Preparation method and application of digestible biological protein fermentation material

Abstract

The invention relates to the technical field of microbial fermentation, in particular to a preparation method and application of a digestible biological protein fermentation material. According to the fermentation method, candida utilis serves as a fermentation strain, industrial and agricultural waste glucose mother liquor and corn steep liquor serve as substrates, high-yield mycoprotein is obtained under the relative low-temperature fermentation condition of 20-25 DEG C, and the obtained fermentation liquor is subjected to single enzymolysis, so that a fermentation material easier to digest is obtained. When weaned piglets are fed with the obtained fermented material, the diarrhea rate of the piglets can be effectively reduced.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a method for preparing and applying an easily digestible biological protein fermentation feed. Background Technology

[0002] In recent years, my country has faced a shortage of protein feed and has often relied on imports. Finding new alternative protein sources has become an urgent priority. The resource utilization of single-cell protein feed has gradually attracted attention. Solid-state fermentation can achieve a protein content of up to 26%, while liquid fermentation can reach as high as 50%, showing potential for research and development in areas such as replacing fishmeal and soybean meal. Adding a certain dose of single-cell protein to the diet or using it as a partial substitute for protein raw materials has no adverse effects on animal growth performance and can maintain healthy development.

[0003] Candida utilis is a commonly used strain for producing single-cell protein feed, rich in nutrients such as protein, vitamins, minerals, and enzymes. Patent CN113455584B discloses a method for producing feed yeast pellets using corn steep liquor. The method includes the following steps: (1) Yeast plate culture: Take 1.0L of 5°Bé malt extract and add 15.0g of agar. Adjust the pH to natural and sterilize in a steam sterilizer at 121℃ for 30 minutes. After cooling, obtain a plate culture medium. Inoculate the yeast strain onto the plate culture medium under aseptic conditions using an inoculation loop. Place the inoculated plate culture medium in a biochemical incubator at 27-33℃ for 20-24 hours to obtain the yeast strain. (2) Preparation of primary seed culture: Stir 60g of tryptone, 40g of yeast extract and 60g of glucose at natural pH and dissolve in 3.8L of distilled water. After complete dissolution, transfer to a steam sterilizer and sterilize at 121℃ for 25min. After cooling, obtain a shake flask liquid culture medium. Under aseptic conditions, inoculate the yeast strain that has grown on the yeast plate in step (1) for 24 hours into the shake flask liquid culture medium and culture at 28-32℃ for 18-20h. After the culture ends, the OD value is 1.3-1.4 to obtain the primary seed culture. (3) Preparation of secondary seed culture: Take 10g of corn steep liquor. Add 20-40 kg of glucose and 100 ml of defoamer to 0-200 kg of the culture medium. Set the pH to natural and sterilize the culture medium by steaming at 121°C for 60 min in a seed tank. After cooling, the secondary seed culture medium is obtained. Inoculate the primary seed liquid prepared in step (2) onto the secondary seed culture medium with an inoculation amount of 1-2%, an aeration ratio of 1:0.3-0.5, a culture temperature of 30-34°C, a seed tank liquid volume of 65%, a fermentation time of 20-22 h, and an OD value of 1.6-1.8 at the end of fermentation to obtain the secondary seed liquid. (4) Fermentation tank production: Pump the corn syrup into a mechanically stirred aerated fermentation tank. In the middle, the natural pH; the secondary seed liquid prepared in step (3) is inoculated into the fermentation tank, the inoculation amount is 2-5%, the ventilation ratio is 1:0.5-1, the culture temperature is 30-40℃, the liquid volume of the fermentation tank is 70%, the fermentation time is 22-24h, and the pH value at the end of fermentation is 7.0-7.2; (5) Multi-effect evaporation and concentration: the fermentation product liquid of step (4) is concentrated by low temperature using a multi-effect evaporator, and the dry matter content of the evaporated liquid is 55-65%; (6) Continuous spray fluidized bed granulation: the evaporated liquid of step (5) is dried by continuous spray fluidized bed granulation to obtain feed yeast granule products.

[0004] Although the technology of producing single-cell protein feed using yeast is constantly developing, its research value still deserves further exploration. Summary of the Invention

[0005] The main objective of this invention is to provide a method for preparing easily digestible biological protein fermentation feed and its application. The method described in this invention can produce high levels of microbial protein, and the resulting fermentation broth, after single-enzymatic hydrolysis, has a high amino nitrogen content, which is more conducive to animal digestion and utilization. This invention further expands the production and application value of single-cell protein.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for preparing an easily digestible biological protein fermentation feed, the method comprising the following steps: activating Candida utilis and preparing a seed culture; inoculating the obtained seed culture into a fermentation medium and fermenting at a temperature of 20 ℃-25 ℃; after fermentation, adjusting the pH of the fermentation broth to 8.5-9.5, and adding alkaline protease for enzymatic hydrolysis, thereby obtaining the feed.

