Condition and flavor optimization for removing zearalenone in corn germ meal by aspergillus niger strain FS-UV-21

Abstract

The invention discloses conditions and flavor optimization for removing zearalenone in corn germ meal by using an aspergillus niger strain FS-UV-21, and belongs to the technical field of microbial fermentation and biodegradation. The aspergillus niger strain FS-UV-21 is used for fermenting the corn germ meal, the removal rate of ZEN in the fermented corn germ meal reaches 63.37%, meanwhile, a pleasant smell is generated, the effects of fully utilizing the corn germ meal and improving the feeding value of the corn germ meal are achieved, and possibility is provided for reasonable utilization of the detoxified corn germ meal in livestock and poultry feed.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation and biodegradation, and in particular to conditions for removing zearalenone from corn germ meal by Aspergillus niger strain FS-UV-21 and flavor optimization. Background Art

[0002] Zearalenone (ZEN) is a weak polar mycotoxin mainly produced by Fusarium and Gibberellin fungi. It is widely present in food and feed and can contaminate food at various stages of production, harvesting, storage and processing. ZEN has estrogenic effects and has a great impact on the reproductive system of animals, which can cause reproductive disorders in the body. A large number of studies have shown that ZEN mainly has four aspects of toxicity: reproductive toxicity, cytotoxicity, genotoxicity and immunotoxicity. In response to the hazards of ZEN toxicity, various countries have established limit standards for the content of ZEN in different foods. The EU stipulates that the ZEN content in cereals and cereal products (except corn by-products) and corn by-products shall not exceed 2ppm and 3ppm. GB13078-2017 "Feed Hygiene Standards" stipulates that the ZEN content in corn and its processed products (except corn husks, corn germ meal, and corn steep powder) and corn husks, corn germ meal, corn steep powder, and corn distiller's grains shall not exceed 0.5mg / kg and 1.5mg / kg.

[0003] At present, the methods for degrading zearalenone include biological, physical and chemical methods. Physical methods include adsorbent adsorption, electron beam irradiation, etc. Chemical methods mainly include ozone treatment, electrochemical oxidation, etc. Both physical and chemical methods have certain defects, either the degradation efficiency is too low, the equipment cost is expensive, or new impurities are introduced, causing the destruction of the nutrients in the product itself. In recent years, biological methods have been widely used in the degradation of fungal toxins because of their green, efficient reaction and mild conditions. For example, Geng Hairong et al. screened out a bacterial strain Y-33 that efficiently degrades ZEN in an acidic and high-temperature hot spring environment. The degradation rate of 2μg / mL ZEN was as high as 93.79% under 36h incubation conditions.

[0004] Corn germ meal, also known as corn navel meal, is a by-product extracted from corn germ by leaching or pressing. It can be used as an energy feed for protein and energy supplementation, and can be applied to the diet of growing and fattening pigs as a protein and energy supplement. At the same time, it has been widely used in the production of poultry such as laying hens, broilers, and geese. Since corn is easily contaminated by ZEN, ZEN residues are also detected in corn germ meal. The probability of ZEN test values ​​in the corn germ meal samples sampled in the first quarter of 2022 being distributed in medium risk is 38%; the probability of ZEN test values ​​in corn germ meal samples being distributed in high risk is 62%. In recent years, a lot of research has been done in China on feeding livestock with corn germ meal as a protein supplement, but corn germ meal contains high levels of cellulose and other substances. ZEN can be closely combined with cellulose and other components in corn germ meal, increasing the difficulty of degradation. At the same time, conventional methods of removing ZEN cannot guarantee the flavor of corn germ meal. Therefore, it is of great significance to find a method that can degrade ZEN toxins in corn germ meal while maintaining or improving the flavor of corn germ meal. Summary of the invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of the present invention is to use a food-grade non-toxigenic Aspergillus niger strain FS-UV-21 as a fermentation strain to degrade zearalenone toxin during the fermentation of corn germ meal.

