A fermentation method of flaxseed meal, diet

Abstract

The present invention belongs to the biological field and discloses a method for fermenting flaxseed meal, using Bacillus subtilis FRI as the fermentation bacterium to ferment flaxseed meal; the preservation number of the Bacillus subtilis FRI is: CGMCC NO. 28734; the preservation date is October 24, 2023, and the preservation unit is the General Microbiology Center of the China Microbial Culture Collection Center. This method uses a strain of Bacillus subtilis FRI with relatively excellent performance isolated by the applicant to ferment flaxseed meal, which can degrade the cyanogenic glycosides in flaxseed cake meal, reduce the content of trypsin inhibitor, and reduce the neutral detergent fiber and / or acid detergent fiber of flaxseed cake, and can effectively partially replace corn and soybean meal in the diet, realizing the resource utilization of flaxseed meal and reducing the feed cost. At the same time, the present invention also provides a diet based on the fermented flaxseed meal.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a flaxseed meal fermentation method and a daily diet. Background Art

[0002] Flaxseed meal, a byproduct of flaxseed oil extraction, is highly nutritious, rich in protein, unsaturated fatty acids, and other nutrients. Studies have confirmed that flaxseed meal has the potential to replace soybean meal as a new protein feed ingredient. Studies have shown that excessive inclusion of flaxseed meal in livestock and poultry diets may cause some negative effects, hindering gastrointestinal digestion and absorption of nutrients, thereby reducing animal feed intake and adversely affecting growth performance. However, the main factor limiting the widespread use of flaxseed meal in livestock and poultry diets is the presence of various anti-nutritional factors, particularly cyanogenic glycosides. Cyanogenic glycosides can be metabolized in animals to form hydrocyanic acid, leading to cell asphyxiation and death. Several studies have shown that microbial fermentation can effectively reduce the levels of anti-nutritional factors and produce organic acids, enzymes, and other beneficial metabolites, enhancing the nutritional value of the ingredient. Therefore, microbial fermentation may be an effective method to degrade cyanogenic glycosides and overcome the bottleneck in the use of flaxseed meal in diets. Currently, no research has been conducted on the effective energy value of fermented flaxseed meal as an ingredient. Assessing the energy requirements of feed ingredients is crucial for precise feeding.

[0003] The technology of using Bacillus subtilis to degrade cyanogenic glycosides in flaxseed cake can be found in patent application CN202010663114.X of another research group of our unit, which discloses Bacillus subtilis and its application in the detoxification of flaxseed cake by fermentation.

[0004] After verifying the strain, the applicant found that the strain needs to be further optimized in terms of degrading cyanogenic glycosides in flaxseed cake, reducing the content of trypsin inhibitors, and neutral detergent fiber and / or acid detergent fiber in flaxseed cake.

[0005] At the same time, CN201210567223.7 discloses a Bacillus subtilis, a screening and culture method thereof, and a method for treating soybean meal. It discloses that the screened Bacillus subtilis can achieve a trypsin inhibitor degradation rate of 55% after fermentation and culture at 35°C for 48 hours.

[0006] CN116396915A discloses a strain of Bacillus subtilis without specific resistance genes and its application. After being mixed with wheat straw and fermented for 48 hours, it can effectively destroy the surface morphology structure of wheat straw, reduce the cellulose crystallinity of wheat straw by about 6%, and reduce the neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents of wheat straw by 6.6% and 12.1%, respectively.

[0007] CN116463238B discloses a cellulose-degradable Bacillus subtilis and its application. Experiments have shown that the cellulose-degradable Bacillus subtilis can significantly destroy the fiber structure of wheat straw, reducing the neutral detergent fiber and acid detergent fiber contents of wheat straw by 5.8% and 11.3%, respectively, and reducing the cellulose crystallinity by 3.3%.

[0008] From the above text records, it can be seen that there have been documents documenting the ability of Bacillus subtilis to degrade cyanogenic glycosides, trypsin inhibitors, neutral detergent fiber, and acid detergent fiber in feed, but most strains only perform well in one aspect and are not excellent in other aspects.

[0009] Therefore, the technical problem solved in this case is: how to prepare a fermented flaxseed meal with a high cyanogenic glycoside degradation rate, low trypsin inhibitor content, and low neutral detergent fiber and / or acid detergent fiber to partially replace corn and soybean meal in the diet, thereby realizing the resource utilization of flaxseed meal and reducing feed costs. Summary of the Invention

[0010] The present invention aims to provide a flaxseed meal fermentation method. The method uses a strain of Bacillus subtilis FRI with relatively excellent performance isolated by the present applicant to ferment the flaxseed meal. The method can degrade cyanogenic glycosides in the flaxseed meal, reduce the trypsin inhibitor content, and reduce the neutral detergent fiber and / or acid detergent fiber in the flaxseed meal. The method can effectively partially replace corn and soybean meal in the diet, realize the resource utilization of the flaxseed meal, and reduce feed costs.

[0011] At the same time, the invention also provides a diet based on the fermented flaxseed meal.

[0012] To achieve the above-mentioned object, the present invention provides the following technical solution: a method for fermenting flaxseed meal, using Bacillus subtilis FRI as a fermentation bacterium to ferment flaxseed meal; the Bacillus subtilis FRI has a deposit number of CGMCC NO.28734; a deposit date of October 24, 2023, and a deposit unit of the General Microbiology Center of the China Culture Collection Administration.