[0008] Furthermore, fermentation was carried out at a temperature of 20 ℃-22 ℃.

[0009] Further, the fermentation medium comprises the following components: 5-10 g / L glucose mother liquor, 0.5-1.5 g / L corn steep liquor, 0.1-0.15 g / L potassium dihydrogen phosphate, 0.1-0.5 g / L magnesium sulfate, 0.05%-0.1 g / L ammonium sulfate, and distilled water as solvent, with a pH value of 5.0-5.5.

[0010] Furthermore, the alkaline protease has an enzyme activity of 200,000-250,000 U / g and is added in an amount of 0.4 wt%-0.6 wt%.

[0011] Furthermore, the enzymatic hydrolysis conditions are: 50 ℃-60 ℃ for 6-8 h.

[0012] Furthermore, the seed liquid inoculation amount is 5 wt%-8 wt%.

[0013] In a second aspect, the present invention provides an easily digestible biological protein fermentation feed prepared by the method described in the first aspect above.

[0014] Furthermore, the weight ratio of amino groups in the fermentation material is >0.1%.

[0015] In a third aspect, the present invention provides an easily digestible biological protein fermented feed prepared by the method described above, and the application of the easily digestible biological protein fermented feed in the prevention of diarrhea in piglets.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] In the first aspect, the present invention has obtained a fermentation method that produces high levels of microbial protein by using Candida utilis as the fermentation strain and glucose mother liquor and corn steep liquor from industrial and agricultural waste as substrates through optimized screening. The fermentation broth is then subjected to single enzymatic hydrolysis to obtain a more easily digestible fermentation material.

[0018] Secondly, the fermented feed obtained by this invention can effectively reduce the diarrhea rate of weaned piglets when fed to them. Attached Figure Description

[0019] Figure 1 Effects of different fermentation conditions on OD value and cell number of Candida utilis fermentation broth.

[0020] Figure 2 3D diagram and contour lines showing the effect of the interaction between the three factors on the OD value.

[0021] Figure 3 3D plot and contour lines showing the effect of the interaction among the three factors on the number of Candida utilis cells.

[0022] Figure 4 The effect of different conditions on the amino nitrogen content in the enzymatic hydrolysis products.

[0023] Figure 5 3D plot and contour plot showing the effect of the interaction among the three factors on amino nitrogen content. Detailed Implementation

[0024] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0025] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0026] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0029] Example 1

[0030] A method for preparing an easily digestible biological protein fermentation feed includes the following steps: activating Candida utilis and preparing a seed culture; inoculating the obtained seed culture at 8 wt% into a fermentation medium and fermenting at 22 ℃ and 160 r / min for 20 h; after fermentation, adjusting the pH of the fermentation broth to 9, adding 0.6 wt% alkaline protease and enzymatically hydrolyzing at 56 ℃ for 6 h to obtain the easily digestible biological protein fermentation feed.

[0031] The *Candida utilis* strain used was *Candida utilis* CICC1801, and the alkaline protease used had an enzyme activity of 240,000 U / g.

[0032] The fermentation medium used was: 8.3 g / L glucose stock solution, 1.2 g / L corn steep liquor, 0.14 g / L potassium dihydrogen phosphate, 0.1 g / L magnesium sulfate, 0.1 g / L ammonium sulfate, and pH 5.5.

[0033] The number of yeast cells in the fermentation broth after fermentation was 4.73 × 10⁻⁶. 8 The amino nitrogen content in the easily digestible biological protein fermentation feed was 0.135% (CFU / mL).

[0034] Example 2

[0035] A method for preparing an easily digestible biological protein fermentation feed includes the following steps: activating Candida utilis and preparing a seed culture; inoculating the obtained seed culture at 5 wt% into a fermentation medium and fermenting at 20 ℃ and 160 r / min for 20 h; after fermentation, adjusting the pH of the fermentation broth to 9, and adding 0.4 wt% alkaline protease for enzymatic hydrolysis at 60 ℃ for 8 h to obtain the easily digestible biological protein fermentation feed.

[0036] The *Candida utilis* strain used was *Candida utilis* CICC1801, and the alkaline protease used had an enzyme activity of 240,000 U / g.

[0037] The fermentation medium used was: 8.3 g / L glucose stock solution, 1.2 g / L corn steep liquor, 0.14 g / L potassium dihydrogen phosphate, 0.1 g / L magnesium sulfate, 0.1 g / L ammonium sulfate, and pH 5.5.

[0038] The number of yeast cells in the fermentation broth after fermentation was 4.21 × 10⁻⁶. 8 The amino nitrogen content in the easily digestible biological protein fermentation feed was 0.125% (CFU / mL).

