RU2111253C1 - Method of preparing biomass - Google Patents

Abstract

FIELD: microbiology. SUBSTANCE: claimed method comprises cultivating bacteria or associations of bacteria containing amylolytic yeast under aeration conditions of culture medium containing, as carbon source, flour and/or cereal waste bran subjected to enzymic treatment with use of complex enzymic agents such as amylosubtilin and/or glucavamorin. The consumption rate of enzymes is from 0.25 % to 1.0% of absolutely dry matter. The claimed method affords one to obtain biomass with protein content of up to 50-60% and with full range of amino acids, vitamins B (including vitamin B12), H, E and F and also micro- and macroelements. Recycle of liquid phase (industrial condensate) to cultivation process affords waste-free technology, smaller amounts of water and mineral salts. EFFECT: more efficient preparation. 4 cl

Description

 The invention relates to the microbiological industry, in particular to methods for producing protein products from the waste of grain and vegetable raw materials used for inclusion in the diet of feed of farm animals and poultry.

 Protein products can be obtained on the basis of various starch and cellulose-containing wastes, in particular on wheat straw, potato starch, soybean meal, peanuts, rapeseed, cereal grain (flour, bran) and others. Known methods for producing protein feed products using yeast, carried out, for example, on a potato fermentolizate (French patent N 2285080, class A 23 J 1/18, 1976) and on a corn fermentolizate (French patent N 2300806, C 12 D 13/06, 1976).

 Previously, the raw materials are subjected to one or another treatment - mechanical, thermal, hydrolyzed with mineral acids or enzymes. Pretreatment of raw materials by enzymatic hydrolysis using amylolytic and proteolytic enzymes has an advantage over acid hydrolysis, since it does not require special equipment from expensive acid-resistant materials and pressurization to accelerate the hydrolysis process. Pre-processed raw materials serve as a source of carbon nutrition when microorganisms are grown on it. As a result, the feed is enriched with microbial protein. To obtain high-protein fodder products, you can use various types of microorganisms that can grow on these substrates - yeast, fungi, algae and bacteria. The latter are the most promising protein producers, as they are able to accumulate in cells 10-15% more protein than other microorganisms.

 A known method of producing protein products based on ground grain of wheat, barley or corn, as well as wheat bran without preliminary hydrolysis processing when growing a mixture of yeast strains on them, including a strain having amylolytic activity (ed. St. USSR N 1601115, class C 12 N 1/16, 1990). However, this method is long, uneconomical and is accompanied by a relatively low accumulation of protein (maximum 28-36% of absolute dry matter).

 A known method of producing a protein feed preparation, which includes pre-treatment with enzymes of starch-containing raw materials based on rye, wheat, barley and potatoes (German patent N 2548641., A 23 J 1/14, publ. 1978). This pre-treatment is carried out in combination with acid hydrolysis and involves the introduction of one or another additional carbon source, for example alcohols, when growing yeast, which can lead to additional costs. However, the protein content in the product in this case is not high enough and does not exceed 44% of ABC.

 The above methods for producing a protein feed preparation do not provide for the reuse of process condensate remaining after isolation of the target product, which ensures non-waste production and saving water consumption.

 A known method of producing biomass based on growing yeast in aeration conditions on a nutrient medium containing hydrocarbons as a carbon source, as well as mineral salts of macro- and microelements, further separation of the yeast suspension into a condensed and clarified fraction, heating the condensed fraction, re-separation into clarified and the condensed fraction, returning the cooled clarified fraction to the stage of growing and drying the condensed fraction (ed. St. USSR N 646798, class C 12 N 1/28, 1975). However, this method only swings the fermentation process on a substrate, which is a highly purified liquid paraffin that is completely assimilated by yeast, and cannot be used for fermentation of such a complex organic substrate as grain waste that is not completely utilized by microorganisms and remaining in the culture liquid as in the form of solid inclusions, and in dissolved form.