[0013] In the above-mentioned flaxseed meal fermentation method, the method specifically comprises: crushing the flaxseed meal and mixing it with water, adding a carbon source, and then inoculating a microbial fermentation liquid containing Bacillus subtilis FRI; and sealing and fermenting.

[0014] In the above-mentioned flaxseed meal fermentation method, the flaxseed meal is crushed and mixed with water at a weight ratio of 1:0.4-0.8, a carbon source is added, and then a microbial fermentation liquid is inoculated; after thorough mixing, the mixture is placed in a fermentation bag, sealed, and stored at 37°C for 13-15 days.

[0015] In the above-mentioned flaxseed meal fermentation method, the flaxseed meal is ground and mixed with water at a weight ratio of 1:0.6, molasses equivalent to 2% by weight of the flaxseed meal is added, and then a microbial fermentation liquid equivalent to 4% by weight of the flaxseed meal is inoculated; after thorough mixing, the mixture is placed in a fermentation bag, sealed, and stored at 37°C for 14 days;

[0016] The content of Bacillus subtilis FRI in the microbial fermentation broth was 1.8×10 8 CFU / mL.

[0017] At the same time, the present invention also discloses a diet containing 1wt% to 50wt% of fermented linseed meal; the fermented linseed meal is prepared by any of the above methods.

[0018] In the above-mentioned diet, the diet contains 25 wt% to 35 wt% of fermented flaxseed meal.

[0019] In the above-mentioned diet, the diet contains the following components:

[0020] Corn 40-70wt%;

[0021] Soybean meal 10-20wt%;

[0022] Fermented linseed meal 25wt% to 35wt%;

[0023] Calcium dihydrogen phosphate 1-2 wt%;

[0024] Salt 0.1-0.5wt%;

[0025] Stone powder 0.1~2wt%;

[0026] Used to provide 0.1-1wt% of premix of vitamins and trace elements.

[0027] In the above-mentioned diet, the diet contains the following components:

[0028] Corn 52-58 wt%;

[0029] Soybean meal 10-13wt%;

[0030] Fermented flaxseed meal 27wt% to 31wt%;

[0031] Calcium dihydrogen phosphate 1.2-1.5wt%;

[0032] Salt 0.2-0.4wt%;

[0033] Stone powder 0.8~1.5wt%;

[0034] Used to provide 0.3-0.7wt% of premix of vitamins and trace elements.

[0035] In the above-mentioned diet, the diet contains the following components:

[0036] Corn 58-64wt%;

[0037] Soybean meal 7-13wt%;

[0038] Bran 8-10wt%;

[0039] Fermented linseed meal 10-20wt%;

[0040] Soybean oil 0.65-0.9wt%;

[0041] Calcium dihydrogen phosphate 0.4-0.5wt%;

[0042] Stone powder 1-1.5wt%;

[0043] Appropriate amounts of amino acids, salt, phytic acid, premix, and chromium trioxide.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] On a dry matter basis, the digestible energy value and metabolizable energy value of flaxseed meal were 14.54 MJ / kg and 12.85 MJ / kg, respectively, and the digestible energy value and metabolizable energy value of fermented flaxseed meal were 16.68 MJ / kg and 15.24 MJ / kg, respectively.

[0046] At the same time, after a more in-depth analysis of the fermented flaxseed meal, it was found that its degradation rate of cyanogenic glycosides CGs was slightly higher than that of the Bacillus subtilis recorded in CN202010663114.X; it was significantly better than the Bacillus subtilis recorded in CN202010663114.X in reducing the content of trypsin inhibitor, neutral detergent fiber and acid detergent fiber.

[0047] Through breeding experiments, it was found that the fermented flaxseed meal of the present invention can significantly improve the apparent digestibility of CP, NDF, ADF, Ca and P in growing pigs after replacing part of the corn and soybean meal in the diet. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 is the Bacillus subtilis FRI plate map;

[0049] Figure 2 is the growth curve of Bacillus subtilis FRI;

[0050] Figure 3 is the Gram staining image of Bacillus subtilis FRI;

[0051] Figure 4 The figure shows the comparison of 16S rRNA sequencing of Bacillus subtilis FRI with the NCBI database;

[0052] Figure 5 The metabolic diagram of Bacillus subtilis FRI; "+" indicates a substrate that can be utilized, "-" indicates a substrate that cannot be utilized, and "w" indicates a substrate that can be metabolized but with a weak metabolic capacity;

[0053] Figure 6A This is a chart showing the effect of solid-state fermentation of flaxseed meal with Bacillus subtilis FRI on the content of cyanogenic glycosides (CGs). FSC stands for flaxseed meal; SFFSC stands for flaxseed meal treated with solid-state fermentation. G-SFFSC is a comparative treatment of the bacteria (CGMCC No. 18229) used in patent CN202010663114.X (all subsequent figures represent this meaning).

[0054] Figure 6B This is a chart showing the effect of solid-state fermentation of flaxseed meal with Bacillus subtilis FRI on reducing the content of cyanogenic glycosides CGs;

[0055] Figure 7A This is a graph showing the effect of trypsin inhibitor content on solid-state fermentation of flaxseed meal by Bacillus subtilis FRI;

[0056] Figure 7B This is a chart showing the effect of solid-state fermentation of flaxseed meal with Bacillus subtilis FRI on reducing the content of trypsin inhibitors;

[0057] Figure 8A This is a graph showing the effect of Bacillus subtilis FRI solid-state fermentation on the neutral detergent fiber (NDF) content of flaxseed meal.