[0039] Example 3

[0040] A method for preparing an easily digestible biological protein fermentation feed includes the following steps: activating Candida utilis and preparing a seed culture; inoculating the obtained seed culture at 8 wt% into a fermentation medium and fermenting at 25°C and 160 r / min for 20 h; after fermentation, adjusting the pH of the fermentation broth to 9, adding 0.6 wt% alkaline protease and enzymatically hydrolyzing at 56°C for 8 h to obtain the easily digestible biological protein fermentation feed.

[0041] The *Candida utilis* strain used was *Candida utilis* CICC1801, and the alkaline protease used had an enzyme activity of 240,000 U / g.

[0042] The fermentation medium used was: 8.3 g / L glucose stock solution, 1.2 g / L corn steep liquor, 0.14 g / L potassium dihydrogen phosphate, 0.1 g / L magnesium sulfate, 0.1 g / L ammonium sulfate, and pH 5.5.

[0043] The number of yeast cells in the fermentation broth after fermentation was 3.95 × 10⁻⁶. 8 The amino nitrogen content in the easily digestible biological protein fermentation feed was 0.105% (CFU / mL).

[0044] Comparative Example 1

[0045] The difference from Example 1 is that after fermentation, the pH of the fermentation broth was adjusted to 7.5 and neutral protease was added. Everything else was the same as in Example 1.

[0046] The amino nitrogen content in the obtained fermentation material was 0.098%.

[0047] Comparative Example 2

[0048] The difference from Example 1 is that after fermentation, the pH of the fermentation broth was adjusted to 8.0, and neutral protease and alkaline protease (the ratio of the two enzymes was 1:1) were added. Everything else was the same as in Example 1.

[0049] The amino nitrogen content in the obtained fermentation material was 0.128%.

[0050] Experimental Example 1

[0051] 1. Materials and Methods

[0052] 1.1 Materials

[0053] Candida utilis (CICC1801) was purchased from the China Industrial Microbial Culture Collection Center. Glucose mother liquor, corn steep liquor, yeast extract, and alkaline protease (240,000 U / g) were provided by Jilin Ruisheng Technology Co., Ltd., Songyuan City, Jilin Province.

[0054] 1.2 Main Instruments and Reagents

[0055] Olympus CX43 inverted fluorescence microscope, N2S visible spectrophotometer, HWS-28 electric thermostatic water bath, FE-28 pH meter, LE3002 / 02 electronic balance, SW-CJ-2FD double-person single-sided clean bench, LEZX-50KBS vertical autoclave, TS-200DC thermostatic shaker, DNP-9052 electric thermostatic incubator, ZBM1520HPE refrigerator.

[0056] Glucose, yeast extract, peptone, yeast extract powder, ammonium sulfate (NH4)2SO4, magnesium sulfate (MgSO4), potassium dihydrogen phosphate (KH2PO4), agar, and formaldehyde were all of analytical grade.

[0057] 1.3 Culture medium

[0058] Yeast extract peptone glucose agar (YPD) medium: yeast extract 1.0 g / L, peptone 2.0 g / L, glucose 2.0 g / L, agar 1.5 g / L, natural pH.

[0059] Slant culture medium: glucose 5.0 g / L, yeast extract 2.0 g / L, potassium dihydrogen phosphate 0.2 g / L, magnesium sulfate 0.1 g / L, ammonium sulfate 0.1 g / L, agar 1.5 g / L, pH 5.5.

[0060] Seed culture medium: glucose 5.0 g / L, yeast extract 2.0 g / L, potassium dihydrogen phosphate 0.2 g / L, magnesium sulfate 0.1 g / L, ammonium sulfate 0.1 g / L, pH 5.5.

[0061] Fermentation medium: 8.3 g / L glucose mother liquor, 1.2 g / L corn steep liquor, 0.14 g / L potassium dihydrogen phosphate, 0.1 g / L magnesium sulfate, 0.1 g / L ammonium sulfate, pH 5.5.

[0062] All culture media were autoclaved at 121 °C for 21 min before use.

[0063] 1.4 Methods

[0064] 1.4.1 Activation of microbial strains

[0065] The *Candida utilis* strain preserved on slant culture was taken and inoculated onto YPD medium using the dilution-spreading method and incubated at 30 °C for 48 h for activation. Colony growth was then observed, and healthy single colonies were inoculated onto slant culture medium and incubated at 30 °C for 48 h. After the colony grew into a mycelial mass, seed culture was prepared in shake flasks.

[0066] 1.4.2 Preparation of Seed Solution

[0067] One loopful of Candida utilis was picked from the slant culture and inoculated into a 500 mL Erlenmeyer flask containing 100 mL of seed culture medium. The flask was shaken at 160 r / min and 30 °C for 20 h and then stored at 4 °C for subsequent determination of various indicators.

