Production of edible protein containing substances

This invention is for improvements in or relating to the production of edible protein containing substances.
It has particular reference to a process for reducing the nucleic acid content of microfungi.
British Pat. 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 a microfungus 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 the assimilable carbohydrate exhausted medium the proliferated organism which constitutes the edible protein-containing substance.
British Pat. No. 1,346,062 describes and claims a process for the production of an edible protein-containing substance which comprises incubating and proliferating, under areobic conditions, a non-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 in proliferation, and separating the proliferated organism comprising the edible protein-containing substance.
British Pat. 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 process of British Patent No. 1,346,062 may be incorporated into a foodstuff for human or animal consumption.
The process of British Pat. No. 1,346,062 are capable of producing an edible protein-containing substance comprising fungal mycelium which possesses a high net 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)/Work Health Organization (WHO) has advised that the nucleic acid content should be reduced 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 microogranisms combined with the minimum loss of protein to render them more acceptable as 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 Protein Advisory Group.
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. This strain has also been deposited with the American Type Culture Collection and assigned the number ATCC 20334.
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 nontoxic microfungus of the class Fungi Imperfecti 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 British Pat. Nos. 1,346,061 and 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 British Patent No. 1,346,062, may be harvested, filtered to remove growth medium 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. for a period of 20 minutes.
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.
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 same temperature 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 a pH 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 commenced (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%.
The cells may be analysed to determine their chemical composition and to evaluate the efficiency of the nucleic acid reduction process.
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.

