 -- 11.34 100 Nucleic Acid 64 1 11.96 -- Reduction Nucleic Acid 64 5 6.12 54 Reduction NucleicAcid 64 10 4.10 36 Reduction Nucleic Acid 64 20 2.70 24 Reduction Nucleic Acid 64 30 2.39 21 Reduction Control 66 -- 8.22 100 Nucleic Acid 66 1 7.03 86 Reduction Nucleic Acid 66 5 3.13 38 Reduction Nucleic Acid 66 10 2.29 28 Reduction Nucleic Acid 66 20 1.99 24 Reduction Nucleic Acid 66 30 2.18 27 Reduction Control 68 -- 8.47 100 Nucleic Acid 68 1 6.78 80 Reduction Nucleic Acid 68 5 3.03 36 Reduction Nucleic Acid 68 10 2.56 30 Reduction Nucleic Acid 68 20 2.44 29 Reduction Nucleic Acid 68 30 2.36 28 Reduction Control 70 -- 7.51 100 Nucleic Acid 70 1 3.29 44 Reduction Nucleic Acid 70 5 2.65 35 Reduction Nucleic Acid 70 10 2.40 32 Reduction Nucleic Acid 70 20 2.33 31 Reduction Nucleic Acid 70 302.22 30 Reduction Control 72 -- 7.62 100 Nucleic Acid 72 1 5.35 70 Reduction Nucleic Acid 72 5 2.74 36 Reduction Nucleic Acid 72 10 2.43 32 Reduction Nucleic Acid 72 20 2.33 31 Reduction Nucleic Acid 72 30 2.33 31 Reduction Control 75-- 9.16 100 Nucleic Acid 75 1 6.02 66 Reduction Nucleic Acid 75 5 5.10 57 Reduction Nucleic Acid 75 10 5.04 55 Reduction Nucleic Acid 75 20 4.73 52 Reduction Nucleic Acid 75 30 4.49 49 Reduction Control 80 -- 8.72 100 Nucleic Acid 80 15.69 65 Reduction Nucleic Acid 80 5 5.26 60 Reduction Nucleic Acid 8O 10 5.11 59 Reduction Nucleic Acid 80 20 4.57 52 Reduction Nucleic Acid 80 30 4.51 52 Reduction ______________________________________
With mould cultivated in the manner described it is possible to reduce the nucleic acid level to acceptably low values within the temperature range 55.degree.-72.degree. C.
The ideal isothermal temperature depends on the extent of RNA removal desired and the duration which can be tolerated on economic grounds.
The preferred conditions for our purposes are pH 6, 62.5.degree. C. for 18 minutes (see also Example B).
Efficiency of Nucleic Acid Reduction Over a pH Range of 4-9.5 at 62.5
F. graminearum IMI 145425, cultivated as described earlier was harvested and washed on a Buchner filtration system. The cells were suspended in tap water at 62.5.degree. C. and a slurry concentration of approximately 10 g/l. The pH wascontrolled at the desired value by automatic addition of either HCl or NH.sub.4 OH. Samples were incubated for 18 minutes.
__________________________________________________________________________ % of the % of original Duration % of % the AN re- pH of of incub- RNA Bio original maining incub- ation in % % mass RNA in the Treatment ation minutes product AN TN Loss remaining product __________________________________________________________________________ Control -- -- 8.24 5.9 7.68 -- 100 100 Isothermal 9.5 18 7.31 6.27 8.60 23.9 67.5 81 at 62.5.degree. C. Isothermal 9.0 18 8.366.36 8.77 21.4 79.7 84.7 at 62.5.degree. C. Isothermal 8.5 18 8.29 6.31 8.52 20.8 79.7 84.7 at 62.5.degree. C. Isothermal 8.0 18 8.28 6.19 8.41 21.4 79.0 82.5 at 62.5.degree. C. Isothermal 7.0 18 2.06 6.34 7.74 28.3 17.9 77.0 at62.5.degree. C. Isothermal 6.0 18 1.01 6.46 7.80 31.4 8.4 75.1 at 62.5.degree. C. Isothermal 5.0 18 1.39 6.48 7.95 32.1 11.4 74.6 at 62.5.degree. C. Isothermal 4.0 18 7.26 6.48 8.49 27.7 63.7 79.4 at 62.5.degree. C. __________________________________________________________________________
(1) The pH optimum for the process carried out at 62.5.degree. C. is in the range pH 4.7-7.0. Maximum reduction was at pH 6.0.