[0068] 1.4.3 Determination of growth indicators

[0069] OD value: The absorbance of the fermentation broth was measured at 600 nm using a spectrophotometer after being diluted 20 times with distilled water.

[0070] Yeast cell count: using a hemocytometer.

[0071] Determination of amino nitrogen: Formaldehyde titration method was used.

[0072] 1.4.2 Single-factor experiment

[0073] 1.4.2.1 Cultivation Conditions

[0074] Culture temperature: The inoculum amount was kept constant at 5% (v / v). Four temperature groups were set: 15 ℃, 20 ℃, 25 ℃, and 30 ℃. The shaking speed was 160 r / min and the fermentation time was 20 h. The OD value of the fermentation broth and the number of yeast cells were used as evaluation indicators to preliminarily screen suitable temperatures.

[0075] Shaking speed: With the inoculum amount remaining constant at 5%, four shaking speeds were set at 130 r / min, 160 r / min, 190 r / min, and 210 r / min. Fermentation was carried out at 20 ℃ for 20 h. The OD value of the fermentation broth and the number of yeast cells were used as evaluation indicators to preliminarily screen suitable shaking speeds.

[0076] Inoculation amount: Four inoculation amounts were set: 2%, 5%, 8%, and 11%. Fermentation was carried out at 20℃ and 160 r / min in a shaker for 20 h. The OD value of the fermentation broth and the number of yeast cells were used as evaluation indicators to preliminarily screen the appropriate inoculation amount.

[0077] 1.4.2.2 Enzymatic hydrolysis conditions

[0078] Experiments were conducted using optimized culture conditions to enzymatically hydrolyze the fermentation broth of Candida utilis protein feed.

[0079] Enzymatic hydrolysis temperature: The pH of the fermentation broth was adjusted to 9, and the amount of alkaline protease added was 0.4%. Four sets of temperatures were set: 45 ℃, 50 ℃, 55 ℃, and 60 ℃. After continuous stirring and enzymatic hydrolysis in a water bath for 4 h, the enzyme was boiled at 100 ℃ for 10 min to inactivate it. The amino nitrogen content of the enzymatic hydrolysate was used as the evaluation index to preliminarily screen suitable enzymatic hydrolysis temperatures.

[0080] Enzymatic hydrolysis time: The pH of the fermentation broth was adjusted to 9, the amount of alkaline protease added was 0.4%, and the water bath was set at 55 ℃. Four groups of enzymatic hydrolysis times were set: 4 h, 6 h, 8 h, and 10 h. After enzymatic hydrolysis, the enzyme was boiled at 100 ℃ for 10 min to inactivate it. The amino nitrogen content of the enzymatic hydrolysate was used as the evaluation index to preliminarily screen the suitable enzymatic hydrolysis time.

[0081] Enzyme addition amount: The pH of the fermentation broth was adjusted to 9, and four groups of alkaline protease addition amounts were set at 0.2%, 0.4%, 0.6%, and 0.8%. After continuous stirring and enzymatic hydrolysis in a water bath at 55 ℃ for 6 h, the enzyme was inactivated by boiling at 100 ℃ for 10 min. The amino nitrogen content of the hydrolysate was used as the evaluation index to preliminarily screen the appropriate enzyme addition amount.

[0082] 1.4.3 Optimization of parameters using response surface methodology

[0083] Based on single-factor experiments, Box-Behnken factor design was used to conduct three-factor, three-level response surface optimization experiments on culture conditions and enzymatic digestion conditions.

[0084] 1.5 Statistical Analysis

[0085] All data were organized using Excel 2021 and analyzed using SPSS (version 20.0) and Design-expert 12.0. A p < 0.05 was considered statistically significant.

[0086] 2 Results and Analysis

[0087] 2.1 Results of Single-Factor Experiment on Culture Conditions of Candida utilis in Protein Feed

[0088] 2.1.1 Screening of culture temperature

[0089] Depend on Figure 1 As shown in Figure A, the OD value and yeast cell number of the fermentation broth first increase and then decrease with increasing temperature. The OD value and yeast cell number reach their maximum values ​​at 20℃, which are 0.806 and 4.56 × 10⁻⁶, respectively. 8 / mL. Temperature changes can affect the activity of metabolic enzymes, cell membrane permeability, and the solubility of nutrients in yeast cells. When the temperature is below or above the optimal growth temperature for yeast cells, their growth and reproduction slow down, and their metabolic capacity is hindered. Yeast cells have the ability to adapt to low-temperature fermentation, and low-temperature fermentation can reduce production costs. Numerous studies have already discovered the potential of using yeast in food and beer production at low temperatures. OLANIRAN Studies have shown that fermenting beer at 22.5 ℃ yields the highest production. Therefore, 20 ℃ was chosen as the suitable cultivation temperature for subsequent experiments.