[0006] In order to achieve the purpose of the present invention, the Aspergillus niger FS-UV-21 of the present invention is separated from Chinese fermented soybeans, and the preservation number of the Aspergillus niger FS-UV-21 is CGMCC No.20751.

[0007] One object of the present invention is to provide a method for improving the quality of corn germ meal, wherein Aspergillus niger FS-UV-21 is inoculated into corn germ meal for fermentation.

[0008] In one embodiment, the improvement of corn germ meal quality is to reduce the content of zearalenone in the corn germ meal and improve the sensory flavor of the corn germ meal.

[0009] In one embodiment, the method of improving the sensory flavor of corn germ meal is to increase the aroma substances in the corn germ meal; the aroma substances include at least one of 3-octanone, isovaleraldehyde, 2,5-dimethylpyrazine, geraniol, lauryl acetate, isobutyric acid, isophorone, and methylcyclopentenolone.

[0010] In one embodiment, corn germ meal is inoculated with a final concentration of (1×10 4 )~(2x10 5 ) CFU / mL of Aspergillus niger FS-UV-21.

[0011] In one embodiment, the fermentation temperature is 20-40° C., and the fermentation lasts for at least 1 day.

[0012] In one embodiment, water is added to corn germ meal for fermentation, and the mass volume ratio of corn germ meal to water is (1:0.5) to (1:2.5) g / mL.

[0013] The invention further discloses a scheme optimization of extracting spores for fermentation and degradation of zeaxenone toxin in corn germ meal by using the Aspergillus niger FS-UV-21 strain.

[0014] In one embodiment, the fermentation conditions were optimized by single factor experimental design, and the concentration of Aspergillus niger spores was controlled at 10 6 CFU / mL, and analyze the effects of inoculation size, fermentation temperature, fermentation time, and feed-water ratio on the ZEN removal rate.

[0015] In one embodiment, the response surface method factor level test design is used to select the optimal fermentation conditions. According to the above single factor test results, the Box-Behnken design method of Design expert11 software is used to select the inoculation amount (A), fermentation temperature (B), fermentation time (C), and material-water ratio (D) as response variables, and the ZEN removal rate is used as the response value for response surface optimization design.

[0016] In one embodiment, it is finally obtained that when the inoculation amount is 15.34%, the fermentation temperature is 29.96°C, the fermentation time is 4.15d, and the material-water ratio is 1:1.93g / mL, the ZEN removal effect is optimal, and the removal rate reaches 63.37%. Considering practical considerations, the fermentation conditions are set to an inoculation amount of 15%, a fermentation temperature of 30°C, a fermentation time of 4d, and a material-water ratio of 1:1.9g / mL. Three groups of parallel fermentation experiments are set up, and the ZEN removal rate is 61.85%, with a relative error of 1.52% from the theoretical value.

[0017] The present invention is achieved through the following technical solutions.

[0018] The Aspergillus niger strain FS-UV-21 was used to optimize the removal conditions of zearalenone from corn germ meal, comprising the following steps:

[0019] The purpose of the present invention is achieved through the following technical solutions:

[0020] (1) Strain culture: Aspergillus niger FS-UV-2 was inoculated into PDA solid medium and cultured at 28°C for 6 days.

[0021] PDA medium: 5 g potato powder, 20 g glucose, 15 g agar, 0.1 g chloramphenicol, natural pH, distilled water to 1000 mL, sterilize at 121°C for 20 min.

[0022] Sterile physiological saline solution containing 0.05% Tween-80: 9g sodium chloride is dissolved in 1000mL distilled water, after dissolution, 500μL Tween-80 is added and mixed, and sterilized at 121℃ for 20min.

[0023] (2) Preparation of spore suspension: Rinse the spores of the strain cultured in step (1) with sterilized physiological saline containing 0.05% Tween-80°C, adjust the spore concentration to 106 CFU / mL, and set aside.

