Method for simultaneously reducing antinutritional factors in safflower seed meal and increasing crude protein content and product thereof
By fermenting safflower seed meal with Ganoderma lucidum, the extracellular enzymes of Ganoderma lucidum degrade the anti-nutritional factor phytic acid, thereby increasing the protein content. This solves the problem of wasted safflower seed meal resources and achieves efficient utilization and nutritional enhancement.
Patent Information
- Application Number
- CN202511949338.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-23
- Publication Date
- 2026-01-23
AI Technical Summary
Existing technologies are insufficient to effectively reduce phytic acid, an anti-nutritional factor, in safflower seed meal and increase its protein content, leading to resource waste and environmental pollution. Traditional physical or chemical treatment methods pose risks of high energy consumption, significant nutrient loss, or chemical residues.
Using Ganoderma lucidum as the fermentation strain, safflower seed meal was fermented in both liquid and solid states. The anti-nutritional factor phytic acid was degraded by the secretion of extracellular enzymes, and the protein content was increased. After fermentation, the product was freeze-dried to obtain freeze-dried mycelium.
It significantly reduces the phytic acid content in safflower seed meal, increases the protein content, and enhances its nutritional value and bioactivity, thus achieving efficient utilization of safflower seed meal. It is suitable for high-value-added protein feed or functional food ingredients.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of agricultural microbial technology, and in particular relates to a method and product for simultaneously reducing anti-nutritional factors and increasing crude protein content in safflower seed meal. Background Technology
[0002] safflower( Carthamus tinctorius Safflower (L.) is an important oilseed crop, and its seeds, after pressing or extraction for oil production, produce a large amount of byproducts—safflower seed meal. Currently, safflower seed meal is generally added to animal feed for pigs and cattle, but a large portion of it is still discarded, causing serious resource waste and environmental pollution. Research results show that safflower seed meal contains various active components, including proteins and polysaccharides. However, it also contains a high content of the anti-nutritional factor phytic acid, which has a bitter and astringent taste, affecting the palatability of the feed. Although traditional physical or chemical treatment methods (such as high-temperature extrusion and acid-base hydrolysis) can partially degrade anti-nutritional components, they have problems such as high energy consumption, significant nutrient loss, or the risk of chemical residues, making it difficult to achieve efficient and value-added utilization of resources.
[0003] In recent years, microbial fermentation technology has played an important role in improving the nutritional characteristics of agricultural and sideline products due to its environmental friendliness, cost-effectiveness, safety, and high efficiency. Currently, the solid-state fermentation strains for plant seed meal mainly utilize Bacillus, lactic acid bacteria, yeast, and mold strains for single-strain fermentation and mixed-strain fermentation. However, there are very few reports on the application of edible and medicinal fungi in the fermentation of seed meals. For example, Wang Mengyang et al. used solid-state fermentation of Pleurotus ostreatus to effectively increase the soluble dietary fiber content of Zanthoxylum bungeanum seed meal (Wang Mengyang, Feng Yinyin, Li Rongchao, et al. Optimization of solid-state fermentation process for producing soluble dietary fiber from Zanthoxylum bungeanum seed meal and its immune activity study [J]. Food Science and Technology, 2024, 49(06):270-277.); In the patent of Zhang Jiachan et al., Ganoderma lucidum was used to ferment sea buckthorn seed meal, and the fermented sea buckthorn seed meal had good antioxidant, anti-aging, blood pressure lowering and immune-enhancing activities (Zhang Jiachan, Wang Changtao, Ma Yuhan, et al. A functional food additive for fermented sea buckthorn seed meal by Ganoderma lucidum and its preparation method). However, these studies did not significantly improve the protein content and anti-nutritional factor levels of plant seed meals before and after fermentation.
[0004] Therefore, how to provide a method for fermenting seed meal using edible and medicinal fungi to improve the protein content and anti-nutritional factor levels in seed meal is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention
[0005] To address the aforementioned technical problems, this invention proposes a method and product for simultaneously reducing anti-nutritional factors and increasing crude protein content in safflower seed meal.
[0006] To achieve the above object, the present application provides the following technical solutions. A method for simultaneously reducing anti-nutritional factors and increasing the content of crude protein in safflower meal, which comprises aerobic fermentation of safflower meal using Ganoderma as the fermentation strain, and then freeze-drying the fermented product to obtain a freeze-dried fungal substance.