(2) Unfortunately, optimum pH for protein retention is not pH 6.0 but pH 8.5-9.0.
(3) Observations on colours: The dried solids, moist filter cakes and slurries at pH's of 6.0 and above were dark grey. Those at pH 5.0 were fawn, and at pH 4.0 the material was white. After the RNA reduction has been accomplished it maytherefore be desirable to adjust the pH to 4.0 to obtain a white product.
Efficiency of Nucleic Acid Reduction Carried Out in Solutions of Varying Logic Ionic Strengths
F. graminearum IMI 145425, cultivated as described earlier was harvested and washed on a Buchner filtration system. The cells were suspended at a slurry concentration of approximately 10 g/l in solutions of varying ionic strengths. The pH wasautomatically maintained at pH 6 and the incubation was carried out at 62.5.degree. C. for a duration of 20 minutes.
______________________________________ Incubation Solution % RNA (maintained in % Amino % Total Treatment at pH 6) product Nitrogen Nitrogen ______________________________________ Control -- 8.58 6.63 8.85 Nucleic Acid Distilled water 1.50 7.12 8.69 Reduction Nucleic Acid 0.01 M NaCl 1.18 6.98 8.81 Reduction Nucleic Acid 0.05 M NaCl 1.05 6.99 9.04 Reduction Nucleic Acid 0.10 M NaCl 1.04 7.06 8.96 Reduction Nucleic Acid 0.20 M NaCl 0.87 6.69 8.64 Reduction Nucleic Acid 0.50 M NaCl 0.91 6.77 8.50 Reduction Nucleic Acid 0.50 M NH.sub.4 Cl 0.62 7.02 8.64 Reduction ______________________________________
In the range studied NaCl and NH.sub.4 Cl had little effect on the nucleic acid reduction process.
Efficiency of Nucleic Acid Reduction at Various Slurry Concentrations
F. graminearum IMI 145425, cultivated as described earlier was harvested and washed on a Buchner filtration system. The cells were suspended at various slurry concentrations in tap water at 63.degree. C. for various durations.
______________________________________ Time of Slurry Incubation % RNA Content Concentration (minutes- (Not corrected Treatment g/l seconds) for biomass loss) ______________________________________ None (Control) None None 8.85 NucleicAcid 1 g/liter 1.00 7.46 Reduction Nucleic Acid 1 g/liter 7.20 3.27 Reduction Nucleic Acid 1 g/liter 15.30 2.37 Reduction Nucleic Acid 1 g/liter 30.30 1.85 Reduction Nucleic Acid 2 g/liter 1.00 6.87 Reduction Nucleic Acid 2 g/liter 7.202.34 Reduction Nucleic Acid 2 g/liter 16.30 1.41 Reduction Nucleic Acid 2 g/liter 31.00 1.31 Reduction Nucleic Acid 4 g/liter 1.00 7.17 Reduction Nucleic Acid 4 g/liter 8.00 2.50 Reduction Nucleic Acid 4 g/liter 16.20 1.34 Reduction Nucleic Acid 4 g/liter 30.10 0.91 Reduction Nucleic Acid 10 g/liter 2.00 7.35 Reduction Nucleic Acid 10 g/liter 9.40 2.02 Reduction Nucleic Acid 10 g/liter 18.00 1.10 Reduction Nucleic Acid 10 g/liter 30.20 0.76 Reduction Nucleic Acid 20g/liter 2.20 6.05 Reduction Nucleic Acid 20 g/liter 6.40 3.13 Reduction Nucleic Acid 20 g/liter 16.20 1.59 Reduction Nucleic Acid 20 g/liter 30.00 0.88 Reduction Nucleic Acid 40 g/liter 1.20 5.31 Reduction Nucleic Acid 40 g/liter 5.10 5.25 Reduction Nucleic Acid 40 g/liter 15.00 1.68 Reduction Nucleic Acid 40 g/liter 30.50 1.02 Reduction ______________________________________
The results show that broadly speaking slurry concentration only affects RNA reduction in as much as heat transfer is affected (i.e. high slurry concentrations may require stirring to ensure rapid temperature equilibration).