[0024] (3) Corn germ meal fermentation treatment: The corn germ meal is crushed, passed through a 50-mesh sieve, mixed evenly, and collected for later use. The crushed corn germ meal is sampled according to the sampling method of GB / T 14699.1-2005. The sterilized corn germ meal is transferred to a fermentation container, sterile water is added to a certain material-water ratio, stirred evenly, and the spore suspension prepared in step (2) is added to a certain inoculation amount, stirred evenly, and then is subjected to constant temperature aerobic fermentation at a certain temperature, and samples are taken at a certain time after fermentation.

[0025] (4) Analysis of ZEN content in corn germ meal

[0026] ZEN extraction method: Dry the fermented corn germ meal at 65℃ overnight. Add chloroform in a ratio of 1:3, mix, ultrasonicate, centrifuge, and absorb the upper liquid. Dry in a vacuum freeze dryer at 1000rpm for 6h, and re-dissolve in 1mL of methanol. Centrifuge, filter with a 0.22μm filter membrane, and place in a chromatographic bottle for testing.

[0027] With reference to GB / T 28716-2012, the determination of ZEN in corn germ meal was optimized and the detection parameters were optimized to prepare a series of ZEN standard solutions: 0.05, 0.1, 0.2, 0.5, 1, 2, 5 μg / mL. Chromatographic column: C18 column (column length 150 mm, diameter 4.6 mm, inner diameter 4 mm); mobile phase: methanol (46%), acetonitrile (46%), water (8%); excitation wavelength: 274 nm; emission wavelength: 440 nm; mobile phase flow rate: 0.8 mL / min; injection volume: 20 μL, column temperature: 30 °C.

[0028] Another object of the present invention is to provide a germ meal product obtained by the method.

[0029] Another object of the present invention is to improve the flavor through fermentation and enhance the palatability of livestock and poultry.

[0030] Further detection of volatile substances in corn germ meal before and after fermentation was carried out, and volatile substances in corn germ meal were extracted by headspace solid phase microextraction flavor substances. 342 volatile substances were identified before and after this fermentation, of which 219 substances were upregulated, 10 substances were downregulated, and 113 substances had no significant changes. The upregulated substances with relatively obvious changes were isomenthol, anisane, 3,3-dimethyl-5-hexanal, and the downregulated substances were longifolene, atractylodesone, and β-ionone.

[0031] Many volatile substances with aromatic smells have been significantly upregulated. Among the upregulated substances, isovaleraldehyde is a food flavoring permitted by my country's GB2760-86 regulations. It has an apple-like aroma when highly diluted and a peach aroma when the concentration is below 10ppm. In addition, 2,5-dimethylpyrazine, geraniol, lauryl acetate, isobutyric acid, isophorone, methylcyclopentenolone and other substances all have a light fragrance, which are in line with my country's food flavoring regulations. The production of these substances can effectively improve the sensory flavor of corn germ meal.

[0032] Another object of the present invention is to provide the use of Aspergillus niger FS-UV-21 in preparing feed with corn germ meal as the main raw material.

[0033] Beneficial effects:

[0034] (1) Corn germ meal samples were fermented using Aspergillus niger FS-UV-21 to achieve the effect of removing zearalenone from zearalenone. The effects of four single factors, namely inoculation amount, fermentation temperature, fermentation time and feed-water ratio, on ZEN removal at five levels were investigated. The optimal fermentation conditions were selected as follows: inoculation amount of 15%, fermentation temperature of 30°C, fermentation days of 4 days and feed-water ratio of 1:2 mg / mL, and the removal rate reached 63.37%.

[0035] (2) Corn germ meal, as a kind of corn by-product, has the nutritional characteristics of high protein and high fiber. After fermentation by Aspergillus niger, the flavor substances in corn germ meal are significantly increased. For example, 3-octanone, isovaleraldehyde, 2,5-dimethylpyrazine, geraniol, lauryl acetate, isobutyric acid, isophorone, methylcyclopentenolone and other substances give corn germ meal an aromatic smell. It is an edible flavoring that complies with the regulations of my country, and its unique smell helps to improve the palatability of livestock and poultry.