[0007] Beneficial effects: The present application mainly aims at the protein and anti-nutritional factor phytic acid level of safflower meal, and selects the medicinal fungus Ganoderma through fermentation, which can improve the protein level of safflower meal and significantly reduce the content of anti-nutritional factor phytic acid. Ganoderma is an important fungus that can secrete a large amount of extracellular enzymes during its growth process, which can be used to degrade the anti-nutritional factors of plants. In addition, Ganoderma fermentation can also synthesize various primary and secondary metabolites such as functional polysaccharides, thereby synergistically improving the nutritional value and biological activity of the product. The application of Ganoderma solid fermentation technology to the modification of safflower meal is expected to simultaneously achieve the improvement of protein content and the elimination of anti-nutritional factors, and provides a new path for the development of high-value protein feed or functional food raw materials.
[0008] Preferably, the inoculation amount of the fermentation strain is 10% (v / v).
[0009] Beneficial effects: Under this inoculation amount, the Ganoderma strain can quickly adapt to the fermentation environment and rapidly grow and reproduce in the safflower meal fermentation substrate. It will neither cause slow fermentation start and prolonged fermentation period due to insufficient inoculation amount, affecting production efficiency, nor cause competition for resources among strains due to excessive inoculation amount, increasing production cost. At the same time, this inoculation amount can make the Ganoderma strain fully secrete extracellular enzymes, effectively degrade the anti-nutritional factor phytic acid in safflower meal, and significantly improve the crude protein content, maximizing the fermentation effect.
[0010] Preferably, the preparation method of the fermentation strain comprises the following steps: The Ganoderma strain is activated and subcultured to obtain a Ganoderma mycelium seed liquid, i.e., the fermentation strain.
[0011] Preferably, the activation culture is as follows: the Ganoderma strain is inoculated into a liquid culture medium and activated at 23-26℃ for 5 days.
[0012] Beneficial effects: Activation culture can restore the activity of Ganoderma strain in a dormant state and enhance its metabolic capacity, preparing for the subsequent rapid growth and fermentation enzyme production.
[0013] Preferably, the subculture is performed once, specifically including the following steps: The activated Ganoderma strain is subcultured into a liquid culture medium at an inoculation amount of 10% (v / v) and subcultured at 23-26℃ for 5 days.
[0014] Beneficial effect: Subculture further expands the number of Ganoderma spores, and obtains sufficient number of Ganoderma mycelium seed liquid with good growth state.
[0015] Preferably, the temperature of the aerobic fermentation is 23-26℃, and the time is 15d.
[0016] Preferably, the fermentation substrate is safflower meal liquid fermentation medium and / or safflower meal-containing solid-state fermentation substrate.
[0017] Preferably, the content of safflower meal in the safflower meal liquid fermentation medium is 240 g / L.
[0018] Preferably, the content of safflower meal in the safflower meal-containing solid-state fermentation substrate is 750 g / kg.
[0019] A freeze-dried fungal substance obtained by a method of simultaneously reducing anti-nutritional factors in safflower meal and increasing the content of crude protein.
[0020] Beneficial effect: The total amino acid and essential amino acid levels of the freeze-dried fungal substance obtained by the fermentation of safflower meal are significantly higher than those of the protein extract of unfermented safflower meal, and the immunomodulatory activity of the protein extract of the obtained freeze-dried fungal substance of fermented safflower meal is significantly stronger than that of the protein extract of unfermented safflower meal.