[0090] 2.1.2 Screening of Shaking Table Speed

[0091] Depend on Figure 1 As shown in Figure B, with the increase of the shaking speed, the OD value and yeast cell number of the fermentation broth first increase and then decrease. When the shaking speed is 160 r / min, the OD value and yeast cell number of the fermentation broth reach their maximum values, which are 0.778 and 4 × 10⁻⁶, respectively. 8 / mL. Shaking speed that is too low or too high will affect bacterial growth. Too low a shaking speed will result in low dissolved oxygen levels in the shake flask, leading to slow bacterial growth; too high a shaking speed will cause significant rotational force that damages bacterial cells, thus inhibiting bacterial growth. Therefore, a shaking speed of 160 r / min was chosen for subsequent experiments.

[0092] 2.1.3 Screening of inoculation volume

[0093] Depend on Figure 1 As shown by -C, with the increase of inoculum size, the OD value and yeast cell number of the fermentation broth showed a trend of first increasing and then decreasing. When the inoculum size was 8%, the OD value and yeast cell number of the fermentation broth reached their maximum values, which were 0.807 and 3.91 × 10⁻⁶, respectively. 8 / mL. Inoculum size is one of the important indicators affecting fermentation efficiency. When the inoculum size is too low, the bacterial count is insufficient and the nutrients in the culture medium cannot be fully utilized, resulting in slow bacterial growth. Conversely, when the inoculum size is too high, excessive consumption of nutrients means the culture medium can no longer supply the energy needed by the bacteria, also leading to slow bacterial growth. Therefore, an 8% inoculation dose was chosen for subsequent trials.

[0094] 2.2 Results of response surface methodology for the culture of Candida utilis on protein diet

[0095] 2.2.1 Results of Response Surface Methodology Tests

[0096] Based on the results of single-factor experiments, a Box-Behnken central composite design in response surface methodology was used. Three factors—culture temperature, shaking speed, and inoculum quantity—and their three levels were selected as the subjects of optimization experiments. OD value and yeast cell count were selected as response values ​​for multi-factor interaction experiments. The data were then analyzed using Design-Expert 12.0 software. The levels of each factor are shown in Table 1, the experimental groups and results are shown in Table 2, and the results of the analysis of variance are shown in Tables 3 and 4. Regression analysis was performed on the data in Table 2 to obtain the quadratic regression equation of the response value OD value on the three factors:

[0097] OD 600=0.8304+0.1884A+0.0379B+0.0068C+0.0107AB-0.0240AC-0.0010BC-0.2401A 2 -0.0471B 2 -0.0428C 2

[0098] The quadratic regression equation for the response value (yeast cell number) on the three factors was obtained:

[0099] Number of yeast cells=4.81+1.09A+0.2325B+0.0750C-0.0075AB-0.2275AC-0.0275BC-1.54A 2 -0.9002B 2 -0.5753C 2

[0100] Table 3 shows that the F-value is 1608.98 and the P-value is <0.0001, indicating that the model difference is highly significant. In the lack-of-fit term, the F-value is 0.3764 and the P-value is 0.7759 >0.05, indicating that the difference is not significant and optimization analysis can be performed. The correlation coefficient R of the model is... 2 =0.9995, indicating that the regression equation fits the changes in OD value in the fermentation broth very well, with 99.95% of the changes in OD value coming from the selected factors. Corrected coefficient of determination R0 2 adj =0.9989, indicating that the actual OD value is close to the predicted value of the regression equation, demonstrating that this equation can effectively predict and analyze the OD value in the fermentation broth. The coefficient of variation (CV%) is 0.9300, and the signal-to-noise ratio (AdeqPrecision) is 104.3816, further indicating that the model has high accuracy and reliability, and can be used to study the fermentation culture conditions of Candida utilis protein feed. The order of influence of each factor on the response value is A>B>C, where A, B, AC, and A are in the equation. 2 B 2 C 2 The effect of C on OD value was extremely significant (P<0.01), and the effects of C and AB on OD value were significant (P<0.05), while the effect of BC on OD value was not significant (P>0.05).