[0036] Therefore, fermentation of corn germ meal by Aspergillus niger FS-UV-21 can not only effectively reduce the ZEN content, but also give it a pleasant smell, which has great application prospects in the feed industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Effect of inoculum size on ZEN removal by Aspergillus niger;

[0038] Figure 2Effect of fermentation temperature on ZEN removal by Aspergillus niger;

[0039] Figure 3 Effect of fermentation time on ZEN removal by Aspergillus niger;

[0040] Figure 4 Effect of feed-water ratio on ZEN removal by Aspergillus niger;

[0041] Figure 5 Response surface diagram of the interaction of various factors on ZEN removal rate;

[0042] Figure 6 Principal component analysis (PCA) score graph of corn germ meal before and after fermentation;

[0043] Figure 7 Changes of volatile substances before and after fermentation;

[0044] Figure 8 Box plots of the up-regulated substances isomenthol, anisane, and 3,3-dimethyl-5-hexanal (A, from left to right) and the down-regulated substances longifolene, atractylodesone, and β-ionone (B, from left to right). DETAILED DESCRIPTION

[0045] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention. The raw materials and reagents used in the present invention are all commercially available.

[0046] The Aspergillus niger FS-UV-21 involved in the following examples has been disclosed in patent CN113736668A, and its deposit number is CGMCC No.20751.

[0047] The ZEN standard product involved in the following examples was purchased from Qingdao Puruibang with the product number of STD#3202.

[0048] The "inoculation amount" involved in the following examples refers to the percentage of the volume of bacterial solution to the mass of corn germ meal (mL:g); the "feed-water ratio" refers to the ratio of the mass of corn germ meal to the volume of sterile water, g:mL.

[0049] The detection methods involved in the following embodiments are as follows:

[0050] Detection of ZEN content in corn germ meal:

[0051] Dry corn germ meal at 65℃ overnight. Extract 1g sample, add chloroform at a ratio of 1:3g / ml, mix by inversion, ultrasonicate, centrifuge (5180×g, 25℃) for 5min, and draw the upper liquid. Repeat the above steps 3 times. Dry in a vacuum freeze dryer at 1000rpm for 6h, and re-dissolve in 1mL of methanol. Vortex mix, centrifuge at 3000xg for 3min, filter through 0.22μm filter membrane, and place in a chromatographic bottle for testing.

[0052] With reference to GB / T 28716-2012, the determination of ZEN in corn germ meal was optimized and the detection parameters were optimized to prepare a series of ZEN standard solutions: 0.05, 0.1, 0.2, 0.5, 1, 2, 5 μg / mL. Chromatographic column: C18 column (column length 150 mm, diameter 4.6 mm, inner diameter 4 mm); mobile phase: methanol (46%), acetonitrile (46%), water (8%); excitation wavelength: 274 nm; emission wavelength: 440 nm; mobile phase flow rate: 0.8 mL / min; injection volume: 20 μL, column temperature: 30 °C.

[0053] The ZEN removal rate was calculated as follows: ZEN removal rate (%) = (ZEN content in unfermented sample - ZEN content in fermented sample) / ZEN content in unfermented sample × 100.

[0054] Example 1: Effect of the inoculum amount of Aspergillus niger FS-UV-21 on the removal rate of ZEN in corn germ meal

[0055] 1. Activation of Aspergillus niger FS-UV-21 strain and preparation of spore suspension:

[0056] Aspergillus niger FS-UV-21 was taken out of the -80℃ refrigerator, inoculated into PDA solid medium and cultured at 28℃ for 6 days, then the spores were washed with sterile saline containing 0.05% Tween-80 and the spore concentration was adjusted to 10 6 CFU / mL, for future use.

