CN117736887A

CN117736887A - Corn byproduct fermentation process, fermentation culture and application thereof in feed

Info

Publication number: CN117736887A
Application number: CN202311696735.8A
Authority: CN
Inventors: 卢煜; 曹国强; 郭依萍; 李爱军; 任道平; 杨海博; 程延庆
Original assignee: Harbin Xiangbai Biotechnology Co ltd
Current assignee: Harbin Xiangbai Biotechnology Co ltd
Priority date: 2023-12-12
Filing date: 2023-12-12
Publication date: 2024-03-22

Abstract

A corn byproduct fermentation process, a fermentation culture and application thereof in feed belong to the technical field of fermentation. Aiming at the technical problems that the utilization rate of corn byproducts is low, the growth of saccharomyces cerevisiae is inhibited by a corn byproduct fermentation process, and the fermentation efficiency is low, the invention provides a corn byproduct fermentation process, which is characterized in that corn byproducts are used as raw materials for fermentation, crystalline sugar mother liquor is added according to a certain flow rate when the dissolved oxygen is 80%, the pH value is raised when the ammonium ion concentration is lower than 0.05%, and the dissolved oxygen, the tank pressure, the air quantity, the temperature and the pH value in a fermentation tank are maintained to be stable; simultaneously provides a yeast culture obtained by the fermentation process and application of the yeast culture in feed. The invention solves the problem that the growth of Saccharomyces cerevisiae is restrained in the corn byproduct fermentation process, so that the corn byproduct is fully utilized, and the provided yeast culture has the function of obviously improving the nutrition components of the corn byproduct fermentation feed.

Description

Corn byproduct fermentation process, fermentation culture and application thereof in feed

Technical Field

The invention belongs to the technical field of fermentation, and particularly relates to a corn byproduct fermentation process, a fermentation culture and application thereof in feed.

Background

Currently, about 2500 ten thousand tons of corn are used for deep processing every year in China, and a large amount of byproducts including corn steep liquor, corn steep water and crystallized sugar mother liquor are generated in the process. The corn soaking water is industrial wastewater generated in the corn processing process, and contains a certain nutrient component for irrigation, animal husbandry production and other purposes; corn steep liquor is a product obtained by concentrating corn steep water, is a byproduct generated in the corn starch processing process, contains a large amount of nutrient substances such as soluble proteins, amino acids, vitamins and the like, and can be used as a low-cost nitrogen source for microbial fermentation; the process of producing crystalline sugar by using corn starch can also produce a large amount of crystalline sugar mother liquor, the dry matter yield can be up to 50%, and the crystalline sugar mother liquor contains abundant material resources and can be used as fermentation raw materials of fermentation products. In the prior art, corn byproducts are used as carbon sources and nitrogen sources in the strain fermentation process, and the growth of saccharomyces cerevisiae is restrained in the fermentation process, so that the fermentation efficiency is greatly reduced, and therefore, development of a fermentation process using the corn byproducts as raw materials is needed to be carried out, so that the corn byproducts are utilized to the maximum extent.

Disclosure of Invention

The invention provides a corn byproduct fermentation process, a fermentation culture and application thereof in feed, which are used for solving the problems of low utilization rate of corn byproducts, growth inhibition of saccharomyces cerevisiae by the corn byproduct fermentation process and low fermentation efficiency in the prior art.

The specific technical scheme of the invention is as follows:

one of the purposes of the present invention is to provide a fermentation process for corn by-products, comprising the steps of:

inoculating yeast into a fermentation tank added with 40% of base material for fermentation, automatically supplementing the crystallized sugar mother liquor for 1s according to the flow rate of 0.5-3g/min when the dissolved oxygen is 80%, and raising the pH value by 0.2 when the ammonium ion concentration is lower than 0.05%, so as to maintain stable dissolved oxygen, tank pressure, air quantity, temperature and pH value in the fermentation tank.

Further defined, the yeast is any one of candida utilis or saccharomyces cerevisiae.

Further defined, the yeast is inoculated in an amount of 10-12%.

Further defined, the base stock includes a nitrogen source, 2g/L magnesium sulfate, 0.05mg/L VB12, a trace element mixed mother liquor, 12g/L monopotassium phosphate, and 2g/L ammonium sulfate.