[0058] Figure 8B This is a chart showing the effect of reducing the acid detergent fiber (ADF) content of flaxseed meal through solid-state fermentation with Bacillus subtilis FRI. DETAILED DESCRIPTION

[0059] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0060] Part I Isolation, Culture, and Identification of Bacteria

[0061] 1.1 Preparation method of Bacillus subtilis

[0062] The present invention uses a plate smear separation method to isolate Bacillus subtilis from flaxseed meal waste. By observing the colony morphology, color, size, shape, edge characteristics, surface roughness, etc., and referring to the "Handbook of Common Bacterial System Identification", a preliminary identification of the selected strains was performed, and one Bacillus subtilis strain was identified. Glycerol (final concentration of 25%) was added to the culture medium and stored at -80°C.

[0063] 1.2 Identification of Bacillus subtilis

[0064] refer to Figure 1 , the test strain was streaked on an LB solid plate (5 g yeast extract, 10 g tryptone, 10 g sodium chloride, 15.0 g agar, NaOH adjusted to pH 7.0 ± 0.2, 1000 mL distilled water. Sterilize at 121 ° C for 15 min, cool and pour the plate) to isolate a single colony, pick a single colony and inoculate it into 5 ml liquid LB medium, incubate it at 37 ° C for 24 h, as the activated first-generation strain; then the first-generation strain was transferred to 5 mL liquid LB medium at a 4% inoculum size, incubated at 37 ° C for 24 h, as the activated second-generation strain, and the activated second-generation strain was used for subsequent experiments.

[0065] The activated Bacillus subtilis strain was inoculated into LB liquid culture medium at a ratio of 3% by mass and cultured at 37° C. for 24 h.

[0066] The morphological, physiological and biochemical characteristics of the above-mentioned preserved bacterial species were identified with reference to the Bergey's Manual of Systematic Bacteriology, and their genetic classification was identified by determining the 16S rRNA gene sequence.

[0067] refer to Figure 2 , the growth curve of the Bacillus subtilis SFFSC of the present invention shows that the strain of the present invention can enter the plateau phase in a shorter time. In the article "Screening of Cyanogenic Glycoside Detoxification Strains and Research on Fermentation of Flaxseed Cakes" written by Professor Guo Baozhu and the Bacillus subtilis G-SFFSC of CN202010663114.X, the plateau phase of this strain appears later than that of the strain of the present invention;

[0068] Morphological characteristics of the strain:

[0069] refer to Figure 3 The strain was identified as a Gram-positive bacterium with a purple Gram stain. The colonies on LB solid medium were off-white, translucent, circular, slightly convex in the middle, with a moist surface and neat edges.

[0070] Strain identification based on 16S rRNA: PCR amplification was performed using Bacillus subtilis FRI genomic DNA as a PCR amplification template in a 25 μL reaction system. Template DNA 0.5 μL, 10 mmoL upstream and downstream primers (27F / 1492R) 0.5 μL each, 2× Taq enzyme 12.5 μL, and ddH2O added to 25 μL. PCR cycle parameters were: 95°C pre-denaturation for 5 min; 95°C denaturation for 3 min, 57°C annealing for 30 s, 72°C extension for 2 min, 30 cycles; 72°C extension for 10 min. The PCR amplification product was sent to BGI for sequencing. The 16S rRNA sequence of the strain was (SEQ ID NO.1):

[0071] CTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACA

[0072] GATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTG

[0073] GGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATAC

[0074] CGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCT

[0075] ACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGG

[0076] CTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACAC

[0077] TGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATC

[0078] TTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAG

[0079] GTTTCCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGA

[0080] ATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACG

[0081] TGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATT

[0082] GGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCC

[0083] GGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGA

[0084] GGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGA

[0085] ACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCG

[0086] AAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAA

[0087] ACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTA

[0088] ACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAA

[0089] AGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGA

[0090] AGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGA

[0091] GATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCG

[0092] TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCC

[0093] TTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGT

[0094] GACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATG

[0095] ACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACC

[0096] GCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCT

[0097] GCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATG

[0098] CCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGT

[0099] The sequence obtained by sequencing was compared with the NCBI GenBank database for homology analysis. The 16S rRNA sequence of the strain was 100% similar to the 16S rRNA sequence of the Bacillus subtilis strain in the NCBI GenBank database, indicating that the strain was Bacillus subtilis and was named Bacillus subtilis FRI (eg Figure 4 shown).

[0100] 1.3 Physiological and biochemical characteristics of Bacillus subtilis

[0101] The strain was cultured at 37°C in a GENIII identification plate 1030 using a Biolg device to determine its metabolic capacity. The results showed that the strain could utilize dextrin, D-trehalose, D-cellobiose, raffinose, glucose, D-mannose, D-fructose, D-sorbitol, D-sorbitol, pectin, lactose, sucrose, gentiobiose, and L-malic acid, but could not utilize D-maltose, D-galactose, etc. (e.g. Figure 5 shown).