[0057] 2. Corn germ meal fermentation process:

[0058] The corn germ meal was crushed, passed through a 50-mesh sieve, mixed evenly, and collected for later use. The crushed corn germ meal was sampled according to the sampling method of GB / T 14699.1-2005. 5 g of the sample was weighed into a 250 mL triangular conical bottle, sealed with a high temperature resistant tissue culture sealing film (12×12 cm), and sterilized by high pressure at 121°C for 20 min.

[0059] The prepared Aspergillus niger FS-UV-21 was inoculated at 1%, 5%, 10%, 15%, and 20% (corresponding to the inoculation concentration of 1×10 4 , 5x104 , 1x10 5 , 1.5x10 5 , 2x10 5 CFU / mL), and after being fully mixed with the prepared sterile water (the total volume of the spore liquid and the sterile water is 5 mL, that is, the material-water ratio is 1:1), poured into the above-mentioned corn germ meal and fully stirred, placed in an incubator at 30°C for constant temperature fermentation for 3 days, and then taken out, the ZEN content in the corn germ meal after fermentation was detected, and the ZEN removal rate was calculated.

[0060] Different concentrations of Aspergillus niger spores inoculated affect the removal of ZEN from corn germ meal. Figure 2 As shown in the figure, when the inoculation amount is in the range of 1-15%, the ZEN removal rate of the sample increases over time; when the inoculation amount is 15%, the ZEN removal rate reaches 52.78% at the highest. At an inoculation amount of 20%, the ZEN removal rate decreases. This may be due to the increase in Aspergillus niger in the system and the rapid consumption of nutrients, which leads to great competition pressure between strains and eventually inhibits growth. When the inoculation amount is 15%, the ZEN content in the fermented corn germ meal is 42.4ng / g.

[0061] Example 2: Effect of fermentation temperature on ZEN removal rate in corn germ meal

[0062] The biological fermentation of corn germ meal was carried out according to the method in Example 1, except that the inoculation amount of the fixed Aspergillus niger FS-UV-21 spore suspension was 15%, the material-water ratio was 1:2, and after inoculation, the mixtures were placed in incubators at 20, 25, 30, 35, and 40° C. for constant temperature fermentation for 3 days, and then taken out. The ZEN content in the corn germ meal after fermentation was detected, and the ZEN removal rate was calculated.

[0063] Temperature has a great influence on the life activities of microorganisms. The temperature determines the growth and fermentation level of Aspergillus niger. Figure 3 It can be seen that the ZEN removal rate first increases and then decreases with the change of temperature. At 30°C, the ZEN removal rate reaches a maximum of 49.74%. At this time, the growth of Aspergillus niger is accelerated, and the activity of the corresponding enzyme increases. When the temperature is greater than 30°C, the activity of the enzyme decreases and the ZEN removal effect becomes worse. Therefore, 30°C is selected as the optimal temperature for Aspergillus niger fermentation under this system. When the fermentation temperature is 30°C, the ZEN content in the corn germ meal after fermentation is 45.1ng / g.

[0064] Example 3: Effect of fermentation time on ZEN removal rate in corn germ meal

[0065] The biological fermentation of corn germ meal was carried out according to the method in Example 1, except that the inoculation amount of the fixed Aspergillus niger FS-UV-21 spore suspension was 15%, the material-water ratio was 1:2, and after inoculation, the mixture was placed in an incubator at 30° C. and fermented at constant temperature for 1 day, 2 days, 3 days, 4 days, and 5 days, and then taken out, the ZEN content in the corn germ meal after fermentation was detected, and the ZEN removal rate was calculated.

[0066] The length of fermentation time is also closely related to the ZEN removal rate. Figure 4 It can be seen that the ZEN removal rate increases with the extension of fermentation time. At 4d and 5d, the ZEN removal rate remains basically unchanged. Previous studies have found that too long fermentation time of Aspergillus niger leads to a decrease in reducing sugar content and filter paper enzyme activity. Therefore, the fermentation time is selected to be around 4d. At 4d of fermentation, the ZEN content in the fermented corn germ meal is 40.1ng / g.

