CN114642239B - Method for improving DDGS nutritional quality and application thereof - Google Patents
Method for improving DDGS nutritional quality and application thereof Download PDFInfo
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- CN114642239B CN114642239B CN202210299864.2A CN202210299864A CN114642239B CN 114642239 B CN114642239 B CN 114642239B CN 202210299864 A CN202210299864 A CN 202210299864A CN 114642239 B CN114642239 B CN 114642239B
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
- A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
- A23K10/38—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material from distillers' or brewers' waste
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/28—Removal of unwanted matter, e.g. deodorisation or detoxification using microorganisms
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Polymers & Plastics (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Physiology (AREA)
- Animal Husbandry (AREA)
- Zoology (AREA)
- Nutrition Science (AREA)
- Botany (AREA)
- Mycology (AREA)
- Sustainable Development (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Fodder In General (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for improving DDGS nutritional quality and application thereof. The method specifically comprises the following steps: mixing and fermenting the bacillus subtilis zymocyte liquid and a fermentation substrate formed by mixing a DDGS (distillers dried grains with soluble), an aspergillus oryzae fermentation product and a fermentation bag to prepare fermented DDGS; wherein the fermentation bag comprises calcium carbonate, enterococcus faecalis and Pichia pastoris. The fermentation method can reduce the content of mycotoxin in DDGS and increase the proportion of DDGS in feed, and can convert NSP, cellulose and macromolecular protein which are difficult to decompose in DDGS into nutrient substances which are easy to absorb, such as monosaccharide, polysaccharide, small peptide and the like, thereby improving the absorption utilization rate of DDGS. In addition, the DDGS is low in price, and DDGS is used for replacing high-price soybean meal, cottonseed meal and the like, so that the cost of the feed can be reduced.
Description
Technical Field
The invention belongs to the technical field of biological fermentation, and particularly relates to a method for improving DDGS nutritional quality and application thereof.
Background
Distillers dried grains with solubles (DDGS) is a byproduct of the corn grain processing industry, and is a vegetable protein feed after extraction of alcohol and oil from corn grains. The DDGS contains high content of protein, fat, available phosphorus and other nutrient components, can replace other expensive feed raw materials such as corn, bean pulp and the like in animal feed, and can promote the healthy growth of animals, improve the quality of animal products and reduce the cost of the feed. However, in practical applications, DDGS still has some problems.
Firstly, the DDGS has high mycotoxin content, and the growth of animals is influenced by the high mycotoxin content, so that the addition amount of the DDGS in the feed is limited. In addition, the nutritional components such as protein, fat and sugar in DDGS are often present in the form of macromolecules, and cannot be absorbed and utilized by monogastric animals. Furthermore, the feed has poor palatability, and therefore, various carbohydrases are required to be added to improve the quality of the feed, which further limits the digestibility of DDGS in the feed.
Therefore, a method for processing DDGS is urgently needed to be found, the mycotoxin content in the DDGS can be reduced, the utilization rate of various nutrient substances in the DDGS can be improved, the DDGS resource can be effectively utilized, the application range is further widened, and the cost of the feed is further reduced.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a method for improving the nutritional quality of DDGS and application thereof, the fermentation method can effectively reduce the content of mycotoxin in DDGS and improve the utilization of non-starch polysaccharides (NSP), cellulose and macromolecular proteins in DDGS, and in addition, the DDGS treated by the fermentation method disclosed by the invention has low water content and good palatability, and the content of DDGS in feed can be further improved.
The invention provides a fermentation method of DDGS, which specifically comprises the following steps:
and (3) mixing and fermenting the bacillus subtilis zymocyte liquid and a fermentation substrate consisting of the DDGS, the aspergillus oryzae fermentation product and a fermentation bag to prepare the fermented DDGS.
According to an aspect of the first aspect of the invention, in some embodiments of the invention, the fermentation bag comprises calcium carbonate, enterococcus faecalis and pichia pastoris.
In some preferred embodiments of the invention, the fermentation substrate consisting of DDGS, aspergillus oryzae fermentation product and fermentation package comprises 60-85% DDGS, 10-35% Aspergillus oryzae fermentation product and 1-5% fermentation package, by weight.