[0102] Part II Performance Testing

[0103] 2.1 Evaluation of the ability of Bacillus subtilis FRI to degrade cyanogenic glycosides

[0104] The frozen Bacillus subtilis FRI strain was inoculated into 30 mL of LB liquid medium and placed in a constant temperature shaker at 37°C and 200 rpm for 24 hours for activation. The activated bacterial solution was set aside for use. The specific fermentation system for solid-state fermentation of flaxseed meal was as follows: 400 g of air-dried flaxseed meal, 8 g of molasses, 216 g of water, 20 mL of Bacillus subtilis FRI bacterial solution (viable cell count of 1.8 × 10 8 CFU / mL). Alternatively, the fermentation system can be scaled up proportionally, for example, by mixing 9 kg of flaxseed meal, 180 g of molasses, 4.86 kg of water, and 450 mL of Bacillus subtilis FRI culture. The mixture is then placed into an anaerobic fermentation bag equipped with a breathing valve, sealed, and placed in a constant-temperature incubator for fermentation at 37°C for 14 days. After fermentation, a sample is taken for determination of CGs content.

[0105] CGs content was determined using the colorimetric method described in GB / T 13084-2006. The specific steps are as follows: 10 g (accurate to 0.001 g) of sample was accurately weighed into a 500 mL conical flat-bottom distillation flask. 250 mL of deionized water was accurately added, and the flask was sealed. After standing at room temperature for 4 hours, 20 mL of zinc acetate solution and 2.0 g of tartaric acid were added. The flask was quickly connected to an HCA-300 multifunctional distiller for distillation. The distillate was collected in a 250 mL conical flask containing 20 mL of NaOH solution (concentration 20 g / L). When the distillate reached the 150 mL mark on the flask, the flask was removed, the distiller was closed, and distillation was terminated. The liquid in the conical flask was completely transferred to a 250 mL volumetric flask and brought to volume. After mixing the liquid in the volumetric flask, accurately pipette 10 mL into a 25 mL colorimetric tube. Add 1 mL of NaOH solution (10 g / L) and 1 drop of phenolphthalein test solution. Slowly add 4% acetic acid solution until the red color disappears. Add 5 mL of phosphate buffer solution (pH = 7) and place in a 37°C constant temperature water bath for 10 minutes. Add 0.25 mL of chloramine T solution (10 g / L) and mix thoroughly. Let it stand for 5 minutes. Add 5 mL of isonicotinic acid-pyrazolone solution, add deionized water to the constant volume line of the colorimetric tube, mix thoroughly, and place in a 37°C constant temperature water bath for 40 minutes. Adjust the zero point with a zero tube and measure the OD value using a UV spectrophotometer at a wavelength of 638 nm. Dilute the "Cyanide Content Analysis Standard Material in Water" with NaOH solution (concentration 2 g / L) to a 1 μg / mL "Cyanide Ion Standard Intermediate Solution." Use a pipette to measure 0.0 mL, 0.3 mL, 0.6 mL, 0.9 mL, 1.2 mL, and 1.5 mL of the Cyanide Ion Standard Intermediate Solution into a 25 mL colorimetric tube. Add water to the 10 mL volume. Measure the OD value according to the above steps. Plot a standard curve with CN- concentration as the abscissa and OD value as the ordinate. Calculate the CGs content (as CN-) and CGs removal rate in the samples using the following formula. CGs content is expressed on a dry matter basis.

[0106]

[0107] Where:

[0108] X—CGs content in sample (in CN - The unit is milligrams per kilogram (mg / kg);

[0109] A—Determine the cyanide mass of the sample solution (in CN - The unit is microgram (μg);

[0110] 1 000—conversion factor;

[0111] m—sample mass, in grams (g);

[0112] V2—volume of distillate used for determination, in milliliters (mL);

[0113] V1—total volume of sample distillate, in milliliters (mL);

[0114] CGs removal rate (%) = (CGs content in FSC - CGs content after fermentation) / CGs content in FSC × 100.

[0115] 2.2 Evaluation of the ability of Bacillus subtilis FRI to reduce trypsin inhibitors

[0116] The frozen Bacillus subtilis FRI strain was inoculated into 30 mL of LB liquid medium and placed in a constant temperature shaker at 37°C and 200 rpm for 24 hours for activation. The activated bacterial solution was set aside for use. The specific fermentation system for solid-state fermentation of flaxseed meal was as follows: 400 g of air-dried flaxseed meal, 8 g of molasses, 216 g of water, 20 mL of Bacillus subtilis FRI bacterial solution (viable cell count of 1.8 × 10 8 CFU / mL). Alternatively, the fermentation system can be scaled up proportionally, for example, by mixing 9 kg of flaxseed meal, 180 g of molasses, 4.86 kg of water, and 450 mL of Bacillus subtilis FRI culture. The mixture is then placed into an anaerobic fermentation bag equipped with a breathing valve, sealed, and placed in a constant-temperature incubator at 37°C for 14 days. After fermentation, a sample is taken for determination of trypsin inhibitor content.

[0117] The trypsin inhibitor was determined using an ELISA kit. The specific determination method was described in the kit instructions.

[0118] 2.3 Evaluation of the ability of Bacillus subtilis FRI to degrade fiber components

[0119] The frozen Bacillus subtilis FRI strain was inoculated into 30 mL of LB liquid medium and placed in a constant temperature shaker at 37°C and 200 rpm for 24 hours for activation. The activated bacterial solution was set aside for use. The specific fermentation system for solid-state fermentation of flaxseed meal was as follows: 400 g of air-dried flaxseed meal, 8 g of molasses, 216 g of water, 20 mL of Bacillus subtilis FRI bacterial solution (viable cell count of 1.8 × 10 8 CFU / mL). Alternatively, the fermentation system can be scaled up proportionally, for example, by mixing 9 kg of flaxseed meal, 180 g of molasses, 4.86 kg of water, and 450 mL of Bacillus subtilis FRI culture. The mixture is then placed into an anaerobic fermentation bag equipped with a breathing valve, sealed, and placed in a constant-temperature incubator at 37°C for 14 days. After fermentation, a sample is taken for determination of trypsin inhibitor content.