[0067] Example 4: Effect of feed-water ratio on ZEN removal rate in corn germ meal

[0068] The biological fermentation of corn germ meal was carried out according to the method in Example 1, except that the inoculation amount of the fixed Aspergillus niger FS-UV-21 spore suspension was 10%, the material-water ratio was adjusted to 1:0.5, 1:1, 1:1.5, 1:2, and 1:2.5, respectively, after inoculation and sufficient mixing, the mixture was placed in an incubator at 30° C. for constant temperature fermentation for 3 days, and then taken out, the ZEN content in the corn germ meal after fermentation was detected, and the ZEN removal rate was calculated.

[0069] The feed-water ratio plays a vital role in the microbial fermentation process, and the water content of the bacteria is also one of the key factors for the normal growth and development of the bacteria. A moderate feed-water ratio can improve the removal rate of ZEN, such as Figure 5 As shown in the figure, when the feed-water ratio is 1:2, the ZEN removal rate is the highest, reaching 44.49%. When the feed-water ratio increases to 1:2.5, the ZEN removal rate is reduced to 41.17%, probably because of the difficulty of oxygen transfer caused by excessive water content. When the feed-water ratio is 1:2, the ZEN content in the fermented corn germ meal is 48.3ng / g.

[0070] Example 5: Response surface methodology optimization of ZEN removal from corn germ meal by Aspergillus niger FS-UV-21

[0071] According to the above single factor test results, the Box-Behnken design method of Design expert11 software was used to select inoculation size (A), fermentation temperature (B), fermentation time (C), and material-water ratio (D) as response variables, and the ZEN removal rate was used as the response value for response surface optimization design. The experimental factors and levels are shown in Table 1.

[0072] Table 1 Response surface experimental factors and levels design

[0073]

[0074] Based on the above single factor test results, a response surface test was designed to obtain the best effect of degrading ZEN, and a four-factor three-level test was designed with the inoculation amount, fermentation temperature, fermentation time, and feed-water ratio interacting with each other. The response surface test design and results are shown in Table 2:

[0075] Table 2 Response surface experimental design and results

[0076]

[0077]

[0078] Establishment of response surface regression model

[0079] The response surface experiment results were analyzed using Design Expert 11, and a quadratic multinomial regression model was obtained with ZEN removal rate (Y) as the response value, inoculation amount (A), fermentation temperature (B), fermentation time (C), and feed-water ratio (D):

[0080] Y=63.05+1.12A-0.4225B+2.23C-1.71D+0.0350AB-0.9AC+0.6975AD+0.3375

[0081] BC-1.48BD+0.8350CD-6.43A2-10.21B2-7.03C2-5.40D2;

[0082] The reliability of the regression model was tested by the results of variance and significance analysis of the regression model. The results are shown in Table 3.

[0083] Table 3 Analysis of variance of quadratic multinomial regression model for ZEN removal rate

[0084]

[0085] Note: NS has no significant effect; *Significant difference P<0.05; **Highly significant difference P<0.01; ***Extremely significant difference P<0.001

[0086] Analysis of response surface regression model

[0087] The results showed that the regression model was highly significant (P<0.0001), the lack of fit term was not significant (P>0.05), and R2 was 0.9330, indicating that the model had a good fit and could accurately predict and analyze and reflect the relationship between the level of each factor and the response value. From the P value in Table 3, it can be seen that the order of the influencing factors of the experiment is as follows: fermentation temperature> feed-water ratio> inoculation amount> fermentation time. Combined with the analysis of Table 3, the significance analysis of the experimental results of AB, AC, AD, BC, BD, and CD was P>0.05, indicating that the results of the pairwise interaction of inoculation amount, fermentation time, fermentation temperature, and feed-water ratio were not significant, such as Figure 6 As shown, the response surface model has a highest point, which is the center of the plane contour map. At the highest point, Aspergillus niger has the best effect in removing ZEN from corn germ meal.