In some preferred embodiments of the present invention, the preparation of the bacillus subtilis zymocyte liquid comprises the following steps: inoculating the bacillus subtilis into a culture medium, wherein the inoculation amount is 0.5-2 wt%, the culture temperature is 35-40 ℃, the rotating speed is 160-200 r/min, and the culture time is 18-24 h.
In some more preferred embodiments of the present invention, the culture medium comprises corn flour 5-15 g/L, soybean meal 10-30 g/L, bran 1-10 g/L, glucose 1-10 g/L, yeast extract 1-10 g/L, sodium chloride 1-5 g/L, dipotassium hydrogen phosphate 1-5 g/L, manganese sulfate 0.1-0.5 g/L and magnesium sulfate 0.2-1 g/L.
In some more preferred embodiments of the invention, the medium is prepared according to conventional techniques in the art.
In some preferred embodiments of the present invention, the Aspergillus oryzae fermentation product is prepared by: inoculating aspergillus oryzae into a fermentation substrate, wherein the inoculation amount is 0.5-2 wt%, the water content of the fermentation substrate is 45-48%, the fermentation temperature is 25-30 ℃, the fermentation time is 36-48 h, and the fermentation mode is shallow fermentation.
In some more preferred embodiments of the present invention, the fermentation substrate for Aspergillus oryzae fermentation comprises 30-70% soybean meal, 10-50% DDGS, and 5-20% carbon source, by weight percentage.
In some more preferred embodiments of the present invention, the carbon source is at least one of soy molasses and corn starch.
In some more preferred embodiments of the invention, the Aspergillus oryzae fermentation product has a number of spores of Aspergillus oryzae of 1X 10 9 ～3×10 9 cfu/g。
In some more preferred embodiments of the invention, the Aspergillus oryzae fermentation product has a protease activity of 800 to 1200U/g.
In some more preferred embodiments of the invention, the xylanase activity in the Aspergillus oryzae fermentation product is from 1000 to 1500U/g.
In some preferred embodiments of the present invention, the amount of the bacillus subtilis zymocyte solution added is 18 to 25% by weight of the fermentation substrate.
In some preferred embodiments of the present invention, the number of viable bacteria in the Bacillus subtilis zymophyte liquid is 1 × 10 9 ～3×10 9 cfu/mL。
In some preferred embodiments of the present invention, the protease activity in the Bacillus subtilis zymocyte fluid is 1200-1500U/mL.
In some preferred embodiments of the present invention, the lipase activity in the Bacillus subtilis zymocyte solution is 40-60U/mL.
In some preferred embodiments of the present invention, the fermentation bag comprises 50 to 70% of calcium carbonate, 10 to 25% of enterococcus faecalis and 10 to 25% of pichia pastoris, by weight percentage.
In some more preferred embodiments of the inventionWherein the effective viable count of enterococcus faecalis is 5 × 10 8 cfu/g。
In some more preferred embodiments of the invention, the effective viable count of the pichia yeast is 1 × 10 9 cfu/g。
In some preferred embodiments of the present invention, the water content of the bacillus subtilis ferment liquid is 26% to 33% after mixing with a fermentation substrate consisting of DDGS, aspergillus oryzae fermentation product and fermentation bag.
In some more preferred embodiments of the present invention, the mixed fermentation is aerobic fermentation.
In some more preferred embodiments of the invention, the mixed fermentation is carried out in a device equipped with a breather valve.
In some preferred embodiments of the present invention, the temperature of the mixed fermentation is 25 to 33 ℃.
In some preferred embodiments of the present invention, the time for the mixed fermentation is 10 to 15 days.
In the fermentation method of DDGS, abundant enzyme systems generated by aspergillus oryzae and bacillus subtilis are utilized to slowly degrade the mycotin the DDGS for a long time, compared with the fermentation method of the DDGS in which protease, xylanase and other enzyme preparations are directly added, the digestive enzyme secreted by the enzyme-producing microorganism in the fermentation method of the DDGS has pertinence to a fermentation substrate, is abundant in enzyme systems, has a synergistic effect among different enzymes, and has a degradation effect on the mycotin. The DDGS prepared by the fermentation method has low mycotoxin content, and the addition proportion of the DDGS in various feeds can be increased, so that the cost of the feeds is further reduced. In addition, the fermentation method of the invention can also decompose the non-decomposable macromolecular substances such as NSP, cellulose and the like into monosaccharides and polysaccharides which can be digested and absorbed by small molecules, degrade macromolecular proteins into easily absorbable nutrient substances such as small peptides and the like, and further promote the growth of animals.