Following is a description by way of example of methods of carrying the invention into effect.
Fusarium graminearum IMI 145425 was cultivated continuously by the following procedure.
______________________________________Medium in tap water.Potato Starch (treated with .alpha.-amylaseand glucamylase) 60 g/lMgSO.sub.4 7H.sub.2 O 0.75 g/lZnSO.sub.4 7H.sub.2 O 10.0 mg/lCuSO.sub.4 5H.sub.2 O 2.0 mg/lNaMoO.sub.4 2H.sub.2 O 2.0 mg/lCoCl 6H.sub.2 O 2.0 mg/lMnSO.sub.4 4H.sub.2 O 10.0 mg/lFeSO.sub.4 7H.sub.2 O 20.0 mg/lNH.sub.4 H.sub.2 PO.sub.4 3.0 g/lK.sub.2 SO.sub.4 2.4 g/lNaCl 0.125 g/lBoric acid 0.5 mg/lBiotin 0.005 mg/lPPG 2000 0.04 mg/lFermenter operation conditionsTemperature 30.degree. CpH 6.0Pressure 15.5 psigStirrer speed 230 rpmAir flow 800 1/minuteDissolved oxygen 6.5 (arbitrary units where air saturation is 80)Sterilization Temperature 135.degree. C by continuous sterilization (90 secs holding time)Dilution rate 0.14 hr.sup.-1Fermenter Volume 1300 l______________________________________Inoculum:- Prior to continuous growth the fermenter wasoperated in a batch fashion from a 20 l inoculum of a -growing culture.When batch growth was complete thefermenter was put on stream under the above conditions. NoteIn some of the examples which follow the % RNA content isnot corrected for "Biomass Loss". ##STR1##The % RNA content not corrected for Biomass Loss is 2%(ie 2 g from the original 100g of cells)Whereas the % RNA corrected for Biomass Loss is 2/70 .times. 100= 2.86% (ie 2g in a final 70g of product)The Isothermal Process like other RNA reduction processesgenerally results in approximately 30% Biomass loss. Howeverbecause the biomass loss is not generally determined oneach sample it becomes necessary to quote RNA content ofthe product as a function of the starting material.______________________________________
Results
Example A
THE NUCLEIC ACID REDUCTION PROCESS STUDIED AT VARIOUS TEMPERATURES
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 various durations.
Results______________________________________ % RNA Content (Not Duration corrected % of the Temperature of Incu- for Bio- original of Incubation bation mass RNATreatment .degree. C.minutes Loss) remaining______________________________________Control -- -- 8.09 100Nucleic Acidreduction 55 5 8.09 100" 55 10 6.58 81" 55 20 3.40 42" 55 40 1.16 14" 55 60 0.80 10Control -- -- 7.56 100Nucleic Acidreduction 58 5 6.26 83" 58 10 3.55 47" 58 20 1.79 24" 58 30 1.21 16" 58 45 0.88 12Control -- -- 10.84 100Nucleic Acidreduction 60 5 8.15 75" 60 10 4.92 45" 60 20 2.39 22" 60 30 1.68 15" 60 45 1.29 12Control -- -- 9.85 100Nucleic AcidReduction 62 1 11.25 --" 62 5 6.37 65" 62 10 4.19 43" 62 20 2.30 23" 62 30 1.83 19Control -- -- 11.34 100Nucleic AcidReduction 64 1 11.96 --" 64 5 6.12 54" 64 10 4.10 36" 64 20 2.70 24" 64 30 2.39 21Control -- -- 8.22 100Nucleic AcidReduction 66 1 7.03 86" 66 5 3.13 38" 66 10 2.29 28" 66 20 1.99 24" 66 30 2.18 27Control -- -- 8.47 100Nucleic AcidReduction 68 1 6.78 80" 68 5 3.03 36" 68 10 2.56 30" 68 20 2.44 29" 68 30 2.36 28Control -- -- 7.51 100Nucleic AcidReduction 70 1 3.29 44" 70 5 2.65 35" 70 10 2.40 32" 70 20 2.33 31" 70 30 2.22 30Control -- -- 7.62 100Nucleic AcidReduction 72 1 5.35 70" 72 5 2.74 36" 72 10 2.43 32" 72 20 2.33 31" 72 30 2.33 31Control -- -- 9.16 100Nucleic AcidReduction 75 1 6.02 66" 75 5 5.10 57" 75 10 5.04 55" 75 20 4.73 52" 75 30 4.49 49Control -- -- 8.72 100Nucleic AcidReduction 80 1 5.69 65" 80 5 5.26 60" 80 10 5.11 59" 80 20 4.57 52" 80 30 4.51 52______________________________________
Conclusions
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).
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 10g/l. The pH was controlled at the desired value by automatic addition of either HCl or NH.sub.4 OH. Samples were incubated for 18 minutes.
Results__________________________________________________________________________ % of the % of original Duration % of % the AN re- pH of of incub- RNA Bio- original maining incub- ation in % % mass RNA in theTreatment ation minutes product AN TN Loss remaining product__________________________________________________________________________Control -- -- 8.24 5.9 7.68 -- 100 100Isothermalat 62.5.degree. C 9.5 18 7.31 6.27 8.60 23.9 67.5 81" 9.0 18 8.36 6.36 8.77 21.4 79.7 84.7" 8.5 18 8.29 6.31 8.52 20.8 79.7 84.7" 8.0 18 8.28 6.19 8.41 21.4 79.0 82.5" 7.0 18 2.06 6.34 7.74 28.3 17.9 77.0" 6.0 18 1.01 6.46 7.80 31.4 8.4 75.1" 5.0 18 1.39 6.48 7.95 32.1 11.4 74.6" 4.0 18 7.26 6.48 8.49 27.7 63.7 79.4__________________________________________________________________________
Conclusions
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 colors: 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 may therefore be desirable to adjust the pH to 4.0 to obtain a white product.
EXAMPLE C
EFFICIENCY OF NUCLEIC ACID REDUCTION CARRIED OUT IN SOLUTIONS OF VARYING 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 was automatically maintained at pH 6 and the incubation was carried out at 62.5.degree. C. for a duration of 20 minutes.
Results______________________________________ Incubation Solution % RNA (maintained in % Amino % TotalTreatment at pH 6) product Nitrogen Nitrogen______________________________________Control -- 8.58 6.63 8.85Nucleic AcidReduction Distilled water 1.50 7.12 8.69Nucleic AcidReduction 0.01M NaCl 1.18 6.98 8.81Nucleic AcidReduction 0.05M NaCl 1.05 6.99 9.04" 0.10M NaCl 1.04 7.06 8.96" 0.20M NaCl 0.87 6.69 8.64" 0.50M NaCl 0.91 6.77 8.50" 0.50M NH.sub.4 Cl 0.62 7.02 8.64______________________________________
Conclusions
