Fermentation method for producing single-cell protein from corn straw

Abstract

The invention provides a fermentation method for producing single-cell protein from corn straw. The fermentation method comprises the following steps: (1) inoculating a microbial seed solution into a crushed corn straw solid culture medium; (2) after the solid-state fermentation is completed, adding water, and reacting at 65-85 DEG C for 4-8 hours; and (3) cooling the liquid culture medium, and then inoculating a microbial seed solution for fermentation. By adopting the method, the yield of single-cell protein can be up to 142.68 g / L, and the yield of crude protein can be up to 44.8%. Compared with direct fermentation of the corn straw, the yield of single-cell protein and the content of crude protein can be remarkably increased, and the fermentation method for achieving efficient utilization of the corn straw is provided.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation, and in particular relates to a fermentation method for producing single-cell protein from corn stalks. Background Art

[0002] Single-cell protein, also known as microbial protein or bacterial protein, is a protein resource obtained by large-scale cultivation of microorganisms using industrial and agricultural by-products as substrates. It has the advantages of low-cost raw materials, small footprint, no time and space restrictions, and large-scale production. It also has high nutritional value and is an excellent substitute for protein feed. At present, the main problem of single-cell protein is that the source of cheap matrix raw materials is unstable and the composition is complex, resulting in unstable purity and impurities of single-cell protein obtained by fermentation; and the cost of using a single raw material to produce single-cell protein is relatively high. Therefore, finding a stable and cheap substrate and a stable fermentation method are key issues in this field.

[0003] Corn straw is a major agricultural and sideline product, with the main components being cellulose, hemicellulose and lignin. It can be converted into sugars, small molecule acids, alcohols and other substances through chemical, physical and biological methods, and is a waste resource with great potential. The key to developing and utilizing straw resources is to increase the degradation rate of cellulose and hemicellulose. Generally, the degradation rate is increased by enzymatic hydrolysis after pretreatment by gas explosion and acid hydrolysis, but gas explosion increases the cost of equipment, and the wastewater from acid hydrolysis pollutes the environment. The cost of commercial enzymes is also an important factor limiting the utilization of straw. The use of microorganisms in the natural environment that have the natural ability to degrade cellulose substances, such as Aspergillus niger, Fusarium, Penicillium, white rot fungi, brown rot fungi, etc., reduces costs and process complexity, but the structure of straw is complex, and pure strains have poor degradation ability, so mixed bacterial fermentation has a better effect. The Chinese patent application with publication number CN113729110A discloses a method for solid-state fermentation of corn stalks using Trichoderma reesei and Aspergillus niger. The crude protein content of corn stalks after gas explosion treatment for 7 days is 28.25%; the Chinese patent application with publication number CN114395490A discloses a method for solid-state fermentation of corn stalks using Neurospora crassa and Aspergillus niger. The crude protein content of corn stalks after 7 days of solid-state fermentation is 28.94%. The Chinese patent application with publication number CN114410487A discloses a dominant yeast strain that produces bacterial protein using rice straw saccharification liquid. The dry weight after liquid fermentation is up to 5.42g / L; the Chinese patent application with publication number CN116333899A discloses a method for producing straw single-cell protein using straw as raw material. After liquid fermentation of Candida utilis and Aspergillus niger using straw polysaccharide liquid as substrate, the protein content is up to 27.7%.

[0004] In summary, the yield of single-cell protein and crude protein content of the existing methods are still relatively low, and new processes need to be developed to increase the yield and content. Summary of the invention

[0005] The inventors of the present application have discovered that corn stalks, under the action of microorganisms, will produce harmful substances such as formic acid and acetic acid that inhibit subsequent fermentation and the growth of microorganisms, resulting in a low efficiency in degrading cellulose-like substances, affecting the biological utilization of stalk-like substances. At the same time, the fibers in the stalks cannot be effectively utilized, and the protein improvement rate is very low. Further research has found that some special microorganisms can continue to grow and ferment in the presence of inhibitors such as acetic acid, thereby developing a combined fermentation process to complete the present invention.

[0006] The object of the present invention is to provide a fermentation method for producing single-cell protein from corn stalks, which can greatly increase the yield and crude protein content of the single-cell protein.

[0007] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0008] A fermentation method for producing single-cell protein from corn stalks, characterized in that it comprises the following steps:

[0009] 1) inoculating a first microbial seed solution into a crushed corn stalk solid medium for solid fermentation;

[0010] 2) After solid-state fermentation is completed, water is added for high-temperature reaction to obtain a liquid culture medium;

[0011] 3) After the liquid culture medium is cooled, the second microbial seed liquid is added to carry out liquid fermentation to obtain single cell protein.