The fermentation method adopts two-stage step-by-step fermentation, can coordinate the growth of anaerobic bacteria and aerobic bacteria, reduces the consumption of aerobic degradation of materials in the fermentation process on the premise of ensuring the degradation rate of mycotoxin, can directly use the fermented product for batching without drying, and has low production cost; the anaerobic fermentation time is long, the fermentation is full, the safety is high, the quality of DDGS is obviously improved, pollution is avoided, and the large-scale use is facilitated.
In a second aspect of the invention, a fermentation product for fermenting DDGS comprises a Bacillus subtilis zymocyte liquid, an Aspergillus oryzae fermentation product and a fermentation bag.
According to the second aspect of the present invention, in some preferred embodiments of the present invention, the fermented product comprises 45-60% by weight of bacillus subtilis zymogen liquid, 35-50% by weight of aspergillus oryzae fermented product and 2-6% by weight of fermentation bag.
In some embodiments of the invention, the fermentation package comprises calcium carbonate, enterococcus faecalis, and pichia pastoris.
In some preferred embodiments of the present invention, the fermentation bag comprises 50 to 70% of calcium carbonate, 10 to 25% of enterococcus faecalis and 10 to 25% of pichia pastoris, in percentage by weight.
In some preferred embodiments of the present invention, the fermentation bag comprises 50 to 70% of calcium carbonate, 10 to 25% of enterococcus faecalis and 10 to 25% of pichia pastoris, in parts by weight.
In a third aspect of the invention, there is provided use of a fermented product according to the second aspect of the invention in the preparation of a feed additive.
According to an aspect of the third aspect of the invention, in some embodiments of the invention, the feed is a pig feed, a poultry feed and an aquaculture feed.
The invention has the beneficial effects that:
(1) The fermentation method can reduce the content of mycotoxin in DDGS and increase the proportion of DDGS in the feed, in addition, the fermentation method can convert NSP, cellulose and macromolecular protein which are difficult to decompose in DDGS into nutrient substances such as monosaccharide, polysaccharide, small peptide and the like which are easy to absorb, the absorption utilization rate of DDGS is improved, the DDGS is low in price, and DDGS is used for replacing bean pulp, cotton pulp and the like with higher price, so that the cost of the feed can be reduced;
(2) The fermentation method can generate mellow fragrance, can improve the palatability of the DDGS, and further improves the ratio of the DDGS in the feed; the DDGS treated by the fermentation method contains abundant probiotics and beneficial metabolites, and can play a probiotic role in the intestinal tracts of animals to achieve the effect of animal health care;
(3) In the fermentation method, the water content in the fermented product is 28-33%, and the fermented product has good free-running property, so that the fermented product can be directly used for producing formula feed without continuously drying, thereby not only saving a large amount of energy consumption and labor in the drying process, but also avoiding the damage of drying on nutrients such as vitamins, amino acids and the like.
Detailed Description
The invention is further described below in conjunction with specific embodiments, and the advantages and features of the invention will become more apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and substitutions are intended to be within the scope of the invention.
The bacillus subtilis in the embodiment is purchased from China center for culture collection and management of industrial microorganisms, and is numbered as CICC 40176.
The aspergillus oryzae in the embodiment is purchased from research center of labor-saving microbial strain engineering technology in Henan, and is numbered as BNCC223758.
Assays for enzymatic activity in this example were tested according to routine techniques in the art, see in particular GBT28715-2012, GBT23874-2009, or GBT5523-2008.
Example 1
(1) Culturing the bacillus subtilis:
the bacillus subtilis is cultured according to the conventional technical means in the field, wherein the culture medium comprises 1L of distilled water, 10g of corn meal, 15g of soybean meal, 5g of bran, 5g of glucose, 3g of yeast extract, 2g of sodium chloride, 2g of dipotassium hydrogen phosphate, 0.2g of manganese sulfate and 0.6g of magnesium sulfate. The inoculation amount of the bacillus subtilis is 1wt%, the culture temperature is 37 ℃, the rotating speed is 180r/min, and the culture time is 20h.