[0021] Compared with the prior art, the present application has the following advantages and technical effects: The present application respectively adopts liquid fermentation and solid-state fermentation means to ferment and treat safflower meal. Through Ganoderma liquid fermentation, the protein content of safflower meal can be increased by 54.20%, and the level of phytic acid can be reduced by 26.91%. Through Ganoderma solid-state fermentation, the protein content of safflower meal can be increased by 51.97%, and the level of phytic acid can be reduced by 34.41%. The obtained fermentation product has good antioxidant and immunomodulatory activity. The present application can greatly improve the utilization rate of safflower meal through simple operation, without the need for physical and chemical means with high cost and great harm, improve the nutritional components of safflower meal, and greenly solve the problem of waste utilization of safflower meal. The present application is easier to be popularized to large-scale production, and has good application prospect. BRIEF DESCRIPTION OF DRAWINGS
[0022] The drawings that form a part of the present application are used to provide further understanding of the present application, and the schematic embodiments of the present application and the description thereof are used to explain the present application, and do not constitute improper limitation on the present application. In the drawings: Figure 1 The liquid fermentation effect diagram and the real object diagram of freeze-dried product after fermentation of Example 1 and Comparative Examples 1-2 are shown in the following figures: In the figures, A and C are fermentation effect diagrams, and B is a real object diagram of freeze-dried product after fermentation; Figure 2 The crude protein content and phytic acid content of safflower seed meal before and after liquid fermentation in Example 1 were detected; Figure 3 The crude protein content and phytic acid content of safflower seed meal before and after solid fermentation in Example 2 were detected; Figure 4 The antioxidant activity of safflower seed meal protein extract before and after fermentation; Figure 5 The in vitro immunomodulatory activity of safflower seed meal protein extract before and after fermentation; Figure 6 The in vivo immunomodulatory activity of safflower seed meal protein extract before and after fermentation. DETAILED DESCRIPTION
[0023] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.
[0024] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the present application will be further described in detail below with reference to the drawings and specific embodiments.
[0025] The embodiments of the present application disclose a method for simultaneously reducing anti-nutritional factors and increasing the crude protein content in safflower seed meal. Ganoderma lucidum is used as a fermentation strain, and safflower seed meal is used as a fermentation substrate for aerobic fermentation. After fermentation, the freeze-dried fungus is obtained.
[0026] In a preferred embodiment, the inoculation amount of the fermentation strain is 10% (v / v).
[0027] In a preferred embodiment, the preparation method of the fermentation strain comprises the following steps: The Ganoderma lucidum strain is activated and subcultured to obtain a Ganoderma lucidum mycelium seed liquid, i.e., a fermentation strain.
[0028] In a preferred embodiment, the activation culture is to inoculate the Ganoderma lucidum strain into a liquid culture medium and activate the culture at 23-26°C for 5 days.
[0029] In a preferred embodiment, the subculture is performed once, and specifically comprises the following steps: The activated Ganoderma lucidum strain is subcultured into a liquid culture medium at an inoculation amount of 10% (v / v) and subcultured at 23-26°C for 5 days.
[0030] In a preferred embodiment, the temperature of the aerobic fermentation is 23-26℃ and the time is 15 days.
[0031] In a preferred embodiment, the fermentation substrate is a liquid fermentation medium of safflower meal and / or a solid state fermentation substrate containing safflower meal.
[0032] In a preferred embodiment, the content of safflower meal in the liquid fermentation medium of safflower meal is 240 g / L.
[0033] In a preferred embodiment, the content of safflower meal in the solid state fermentation substrate containing safflower meal is 750 g / kg.
[0034] The present application also discloses a freeze-dried fungal substance obtained by the method for simultaneously reducing the anti-nutritional factors and increasing the content of crude protein in safflower meal.
[0035] Unless otherwise specified, the raw materials in the embodiments of the present application are commercially available products. In the embodiments of the present application and the comparative examples, the Ganoderma lucidum strain is Ganoderma lucidum CGMCC 5.26. The liquid fermentation medium of safflower meal is composed of 20 g / L of glucose, 2 g / L of potassium dihydrogen phosphate, 1.5 g / L of magnesium sulfate and 240 g / L of safflower meal, wherein the content of crude protein is 223.38±13.13 g / kg, and the content of phytic acid is 480.51±61.80 nmol / g. The solid state fermentation substrate containing safflower meal is composed of 50 g / kg of glucose, 750 g / kg of safflower meal, 250 g / kg of bran and 80% of water content, wherein the content of crude protein is 206.688±4.961 g / kg, and the content of phytic acid is 849.736±45.187 nmol / g. The liquid medium is composed of the following raw materials: 20 g / L of glucose, 10 g / L of proteose peptone, 10 g / L of yeast powder, 2 g / L of potassium dihydrogen phosphate and 1.5 g / L of magnesium sulfate.
[0036] Unless otherwise specified, the room temperature or normal temperature in the embodiments of the present application is 20-30℃, and the inoculation amount refers to the volume ratio of strain culture to medium.