Closest to the proposed one is a method for producing biomass by growing yeast cultures under aeration conditions on a nutrient medium containing bran and / or cereal flour, subjected to preliminary enzymatic treatment, as well as mineral salts and trace elements, followed by isolation of the target product. The enzymatic treatment is carried out with amylolytic or proteolytic enzymes taken in an amount of 0.5-1.0% on ABC (absolutely dry substances) raw materials, at 50-70 ^o C, pH 7.0-7.4 for 1-1.5 hours, and the cultivation of yeast p. Candida is carried out under conditions of an excess of iron and phosphate ions (Russian patent N 2041946, class C 12 N 1/16, 1995).

 In this method, for the enzymatic treatment of bran and / or flour, mainly individual amylolytic or proteolytic enzymes, such as α-amylase, protease, and Candida guilliermondii or Candida tropicalis yeast biomass are used.

 As a result, the protein content in the finished product can, in optimal cases, reach 46% on wheat bran and 50% on flour. However, as the authors of the method indicate, in the average sample of the finished product on wheat bran, the protein concentration is significantly lower and is only 30% of crude protein or 28% of protein according to Barnstein.

 The disadvantage of this method is the reduced protein content in the resulting product, which is explained by the use of yeast as protein producers, capable of accumulating protein in cells up to 55-65% of ABC and inferior to bacteria to this extent. In addition, a deep enzymatic pretreatment of the substrate due to the action of non-complex enzyme preparations does not provide a sufficient amount of carbohydrate and other compounds available for microorganisms, which limits the reproduction and leads to a low protein content in the finished product.

 The disadvantages of the method should also include the lack of return during the cultivation of the remaining after the biomass separation of the aqueous phase, the so-called "process condensate".

 The challenge facing the authors of this method is to increase the protein content in the target product and to increase the efficiency of production.

 The technical result is determined by increasing the nutritional value of grain waste bran and flour by enriching them with protein and obtaining high protein biomass, which is used as a feed additive for farm animals and poultry.

 The essence of the method is as follows: from a crushed karmal-containing raw material - bran and / or flour or a mixture thereof in a ratio of 1: 1, an aqueous suspension is prepared with a dilution module of at least Mp = 1: 8. The suspension is subjected to enzymatic treatment using enzyme preparations of Amylosubtilin GX and / or Glucavamorin GC, which are complexes of various enzymes. Amylosubtilin GX contains, in addition to the main enzyme, α-amylase, a wide range of enzymes, namely glucoamylase, lipase, protease, cellulose, deaminase, amylase, ribonuclease. Glucavamorin GC consists mainly of glucoamylase with a sufficiently high content of α-amylase, glucoisomerase, CMCase, cellulose, protease and provides a more complete decomposition of substrate components than only α-amylase or protease, or amylosubtiline. As a result of the action of the complex of enzymes, mono- and disaccharum compounds, amino sugars, dextrins, organic acids, accessible to the base by microorganisms, are formed.

 Amylosubtilin GX and Glucavamorin GC can be used for fermentolysis of substrates. However, the use of Glucavamorin GC or its mixture with Amilosubtilin GZH is more economical.

 Enzymes are added to the aqueous suspension in an amount of 0.25-1.0% by absolutely dry weight of the feed, preferably 0.5%. When enzymes are added in an amount of 0.25%, the quality of the final product is slightly reduced due to a decrease in protein content by 2-3%, and at a cost of 1%, a high-protein but more expensive product is obtained (3-5% more protein than at 0 ,5%).

Fermentolysis is carried out for 1-1.5 hours at 50-70 ^o C (preferably at 60 ^o C), pH 5.0-7.4, depending on the enzyme used: for Amylosubtilin GZH pH 7.0-7, 4, for Glucavamorin GC pH 5.0-5.5 for a mixture of both enzyme preparations pH 6.0-6.4.