Efficiency of Nucleic Acid Reduction Under Various Agitation Conditions
F. graminearum IMI 145425, cultivated as described earlier was harvested and washed on a Buchner filtration system. The cells were suspended at approximately 10 g/liter in tap water at 63.degree. C. under various agitation conditions.
______________________________________ Agitation in Grant % RNA Content Water Bath Duration of (Not corrected strokes/ incubation for Biomass Treatment minute minutes Loss) ______________________________________ None (Control) None None8.30 Nucleic Acid Zero 1 7.31 Reduction Nucleic Acid Zero 5 2.33 Reduction Nucleic Acid Zero 10 1.04 Reduction Nucleic Acid Zero 20 0.67 Reduction Nucleic Acid Zero 30 0.59 Reduction Nucleic Acid 50 1 7.44 Reduction Nucleic Acid 50 52.59 Reduction Nucleic Acid 50 10 1.23 Reduction Nucleic Acid 50 20 0.84 Reduction Nucleic Acid 50 30 0.74 Reduction Nucleic Acid 250 1 6.26 Reduction Nucleic Acid 250 5 2.10 Reduction Nucleic Acid 250 10 1.37 Reduction Nucleic Acid 250 20 1.13 Reduction Nucleic Acid 250 30 1.13 Reduction ______________________________________
The results show that it is not necessary to stir the slurry during the Isothermal process. The effect of shaking at this slurry concentration is negligible. This has tremendous implications from the chemical engineering point of view whenscale-up to larger plant is carried out.
Typical Nucleic Acid Reduction Experiment
F. graminearum IMI 145425, cultivated as described earlier was harvested and washed on a Buchner filtration system. The cells were resuspended at a slurry concentration of approximately 10 g/l in a solution of 0.1M NaCl. The pH wasautomatically maintained at pH 6 and the incubation was carried out at 62.5 C. for a duration of 20 minutes.
______________________________________ 31.9 g Biomass 100 g of fungus containing 68.1 g product Lost ______________________________________ 37.30 g protein 30.00 g protein + ##STR3## -- 8.58 g RNA 0.71 g RNA ______________________________________
The product contains 44% protein and 0.8% ribonucleic acid i.e. a protein to nucleic acid ratio of 55:1. The starting material contains 37.3% protein and 8.58% ribonucleic acid i.e. a protein to nucleic acid ratio of 4.35:1.
An Optional Prior Process Designed to Destroy the Protease Activity Without Substantially Destroying the Ribonuclease Activity
F. graminearum IMI 145425, cultivated as described earlier was harvested and washed on a Buchner filtration system. The cells were suspended in distilled water at 65.degree. C. and a slurry concentration of approximately 10 g/l. The pH wascontrolled at 8.5 for various times.
After this prior treatment the cells were subjected to RNA removal by the isothermal process at 65.degree. C., pH 6 for 20 minutes.
______________________________________ % of % AN % TN RNA in in in % Biomass Treatment product product product Loss ______________________________________ Control 8.37 6.26 8.31 Zero 1/2 minutes at pH 1.09 7.03 9.12 43.4 8.5 + 20 min pH 6 1 minute at pH 1.31 7.23 9.49 35.0 8.5 + 20 min pH 6 2 minutes at pH 1.52 7.21 9.38 35.4 8.5 + 20 min pH 6 5 minutes at pH 2.52 7.24 9.38 31.4 8.5 + 20 min pH 6 ______________________________________
The optimal example which removes the maximum quantity of RNA but at the same time retains maximal Biomass and amino nitrogen is fulfilled by conditions of 1 minutes at 65.degree. C. and pH 8.5 followed by the isothermal process at 65.degree. C. pH 8.5 for 20 minutes.