[0037] Embodiment 1 A liquid fermentation method of safflower meal, comprising the following steps: (1) Preparation of Ganoderma lucidum mycelium seed liquid: The Ganoderma mycelium seed liquid was prepared as follows: a PDA (potato dextrose agar medium) slant culture of Ganoderma sp. was inoculated with a loopful of mycelium into a liquid medium, and then incubated at 25°C with shaking at 150 r / min for 5 days. The activated Ganoderma culture was subcultured once, i.e., the Ganoderma culture was subcultured into a liquid medium (100 mL liquid in a 250 mL flask, inoculation amount 10 vol%), and then incubated at 23-26°C with shaking at 150 r / min for 5 days. After the subculture, the Ganoderma mycelium seed liquid was obtained.
[0038] (2) Liquid fermentation of safflower meal The Ganoderma mycelium seed liquid obtained in step (1) was inoculated into 30 mL of safflower meal liquid fermentation medium (in a 100 mL flask) at an inoculation amount of 10% (V / V), and then incubated at 23-26°C with shaking at 150 r / min for 15 days. After the fermentation, the Ganoderma-safflower meal fermentation medium was filtered, washed twice with deionized water, and then freeze-dried to obtain the freeze-dried medium. Figure 1 Part B of Section 1).
[0039] Example 2 A solid fermentation method of safflower meal, comprising the following steps: (1) Preparation of Ganoderma mycelium seed liquid The Ganoderma mycelium seed liquid was prepared as follows: a PDA (potato dextrose agar medium) slant culture of Ganoderma sp. was inoculated with a loopful of mycelium into a liquid medium, and then incubated at 25°C with shaking at 150 r / min for 5 days. The activated Ganoderma culture was subcultured once, i.e., the Ganoderma culture was subcultured into a liquid medium (100 mL liquid in a 250 mL flask, inoculation amount 10 vol%), and then incubated at 23-26°C with shaking at 150 r / min for 5 days. After the subculture, the Ganoderma mycelium seed liquid was obtained.
[0040] (2) Solid fermentation of safflower meal The Ganoderma mycelium seed liquid obtained in step (1) was inoculated into 25 g of safflower meal-containing solid fermentation medium (fermentation bag, water content 80%) at an inoculation amount of 10% (V / V), and then incubated at 23-26°C with a humidity of 70-80% for 30 days. After the fermentation, the Ganoderma-safflower meal fermentation medium was collected and freeze-dried to obtain the freeze-dried medium.
[0041] Comparative Example 1 A liquid fermentation method of safflower meal, which is different from Example 1 only in that the Ganoderma mycelium seed liquid is replaced by sterilized water. The method comprises the following steps: (1) Sterilized water was obtained by high temperature and high pressure sterilization treatment of ultra-pure water.
[0042] (2) Liquid fermentation of safflower meal The sterilized water obtained in step (1) was inoculated into 30 mL of safflower meal liquid fermentation medium (in a 100 mL shake flask) at an inoculation amount (V / V) of 10%, and the culture was continued to be shaken at 25°C and 150 r / min for 15 days. After the fermentation was completed, the safflower meal was filtered and washed with deionized water twice, and then freeze-dried to obtain freeze-dried safflower meal.
[0043] Comparative Example 2 A solid fermentation method of safflower meal, which is different from Example 2 only in that the Ganoderma lucidum mycelium seed liquid is replaced by sterilized water. Specifically, it comprises the following steps: (1) Sterilized water was obtained by high temperature and high pressure sterilization treatment of ultra-pure water.
[0044] (2) Solid fermentation of safflower meal The sterilized water obtained in step (1) was inoculated into 25 g of solid fermentation medium containing safflower meal (fermentation fungus bag, water content 80%) at an inoculation amount (V / V) of 10%, and the culture was continued to be incubated at 23-26°C and humidity of 70-80% for 30 days. After the fermentation was completed, the safflower meal medium was collected and freeze-dried to obtain freeze-dried safflower meal.
[0045] Technical effects: 1. Content of substances in freeze-dried fungus The content of crude protein in freeze-dried fungus was determined by Kjeldahl nitrogen determination method, and the content of tannin and phytic acid in freeze-dried fungus was determined by colorimetry. Three parallel tests were performed, and the average value was taken as the result. The results are shown in Table 1 and Figures 2-3 Table 1 As can be seen from Table 1 and Figures 2-3 , the crude protein content of the freeze-dried fungus obtained in Example 1 was increased by 54.20% and the phytic acid content was reduced by 26.91% compared with Comparative Example 1. The crude protein content of the freeze-dried fungus obtained in Example 2 was increased by 51.97% and the phytic acid content was reduced by 34.41% compared with Comparative Example 2.