 As a result of fermentolysis, the content of carbohydrates (reducing substances - PB) acceptable for microorganisms in the suspension increases from 0.5% to 2.5% and higher depending on the enzyme used. With Glucavamorin, GC PB is approximately two times higher than PB with Amilosubtilin GC. The amount of organic acids also increases from 0.1% to 0.5%, which are also well consumed for cell growth.

The obtained fermentolizate is then cooled to 35-40 ^o C and mineral salts are added to it in an amount, g / l:
(NH ₄ ) ₂ SO ₄ - 3.5 - 5.0 (depending on the content of nitrogen and potassium in the substrate)
KH ₂ PO ₄ - 0.1 - 0.5
MnSo ₄ • 5H ₂ O - 0.05
CuSO ₄ • 5H ₂ O - 0.008
ZnSO ₄ • 7H ₂ O - 0.05
H ₃ BO ₃ - 0.002
Na ₂ MoSO ₄ • 2H ₂ O - 0.005
Iron salts are not added to the fermentolizate, since the substrate itself contains it in excess.

 Automatically, to maintain the pH at a given level during the growth process, an ammonia solution is introduced into the fermenter, which is also a source of nitrogen.

The pH of the medium is maintained at 6.5 - 6.8, the temperature at 32 - 39 ^o C (depending on the type of bacteria).

Bacterial strains known as methanol-based protein producers and belonging to the genus Acetobacter are used as protein and biomass producers:
Acetobacter methylicum VSB-924 TsMPM B-2942 (RF patent N 116363, C 12 N 15/00, 1984);
Acetobacter methylicum VSB-867 TsMPM B-1947 (RF patent N 925112, C 12 N 15/00, 1982);
Acetobacter methylovorans VSB-914 TsMPM B-2479 (RF patent N 1070916, C 12 N 15/00, 1983);
All strains are characterized by a high protein content - up to 76% to ACB. They are very close in their basic morphological, physiological and biochemical properties, are optional acidophilic methylotrophs, but in addition to methanol, they also use other compounds, in particular mono- and disaccharides, organic acids, and others. Strains differ in sensitivity to typical phages and thermal stability : optimal growth for Acetobacter methylicum VSB-867 at 28-30 ^o C, for Acetobacter methylicum VSB-924 - 30-36 ^o C and Acetobacter methylovorans VSB-914 at 36-40 ^o C. If necessary, the strains can be interchanged.

 As a result of numerous experiments conducted by the authors of this method, it was possible to establish an important property of the above strains, the ability to actively develop on carbohydrate substrates and, in particular, on bran and / or flour fermetolizate, not only at low pH values, but at pH 6.5 -6.8. During non-sterile cultivation in the enzyme on bran and / or flour fermentolysates in an acidic environment at a pH below 6.0, experiments have shown that rapid infection with yeast and fungi and displacement of the producer from the process by 30-40% or more of the total number of cells. As a result, the protein content in the resulting product is greatly reduced.

 When the pH of the medium is increased to 6.5-6.8, the infection with yeast and fungi decreases to 5-10% and this makes it possible to obtain a high-quality product with a sufficiently high protein content.

 The authors of the method have verified the possibility of using yeast strains similar to the prototype strains instead of bacteria as protein producers. Yeast was grown on bran and a mixture of bran and flour treated with Amylosubtilin GLC in accordance with the claimed method, as well as α-amylase according to the prototype method. The content of PB in the fermetholysate according to the proposed method is 2.0, and according to the prototype method - 1.7. The protein content in the final product was 3% higher than by the prototype method, but significantly lower than when using bacterial strains as producers.

 Bacteria Acetobacter methylicum VSB-924, Acetobacter methylicum VSB-867 and Acetobacter methylovorans VSB-914 are grown on a fermentolizate both in pure culture and in a mixture with Saccharomyces cerenisiae VKPM U-1218 Saccharomyces cereum yeast. Yeast provides, due to its own enzymes, in addition to the enzyme preparations introduced, hydrolysis of the starch substrate, and as a result of this, the protein content in the final product increases.