[0101] Table 4 shows that the F-value is 83.92 and the P-value is <0.0001, indicating that the model difference is highly significant. In the lack-of-fit term, the F-value is 1.51 and the P-value is 0.3413 >0.05, indicating that the difference is not significant and optimization analysis can be performed. The correlation coefficient R of this model is... 2=0.9908, indicating that the regression equation fits the changes in yeast cell number in the fermentation broth very well, with 99.08% of the changes in yeast cell number attributable to the selected factors. Corrected coefficient of determination R0 2 adj =0.9790, indicating that the actual yeast cell count is close to the predicted value of the regression equation, demonstrating that this equation can effectively predict and analyze the yeast cell count in the fermentation broth. The coefficient of variation (CV%) is 5.51, and the signal-to-noise ratio (Adeq Precision) is 26.2964, further indicating that the model has high accuracy and reliability, and can be used to study the fermentation culture conditions of Candida utilis protein feed. The order of influence of each factor on the response value is A>B>C, where A, B, and A are in the equation. 2 B 2 C 2 The effect of AC on yeast cell number was extremely significant (P<0.01), while the effect of AC on yeast cell number was significant (P<0.05). The effects of C, AB and BC on yeast cell number were not significant (P>0.05).

[0102] 2.2.2 Interaction Analysis of Various Factors

[0103] To further investigate the effects of culture temperature, shaking speed, and inoculum dosage on the OD value and yeast cell count of the fermentation broth, 3D plots and contour plots of the interactions between the factors were constructed based on the model regression equations. A steeper response surface curve indicates a more significant impact of the interaction between factors on the response value, while a flatter curve indicates a smaller impact. The shape of the contour lines also reflects the interaction between factors; a more elliptical shape indicates a stronger interaction and a more significant impact on the response value. .

[0104] Depend on Figure 2 -A and Figure 2 -B indicates that the slope in the 3D graph is quite steep, and the contour lines are close to an ellipse, suggesting a significant interaction between culture temperature and shaking speed, which greatly affects the OD value of the fermentation broth; Figure 2 -C and Figure 2 -D indicates that the slope of the 3D plot is significantly steep, and the contour lines are elliptical and closely spaced, indicating a highly significant interaction between culture temperature and inoculum quantity, which greatly affects the OD value of the fermentation broth; Figure 2 -E and Figure 2 As can be seen from -F, the slope of the 3D graph is gentle and the contour lines are approximately circular, indicating that the interaction between the shaking speed and the amount of bacteria added is not significant and has little impact on the OD value of the fermentation broth.

[0105] Depend on Figure 3 -A and Figure 3 -B、 Figure 3 -E and Figure 3 -F indicates that the slope of the 3D plot is gentle and the contour lines are approximately circular, suggesting that the interactions between shaking speed and inoculum addition, and between shaking speed and culture temperature, are not significant, and have little impact on the yeast cell count in the fermentation broth. Figure 3 -C and Figure 3 As indicated by -D, the slope in the 3D plot is quite steep, and the contour plot is nearly elliptical, suggesting a significant interaction between culture temperature and the amount of inoculum added, which greatly affects the yeast cell count in the fermentation broth. These results are consistent with the regression analysis of variance in the model.

[0106] The optimal fermentation conditions, predicted by quadratic regression equation and response surface variance analysis, are: a culture temperature of 22℃, a shaking speed of 160 r / min, and a strain addition of 8%. The model predicts an OD value of 0.872 and a yeast cell count of 5.02 × 10⁻⁶. 8 / mL. The above-mentioned optimal scheme was used for experimental verification. The average value of three parallel experiments was taken, resulting in an OD value of 0.848 and a yeast cell count of 4.73 × 10⁹ / mL. 8 The value was approximately 0.5 mL, which is close to the predicted value. Therefore, this model can be used to optimize the fermentation culture conditions for Candida utilis protein feed.

[0107] Table 1. Response surface methodology for optimizing fermentation culture conditions of *Candida utilis* protein feed.

[0108]

[0109] Table 2 Box-Behnken Experimental Design and Results

[0110]

[0111] Table 3. Results of ANOVA for the regression model using OD values ​​as response values.

[0112]

[0113] R 2 =0.9995, R 2 adj=0.9989, coefficient of variation (CV%)=0.9300, signal-to-noise ratio (Adeq Precision)=104.3816, ***p<0.001, **p<0.01, *p<0.05, ns: not significant.

[0114] Table 4. Results of ANOVA for the regression model with yeast cell count as the response value.

[0115]

[0116] R2 =0.9908, R 2 adj=0.9790, coefficient of variation (CV%)=5.51, signal-to-noise ratio (Adeq Precision)=26.2964, ***p<0.001, **p<0.01, *p<0.05, ns: not significant.