Examples of Successful Reduction of the Nucleic Acid Levels in Various Micro-organisms Other Than A3/5
Fusarium solani IMI 154217 was cultivated by the following procedure:
______________________________________ MgSO.sub.4.7H.sub.2 O 0.25 g/liter KH.sub.2 PO.sub.4 15.0 g/liter (NH.sub.4).sub.2 SO.sub.4 2.83 g/liter Biotin 0.05 ml/liter (of stock solution 1 mg/ml) Choline 50 mg/liter Trace elements 5ml/liter (STOCK SOLUTION) NaOH 1 g/liter.fwdarw. pH 6.0 Glucose 10% solution (20 mls added after sterilization) ______________________________________
Minimal Salts or Trace element stock solution
______________________________________ ZnCl.sub.2 1 g/liter MnCl.sub.2 4H.sub.2 O 1 g/liter FeCl.sub.3 6H.sub.2 O 2 g/liter CuCl.sub.2 2H.sub.2 O 0.2 g/liter NaMnO.sub. 4 2H.sub.2 O 0.2 g/liter CoCl.sub.2 6H.sub.2 O 0.2 g/liter CaCl.sub.2 2H.sub.2 O 2 g/liter ______________________________________
All components with the exception of glucose were sterilised together, and the quantity of these materials required for 1 liter of medium were dissolved, made up to 850 ml and distributed in 5 1 liter conical flasks, each containing 170 ml. A0.10% w/v solution of glucose was prepared and sterilized in 20 ml portions in universal bottles. Sterilisation was effected in an autoclave at 15 p.s.i. for 15 minutes.
Before inoculation with 10 ml of a spore suspension, the contents of one bottle of sterile glucose solution were added to each flask. Culture of the organism then proceeded on an Orbital Shaker, with 2 inch throw, at 160 r.p.m and a temperatureof 30.degree. C. The culture was harvested after 48 hours.
Nucleic Acid Reduction Process
Cells were collected and washed on a Buchner filtration system and suspended at a slurry concentration of approximately 10 g/liter in tap water at 64.degree. C. and the pH adjusted to 6 with NaOH and H.sub.2 SO.sub.4.
______________________________________ Time of % RNA % amino Microfungi Treatment incubation content nitrogen ______________________________________ F. solani None None 5.15 5.4 Nucleic 10 2.95 5.92 Acid Reduction Nucleic 20 0.67 6.11 Acid Reduction Nucleic 30 0.55 6.00 Acid Reduction ______________________________________
Fusarium oxysporum IMI 154214 was cultivated in a similar manner to that described for F. solani except that the growth medium contained 0.5 g/liter oxoid yeast extract and 0.5 g/liter mycological peptone in addition to the chemicals listed inExample H.
The cells were harvested after 72 hours and the nucleic acid reduction process conducted as in the previous example.
______________________________________ Time of incubation % RNA % amino Microfungi Treatment minutes content nitrogen ______________________________________ F. oxysporum None None 6.57 6.28 Nucleic 10 1.00 7.47 Acid Reduction Nucleic 200.65 7.45 Acid Reduction Nucleic 30 0.54 7.53 Acid Reduction ______________________________________
The Nucleic Acid Reduction Process as Carried Out in Pilot Plant
F. graminearum IMI 145425, cultivated as described earlier was processed without separation from the growth medium as follows:
1. Mycelium slurry at a concentration of 20 grams per liter exists from the fermenter at a temperature of 30.degree. C. and a pH of 6 and enters a mono-pump.
2. The mycelial slurry is pumped to a steam injector and the temperature of the material raised from 30.degree. C. to 64.degree. C. rapidly, the duration of the temperature rise, preferably being instantaneous (in practice being less than 5seconds).
3. The material now at 64.degree. C. and pH 6 is moved through a pipe and its temperature maintained for a duration of 45 minutes.
4. The material is passed through a heat exchanger to cool to approximately 20.degree. C. (to reduce the possibility of later microbial infection).
5. The slurry is passed into the trough of a rotary vacuum filter.
6. Liquid is drawn through a filter belt and the mycelium accumulates on the filter. The filter drum rotates above the liquid level carrying the mycelial cake.
7. The filter cake is washed with about 2 bed volumes of water. The filter drum continues to rotate and a vacuum pulls the cake to about 70% moisture by weight.
8. The mycelial cake is scraped off the drum.
9. The cake is reslurried in water and spray dried.
______________________________________ % Amino % RNA Nitrogen % Total Nitrogen Treatment Content Content Content ______________________________________ Dry untreated 8.22 6.45 8.74 material Dry nucleic 0.43 6.86 8.30 acid reduced material ______________________________________
The nucleic acid content is effectively reduced by the process described.
In the fermentation operation conditions it is possible to employ a higher dilution rate of up to 0.20 hrs.sup.-1, for example 0.17 hrs.sup.-1.
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