[0046] The amino acid composition was determined by HPLC-OPA / FMOC method, and the changes of amino acid composition of safflower meal caused by Ganoderma lucidum fermentation were compared. The results are shown in Table 2.
[0047] Table 2 Analysis of amino acid composition of safflower meal before and after fermentation From Table 2, it can be seen that after fermentation, the content of each amino acid component is significantly improved. The total amino acid content of the freeze-dried mycoderm obtained in Example 1 is increased by 134.72% and the essential amino acid content is increased by 621% compared with Comparative Example 1.
[0048] The total amino acid content of the freeze-dried mycoderm obtained in Example 2 is increased by 1923.72% and the essential amino acid content is increased by 6579.8% compared with Comparative Example 2.
[0049] 2. Study on the antioxidant activity of safflower meal solid fermentation protein (1) Preparation of protein extract of safflower meal fermentation mycoderm The safflower meal protein (SP) and the ganoderma-safflower meal fermentation mycoderm protein (GSP) obtained in Examples 1-2 were prepared by alkali extraction and acid precipitation, which included the following steps: Alkaline extraction: 1.0 mol / L NaOH solution was added to the safflower meal powder (or two kinds of freeze-dried mycoderm, hereinafter referred to as sample) according to the solid-liquid ratio of 1:25 (g / mL), and the pH was adjusted to 10. The sample was extracted at 50°C under ultrasonic power of 450W for 60min to obtain the alkaline extract of the sample protein.
[0050] Solid-liquid separation: The above-mentioned alkaline extract was placed in a high-speed centrifuge and centrifuged at a speed of 6000r / min for 20min. The supernatant was collected and the precipitate residue was discarded.
[0051] Acid precipitation of protein: 1.0 mol / L HCl solution was used to adjust the pH value of the supernatant to 4.5, and the mixture was allowed to stand at 4°C overnight to precipitate the protein.
[0052] Protein separation and purification: The mixture after acid precipitation was centrifuged at a speed of 5000r / min for 15min, and the precipitate was collected. The protein was redissolved with distilled water, and the pH value was adjusted to 7.5 to obtain a protein solution.
[0053] The protein solution was detected by BCA method to detect the protein concentration, and different concentrations of protein solution were prepared for standby.
[0054] (2) Study on the antioxidant activity of safflower meal fermentation mycoderm protein The DPPH and ABTS free radical scavenging activity of the protein extract of safflower meal fermentation mycoderm was investigated by in vitro experiment, which included the following steps: DPPH free radical scavenging activity determination Sample group determination: take 2 mL of protein sample solution of series concentration, add 2 mL of 0.1 mmol / L DPPH ethanol solution, mix thoroughly, and then stand for 30 min at 25°C in the dark; after the reaction is completed, measure the absorbance of the mixed solution at 517 nm (recorded as A sample) with anhydrous ethanol as the reference.
[0055] Blank group determination: take 2 mL of protein sample solution, add 2 mL of anhydrous ethanol, mix, and then stand for 30 min under the same conditions; measure the absorbance at 517 nm (recorded as A blank).
[0056] Control group determination: take 2 mL of 0.1 mmol / L DPPH ethanol solution, add 2 mL of 0.01 mol / L PBS buffer (pH 7.4), mix, and then stand for 30 min; measure the absorbance at 517 nm (recorded as A control).
[0057] Parallel experiment: 3 sets of parallel experiments are set for each concentration gradient, and the average absorbance is used for calculation.
[0058] Clearance rate calculation: the DPPH free radical clearance rate is calculated according to the following formula: DPPH free radical clearance rate (%) = [1 - (A sample - A blank) / A control] x 100% ABTS free radical scavenging activity determination Sample group determination: take 0.2 mL of protein sample solution of series concentration, add 2 mL of ABTS working solution, mix thoroughly, and then stand for 6 min at 25°C in the dark; measure the absorbance of the mixed solution at 734 nm (recorded as B sample) with anhydrous ethanol as the reference.
[0059] Blank group determination: take 0.2 mL of protein sample solution, add 2 mL of anhydrous ethanol, mix, and then stand for 6 min; measure the absorbance at 734 nm (recorded as B blank).
[0060] Control group determination: take 0.2 mL of 0.01 mol / L PBS buffer (pH 7.4), add 2 mL of ABTS working solution, mix, and then stand for 6 min; measure the absorbance at 734 nm (recorded as B control).