[0117] 2.3 Results of Single-Factor Experiment on Enzymatic Hydrolysis Conditions of Candida utilis Protein Feed

[0118] 2.3.1 Screening of enzyme addition amount

[0119] Alkaline protease was used to break down the macromolecular nutrients in *Candida utilis* protein feed into smaller, more easily digestible nutrients for animals through enzymatic hydrolysis, thereby improving protein utilization, enhancing the nutritional value of the finished product, and increasing its flavor. The amino nitrogen content indirectly indicates the degree of hydrolysis in the hydrolysate; a higher content indicates a more complete hydrolysis reaction, a greater degree of hydrolysis, and higher protein utilization of the substrate. Alkaline protease is an endonuclease that can recognize multiple amino acids and promote protein breakdown. The effect of enzyme dosage on amino nitrogen content was investigated at a hydrolysis temperature of 55 °C and a hydrolysis time of 4 h. Figure 4 As shown in Figure A, the amino nitrogen content first increases and then decreases with increasing enzyme dosage, reaching its maximum when the enzyme dosage is 0.6%. An appropriate enzyme dosage ensures sufficient contact between the enzyme and substrate for the enzymatic hydrolysis reaction. Excessive enzyme dosage leads to supersaturation of the enzyme and substrate, affecting the hydrolysis reaction. Therefore, a 0.6% enzyme dosage was chosen for subsequent experiments.

[0120] 2.3.2 Screening of enzymatic hydrolysis time

[0121] The effect of enzyme addition time on amino nitrogen content was investigated when the enzyme dosage was 0.6% and the enzymatic hydrolysis temperature was 55 ℃. Figure 4 As shown in section -B, the amino nitrogen content first increases and then decreases with increasing enzymatic hydrolysis time, reaching its maximum at 6 hours. An appropriate hydrolysis time ensures the complete enzymatic reaction; if the hydrolysis time is too long, the substrate is completely hydrolyzed, and the reaction stops. Therefore, a hydrolysis time of 6 hours was chosen for subsequent experiments.

[0122] 2.3.3 Screening of enzymatic hydrolysis temperature

[0123] The effect of enzyme dosage of 0.6% and enzymatic hydrolysis time of 6 h were investigated on the effect of enzymatic hydrolysis temperature on amino nitrogen content. Figure 4As shown by -C, the amino nitrogen content initially increases and then gradually decreases with increasing enzymatic hydrolysis temperature, reaching its maximum at 55 ℃. Alkaline protease is highly sensitive to temperature; below the optimal temperature, the enzymatic hydrolysis reaction conditions are not met; above the optimal temperature, enzyme activity decreases, the reaction rate slows, and the amino nitrogen content decreases. Therefore, an enzymatic hydrolysis temperature of 55 ℃ was chosen for subsequent experiments.

[0124] 2.4 Results of Conditional Response Surface Methodology Experiment on Enzymatic Hydrolysis of Candida utilis Protein Feed

[0125] 2.4.1 Results of Response Surface Methodology Tests

[0126] Based on the results of single-factor experiments, a Box-Behnken central composite design in response surface methodology was used. Three factors—enzyme dosage, hydrolysis time, and hydrolysis temperature—and their three levels were selected as the subjects of optimization experiments. Amino nitrogen content was chosen as the response value for a three-factor interaction experiment. The data were then analyzed using Design-Expert 12.0 software. The levels of each factor are shown in Table 5, the experimental groups and results are shown in Table 6, and the results of the analysis of variance are shown in Table 7. Regression analysis was performed on the data in Table 6, yielding the quadratic regression equation for the response value (amino nitrogen content) on the three factors:

[0127] Amino nitrogen content =0.1282+0.0018A+0.0046B+0.0106C-0.0060AB+0.0065AC-0.0042BC-0.0100A 2 -0.0192B 2 -0.0207C 2

[0128] Table 7 shows that the F-value is 30.07 and the P-value is <0.0001, indicating that the model difference is highly significant. In the lack-of-fit term, the F-value is 0.1381 and the P-value is 0.9322 >0.05, indicating that the difference is not significant and optimization analysis can be performed. The correlation coefficient R of this model is... 2 =0.9748, indicating that the regression equation fits the changes in amino nitrogen content in the enzymatic hydrolysis products very well, with 97.48% of the changes in amino nitrogen content attributable to the selected factors. Corrected coefficient of determination R0 2 adj=0.9424, indicating that the actual value of amino nitrogen content is close to the predicted value of the regression equation, demonstrating that this equation can effectively predict and analyze the amino nitrogen content in enzymatic hydrolysis products. The coefficient of variation (CV%) is 4.37, and the signal-to-noise ratio (Adeq Precision) is 16.9407, further indicating that the model has high accuracy and reliability and can be used to study the enzymatic hydrolysis conditions of Candida utilis protein feed. The order of influence of each factor on the response value is C>B>A, where C and A in the equation... 2 B 2 C 2 The effect of B on amino nitrogen content was extremely significant (P<0.01), and the effects of B, AB, and AC on amino nitrogen content were significant (P<0.05), while the effects of A and BC on amino nitrogen content were not significant (P>0.05).