Further defined, the mass percentages of the magnesium sulfate, VB12, the microelement mixed mother solution, the monopotassium phosphate and the ammonium sulfate are (0.1-0.5%): (0.2-0.5%): (0.2-0.6%): (0.3-0.7%): (0.1-0.5%).

Further defined, the nitrogen source is any one of corn steep liquor or corn steep liquor.

Further defined, the corn steep liquor concentration is 20% and the addition is 75-150g/L.

Further defined, the corn steep water is added at 90%.

Further defined, the microelement mixed mother liquor comprises 0.15232g/L ferrous sulfate heptahydrate, 0.009478g/L manganese sulfate monohydrate, 0.04g/L sodium sulfate, 0.0128g/L zinc sulfate, 0.008g/L cobalt chloride hexahydrate and 0.0012g/L copper sulfate pentahydrate with the mass percentage of 0.015232%:0.000948%:0.004%:0.00128%:0.0008%:0.00012%.

Further defined, the crystallized sugar mother liquor concentration is 50%

Further limited, the dissolved oxygen amount is stabilized at 47-60%, the tank pressure is stabilized at 0.04-0.09MPa, the air quantity is stabilized at 2-8L/h, and the temperature is stabilized at 28-30 ℃.

Further defined, the pH is stabilized between 4.5 and 5.5 during the fermentation process.

Further defined, the pH control method is: the supplemental lowering of the pH of ammonium sulfate is performed when the pH is raised to or above 5.5, and the supplemental raising of the pH of ammonia is performed when the pH is lowered to or below 4.5.

It is a second object of the present invention to provide a yeast culture prepared by the above fermentation process.

The invention also aims to provide an application of the yeast culture in preparing fermented feed.

The invention has the beneficial effects that:

(1) The fermentation process provided by the invention controls the dosage of the nitrogen source and the flow acceleration of the carbon source crystallization sugar mother liquor, on one hand, because the corn steep liquor contains a large amount of inorganic salts and toxins, the growth of saccharomycetes can be inhibited under the condition of too high content; on the other hand, the carbon source can be utilized in the growth of the strain in the fermentation process to generate a large amount of ethanol, so that the growth of saccharomycetes is inhibited, and the flow acceleration of the carbon source crystal sugar mother liquor is controlled to avoid the utilization of a large amount of carbon source in the growth of the strain, so that the low-sugar fermentation is kept, the conversion efficiency of the carbon source is ensured to be maximized, the generation of ethanol is reduced, and the problem that the growth of the strain is inhibited in the corn byproduct fermentation process is solved.

(2) The yeast culture obtained by the fermentation process provided by the invention can effectively improve the crude protein and crude fat content in the feed, reduce the crude fiber content in the feed, has the function of obviously improving the nutrition components of the corn byproduct fermented feed, and can be applied to the preparation of the corn byproduct fermented feed.

(3) The corn byproduct fermentation process provided by the invention has the advantages of low cost and higher fermentation efficiency, so that the corn byproduct is fully utilized, and the resource waste is avoided.

Detailed Description

Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included herein. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the methods and applications described herein can be modified or adapted and combined to implement and utilize the technology of this invention without departing from the spirit and scope of this invention.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to specific embodiments. The experimental methods used in the examples below were conventional, and the materials, reagents, methods and apparatus used, unless otherwise indicated, were all conventional in the art and commercially available to those skilled in the art.

(1) Preparing a microelement mixed mother solution: the proportion of 0.15232g/L ferrous sulfate heptahydrate, 0.009478g/L manganese sulfate monohydrate, 0.04g/L sodium sulfate, 0.0128g/L zinc sulfate, 0.008g/L cobalt chloride hexahydrate and 0.0012g/L copper sulfate pentahydrate is 0.015232 percent: 0.000948%:0.004%:0.00128%:0.0008%:0.00012%.

(2) Seed culture medium: peptone 20g/L, glucose 20g/L, yeast extract 10g/L, pH=6.5.

(3) Shake flask medium: 5g/L yeast extract powder, 10g/L peptone, 2g/L trisodium citrate, 2g/L magnesium sulfate, 12g/L anhydrous dipotassium hydrogen sulfate, 2 drops/L dichlord, and pH=8.2.

(4) The corn steep liquor, crystallized sugar liquor and the above culture medium used in the examples were sterilized at 121℃under pressure of 103.4kPa for 20 minutes, and cooled to room temperature after sterilization.