[0061] Parallel experiment: 3 sets of parallel experiments are set for each concentration gradient, and the average absorbance is used for calculation.
[0062] Clearance rate calculation: the ABTS free radical clearance rate is calculated according to the following formula: ABTS radical scavenging rate (%) = [1 - (B sample - B blank) / B control] x 100%.
[0063] The concentration of protein solution was taken as the abscissa, and the corresponding radical scavenging rate was taken as the ordinate to draw the concentration-scavenging rate curve.
[0064] The results are shown in Table 1. Figure 4 As can be seen from Table 1, the DPPH and ABTS radical scavenging abilities of GSP and SP both showed a concentration-dependent increase. When the protein concentration was 10 mg / mL, the scavenging activities of both reached the highest value, in which the DPPH scavenging rate of GSP was 67.68 ± 0.8815%, and the ABTS scavenging rate was 60.69 ± 0.4521%. Notably, the DPPH and ABTS scavenging rates of GSP obtained by fermentation with Ganoderma lucidum were significantly higher than those of SP.
[0065] 3. Study on the immunomodulatory activity of solid-state fermented protein of safflower meal (1) Preparation of protein extract of fermented mycelium of safflower meal The safflower meal protein (SP) and the Ganoderma lucidum-safflower meal fermented mycelium protein (GSP) obtained in Example 1-2 were prepared by the alkali extraction and acid precipitation method, which included the following steps as described above.
[0066] (2) Study on the immunomodulatory activity of protein of fermented mycelium of safflower meal The immunomodulatory activity of the protein extract of fermented mycelium of safflower meal was investigated by in vitro and in vivo experiments, which included the following steps: Determination of in vitro immunomodulatory activity Cell proliferation assay: After the RAW264.7 macrophages were recovered, they were subcultured in DMEM medium containing 10% FBS at 37 °C and 5% CO2, and the cell density was adjusted to the logarithmic phase. Then the RAW264.7 cells were placed in a 96-well plate, and the cell concentration was 5 x 10 4 6 / mL per well. The cells were incubated at 37 °C and 5% CO2 for 16 h to allow the cells to adhere to the bottom of the wells. Then the supernatant in the wells was aspirated, and the cells were treated with different concentrations (25, 50, 100, 200, and 300 μg / mL) of GSP and SP, and then incubated in the incubator for another 24 h. At the same time, medium blank and LPS (1.0 μg / mL) were set as positive controls. After 24 h, the supernatant in the wells was aspirated, and 10 μL of CCK-8 reagent was added to each well, and the incubation was continued for another 4 h. After the incubation, the absorbance (OD value) at 450 nm was measured by a microplate reader.
[0067] Griess method for detecting NO release: After administration according to the above method, 50 μL of supernatant was accurately taken and added to a new enzyme-labeled plate, and then 50 μL of Griess I and Griess II reagent was respectively taken and added, mixed uniformly, and the absorbance value was measured at a wavelength of 540 nm using an enzyme-labeled instrument.
[0068] Inflammatory factor determination: After administration according to the above method for 24 hours, the culture medium of each well was taken out, centrifuged at 2000 r / min at 4 ℃ for 10 min, and the supernatant after centrifugation was collected. The concentration of TNF-α and IL-6 was measured according to the instruction of the ELISA kit. The concentration of cytokines in the sample was calculated using the standard curve.
[0069] In vivo immunomodulatory activity determination Animal grouping: Male BALB / c mice (18-20 g, 6 weeks old). Before the experiment, the mice were adaptively fed for one week, and the environmental conditions were maintained at a temperature of about 25°C, a relative humidity of 50-55%, and a 12 h light / dark cycle. During the adaptation period, the mice could freely drink sterile water and standard laboratory diet. Male mice were randomly divided into six groups: normal control group (Con), model control group (MC), positive control group (PC), GSP-1 group, GSP-2 group, SP-1 group and SP-2 group. Specifically, the MC group, PC group, GSP-1 group, GSP-2 group, SP-1 group and SP-2 group of mice were intraperitoneally injected with 80 mg / kg of CTX for 3 consecutive days, and the Con group of mice was given normal saline in the same way. Subsequently, the Con group and MC group of mice were given 0.2 mL of sterile water by gavage every day. The mice in the PC group were given 60 mg / kg of levamisole hydrochloride by gavage every day, and the mice in the GSP-1 group, GSP-2 group, SP-1 group and SP-2 group were given 80 mg / kg and 120 mg / kg of GSP and SP by gavage every day, for 14 consecutive days. The body weight of each group of mice was measured from the first day of feeding. The animals were euthanized 24 h after the last administration. Serum was collected, and the thymus and spleen were isolated.