[0120] Neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents were determined according to the method of Van Soest et al. (1991).

[0121] Part III Results Analysis

[0122] Under the solid-state fermentation conditions of temperature: 37 ° C, material-water ratio: 1:0.6, fermentation time: 14 days, and Bacillus subtilis FRI inoculation amount of 4%, the content of cyanogenic glycosides CGs in flaxseed meal can be effectively reduced (reduced by 55.0%) (such as Figure 6A As shown), it also significantly reduced the trypsin inhibitor content in flaxseed meal (reduced by 66.18%) (as shown Figure 7A As shown), reduce the fiber components in flaxseed meal that are not easily digested and utilized by animals, such as neutral detergent fiber (reduced by 34.1%) (as shown Figure 8A ) and acid detergent fiber content (reduced by 12.7%) (as shown in Figure 8B In addition, the degradation rate of cyanogenic glycosides CGs was significantly improved under the fermentation conditions (as shown in Figure 2). Figure 6B As shown), the patented bacteria have certain advantages. In addition, in reducing trypsin inhibitors (such as Figure 7B As shown), neutral detergent fiber (such as Figure 8A As shown) and acid detergent fiber aspects (as Figure 8B also has obvious advantages.

[0123] According to the records in CN202010663114.X, the content of cyanogenic glycoside CGs is 38-86 mg / kg. For this indicator, the Bacillus subtilis SFFSC of the present invention is relatively similar to the Bacillus subtilis G-SFFSC in this case. At the same time, the results of the article published by Professor Guo Baozhu entitled "Screening of Cyanogenic Glucoside Detoxification Strains and Research on Fermented Flaxseed Cake" are also similar. The performance of the present invention is slightly better.

[0124] The unique advantage of this case is that the Bacillus subtilis SFFSC in this case has a very significant advantage in reducing the content of trypsin inhibitors compared to Bacillus subtilis G-SFFSC;

[0125] At the same time, the present invention is superior to Bacillus subtilis G-SFFSC in reducing the content of neutral detergent fiber and acid detergent fiber, especially in reducing the content of neutral detergent fiber more significantly.

[0126] Part 4 Determination of the effective energy value of fermented flaxseed meal in growing pigs

[0127] 4.1 Experimental Materials

[0128] The flaxseed meal in this experiment was purchased from Hebei Kaikuo Food Group Co., Ltd., and the fermented flaxseed meal was fermented and prepared in this experiment. The specific operation is as follows: 1) The flaxseed meal was crushed and sieved. Subsequently, the flaxseed meal was mixed with water in a ratio of 1:0.6, and 2% (dry weight of flaxseed meal) molasses was added, and then 4% (dry weight of flaxseed meal) of microbial fermentation liquid was inoculated. After thorough mixing, the resulting fermentation sample was placed in a fermentation bag, sealed and stored at 37°C for 14 days. The Bacillus subtilis strain (1.8×10 8 Flaxseed meal was subjected to solid-state fermentation using flaxseed meal (CFU / mL). The strain used in this experiment, Bacillus subtilis FRI (CGMCC No. 28734), was deposited at the General Microbiology Center of the China Culture Collection Administration. The nutritional composition of flaxseed meal and fermented flaxseed meal is shown in Table 1.

[0129] Table 1. Nutritional composition of flaxseed meal and fermented flaxseed meal (%, dry matter basis)

[0130]

[0131] Note: FSC, flaxseed meal; SFFSC, fermented flaxseed meal.

[0132] 4.2 Experimental animals and experimental diets

[0133] In this study, 18 three-way hybrid growing pigs (53.64 ± 5.04 kg initial weight) were randomly divided into three treatment groups, with six replicates per treatment group and one pig per replicate. The control group was fed a corn-soybean meal-based diet, while the experimental diets consisted of a flaxseed meal diet and a fermented flaxseed meal diet. The entire experimental period lasted 10 days, with the first 7 days being a diet adaptation period and the last 3 days being a feces and urine collection period. The experimental diet formulations and nutrient levels are shown in Table 2.

[0134] Table 2. Composition and nutrient levels of experimental diets (%, feeding basis)

[0135]

[0136] Note: 1 The premix provides the following per kilogram of feed: Vitamin A, 35.2 mg; Vitamin D3, 7.68 mg; Vitamin E, 128 mg; Vitamin K3, 8.16 mg; Vitamin B1, 4 mg; Vitamin B2, 12 mg; Vitamin B6, 8.32 mg; Vitamin B12, 4.8 mg; Niacin, 38.4 mg; Calcium pantothenate, 25 mg; Folic acid, 1.68 mg; Biotin, 0.16 mg; Zinc (ZnSO4·H2O), 110 mg; Copper (CuSO4·5H2O), 125 mg; Iron (FeSO4·H2O), 171 mg; Cobalt (CoCl2), 0.19 mg; Manganese (MnSO4·H2O), 42.31 mg; Iodine (Ca(IO3)2), 0.54 mg; Selenium (Na2SeO3), 0.19 mg.