In the range studied NaCl and NH.sub.4 Cl had little effect on the nucleic acid reduction process.
EXAMPLE D
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.
Results______________________________________ Time of % RNA Content Slurry Incubation (Not corrected Concentration (minutes- for biomassTreatment g/l seconds) loss)______________________________________None(Control) None None 8.85Nucleic AcidReduction 1 1.00 7.46" 1 7.20 3.27" 1 15.30 2.37" 1 30.30 1.85" 2 1.00 6.87" 2 7.20 2.34" 2 16.30 1.41" 2 31.00 1.31" 4 1.00 7.17" 4 8.00 2.50" 4 16.20 1.34" 4 30.10 0.91" 10 2.00 7.35" 10 9.40 2.02" 10 18.00 1.10" 10 30.20 0.76" 20 2.20 6.05" 20 6.40 3.13" 20 16.20 1.59" 20 30.00 0.88" 40 1.20 5.31" 40 5.10 5.25" 40 15.00 1.68" 40 30.50 1.02______________________________________
Conclusion
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).
EXAMPLE E
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.
Results______________________________________ Agitation in Grant % RNA Content Water Bath Duration of (Not corrected strokes/ incubation for BiomassTreatment minute minutes Loss)______________________________________None(Control) None None 8.30Nucleic AcidReduction Zero 1 7.31" " 5 2.33" " 10 1.04" " 20 0.67" " 30 0.59" 50 1 7.44" " 5 2.59" " 10 1.23" " 20 0.84" " 30 0.74" 250 1 6.26" " 5 2.10" " 10 1.37" " 20 1.13" " 30 1.13______________________________________
Conclusion
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 when scale-up to larger plant is carried out.
EXAMPLE F
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 was automatically maintained at pH 6 and the incubation was carried out at 62.5.degree. C for a duration of 20 minutes.
Results______________________________________100 g of fungus 68.1 g 31.9 g Biomasscontaining product Lost______________________________________37.30 g protein 30.00 g protein + ##STR2## --8.58 g RNA 0.71 g RNA______________________________________
conclusion
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.
EXAMPLE G
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 was controlled 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.
Results______________________________________ % of % AN % TN % RNA in in in BiomassTreatment product product product Loss______________________________________Control 8.37 6.26 8.31 Zero1/2 minutes at pH 8.5 1.09 7.03 9.12 43.4+ 20 min pH61 minutes at pH 8.5 1.31 7.23 9.49 35.0+ 20 min pH62 minutes at pH 8.5 1.52 7.21 9.38 35.4+ 20 min pH65 minutes at pH 8.5 2.52 7.24 9.38 31.4+ 20 min pH6______________________________________
Conclusions
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 FUSARIUM GRAMINEARUM IMI 145425
Example H
Fusarium solani IMI 154217 (also deposited in the American Type Culture Collection and assigned the number ATCC 20327) was cultivated by the following procedure:
______________________________________Medium in distilled water:Mg SO.sub.4 . 7H.sub.2 O 0.25 g/literKH.sub.2 PO.sub.4 15.0 g/liter(NH.sub.4 ).sub.2 SO.sub.4 2.83 g/literBiotin 0.05 ml/liter (of stock solution 1 mg/ml)Choline 50 mg/literTrace elements 5 ml/liter (STOCK SOLUTION)NaOH 1 g/liter .fwdarw. pH6.0Glucose 10% solution (20 mls added after sterilization)Minimal Salts or Trace element stock solutionZnCl.sub.2 1 g/literMnCl.sub.2 4H.sub.2 O 1 g/literFeCl.sub.3 5H.sub.2 O 2 g/literCuCl.sub.2 2H.sub.2 O 0.2 g/literNaMn O.sub.4 2H.sub.2 O 0.2 g/literCoCl.sub.2 6H.sub. 2 O 0.2 g/literCaCl.sub.2 2H.sub.2 O 2 g/liter______________________________________
Sterilisation
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. A 0.10% 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.
Growth Conditions
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 temperature of 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.
Results______________________________________ Treat- Time of % RNA % aminoMicrofungi ment incubation content nitrogen______________________________________F.solani None None 5.15 5.4 Nucleic Acid 10 2.95 5.92 Reduction " 20 0.67 6.11 " 30 0.55 6.00______________________________________
Conclusion
The level of Nucleic acid was effectively reduced by the treatment described.
EXAMPLE I
Fusarium oxysporum IMI 154214 (also deposited in the American Type Culture Collection and assigned the number ATCC 20328 ) 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 in Example H.
The cells were harvested after 72 hours and the nucleic acid reduction process conducted as in the previous example.
Results______________________________________ Time of Treat- incubation % RNA % aminoMicrofungi ment minutes content nitrogen______________________________________F. oxysporum None None 6.57 6.28 Nucleic Acid 10 1.00 7.47 Reduction " 20 0.65 7.45 " 30 0.54 7.53______________________________________
Conclusion
The level of Nucleic acid was effectively reduced by the treatment described.
EXAMPLE J
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 stream 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 5 seconds).
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.
Results______________________________________ % Amino % RNA Nitrogen % Total NitrogenTreatment Content Content Content______________________________________Dry untreatedmaterial 8.22 6.45 8.74Dry nucleicacid reducedmaterial 0.43 6.86 8.30______________________________________
Conclusion
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.

Number	Name	Date
2450055	Nord	Sep 1948
3775393	Akin et al.	Nov 1973
3784536	Akin et al.	Jan 1974
3809776	Chao	May 1974
3937654	Solomons et al.	Feb 1976

Production of edible protein containing substances

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