 A mixture of bacteria and yeast is prepared in a ratio of 10: 1 - 10: 0.5 (100 bacterial cells per 5-10 yeast cells).

 To obtain a mixed culture, bacteria and yeast are pre-grown separately by conventional methods provided for the cultivation of seed, and then introduced into the nutrient medium at the same time. The amount of yeast seeded can vary widely, for example, from 0.5 to 20% of the total number of cells. In the process of co-cultivation with bacteria at pH 6.5-6.8, the content of yeast cells regardless of seeding will be at the same level, about 5-10% (i.e. 5-10 cells per 100 bacterial cells). Such self-regulation of the mixed culture system is ensured mainly by a pH value unfavorable for the active development of yeast.

 Due to the use of a mixed culture of bacteria and amylolytic yeast, the protein content in the product increases by 2-3% compared with bacteria.

 The cultivation of bacteria or bacteria mixed with amylolytic yeast is carried out in a fermenter with aeration in batch and / or continuous methods.

 Periodic cultivation continues for 8 hours until almost complete consumption of sugar, which is controlled by the amount of PB in the medium. (PB should be below 0.05%). Then the biomass is kept in the fermenter for 2-3 hours during aeration (stage of "ripening"). During this stage, not only the sugar residue is recycled, but also other carbon components of the substrate, in particular organic acids, the content of which during this period is reduced by 30-50%. g As a result, the medium is alkalized to a pH of 7.0-7.5, which serves as an indicator of the completion of the process.

 The producer during the process is not replaced by extraneous microorganisms and in the biocenosis of the fermenter is 95-98% of the total number of cells.

 At the end of the cultivation process, the grown biomass in the form of a suspension, together with the residues of the substrate, is subjected to plasmolysis, evaporation to ABC 7.0-12% and spray drying.

 The liquid phase (process condensate) after evaporation is completely sent back to the growing stage and is used instead of water to prepare a suspension of substrate and fermetolizate. This saves 50-80% of the water spent on growing and provides an environmentally friendly process.

 Example 2

The cultivation of bacteria Acetobacter methylicum VSB-924 is carried out in a fermenter with a working volume of 3.5 m ³ with aeration of 1 nm ³ / m ³ / min.

 As a substrate, a mixture of wheat and rye bran in a ratio of 1: 1, (i.e., 192.5 kg of each species) is used, from which an aqueous suspension with a dilution modulus of at least Mp = 1: 8 is prepared, i.e. 385 kg of bran contribute to 3080 liters of water. The content of absolutely dry substances in the suspension is 11.1%.

Fermentolysis is carried out for 1.5 hours at 60 ^o C and a pH of 6.7-6.8. Amylosubtilin GLC in the amount of 0.5% of the ABC substrate, i.e. 1.93 kg, is taken as an enzyme.

The obtained fermentolizate is cooled to 40 ^o C. The content of reducing substances (PB) in it is 2.03%, organic acids - 0.25%.

Mineral salts are added to the finished fermentolizate, g / l:
(NH ₄ ) ₂ SO ₄ - 3,5
KH ₂ PO ₄ - 0.1
MnSO ₄ • 5H ₂ O - 0.05
CuSO ₄ • 5H ₂ O - 0.008
ZnSO ₄ • 7H ₂ O - 0.05
H ₃ BO ₃ - 0.002
Na ₂ MoSO ₄ • 2H ₂ O - 0.005
Bacteria are grown in a batch manner for 8 hours. The medium is maintained at a temperature of 32-35 ^o C, pH 6.5-6.8. To maintain the pH at a predetermined level during the growth of the culture, an ammonia solution (6%) is automatically introduced.

 The cultivation process is controlled by the content of carbohydrates. After consumption of carbohydrates to PB = 0.02%, the culture in the fermenter is kept for another two hours with aeration and stirring. During this period ("dosing") they consume carbohydrate residues and intensive use of organic acids takes place (their content is almost halved, to 0.12%). The level of protein accumulation in two hours of "dosing" rises by 3% and by the end of the process is 50.0% of ABC.