In the obtained Bacillus subtilis culture solution, the effective viable count of Bacillus subtilis is 2.1 × 10 9 cfu/g, the protease activity was 1360u/mL, and the lipase activity was 52u/mL.
(2) Preparing an aspergillus oryzae primary fermented product:
aspergillus oryzae was fermented according to conventional techniques in the art, wherein the fermentation substrate included 30kg of soybean meal, 15kg of DDGS, and 5kg of soy molasses. Inoculating Aspergillus oryzae into a fermentation substrate for primary fermentation, wherein the inoculation amount is 1wt%, adjusting the water content of the fermentation substrate to 45-48% by using clean water, and performing shallow fermentation for 45h at 28 ℃.
The number of Aspergillus oryzae spores in the Aspergillus oryzae fermented product was 1.2X 10 9 cfu/g, 960U/g protease activity, 1120U/g xylanase activity.
(3) Mixed bacteria secondary fermentation:
and (3) taking DDGS as a main secondary fermentation substrate, and adding the primary fermentation product of aspergillus oryzae in the step (2) and calcium carbonate, pichia pastoris and enterococcus faecalis into the DDGS to form the secondary fermentation substrate. Wherein, the DDGS is 70kg, the primary fermentation product of Aspergillus oryzae is 13.5kg, calcium carbonate is 0.9kg, yeast for hypo-yeast is 0.3kg, and enterococcus faecalis is 0.3kg. Adding 20kg of the bacillus subtilis culture solution obtained in the step (1), adjusting the water content of the secondary fermentation substrate to be 30%, uniformly mixing by a ribbon mixer, then using a scattering machine to scatter, filling into a breathing bag, sealing and fermenting, wherein the fermentation temperature is 28 ℃, and the fermentation time is 14 days.
Comparative example 1
The preparation method in comparative example 1 is the same as that in example 1 except that in the mixed-strain secondary fermentation in step (3), the primary fermentation product of Aspergillus oryzae was replaced with 13.5kg of a fermentation substrate of non-inoculated Aspergillus oryzae.
Comparative example 2
Comparative example 2 was prepared as in example 1 except that in the mixed secondary fermentation of step (3), the A.oryzae primary fermentation product was replaced with a mixture of 13.5kg of a fermentation substrate not inoculated with A.oryzae, 0.2kg of a protease and 0.3kg of a xylanase, wherein the protease activity was 10 ten thousand U/g and the xylanase activity was 5 ten thousand U/g.
Comparative example 3
The preparation method in comparative example 3 is the same as that in example 1 except that in the mixed-bacteria secondary fermentation in step (3), the culture solution of Bacillus subtilis was replaced with 20kg of a culture medium that was not inoculated with Bacillus subtilis.
Comparative example 4
The preparation method of comparative example 4 is the same as that of example 1 except that in the mixed secondary fermentation of step (3), the Bacillus subtilis culture solution is replaced with a mixture of 20kg of a Bacillus subtilis non-inoculated medium, 0.3kg of a protease and 0.02kg of a lipase, wherein the activity of the protease is 10 ten thousand U/g and the activity of the lipase is 10 ten thousand U/g.
Comparative example 5
Comparative example 5 is the same as example 1 except that in the mixed secondary fermentation of step (3), the primary fermentation product of Aspergillus oryzae is replaced with a mixture of 13.5kg of a fermentation substrate of non-inoculated Aspergillus oryzae, 0.2kg of a protease and 0.3kg of a xylanase, wherein the activity of the protease is 10 ten thousand U/g and the activity of the xylanase is 5 ten thousand U/g; the Bacillus subtilis culture solution is replaced by a mixture of 20kg of a culture medium without inoculated Bacillus subtilis, 0.3kg of protease and 0.02kg of lipase, wherein the activity of the protease is 10 ten thousand U/g, and the activity of the lipase is 10 ten thousand U/g.