[0088] Determination of the optimal conditions for response surface analysis

[0089] According to the above response surface analysis, the removal effect of ZEN is optimal when the inoculation amount is 15.34%, the fermentation temperature is 29.96℃, the fermentation time is 4.15d, and the material-water ratio is 1:1.93g / mL, and the removal rate reaches 63.37%. Considering the actual situation, the fermentation conditions are set to 15% inoculation amount, 30℃ fermentation temperature, 4d fermentation days, and 1:1.9g / mL material-water ratio. Three groups of parallel fermentation experiments are set to obtain a ZEN removal rate of 61.85%, with a relative error of 1.52% with the theoretical value, indicating that the optimized parameters are feasible and have practical application value. At this time, the ZEN content in the fermentation product is 34.22ng / g.

[0090] Example 6: Analysis and evaluation of volatile flavor substances in corn germ meal under optimal fermentation parameters

[0091] Specific implementation method Referring to Example 1, the difference is that fermentation is carried out under the conditions of inoculation amount 15%, fermentation temperature 30°C, fermentation days 4d, and material-water ratio 1:1.9g / mL, and the headspace solid phase microextraction flavor substance method is used to extract volatile substances in corn germ meal. The corn germ meal samples before and after fermentation are dried for 12h until the water evaporates, 3g of sample is weighed and placed in a headspace injection bottle, and heated for 30min. The extraction head of the solid phase microextraction is inserted into the headspace injection bottle for adsorption for 30min. After the end, the extraction head is taken out and inserted into the gas chromatography injection port for analysis for 15min.

[0092] The method for detecting volatile substances before and after fermentation by gas chromatography-mass spectrometry (GC-MS) is as follows:

[0093] Chromatographic conditions: the chromatographic capillary column was an Equity-5 column (30 m × 0.32 mm × 0.25 μm), the injection port temperature was 250 °C; the carrier gas was high-purity helium with a purity of ≥99.999%; the flow rate was 2.5 mL / min, without splitting, and the programmed temperature was as follows: initial temperature 80 °C, maintained for 2 min, increased to 200 °C at 10 °C / min, maintained for 2 min, increased to 260 °C at 8 °C / min, and maintained for 5 min.

[0094] Mass spectrometry conditions: ionization mode was electron impact source (EI), electron energy was 70 eV, detector voltage was -1499 V, and m / z range was 15-350.

[0095] Analysis and evaluation of volatile flavor substances:

[0096] Corn germ meal produces a unique aroma after being fermented under optimal conditions. Flavor substances play an important role in seasoning feed. They give feed a special taste and aroma, which can improve the palatability of livestock and poultry. Figure 6 As shown, under the optimal fermentation conditions, the contribution rates of PC1 and PC2 were 76.5% and 12.4% respectively, and the total contribution rate was 88.9%. There was some overlap between the first and second groups after fermentation, and the first and second groups before fermentation were relatively close. There was an obvious separation trend between the groups before and after fermentation.

[0097] A total of 342 volatile substances were identified before and after the fermentation. The detected volatile substances were plotted into a volcano map based on the fold change (FC) and the significant difference P value to reflect the overall distribution of the two groups of different substances before and after the fermentation ( Figure 7 ); P < 0.05 and log 2 (FC) > 1 or < -1 were selected as the analysis criteria for the differences in volatile substances before and after fermentation. Red indicated up-regulated substances, and blue indicated down-regulated substances. Among them, 219 substances were up-regulated, 10 substances were down-regulated, and 113 substances had no significant changes. The up-regulated substances with relatively obvious changes were isomenthol, anisane, 3,3-dimethyl-5-hexanal, and the down-regulated substances were longifolene, atractylodesone, and β-ionone ( Figure 8 , Table 4); 3,3-dimethyl-5-hexanal compounds can provide a fresh, herbaceous aroma in feed; isomenthol has a fresh minty aroma and can provide a cool and refreshing feeling. The increase in these two substances can improve the palatability of feed. Atractylodes ketone has a certain spicy aroma and may produce a pungent smell, resulting in a decrease in the palatability of animals. The content of these flavor substances regulates the sensory flavor of corn germ meal, making it easier for animals to accept it as feed.