[0012] In some specific embodiments, the crushed corn stalks in step 1) are sieved through a 20-mesh sieve; preferably, the particle size of the crushed corn stalks is 12-16 mesh, such as 13 mesh, 14 mesh, 15 mesh, etc.

[0013] In some specific embodiments, the moisture content of the crushed corn stalk solid culture medium is in the range of 40wt%-60wt%, such as 45wt%, 50wt%, 55wt%, etc., preferably 40-45wt%.

[0014] In some specific embodiments, the first microbial seed liquid activated with LB medium is inoculated in the crushed corn stalk solid medium, and the inoculation method can refer to the prior art and is not particularly limited. In the present invention, the microorganism of the inoculated first microbial seed liquid is selected from one or more of Aspergillus niger, Trichoderma, Fusarium, Penicillium, white rot fungi, brown rot fungi, and soft rot fungi, preferably selected from any one of Aspergillus niger, Trichoderma, and white rot fungi. There is no particular limitation on the preparation method of the microbial seed liquid, for example, it can be prepared by using the conventional method of activating strains with LB medium in the fermentation field.

[0015] In some specific embodiments, solid-state fermentation is carried out under the following conditions: the inoculation amount is 2-20wt%, for example 5-10wt%, 5-15wt% or 10-20wt%, etc., preferably 10-15wt%; the fermentation temperature is 25-40°C, for example 25-35°C or 28-38°C, etc., preferably 30-37°C; the fermentation time is 48-96h, for example 50h, 70h or 90h, preferably 60-72h.

[0016] In some specific embodiments, after step 1) solid-state fermentation is completed, water is added for further high-temperature reaction to obtain a liquid culture medium. In the present invention, the high-temperature reaction is carried out under the following conditions: the amount of water added is 8 to 15 times the weight of the solid culture medium, such as 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, etc., preferably 10 to 12 times; the reaction temperature is 65 to 85°C, such as 70°C, 75°C, 80°C, etc., preferably 70 to 75°C; the reaction time is 4 to 8h, such as 5h, 6h, 7h, etc., preferably 5 to 6h.

[0017] In some specific embodiments, the liquid culture medium obtained by the high temperature reaction is cooled and then inoculated with a second microbial seed liquid activated with LB medium for liquid fermentation to obtain single cell protein. In the present invention, the microorganism of the inoculated second microbial seed liquid is at least any one of Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, Kluyveromyces or Candida utilis, preferably any one of Candida utilis, Yarrowia lipolytica and Saccharomyces cerevisiae.

[0018] In some specific embodiments, liquid fermentation is carried out under the following conditions: the inoculation amount is 1-10%, for example, 1-5%, 3-8% or 5-10%, etc., preferably 4-6wt%; the fermentation temperature is 25-35°C, for example, 25-30°C or 30-35°C, etc., preferably 28-32°C; the fermentation time is 48-72h, for example, 50h, 60h or 70h, etc., preferably 55-60h.

[0019] The method of the present invention firstly performs solid-state fermentation to preliminarily enzymatically hydrolyze corn stalks to produce acid, then further decomposes cellulose-like substances by high temperature, converts them into small-molecule sugars and acids, and finally converts them into single-cell proteins by liquid fermentation. Although the present invention combines three steps of solid-state fermentation, high-temperature hydrolysis, and liquid fermentation, and the process is slightly complicated, the single-cell protein yield and crude protein produced by this method are significantly improved, providing a new method for high-value utilization of corn stalks.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The method of the present invention first solid-state ferments and acidifies the crushed corn stalks, then further hydrolyzes the stalks at 65-85°C, and then uses the stalks as a liquid fermentation substrate for liquid fermentation to prepare single-cell protein. The single-cell protein yield and crude protein content obtained by this method are significantly improved, which has positive significance for the utilization of corn stalks. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited to the above contents. In addition, the preferred ranges of the various technical means described in the text can be combined with each other at will.

[0023] Unless otherwise specified, the reagents and instruments used in the following methods are commonly used reagents and instruments in the art and can be obtained commercially; the methods used are conventional methods in the art, and those skilled in the art can undoubtedly perform the methods and obtain corresponding results based on the contents described in the embodiments.

[0024] Embodiment 1:

[0025] (1) crush corn stalks and pass them through a 16-mesh sieve;

[0026] (2) adjusting the moisture content of 100 g of the corn stalks crushed in step (1) to 40%;

[0027] (3) The solid culture medium in step (2) is inoculated with Aspergillus niger, Trichoderma spp., and white rot fungi, with the inoculation amounts being 2%, 5%, and 3%, respectively, and the fermentation temperature being controlled at 37° C. for 60 h.