[0129] 2.4.2 Interaction Analysis of Various Factors

[0130] To further investigate the effects of enzyme dosage, hydrolysis time, and hydrolysis temperature on the amino nitrogen content in the hydrolysis products, 3D response surface plots and contour plots of the interactions among the factors were drawn based on the model regression equation. Figure 5 -A, B and Figure 5 - As shown in C and D, the slope of the 3D response surface plot is relatively steep and the contour plot is also close to an ellipse, indicating that there is a significant interaction between the amount of enzyme added and the hydrolysis time, and between the amount of enzyme added and the hydrolysis temperature, which has a large impact on the amino nitrogen content. Figure 5 The 3D response surface plots of -E and F have gentle slopes and the contour plots are approximately circular, indicating that the interaction between enzymatic hydrolysis time and enzymatic hydrolysis temperature is not significant and has little impact on amino nitrogen content, which is consistent with the results of the model regression analysis of variance.

[0131] The optimal enzymatic hydrolysis conditions, predicted by quadratic regression equation and response surface variance analysis, are: enzyme dosage 0.6%, hydrolysis time 6 h, and hydrolysis temperature 56 ℃. The model predicts an amino nitrogen content of 0.130%. Experimental verification was conducted using the above optimal scheme. The average of three parallel experiments yielded an amino nitrogen content of 0.135%, which is close to the predicted value. Therefore, this model can be used to optimize the enzymatic hydrolysis conditions for Candida utilis protein feed.

[0132] Table 5. Optimization of Enzymatic Hydrolysis Conditions and Response Surface Methodology Factors and Levels for Candida utilis Protein Feed.

[0133]

[0134] Table 6 Box-Behnken Experimental Design and Results

[0135]

[0136] Table 7. Results of the model variance analysis using amino nitrogen content as the response value.

[0137]

[0138] R 2 =0.9748, R 2 adj=0.9424, coefficient of variation (CV%)=4.37, signal-to-noise ratio (Adeq Precision)=16.9407, ***p<0.001, **p<0.01, *p<0.05, ns: not significant

[0139] Experimental Example 2

[0140] 1.1 Experimental grouping and feeding management

[0141] Thirty healthy sows, one month before farrowing, were randomly divided into three groups of 10 sows each. The number of piglets born to each sow was a replicate. Piglets started eating creep feed at 7 days of age and were weaned at 21 days of age. After weaning, the sows were removed and the piglets were kept. The specific groupings are shown in Table 8.

[0142] Table 8 Experimental Groups

[0143]

[0144] 1.2 Methods

[0145] Feed from 7 days after birth until 10 days after weaning (21 days). Record growth and diarrhea from weaning to 10 days after weaning.

[0146] 2. Test Results

[0147] The diarrhea status of piglets in each group is shown in Table 9 below.

[0148] Table 9 Growth and diarrhea status of weaned piglets

[0149]

[0150] As shown in Table 9, the fermented feed described in the embodiments of the present invention helps the growth of weaned piglets and can reduce piglet diarrhea.

[0151] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing an easily digestible biological protein fermentation feed, characterized in that, Includes the following steps: The Candida utilis was activated and a seed culture was prepared. The obtained seed culture was inoculated into a fermentation medium and fermented at a temperature of 20 ℃-25 ℃. After fermentation, the pH of the fermentation broth was adjusted to 8.5-9.5, and alkaline protease was added for enzymatic hydrolysis to obtain the final product.

2. The preparation method according to claim 1, characterized in that, Fermentation is carried out at a temperature of 20 ℃-22 ℃.

3. The preparation method according to claim 1, characterized in that, The fermentation medium comprises the following components: 5-10 g / L glucose mother liquor, 0.5-1.5 g / L corn steep liquor, 0.1-0.15 g / L potassium dihydrogen phosphate, 0.1-0.5 g / L magnesium sulfate, 0.05%-0.1 g / L ammonium sulfate, and distilled water as solvent, with a pH of 5.0-5.

5.

4. The preparation method according to claim 1, characterized in that, The alkaline protease has an enzyme activity of 200,000-250,000 U / g and is added at a rate of 0.4 wt%-0.6 wt%.

5. The preparation method according to claim 1, characterized in that, Enzymatic hydrolysis was performed at 50 ℃-60 ℃ for 6-8 h.

6. The preparation method according to claim 1, characterized in that, The seed culture inoculation amount is 5 wt%-8 wt%.

7. The easily digestible biological protein fermentation feed prepared by the method according to any one of claims 1-6.

8. The easily digestible biological protein fermentation feed according to claim 7, characterized in that, The weight ratio of amino groups in the fermentation feed is >0.1%.

9. The easily digestible biological protein fermented feed prepared by the method according to any one of claims 1-6, and the application of the easily digestible biological protein fermented feed according to claim 7 or 8 in the prevention of diarrhea in piglets.

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