[0137] 4.3 Feeding and management

[0138] The experiment was conducted at Tianpeng Animal Husbandry Co., Ltd. in Langfang City, Hebei Province.

[0139] All experimental pigs were individually housed in stainless steel digestion and metabolism cages for 7 days. During the adaptation period, they were fed a complete diet, and the amount of test feed was gradually increased until the end of the adaptation period, when all the feed was replaced with the test diet. The ambient temperature was maintained at 22±2°C, and drinking water was free. The daily feeding amount of the experimental pigs was 4% of their body weight, and the total feeding amount was divided into two parts, fed at 8:00 and 16:00 every day, and the daily feed intake was recorded. After feeding every day, the shed was cleaned to keep the environment clean and hygienic. The animal experiment protocol involved in this study has been approved by the Animal Feed and Use Professional Committee of the Feed Research Institute of the Chinese Academy of Agricultural Sciences (IFR-CAAS20221025).

[0140] 4.4 Sample collection

[0141] During the experimental collection period, in order to accurately record the feed intake, the feed residue was collected, dried and weighed every day. The last three days of the experiment were the feces and urine collection period. The 24-hour feces of each experimental pig were collected in a metabolic box using the full feces collection method and immediately stored in a refrigerator at -20°C to prevent fermentation. Finally, all the collected feces were mixed evenly and weighed and recorded. Then, about 500g of sample was taken from each pig and placed in a drying manure tray. It was dried in an oven at 65°C for 3 days to constant weight. After crushing and passing through a 40-mesh sieve, the feces sample was placed in a well-labeled sealed bag for testing.

[0142] In addition, urine samples were collected completely during the final three days. 50 ml of 6N HCl was added beforehand and placed under the metabolic chamber. The total urine volume of the experimental pigs over a 24-hour period was accurately measured and recorded. After thorough mixing, 1 / 20 of the daily urine volume was stored in a -20°C refrigerator. At the conclusion of the experiment, urine from each pig was thawed, pooled, and subsampled for further chemical analysis.

[0143] 4.5 Detection indicators and measurement methods

[0144] (1) Detection indicators

[0145] Diet: dry matter, crude protein, crude fat, crude ash, calcium, phosphorus, neutral detergent fiber, acid detergent fiber, gross energy

[0146] Fecal sample: dry matter, crude protein, crude fat, crude ash, calcium, phosphorus, neutral detergent fiber, acid detergent fiber, gross energy

[0147] Urine sample: crude protein, total energy

[0148] (2) Detection method

[0149] Dry matter (DM), crude protein (CP), crude fat (EE), calcium (Ca), phosphorus (P), and crude ash (Ash) were analyzed according to AOAC standards. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined according to the methods of Van Soest et al. (1991). Energy content in fecal and urine samples was measured using an automatic oxygen-nitrogen calorimeter. CGs content was determined using the colorimetric method described in GB / T 13084-2006.

[0150] 4.6 Calculation formula

[0151] The apparent digestibility of DE and ME is calculated according to the formula [1]:

[0152] Dietary apparent DE (MJ / kg) = (GE ingested - GE in feces) / Dietary feed intake

[0153] Dietary apparent ME (MJ / kg) = (GE ingested - GE in feces - GE in urine) / Dietary feed intake

[0154] Apparent DE of raw material (MJ / kg) = (apparent DE of diet - (100 - X%) × DE of basal diet) / X%

[0155] Raw material apparent ME (MJ / kg) = (diet apparent ME - (100 - X%) × basal diet ME) / X%

[0156] ATTD GE (%) = (ingested GE - fecal GE) / ingested GE × 100%

[0157] Among them, X% is the percentage of the energy supply of the diet replaced by the raw material to be tested, which is 30% in this experiment.

[0158] 4.7 Statistical analysis

[0159] All experimental data were tested for normality using tests for normality and homogeneity of variance. All data sets were normally distributed. A one-way analysis of variance was performed using SPSS 25.0 for Windows (SPSS Inc., Chicago, IL, USA). All indicators are expressed as mean ± standard error of the mean (SEM). P < 0.05 indicated statistical significance.

[0160] 4.8 Results

[0161] As shown in Tables 3 and 4, on a dry matter basis, the digestible and metabolizable energy values ​​of flaxseed meal were 14.54 MJ / kg and 12.85 MJ / kg, respectively, while those of fermented flaxseed meal were 16.68 MJ / kg and 15.24 MJ / kg, respectively. The digestible and metabolizable energy values ​​of the fermented flaxseed meal group were significantly higher than those of the flaxseed meal group (P < 0.01). Furthermore, the digestibility of CP, ADF, NDF, Ca, P, and GE in the fermented flaxseed meal group was significantly higher than that in the flaxseed meal group (P < 0.05), and the apparent digestibility of organic matter showed an upward trend (P < 0.10). Regarding dietary nitrogen balance, nitrogen intake, fecal nitrogen, urinary nitrogen, and deposited nitrogen in the flaxseed meal and fermented flaxseed meal groups were all higher than those in the basal diet (P < 0.01), but no significant differences were found between the two groups. Among the raw materials, the SFFSC group showed a trend toward increased nitrogen deposition compared to the FSC group (P = 0.078).

[0162] Table 3. Effects of flaxseed meal and fermented flaxseed meal diets on apparent digestibility of nutrients and nitrogen balance in growing pigs (dry matter basis)

[0163]

[0164] Note: a, b, c indicate that the same letters in the same column represent no significant difference between the two groups (P>0.05), and different letters represent significant difference between the two groups (P<0.05).