(5) Strain sources: saccharomyces cerevisiae 1 was purchased from the Beijing challenge group;

saccharomyces cerevisiae 2 was purchased from China center for type culture Collection of microorganisms, and is numbered CICC 1558;

candida utilis is purchased from the China center for type culture collection of industrial microorganisms, and is numbered as cic 31170.

Example 1:

inoculating strains stored in a 200 mu glycerol tube into a seed culture medium, wherein the strains are Saccharomyces cerevisiae 1, saccharomyces cerevisiae 2 and candida utilis respectively, and shake culturing at 290rpm for 16 hours at 30 ℃ to obtain corresponding seed solutions respectively; then, 10% seed solution was inoculated into the shake flask medium, 0.5% corn steep liquor was added to each shake flask, and after 24 hours of cultivation.

In order to compare the growth potential of different strains in corn steep liquor, the corn steep liquor was used as the only limiting factor, and the absorbance at 660nm was measured by an ultraviolet spectrophotometer, and the result is shown in Table 1, wherein the higher the OD value is, the higher the strain concentration, the more vigorous the growth, and the absorbance at 660nm is, wherein Saccharomyces cerevisiae 2 and Candida utilis are the highest, so Saccharomyces cerevisiae 2 and Candida utilis are selected for the subsequent fermentation test, and the following examples use Saccharomyces cerevisiae 2.

TABLE 1 corn steep liquor shaking flask strain preliminary screening results

Strain name	OD ₆₆₀
		Saccharomyces cerevisiae 1	9.9
Saccharomyces cerevisiae 2	11.39
		Candida utilis	14.93

Example 2:

inoculating 10% candida utilis seed solution into a fermentation tank added with 40% base material for fermentation, (adding 75g/L corn steep liquor, 0.3% magnesium sulfate, 0.35% vitamin mixed mother liquor, 0.45% trace element mixed mother liquor, 0.6% potassium dihydrogen phosphate and 0.4% ammonium sulfate into the base material), automatically supplementing crystalline sugar mother liquor for 1s according to the flow rate of 0.5-3g/min when the dissolved oxygen is 80%, raising the pH value to 0.2 when the ammonium ion concentration is lower than 0.05%, carrying out ammonium sulfate supplementation to reduce the pH value when the pH value is raised to or above 5.5, carrying out ammonia water supplementation to raise the pH value when the pH value is lowered or lower than 4.5, stabilizing the dissolved oxygen amount at 47-60%, stabilizing the tank pressure at 0.04-0.09MPa, stabilizing the air amount at 2-8L/h, and continuously monitoring the wet weight and dry weight of the strain in the fermentation culture process.

Example 3:

this example differs from example 2 in that 150g/L corn steep liquor was added to the base stock, the other things being the same as in example 2.

Example 4:

this example differs from example 2 in that 150g/L corn steep liquor was added to the base stock, and in that corn steep liquor was continuously added during fermentation at a flow rate of 0.5mL/min, the total amount of corn steep liquor added was 150-200g/L, otherwise the same as in example 2.

Example 5:

this example differs from example 2 in that the corn steep liquor was replaced with corn steep water in the base stock in an amount of 90% and the other things were the same as in example 2.

Example 6:

this example differs from example 5 in that the added yeast is Saccharomyces cerevisiae, and the other is the same as example 5.

Example 7:

the corn byproduct was crushed and sieved with a 30-mesh sieve to be used as a fermented feed substrate, and the fermentation culture obtained in example 2 was sprayed onto the fermented feed substrate at a mass ratio of 10%, uniformly mixed, and fermented at 30 ℃ for 5d.

After fermentation, the crude protein content is measured by a Kjeldahl nitrogen method according to the national standard of GB/T6432-2018, the crude fat content in the feed is measured according to the standard of GB 2906-82, and the crude fiber content is measured according to the method of GB/T6463-2006.

Example 8:

this example differs from example 7 in that the added fermentation culture was obtained in example 5, the other being the same as in example 7.

Example 9:

this example differs from example 7 in that the added fermentation culture was obtained in example 6, the other being the same as in example 7.