 The resulting biomass, together with the remainder of the substrate, is subjected to plasmolysis, evaporation and spray drying.

The finished product in addition to protein contains B vitamins (mg / kg):
B ₁ - 18.3
B ₂ - 39.5
B ₃ - 49.8
B ₅ - 193.0
B ₆ - 7.7
B _c - 0.25
B ₁₂ - 1.02,
as well as vitamins
H - 0.85
E - 90.7 and others.

 In addition, the product has a wide range of micro and macro elements: Ca, K, Mg, Na, Fe, Zn, Mn, Cu, Co, Mo, B, amino acids (total amino acids 37% of ABC), including such as tryptophan, lysine, methionine, cystine, aspartic acid, etc.

 Example 2

 The bacteria Acetobacter merhylicum BSC-924 are grown according to the same regimen as in Example 1.

 The fermentolizate is prepared in the same manner as in example 1, but wheat flour is used as a substrate (385 kg) instead of bran.

 The resulting fermentolizate contains PB = 4.8%.

 The quality of the final product as in example 1.

 Crude protein content 60% of ABC.

 Example 3

The bacteria Acetobacter merhylicum VSB-867 are grown according to the same regimen as in Example 1, but at 28-30 ^o C.

 The fermentolizate is prepared in the same manner as in Example 1, but a mixture of rye bran and wheat flour in a ratio of 1: 1 (i.e., 192.5 kg of each species) is used as a substrate.

 The resulting fermentolizate contains PB = 3.6%.

 The quality of the final product, as in example 1.

 Crude protein content 54% of ABC.

 Example 4

 The bacteria Acetobacter merhylicum BSC-924 are grown according to the same regimen as in Example 1.

 The fermentolizate is prepared in the same manner as in example 1, but the enzyme Glucavamorin GC is added in an amount of 0.5% of ABC.

 The resulting fermentolizate contains PB = 4.54%.

 The quality of the final product as in example 1.

 Crude protein content 56% of ABC.

 Example 5

The bacteria Acetobacter merhylovorans VSB-914 are grown according to the same regimen as in Example 1, but at 36-38 ^o C.

 The fermentolizate is prepared in the same manner as in example 1, but a mixture of the enzymes Amilosubtilin GX and Glucavamorin GC is added in a ratio of 1: 1 (0.25% of each of the ABC substrate, i.e. 0.96 kg).

 The resulting fermentolizate contains PB = 3.56%.

 The quality of the final product, as in example 1.

 Crude protein content 53.5%.

 Example 6

 The bacteria Acetobacter methylicum BSC-924 are grown according to the same regimen as in Example 1.

 The fermentolizate is prepared in the same manner as in example 1, but a mixture of the enzymes Amilosubtilin GX and Glucavamorin GC is added in a ratio of 3: 1 (0.375% Amylosubtilin GX and 0.125% Glucavamorin GC from the ABC substrate, i.e. 1.44 kg and 0 , 48 kg, respectively).

 The resulting fermentolizate contains PB = 3.06%.

 The quality of the final product, as in example 1.

 Crude protein content 51.0%.

 Example 7

 The bacteria Acetobacter methylicum VSB-924 are grown in a mixture with amylolytic yeast Saccharomyces cerevisiae VKPM U-1218 according to the same regimen in Example 1. Yeast is introduced into the inoculum in an amount of 10% of the total number in the inoculum (90% of bacterial cells and 10% of cells yeast).

 The fermentolizate is prepared in the same manner as in example 1. The resulting fermenolysate contains PB = 2.10%.

 At the end of the cultivation, the ratio of the number of bacterial cells and yeast remains practically unchanged and remains at the level of inoculum culture.

 The quality of the final product is close to example 1, but the amount of vitamin H is slightly lower. Crude protein content is 52%.

 Example 8

 The fermentolizate is prepared in the same manner as in example 1.