[0070] Immunological organ index analysis: After 24 h after the last gavage, the mice were weighed and euthanized, and the thymus and spleen of the mice were removed. The organ index was calculated according to the following formula: Organ index = organ weight (mg) / mouse weight (g) Serum cytokine and immunoglobulin determination: After eyeball blood collection, the blood sample was placed at 4℃ for 24 h, and then centrifuged at 4℃, 3000 r / min for 10 min to obtain a serum sample. According to the requirements in the ELISA kit instructions, a standard curve including different concentrations of standard samples was prepared. According to the detailed operation steps in the ELISA kit instructions, the prepared kit was used to determine the contents of INF-γ, TNF-α, IL-6, Ig-A and Ig-G in the serum sample. The determination results were recorded, and data analysis and statistical processing were performed as needed.
[0071] In vitro experimental results Figure 5 It can be seen that compared with SP, GSP can significantly promote the proliferation of macrophages and induce the production of higher levels of nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), indicating that the fermentation of ganoderma lucidum effectively improves the in vitro immune activity of safflower meal protein. In vivo experimental results Figure 6 It can be seen that compared with SP, GSP can significantly improve the thymus index and spleen index of mice, and promote the levels of TNF-α, IL-6, IFN-γ and IgG, IgA in serum. These results comprehensively show that the fermented protein has the effect of enhancing the immune ability of the body. It is worth noting that the immune regulation ability of GSP obtained by ganoderma lucidum fermentation is obviously better than that of safflower meal protein (SP).
[0072] The above is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.
Claims
1. A method for simultaneously reducing anti-nutritional factors and increasing crude protein content in safflower seed meal, characterized in that, Aerobic fermentation was carried out using Ganoderma lucidum as the fermentation strain and safflower seed meal as the fermentation substrate. After fermentation, the culture was freeze-dried to obtain freeze-dried mycelium.
2. The method for simultaneously reducing anti-nutritional factors and increasing crude protein content in safflower seed meal according to claim 1, characterized in that, The inoculation amount of the fermentation strain is 10% (v / v).
3. The method for simultaneously reducing anti-nutritional factors and increasing crude protein content in safflower seed meal according to claim 1, characterized in that, The method for preparing the fermentation strain includes the following steps: After activation and subculturing, Ganoderma lucidum mycelium seed liquid, i.e., fermentation strain, is obtained.
4. The method for simultaneously reducing anti-nutritional factors and increasing crude protein content in safflower seed meal according to claim 3, characterized in that, The activation culture is as follows: Ganoderma lucidum strains are inoculated into liquid culture medium and activated cultured at 23-26℃ for 5 days.
5. The method for simultaneously reducing anti-nutritional factors and increasing crude protein content in safflower seed meal according to claim 3, characterized in that, The number of subcultures is 1, and specifically includes the following steps: The activated Ganoderma lucidum strain was subcultured into liquid culture medium at an inoculation rate of 10% (v / v) and cultured at 23-26℃ for 5 days.
6. The method for simultaneously reducing anti-nutritional factors and increasing crude protein content in safflower seed meal according to claim 1, characterized in that, The aerobic fermentation was carried out at a temperature of 23-26℃ for 15 days.
7. The method for simultaneously reducing anti-nutritional factors and increasing crude protein content in safflower seed meal according to claim 1, characterized in that, The fermentation substrate is a safflower seed meal liquid fermentation medium and / or a solid fermentation substrate containing safflower seed meal.
8. The method for simultaneously reducing anti-nutritional factors and increasing crude protein content in safflower seed meal according to claim 7, characterized in that, The safflower seed meal content in the liquid fermentation medium is 240 g / L.
9. The method for simultaneously reducing anti-nutritional factors and increasing crude protein content in safflower seed meal according to claim 7, characterized in that, The solid fermentation substrate containing safflower seed meal contains 750 g / kg of safflower seed meal.
10. Freeze-dried mycelium obtained by the method described in any one of claims 1-9, which simultaneously reduces anti-nutritional factors in safflower seed meal and increases crude protein content.