Testing of DDGS degradation
Nutritional indexes such as crude protein (GBT 6432-2018), crude fiber (GBT 6434-2006), true protein (T/NAIA 060-2021), crude fat (GB/T6433-2006), acid soluble protein (NY/T3801-2020), NSP (see "method for measuring non-starch polysaccharide in feed" by Heyue et al) and the content of aflatoxin B1 (GB/T36858-2018) in the fermented DDGS, the DDGS products prepared in example 1 and comparative examples 1-5 were examined.
As shown in Table 1, it is evident from Table 1 that the crude protein content in the DDGS product after fermentation using the fermentation method in the present example increased from 27.3% to 36.8%, the true protein content increased from 76.4% to 95.1%, the crude fat decreased from 8.4% to 1.6%, the crude fiber decreased from 7.8% to 3.3%, the acid soluble protein increased from 12.9% to 27.2%, the NSP decreased from 32.6% to 13.1%, and the aflatoxin B1 decreased from 49.5. Mu.g/kg to 12.8. Mu.g/kg, compared to the unfermented DDGS. The content of crude, true and prolamin in the fermented DDGS prepared in comparative examples 1-5 were all lower than the DDGS prepared in inventive example 1, while the content of crude fat, crude fiber, NSP and aflatoxin were significantly higher than the DDGS in example 1. The fermentation method in the embodiment 1 of the invention can effectively degrade macromolecular substances in the DDGS, is beneficial to the absorption of nutrient substances in the DDGS, greatly reduces the aflatoxin B1, and makes the large-proportion application of the fermented DDGS in the pig poultry feed possible.
TABLE 1 comparison of the content of substances before and after fermentation of DDGS (in terms of air-dried substance)
Testing of digestibility before and after DDGS fermentation
The digestibility before and after DDGS fermentation was determined by mimicking the digestion of monogastric animals.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Specific sources of materials used in the following examples are shown in table 2.
TABLE 2 statistical table of sources of materials used in the testing of digestibility before and after DDGS fermentation
1. Preparation of samples
1. Treatment of test samples
Taking 200g of unfermented DDGS, the DDGS product fermented in the embodiment 1 of the invention and the DDGS products fermented in the comparative examples 1-5, respectively, placing the products in an electric heating thermostat, drying, wherein the drying temperature is (65 +/-1) DEG C, the drying time is 24h, crushing the dried sample by using a plant crusher or a mortar, sieving by using a test sieve after crushing, and sealing and storing the treated sample for later use.
2. Preparation of simulated samples
(1) Preparation of gastric buffer
10.36g of sodium chloride, 0.98g of potassium chloride and 4g of digestion stabilizer are weighed into a 2000mL beaker, 1800mL of deionized water are added for dissolution, and the pH of the solution is adjusted to 2 (39 ℃) with 2mol/L hydrochloric acid. After cooling, the solution was transferred to a 2000mL volumetric flask and made to volume with deionized water.
(2) Preparation of Small intestine buffer
8.32g of anhydrous disodium hydrogenphosphate, 40.96g of anhydrous sodium dihydrogenphosphate, 11.55g of sodium chloride, 2.45g of potassium chloride, 160 million U of penicillin (0.969 g of penicillin in the test of the present invention), and 8g of a digestion stabilizer were weighed into a 2000mL beaker, and 1800mL of deionized water was added to dissolve the solution, and the pH of the solution was adjusted to 6.44 (39 ℃ C.) with 1mol/L of phosphoric acid or 1mol/L of sodium hydroxide. After cooling, the solution was transferred to a 2000mL volumetric flask and made to volume with deionized water.
(3) Preparation of Large intestine buffer solution
7.99g of anhydrous disodium hydrogenphosphate, 41.23g of anhydrous sodium dihydrogenphosphate, 11.98g of sodium chloride, 1.82g of potassium chloride, 160 million U of penicillin (0.969 g of penicillin in the test of the present invention), and 8g of a digestion stabilizer were weighed into a 2000mL beaker, and 1800mL of deionized water was added to dissolve them, and the pH of the solution was adjusted to 6.42 (39 ℃) with 1mol/L of phosphoric acid or 1mol/L of sodium hydroxide. After cooling, the solution was transferred to a 2000mL volumetric flask and made to volume with deionized water.