[0165] Table 4. Effects of flaxseed meal and fermented flaxseed meal on available energy, apparent digestibility of nutrients, and nitrogen balance in growing pigs (%, dry matter basis)

[0166]

[0167]

[0168] 4.9 Summary

[0169] On a dry matter basis, the digestible and metabolizable energy values ​​of flaxseed meal were 14.54 MJ / kg and 12.85 MJ / kg, respectively, while those of fermented flaxseed meal were 16.68 MJ / kg and 15.24 MJ / kg, respectively. Furthermore, fermented flaxseed meal significantly increased the apparent digestibility of crude protein, neutral detergent fiber, acid detergent fiber, calcium, and phosphorus in growing pigs (P < 0.05).

[0170] Part V Determination of the Effective Energy Value of Fermented Flaxseed Meal in Piglets

[0171] This experiment mainly verifies the effects of different amounts of fermented flaxseed meal on the growth performance of piglets.

[0172] 5.1 Experimental Materials

[0173] The fermentation of flaxseed meal in this experiment is the same as that in Part 4.

[0174] 5.2 Experimental animals and experimental diets

[0175] In this study, 96 growing pigs of the Duroc × Chang × Da strain (9.57 ± 3.57 kg initial weight) were randomly assigned to four treatment groups, each with eight replicates (pens) and three pigs per replicate. The amount of fermented flaxseed meal in treatments 1 to 4 was increased from 0% to 20%. The experimental diets lasted 30 days. The experimental diet formula and nutrient levels are shown in Table 5.

[0176] Table 5 Composition and nutritional level of experimental diets (%, feeding basis)

[0177]

[0178] Note: Premix components are the same as the test formula table in Part 4.

[0179] 5.3 Feeding and management

[0180] The experiment was conducted at the experimental base of Tianpeng Animal Husbandry Co., Ltd. in Langfang, Hebei Province. Pigs were housed in 1.5 m x 1.7 m slatted-floor pens, each equipped with two nipple drinkers and a stainless steel adjustable feed trough. During the experiment, piglets had free access to food and water. Lighting and ventilation conditions were checked daily, and the piggeries were cleaned regularly. The animal experimental protocol for this study was approved by the Animal Husbandry and Use Committee of the Institute of Feed Research, Chinese Academy of Agricultural Sciences (IFR-CAAS20221025).

[0181] 5.4 Sample collection

[0182] From Days 28 to 30 of the experimental period, approximately 300g of fresh fecal samples were randomly collected from each pen. After mixing thoroughly, the samples were dried in a 65°C oven for 72 hours. After 24 hours of moisture recovery, the samples were crushed through a 40-mesh sieve and placed in labeled ziplock bags for testing. On Day 30, 10mL of blood was collected from the anterior vena cava of eight pigs per replicate, one from each group. After the collected blood samples were allowed to rest for 30 minutes, they were centrifuged at 3000r / min at 4°C for 10 minutes to obtain serum samples, which were then stored at 80°C for analysis. On Day 30, 5g of fresh feces were collected from each pen, placed in 2ml cryovials, and stored at -80°C for testing.

[0183] 5.5 Detection indicators and measurement methods

[0184] 5.5.1 Growth performance

[0185] The experimental pigs were weighed in the mornings of day 0 and day 30, and average daily gain (ADG) was calculated. During the experimental period, the piglets' feed intake was recorded daily, and average daily feed intake (ADFI) was calculated. The specific calculation formula is as follows:

[0186] ADFI = [(feed amount during the experimental period - feed remaining amount during the experimental period) / number of heads] / number of experimental days;

[0187] ADG = [(weight at the end of the test - weight at the beginning of the test) / number of heads] / number of test days;

[0188] 5.5.2 Nutrient digestibility

[0189] Dry matter (DM), crude protein (CP), crude fat (EE), calcium (Ca), phosphorus (P), and crude ash (Ash) in diets and fecal samples were determined according to AOAC standards. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined according to the methods of Van Soest et al. (1991). Chromium was determined by atomic absorption spectrometry.

[0190] The calculation formula for the apparent digestibility of each nutrient is as follows:

[0191] Apparent terminal ileal digestibility of dietary nutrients (%) = [100 – (diet Cr content × fecal nutrient content) / (fecal Cr content × dietary nutrient content)] × 100%

[0192] 5.5.3 Serum immunochemical indicators

[0193] Immune indicators: Immunoglobulins (IgA, IgG, and IgM) were measured using the ELISA kit as described in the detailed procedures. Levels were calculated using ELISAcalc fitting of the logistic curve (four-parameter equation). Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), glucose (GLU), total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), total protein (TP), and albumin (ALB) were analyzed using a fully automated biochemical analyzer.

[0194] 5.5.4 Volatile fatty acids

[0195] Weigh 1.5 grams of fresh stool sample into a centrifuge tube. Add 1.5 mL of sterile water to the tube and centrifuge at 15,000 × g for 15 minutes at 4°C. Carefully transfer the resulting supernatant to a gas chromatography vial. Volatile fatty acid content is measured using gas chromatography.

[0196] 5.6 Results

[0197] Please refer to Tables 6 to 10 for the results.