[0028] (4) After solid-state fermentation, add 1000 g of water and heat to 70 °C for 5 h;

[0029] (5) cooling the liquid culture medium in step (4) to 30° C. and inoculating Saccharomyces cerevisiae at an inoculation amount of 2%;

[0030] (6) Liquid fermentation pH was controlled at 6.5, temperature was controlled at 30°C, and fermentation was carried out for 50 h;

[0031] (7) Fermentation broth OD in step (6) 600 It reached 312, and after centrifugation at 4000 rpm for 3 min, the precipitate was dried at 80 °C to constant weight, and the dry weight was measured to be 134.67 g / L;

[0032] (8) The crude protein content of the dried sample in step (7) was determined by Kjeldahl method, and the crude protein content was 40.3%.

[0033] Embodiment 2:

[0034] (1) crush corn stalks and pass them through a 12-mesh sieve;

[0035] (2) adjusting the moisture content of 100 g of the corn stalks crushed in step (1) to 50%;

[0036] (3) The solid culture medium in step (2) is inoculated with Trichoderma, Fusarium, and soft rot fungi, with the inoculation amounts being 8%, 2%, and 5%, respectively, and the fermentation temperature being controlled at 35° C. for 72 hours.

[0037] (4) After solid-state fermentation, add 1200 g of water and heat to 80 °C for 4 h;

[0038] (5) cooling the liquid culture medium in step (4) to 25° C. and inoculating Pichia pastoris and Kluyveromyces in an inoculum amount of 2% and 5%, respectively;

[0039] (6) Liquid fermentation pH was controlled at 6.8, temperature was controlled at 25°C, and fermentation was carried out for 60 h;

[0040] (7) Fermentation broth OD in step (6) 600 reached 284, and after centrifugation at 4000 rpm for 3 min, the resulting precipitate was dried at 80 °C to constant weight, and the dry weight was measured to be 117.39 g / L;

[0041] (8) The crude protein content of the dried sample in step (7) was determined by Kjeldahl method, and the crude protein content was 38.2%.

[0042] Embodiment 3:

[0043] (1) crush corn stalks and pass them through a 12-mesh sieve;

[0044] (2) adjusting the moisture content of 100 g of the corn stalks crushed in step (1) to 45%;

[0045] (3) The solid culture medium in step (2) is inoculated with Aspergillus niger, Trichoderma spp., and white rot fungi, with the inoculation amounts being 6%, 5%, and 2%, respectively, the fermentation temperature being controlled at 30° C., and the fermentation is performed for 72 hours.

[0046] (4) After solid-state fermentation, add 1200 g of water and heat to 75 °C for 5 h;

[0047] (5) cooling the liquid culture medium in step (4) to 30° C. and inoculating Yarrowia lipolytica with an inoculation amount of 5%;

[0048] (6) Liquid fermentation pH was controlled at 6.5, temperature was controlled at 30°C, and fermentation was carried out for 55 h;

[0049] (7) Fermentation broth OD in step (6) 600 reached 336, and after centrifugation at 4000 rpm for 3 min, the resulting precipitate was dried at 80 °C to constant weight, and the dry weight was measured to be 142.68 g / L;

[0050] (8) The crude protein content of the dried sample in step (7) was determined by Kjeldahl method, and the crude protein content was 44.8%.

[0051] Comparative Example 1: Solid-state fermentation only

[0052] (1) crush corn stalks and pass them through a 12-mesh sieve;

[0053] (2) adjusting the moisture content of 100 g of the corn stalks crushed in step (1) to 45%;

[0054] (3) The solid culture medium in step (2) is inoculated with Aspergillus niger, Trichoderma spp., and white rot fungi, with the inoculation amounts being 6%, 5%, and 2%, respectively, the fermentation temperature being controlled at 30° C., and the fermentation is performed for 72 h.

[0055] (4) After the fermentation product in step (3) was dried to constant weight at 80°C, the dry weight was measured to be 88.78 g / L;

[0056] (5) The crude protein content of the corn stalks from step (1) and the dried sample from step (4) was determined by Kjeldahl method, and the crude protein content was 3.64% and 20.5%, respectively.