(4) Preparation of simulated gastric fluid
Simulated gastric fluid (pepsin activity 737.5U/mL): the activity of pepsin was determined according to the pepsin activity assay described by KLANS/X1001-2010. Then, 84.38KU of pepsin was dissolved in 250mL of a hydrochloric acid solution having a pH of 2.0 (pH value was measured at 39 ℃) in accordance with the concentration of 737.5U/mL of pepsin in the simulated gastric fluid, and the solution was slowly stirred until dissolved and prepared immediately before use.
(5) Preparation of simulated small intestine solution
Simulated intestinal fluid (amylase activity 221.43U/mL, trypsin activity 69.10U/mL, chymotrypsin activity 8.68U/mL): the activities of corresponding digestive enzymes in the pig intestine liquid enzyme powder reagent-grade amylase, trypsin and chymotrypsin are determined according to an alpha-amylase activity determination method described by KLANS/X1002-2010, a trypsin activity determination method described by KLANS/X1003-2010 and a chymotrypsin activity determination method described by KLANS/X1004-2010. Then, according to the activity of the three digestive enzymes in the simulated intestinal fluid, 53.59KU of amylase, 16.72KU of trypsin and 2.10KU of chymotrypsin are respectively weighed and dissolved in 22mL deionized water, and are slowly stirred until dissolved, and are prepared before use.
(6) Preparation of simulated large intestine solution
According to the method for measuring the activity of the cellulase described in GB/T23881-2009, the activity of the corresponding digestive enzyme in the reagent grade cellulase is measured. 11U of cellulase was weighed out and dissolved in 22mL and stirred slowly until dissolved. The concentration of the finally prepared cellulase is 0.04U/mL, and the cellulase is prepared before use.
2. Design of the Experimental protocols
Dialysis bags used in the testing of digestibility before and after DDGS fermentation were purchased from Viskase, USA under the model of MEMBRA-CEL MD34-14, and had a molecular weight cut-off of 14000 daltons and a flat diameter of 44mm. The pretreatment steps of the dialysis bag are as follows: cut the dialysis bag into 25cm pieces. The dialysis bag was boiled in 2000mL of a mixture solution of sodium bicarbonate with a mass-volume fraction of 2% and 1mmol/L of disodium ethylenediaminetetraacetate solution (pH = 8) for 10 minutes. After the mixed solution was poured off, the mixture was washed with distilled water 3 to 5 times. The washed dialysis bag was placed in a 1mmol/L disodium ethylenediaminetetraacetate solution (pH = 8) and boiled for 10 minutes. After the waste liquid was discarded, deionized water was added and stored in a refrigerator at 4 ℃ for further use. Before use, the dialysis tube is filled with water and then discharged, and the dialysis bag is thoroughly cleaned.
1000mL of gastric buffer solution, 1000mL of buffer solution at the front section of the small intestine and 1000mL of buffer solution at the rear section of the small intestine are respectively poured into a buffer solution bottle, the buffer solution bottle is placed into a buffer solution temperature-controlled storage chamber of the bionic digestive system of the monogastric animals, and a pipeline of the system is well connected with the buffer solution bottle. In the control software of the bionic digestion system, the preheating time of the bionic digestion system of the monogastric animals is set to be 60min; the mixing frequency was set to 180rpm and the buffer flow rate was set to 120mL/min; the temperatures of the simulated digestion chamber, the buffer solution control chamber and the pipeline heat preservation chamber are set to be 39 ℃; the temperature of the digestive juice heat preservation chamber is set to be 5 ℃; (the gastric digestion time is set to 4h, the gastric buffer emptying time is set to 4min, the volume of cleaning solution (deionized water) in the gastric digestion stage is set to 1500 mL/time, the cleaning time is set to 40min, the cleaning frequency is set to 3 times, the switching cycle time of the gastric-small intestine buffer is set to 30min, the digestion time of the small intestine is set to 16h, the emptying time of the small intestine buffer is set to 4min, the injection volume of the concentrated simulated small intestine solution is set to 2.2mL, the volume of the supplemented concentrated simulated small intestine solution is set to 2mL, the volume of the cleaning solution (deionized water) is set to 1500 mL/time, the cleaning time is set to 40min, and the cleaning frequency is set to 3 times.