[0098] Table 4 Changes of special aroma substances in corn germ meal before and after fermentation

[0099]

[0100] Table 5 Classification of compounds produced by fermentation of corn germ meal

[0101]

[0102] Example 7: Preparation of feed with low ZEN content and high flavor substance content

[0103] Specific implementation method Referring to Example 6, the corn germ meal is fermented and sterilized in a high-pressure steam autoclave at 121°C and 101kPa for 20 minutes. The sterilized fermented germ meal is dried in a constant temperature hot air drying phase at 80°C for two hours to obtain a fermented corn germ meal feed with low ZEN content and high flavor.

[0104] Comparative Example 1: Effects of different fermentation parameters on the content of main volatile flavor substances

[0105] Method 1: Specific implementation method Refer to Example 6, except that the fermentation conditions are changed to fermentation under the conditions of inoculation amount 10%, fermentation temperature 32°C, fermentation days 3d, and material-water ratio 1:2g / mL.

[0106] Method 2: Specific implementation method: Refer to Example 6, except that the fermentation conditions are changed to fermentation under the conditions of inoculation amount 12%, fermentation temperature 35°C, fermentation days 3.5d, and material-water ratio 1:1.5g / mL.

[0107] Method 3: Specific implementation method Refer to Example 6, except that the fermentation conditions are changed to fermentation under the conditions of inoculation amount 15%, fermentation temperature 28°C, fermentation days 5d, and material-water ratio 1:1g / mL.

[0108] Table 6 Effect of different fermentation parameters on the content of volatile flavor substances

[0109]

[0110] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for improving the quality of corn germ meal, characterized in that: The method comprises the following steps: inoculating Aspergillus niger FS-UV-21 into corn germ meal for fermentation; the preservation number of Aspergillus niger FS-UV-21 is CGMCC No.20751.

2. The method according to claim 1, characterized in that The improvement of corn germ meal quality is to reduce the content of zearalenone in the corn germ meal and improve the sensory flavor of the corn germ meal.

3. The method according to claim 2, characterized in that The method of improving the sensory flavor of corn germ meal is to increase the aroma substances in the corn germ meal; the aroma substances include at least one of 3-octanone, isovaleraldehyde, 2,5-dimethylpyrazine, geraniol, lauryl acetate, isobutyric acid, isophorone, and methylcyclopentenolone.

4. The method according to any one of claims 1 to 3, characterized in that: The corn germ meal was inoculated with a final concentration of (1x10 4 )~(2x10 5 ) CFU / mL of Aspergillus niger FS-UV-21.

5. The method according to claim 4, characterized in that The fermentation temperature is 20-40° C. and the fermentation lasts at least 1 day.

6. The method according to claim 5, characterized in that Water is added to corn germ meal for fermentation, and the mass volume ratio of corn germ meal to water is (1:0.5) to (1:2.5) g / mL.

7. A corn germ meal product obtained by the method described in any one of claims 1 to 6.

8. Application of Aspergillus niger FS-UV-21 in improving the flavor of corn germ meal.

9. The use according to claim 8, characterized in that: The method for improving the flavor of corn germ meal comprises increasing the content of at least one substance selected from the group consisting of 3-octanone, isovaleraldehyde, 2,5-dimethylpyrazine, geraniol, lauryl acetate, isobutyric acid, isophorone and methylcyclopentenolone in the corn germ meal.

10. Application of Aspergillus niger FS-UV-21 in the preparation of feed with corn germ meal as the main raw material.

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