The bacteria Acetobacter methylicum VSB-924 are grown under the same conditions as in Example 1, but not in a batch, but in a continuous mode at a dilution rate of 0.2 h ^-1 at an initial concentration in medium PB of up to 2.0%, ACB = 8.0-10%.

Crude protein content 48-50%
The average sample of the finished product obtained under continuous growing conditions at a dilution rate of 0.12 to 0.2 h ^-1 has the following characteristic:
Content of components in% of ABC:
Crude protein - 48.5
Lipids - 2.5
Nucleic acids - 5.6
Carbohydrates - 12.5
Amount of amino acids - 37.2
(including tryptophan - 1.0; lysine - 2.1; methionine + cystine - 1.3, etc.).

 In addition, the sample contains vitamins of groups B, D, A, E, H, a set of micro and macro elements.

 Heavy metals Hg, Pb, Cd are absent.

 Example 9

 After growing and "dosing the biomass" obtained according to the regime of example 1 or according to the regime of example 8, it is separated from the culture fluid during evaporation. The separated liquid phase (process condensate) is completely returned to prepare the fermentolizate.

 Fereretolizate is prepared on a mixture of water and process condensate (in a 1: 1 ratio) in the same way as in example 1, but mineral salts are added in a smaller amount than in example 1, i.e.

(NH ₄ ) ₂ SO ₄ - 2.0
KH ₂ PO ₄ - 0.05
MnSO ₄ • 5H ₂ O - 0.03
CuSO ₄ • 5H ₂ O - 0.004
ZnSO ₄ • 7H ₂ O - 0.03
H ₃ BO ₃ - 0.001
Na ₂ MoSO ₄ • 2H ₂ O - 0.003
The bacteria Acetobacter methylicum VSB-924 are grown under the same conditions as in Example 1. The crude protein content is 51.0%.

 Thus, the proposed method allows due to the pre-treatment of grain production waste - bran and / or flour, etc. enzyme preparations containing in the complex a set of specific individual enzymes that allow the maximum destruction of carbon-containing raw materials and obtain a fermentolizate with nutrients available for assimilation by microorganisms. At the same time, the level of reducing substances will increase significantly to 4.8-5.0 from ABC.

 Growing high-protein bacterial strains rich in carbohydrates and other easily digestible mono-and dis-compounds makes it possible to obtain the final product with a high protein content of 50-60% (depending on the type of substrate) and a wide range of vitamins B, H, E, D, etc. , micro and macro elements.

 Returning process condensate to the production line saves water, reduces emissions into the environment and reduces the supply of mineral salts to the culture medium.

Claims (4)

1. A method of producing biomass, including the cultivation of microorganisms producing protein under conditions of aeration on a nutrient medium containing bran and / or cereal flour as a carbon source, treated with enzymes at 50 -70 ^o C for 1.0 - 1.5 h with the addition of trace elements, mineral salts and water, followed by isolation of the target product, characterized in that microorganisms use strains of methylotrophic bacteria or their associations with yeast strains having amylolytic activity.  2. The method according to p. 1, characterized in that the microorganisms producing the protein use strains of bacteria Acetobacter methylicum CMPM B-2942, or Acetobacter methylicum CMPM B-1947, or Acetobacter methylovorans CMPM B-2479 individually, or in a mixture with yeast strains with amylolytic activity, Saccharomyces cerevisiae VKPM U-1218 or Saccharomyces cerevisiae VKPM U-446.  3. The method according to claim 1, characterized in that the enzymes used are taken in the amount of 0.25 - 1.0% for the absolutely dry weight of the raw materials complex enzyme preparations Amilosubtilin GXX at pH 6.5 - 7.4, or Glucavamorin GC at pH 5.0 - 5.5, or a mixture thereof in a ratio of 1: 1 - 3: 1, respectively, at pH 5.0 - 7.4.  4. The method according to claim 1, characterized in that the liquid phase remaining after isolation of the target product is returned to the growing process.