[0057] Comparative Example 2: Solid-state fermentation plus high-temperature hydrolysis

[0058] (1) crush corn stalks and pass them through a 12-mesh sieve;

[0059] (2) adjusting the moisture content of 100 g of the corn stalks crushed in step (1) to 45%;

[0060] (3) The solid culture medium in step (2) is inoculated with Aspergillus niger, Trichoderma spp., and white rot fungi, with the inoculation amounts being 6%, 5%, and 2%, respectively, the fermentation temperature being controlled at 30° C., and the fermentation is performed for 72 h.

[0061] (4) After solid-state fermentation, add 1200 g of water and heat to 75 °C for 5 h;

[0062] (5) The liquid in step (6) was centrifuged at 4000 rpm for 3 min, and the resulting precipitate was dried at 80° C. to a constant weight, and the dry weight was measured to be 68.23 g / L;

[0063] (6) The crude protein content of the dried sample in step (5) was determined by Kjeldahl method, and the crude protein content was 28.9%.

[0064] Comparative Example 3: High temperature hydrolysis plus liquid fermentation

[0065] (1) crush corn stalks and pass them through a 12-mesh sieve;

[0066] (2) taking 100 g of the corn stalks crushed in step (1), adding 1200 g of water, heating to 75° C. and keeping warm for 5 h;

[0067] (3) cooling the liquid culture medium in step (2) to 30° C. and inoculating Yarrowia lipolytica at an inoculation amount of 5%;

[0068] (4) Liquid fermentation pH was controlled at 6.5, temperature was controlled at 30°C, and fermentation was carried out for 55 h;

[0069] (5) Fermentation broth OD in step (4) 600 reached 236, and after centrifugation at 4000 rpm for 3 min, the resulting precipitate was dried at 80 °C to constant weight, and the dry weight was measured to be 86.21 g / L;

[0070] (6) The crude protein content of the dried sample in step (5) was determined by Kjeldahl method, and the crude protein content was 6.7%.

[0071] The above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the embodiments here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A fermentation method for producing single-cell protein from corn stalks, characterized in that: The following steps are involved: 1) inoculating a first microbial seed solution into a crushed corn stalk solid medium for solid fermentation; 2) After solid-state fermentation is completed, water is added for high-temperature reaction to obtain a liquid culture medium; 3) After the liquid culture medium is cooled, the second microbial seed liquid is added to carry out liquid fermentation to obtain single cell protein.

2. The fermentation method according to claim 1, characterized in that The crushed corn stalks in step 1) are passed through a 20-mesh sieve; Preferably, the particle size of the crushed corn stalks is between 12 and 16 meshes.

3. The fermentation method according to claim 1 or 2, characterized in that: The water content of the solid culture medium in step 1) is in the range of 40-60wt%, preferably 40-45wt%.

4. The fermentation method according to any one of claims 1 to 3, characterized in that The microorganisms of the first microorganism seed liquid inoculated in step 1) are selected from one or more of Aspergillus niger, Trichoderma, Fusarium, Penicillium, white rot fungi, brown rot fungi, and soft rot fungi.

5. The fermentation method according to claim 4, characterized in that The microorganism of the first microorganism seed liquid inoculated in step 1) is selected from any one of Aspergillus niger, Trichoderma, and white rot fungi.

6. The fermentation method according to any one of claims 1 to 5, characterized in that In step 1), solid-state fermentation is carried out under the following conditions: The inoculum amount is 2 to 20 wt%, preferably 10 to 15 wt%; and / or The fermentation temperature is 25-40°C, preferably 30-37°C; and / or The fermentation time is 48 to 96 hours, preferably 60 to 72 hours.

7. The fermentation method according to claim 1, characterized in that In step 2), a high temperature reaction is carried out under the following conditions: The amount of water added is 8 to 15 times, preferably 10 to 12 times, the weight of the solid culture medium in step 1); and / or The reaction temperature is 65 to 85°C, preferably 70 to 75°C; and / or The reaction time is 4 to 8 hours, preferably 5 to 6 hours.

8. The fermentation method according to claim 1, characterized in that The microorganism of the second microorganism seed solution introduced in step 3) is selected from at least any one of Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, Kluyveromyces or Candida utilis.

9. The fermentation method according to claim 8, characterized in that The microorganism of the second microorganism seed solution introduced in step 3) is selected from any one of Candida utilis, Yarrowia lipolytica and Saccharomyces cerevisiae.

10. The fermentation method according to claim 7, characterized in that In step 3), liquid fermentation is carried out under the following conditions; The inoculation amount is 1 to 10 wt%, preferably 4 to 6 wt%; and / or The fermentation temperature is 25-35°C, preferably 28-32°C; and / or The fermentation time is 48 to 72 hours, preferably 55 to 60 hours.