Comparative example 1:

the embodiment is a high-density culture method of saccharomyces cerevisiae provided in patent CN 108220175B, which specifically comprises the following steps: removing residues from the molasses, precipitating, acidifying and hydrolyzing with sulfuric acid to obtain fermented molasses with 25% of reducing sugar weight, and adding water into yeast extract powder to obtain 25% solution; then 10% of saccharomyces cerevisiae seed liquid is inoculated into a fermentation tank added with 60% of base material, the fermentation temperature is stabilized at 30 ℃, the tank pressure is stabilized at 0.03-0.15MPa, the ventilation ratio is stabilized at 1.6-2.5VVM, and the dissolved oxygen is stabilized at 45-70%; the pH is stabilized at 4.2-6.0 by regulating and controlling the flow rate of carbon source molasses and nitrogen source YE in the fermentation process, the flow rate of molasses is 5-35mL/min, the flow rate of YE is 0.5-2mL/min, and the wet weight/dry weight of strains is continuously monitored in the fermentation culture process;

comparative example 2:

this example differs from comparative example 1 in that YEs was replaced with corn steep liquor as the nitrogen source, molasses was replaced with crystallized sugar mother liquor as the carbon source, and the other is the same as in example 2.

Comparative example 3:

this example differs from example 7 in that the added fermentation culture is a commercially available Angel brand yeast culture, which is added to the fermented feed substrate in a mass ratio of 5% and mixed uniformly, otherwise in the same manner as in example 7.

Comparative example 4:

this example differs from example 7 in that the fermentation culture added was the fermentation culture obtained in comparative example 1, except that it was the same as in example 7.

1. Detection of yeast fermentation efficiency

(1) The wet weight and dry weight of the strain in the culture process are continuously monitored, and the results are shown in table 2, wherein the fermentation process in comparative example 1 and comparative example 2 has a large difference in wet weight of the strain after 24 hours of culture due to different nitrogen sources and carbon sources; it is observed that the fermentation process of the strain of comparative example 2 consumes sugar, does not split (does not bud), and the trace elements, the compound vitamins, the phosphate, the magnesium salt, the calcium salt, the zinc sulfate and the like are added in the fermentation process, so that the fermentation of the strain is promoted, and the fermentation tail gas contains a large amount of volatile smell of alcohol.

Analysis of results: example 2 and comparative example 2 prove that the fermentation process provided by the invention stops continuous flow of nitrogen source and controls the flow rate of carbon source at the same time, and under the condition of ensuring effective replenishment of nitrogen source, the normal growth of saccharomycetes is not affected, a large amount of carbon source is prevented from being utilized to generate metabolic product ethanol in the growth of the strain, the growth of saccharomycetes is inhibited, and the conversion efficiency of the carbon source is maximized.

(2) The fermentation process provided by the invention is used for fermenting candida utilis, the strain fermentation result is shown in table 2, and compared with example 2, the nitrogen source corn steep liquor addition amount is increased in example 3, the dry weight content of the saccharomycetes is reduced by 27.5% and the wet weight content is reduced by 28.7% during fermentation for 60 hours; example 4 continuous feeding of nitrogen source corn steep liquor during fermentation, and strain autolysis during 29h fermentation, one reason may be that the high extracellular osmotic pressure of yeast caused by too high inorganic salt content in corn steep liquor, has an inhibitory effect on growth of yeast; another reason may be that the corn steep liquor contains toxins such as vomitoxin, which have a certain inhibiting effect on the growth of yeast cells.

(3) Example 5 selects cheaper corn steep water instead of corn steep liquor as nitrogen source, and compared with example 2, the method finds that the strain wet weight can reach more than 500g/L when fermenting for 60 hours, and the difference is not obvious, which shows that the method has higher fermentation efficiency, but the corn steep water is cheaper and has lower cost.

(4) Example 6 the substitution of candida utilis in example 5 for saccharomyces cerevisiae resulted in the finding that under the same inoculation amount and fermentation conditions, the wet weight of the strain after 60h fermentation in example 5 and example 6 reached 500g/L, whereas the strain did not grow any more when the fermentation was completed for 60h in comparative example 1; the corn byproduct fermentation process provided by the invention is suitable for candida utilis and saccharomyces cerevisiae, and solves the problem that the growth of the saccharomyces cerevisiae is inhibited in the corn byproduct fermentation process.

TABLE 2 detection of fermentation results of bacterial species

2. Detection of nutrient content of corn byproduct fermented feed

(1) Crude protein, crude fiber, crude fat content in commercial corn byproduct feed was measured and used as a blank.