[0198] Table 6. Effects of adding different doses of fermented flaxseed meal to the diet on the growth performance of growing pigs

[0199]

[0200]

[0201] Table 7. Effects of different levels of fermented flaxseed meal in the diet on the apparent digestibility of nutrients in growing pigs

[0202]

[0203] Table 8. Effects of adding different doses of fermented flaxseed meal to the diet on serum immunoglobulins in growing pigs (μg / mL)

[0204]

[0205]

[0206] Note: IgA, immunoglobulin A; IgG, immunoglobulin G; IgM, immunoglobulin M.

[0207] Table 9. Effects of adding different levels of fermented flaxseed meal to the diet on serum biochemical parameters of growing pigs

[0208]

[0209] Table 10. Effects of adding different doses of fermented flaxseed meal to the diet on volatile fatty acids in feces of growing pigs (mmol / kg)

[0210]

[0211]

[0212] 5.7 Summary

[0213] During the growing stage of piglets, fermented flaxseed meal was added to replace soybean meal in the diet at 10%, 15%, and 20%, respectively, to maintain a consistent crude protein level in the diet. The study found that the addition of 10%, 15%, and 20% fermented flaxseed meal had no effect on pig growth performance (Table 6). However, the apparent digestibility of neutral detergent fiber (NDF) (an average increase of 17.89%) and acid detergent fiber (ADF) (an average increase of 15.66%) showed a significant linear increase (Table 7). The addition of fermented flaxseed meal to the diet linearly increased serum immunoglobulin IgG (an average increase of 46.36%) and IgM (an average increase of 116.82%) (Table 8), improving the pigs' immune capacity. The addition of fermented flaxseed meal to the diet linearly decreased serum alanine aminotransferase (ALT) levels (Table 9). Since ALT represents an indicator of liver damage, a lower ALT value indicates better liver function. At the same time, feeding fermented flaxseed meal also linearly increased the production of microbial metabolites (butyric acid) in the intestine that are beneficial to intestinal health (Table 10), improving the beneficial intestinal metabolism of pigs.

[0214] Comprehensive analysis:

[0215] 1. The Bacillus subtilis SFFSC of the present invention has a very significant advantage in reducing the content of trypsin inhibitor compared to Bacillus subtilis G-SFFSC; at the same time, the present invention is superior to Bacillus subtilis G-SFFSC in reducing the content of neutral detergent fiber and acid detergent fiber, especially the reduction of neutral detergent fiber is more obvious.

[0216] 2. The fermented flaxseed meal of the present invention can replace part of the corn and soybean meal in the diet of growing pigs and piglets, thereby improving the growth level of growing pigs, increasing the digestibility of neutral detergent fiber (NDF) and acid detergent fiber (ADF), increasing the immune ability of piglets, enhancing the liver function of pigs, and improving the beneficial intestinal metabolism of piglets.

Claims

1. A method for fermenting flaxseed meal, It is characterized in that Using Bacillus subtilis ( Bacillus subtilis FRI ) as the fermentation bacterium to ferment flaxseed meal; the preservation number of the said Bacillus subtilis ( Bacillus subtilis FRI ) is: CGMCC NO. 28734; preservation date: October 24, 2023, preservation unit: China General Microbiological Culture Collection Center.

2. The fermentation method of flaxseed meal according to claim 1, It is characterized in that The specific method is as follows: After crushing linseed meal, it is mixed with water, then a carbon source is added, and then a microbial fermentation broth containing Bacillus subtilis ( Bacillus subtilis FRI ) is inoculated; it is fermented under sealed conditions.

3. The fermentation method of flaxseed meal according to claim 2, It is characterized in that The flaxseed meal is crushed and mixed with water in a weight ratio of 1:0.4-0.8, a carbon source is added, and then a microbial fermentation liquid is inoculated; after being fully mixed, it is put into a fermentation bag, sealed and stored at 37°C for 13-15 days.

4. The fermentation method of flaxseed meal according to claim 3, It is characterized in that The flaxseed meal is crushed and mixed with water in a weight ratio of 1:0.6, and then molasses equivalent to 2% of the weight of the flaxseed meal is added, and then a microbial fermentation liquid equivalent to 4% of the weight of the flaxseed meal is inoculated; after being fully mixed, it is put into a fermentation bag, sealed and stored at 37°C for 14 days; The content of Bacillus subtilis ( Bacillus subtilis FRI ) in the microbial fermentation broth is 1.8 × 10 8 CFU / mL.

5. A diet, It is characterized in that The diet contains 1wt% to 50wt% of fermented linseed meal; the fermented linseed meal is prepared by the method as claimed in any one of claims 1 to 4.

6. The diet according to claim 5, It is characterized in that The diet contains 25 wt% to 35 wt% of fermented linseed meal.

7. The diet according to claim 6, It is characterized in that The diet contained the following ingredients: Corn 40-70wt%; Soybean meal 10-20wt%; Fermented flaxseed meal 25wt%~35wt%; Calcium dihydrogen phosphate 1~2wt%; Salt 0.1~0.5wt%; Stone powder 0.1~2wt%; Used to provide 0.1-1wt% premix of vitamins and trace elements.

8. The diet according to claim 7, It is characterized in that The diet contained the following ingredients: Corn 52-58wt%; Soybean meal 10-13wt%; Fermented flaxseed meal 27wt%~31wt%; Calcium dihydrogen phosphate 1.2~1.5wt%; Salt 0.2~0.4wt%; Stone powder 0.8~1.5wt%; Premix used to provide 0.3-0.7wt% of vitamins and trace elements.

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