(2) The crude protein detection results show that the crude protein content in comparative examples 3-4 and examples 7-9 are significantly different from that in a control group, wherein the crude protein content in comparative example 3 is not significantly different from that in examples 7-9, but is higher than that in the control group, and the yeast culture obtained by the fermentation process provided by the invention is proved to be capable of obviously improving the crude protein nutrition component of the corn byproduct feed.

(3) The crude fat detection results show that the crude fat contents in comparative examples 3-4 and examples 7-9 are not significantly different from the control group, but are higher than the control group, wherein the crude fat contents in comparative example 3 and example 9 are not significantly different, but are increased by 42.62% and 4.16% respectively compared with the control group, and the yeast culture obtained by the fermentation process provided by the invention can obviously improve the crude fat nutrition components of the corn byproduct type feed.

(4) The crude fiber detection results show that the crude fiber content in the comparative examples 3-4 and the examples 7-9 are obviously different from that in the control group, wherein the crude fiber content in the comparative example 3 is not obviously different from that in the examples 7-9, but is higher than that in the control group, and the yeast culture obtained by the fermentation process provided by the invention can obviously improve the crude fiber nutrition component of the corn byproduct feed.

Analysis of results: the content of crude protein and crude fat in the corn byproduct type fermented feed prepared by the yeast culture obtained by the fermentation process provided by the invention is improved compared with that of the corn byproduct type feed on the market, the content of crude fiber is reduced, and compared with the nutritional ingredient data in the feed (comparative example 3) prepared by the yeast with better performance on the market, the content of the crude protein and the crude fiber is not significantly different, so that the yeast culture provided by the invention has the same fermentation effect as the yeast culture provided by the invention, and therefore, the yeast culture obtained by the fermentation process provided by the invention can be proved to be capable of obviously improving the nutritional ingredients of the corn byproduct type feed.

TABLE 3 corn byproduct fermented feed nutrient detection results

Note that: the ratio of the variation is the same as that of the blank control group

The details of the present invention which are not described in detail in the present specification are known to those skilled in the art. While the invention has been described in terms of preferred embodiments, it is not intended to be limited thereto, but rather to enable any person skilled in the art to make various changes and modifications without departing from the spirit and scope of the present invention, which is therefore to be limited only by the appended claims.

Claims

1. A corn byproduct fermentation process, comprising the steps of:

2. The fermentation process of claim 1, wherein the yeast is either candida utilis or saccharomyces cerevisiae and the inoculum size of the yeast is 10-12%.

3. The fermentation process of claim 1, wherein the base stock comprises a nitrogen source, 2g/L magnesium sulfate, 0.05mg/L VB12, a trace element mixed mother liquor, 12g/L potassium dihydrogen phosphate, and 2g/L ammonium sulfate, wherein the mass percentages of magnesium sulfate, VB12, trace element mixed mother liquor, potassium dihydrogen phosphate, and ammonium sulfate are (0.1-0.5%): (0.2-0.5%): (0.2-0.6%): (0.3-0.7%): (0.1-0.5%).

4. A fermentation process according to claim 3, wherein the nitrogen source is any one of corn steep liquor or corn steep liquor, the corn steep liquor concentration is 20%, and the addition amount is 75-150g/L; the addition amount of the corn soaking water is 90%.

5. A fermentation process according to claim 3, wherein the trace element mixed mother liquor comprises 0.15232g/L ferrous sulphate heptahydrate, 0.009478g/L manganese sulphate monohydrate, 0.04g/L sodium sulphate, 0.0128g/L zinc sulphate, 0.008g/L cobalt chloride hexahydrate and 0.0012g/L copper sulphate pentahydrate.

6. The fermentation process of claim 1, wherein the concentration of the crystallized sugar liquor is 50%.

7. The fermentation process of claim 1, wherein the dissolved oxygen is stabilized at 47-60%, the tank pressure is stabilized at 0.04-0.09MPa, the air volume is stabilized at 2-8L/h, and the temperature is stabilized at 28-30 ℃.

8. The fermentation process according to claim 1, wherein the pH is stabilized at 4.5-5.5 during the fermentation process; the control method comprises the following steps: the supplemental lowering of the pH of ammonium sulfate is performed when the pH is raised to or above 5.5, and the supplemental raising of the pH of ammonia is performed when the pH is lowered to or below 4.5.

9. A yeast culture, characterized in that it is prepared by the fermentation process of claims 1-8.

10. Use of a yeast culture according to claim 9 for the preparation of a fermented feed.