The switching cycle time of the small intestine-large intestine buffer solution is set to 30min, the digestion time of the large intestine is set to 6h, the injection volume of the concentrated simulated large intestine solution is set to 2.2mL, the volume of the supplemented concentrated simulated large intestine solution is set to 2mL, the volume of the washing liquid (deionized water) is set to 1500 mL/time, the washing time is set to 40min, and the washing times are set to 6 times).
After digestion is complete, undigested debris in the dialysis bag is transferred without loss to a 90mm petri dish of known absolute dry weight (this process requires the dialysis bag to be removed from the simulated digester and the bag to be longitudinally cut and rinsed with deionized water). Drying the culture dish filled with undigested residues at 65 ℃ until no water mark exists, then drying the culture dish at 105 ℃ until the weight is constant, weighing the dried weight (namely the mass of the dry matter after the sample is digested), and calculating the dry matter digestibility. The digestion residues in the petri dish were scraped off completely and transferred to a petri dish of known absolute dry weight for protein content detection (recorded as protein content after digestion of the sample).
3. Detecting the index
(1) Dry matter digestibility:
(2) Digestibility of protein:
4. analysis of results
The results of dry matter digestibility and protein digestibility of DDGS and DDGS fermented in example 1 and comparative examples 1 to 5 are shown in Table 3. As can be seen from Table 3, the DDGS after fermentation treatment in example 1 showed an increase in dry matter digestibility of 27.79% compared to DDGS without fermentation treatment. The protein digestibility is improved by 23.2%. In comparison, the DDGS in comparative examples 1 to 5 had lower dry matter digestibility and lower protein digestibility than the DDGS in example 1. The results show that the DDGS is fermented by adopting the fermentation method in the embodiment of the invention, so that the digestion utilization rate of nutrient substances in the DDGS can be greatly improved, the cost is reduced, and the resources are saved.
TABLE 3 statistical Table of dry matter and protein digestibility of unfermented DDGS and DDGS treated in example 1 and comparative examples 1 to 5
The embodiment 1 is a preferred embodiment of the present invention, but the present invention is not limited to the above embodiment, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, and equivalents thereof are intended to be included in the scope of the present invention.
Claims (3)
1. A fermentation process for reducing aflatoxin B1 in DDGS, comprising the steps of:
(1) Culturing the bacillus subtilis:
inoculating the bacillus subtilis into a culture medium, wherein the inoculation amount is 0.5-2wt%, the culture temperature is 35-40 ℃, the rotation speed is 160-200r/min, and the culture time is 18-24h, so as to obtain a bacillus subtilis zymocyte liquid;
wherein the culture medium comprises 5-15g/L corn flour, 10-30g/L soybean meal, 1-10g/L wheat bran, 1-10g/L glucose, 1-10g/L yeast extract, 1-10g/L sodium chloride, 1-5g/L dipotassium hydrogen phosphate, 0.1-0.5g/L manganese sulfate and 0.2-11g/L magnesium sulfate;
(2) Preparation of aspergillus oryzae fermentation product:
inoculating aspergillus oryzae into a fermentation substrate, wherein the inoculation amount is 0.5 to 2wt%, the water content of the fermentation substrate is 45 to 48%, the fermentation temperature is 25 to 30 ℃, the fermentation time is 36 to 48h, and the fermentation mode is shallow fermentation to obtain an aspergillus oryzae fermentation product;
wherein the fermentation substrate of the aspergillus oryzae comprises 30 to 70 percent of bean pulp, 10 to 50 percent of DDGS and 5 to 20 percent of carbon source;
(3) Mixed fermentation:
mixing and fermenting the bacillus subtilis zymocyte liquid and a fermentation substrate consisting of DDGS, aspergillus oryzae fermentation products and a fermentation bag to prepare fermented DDGS;
wherein the mixed fermentation is aerobic fermentation, the temperature of the mixed fermentation is 25 to 33 ℃, and the time of the mixed fermentation is 10 to 15 days;
the fermentation substrate comprises 60 to 85 percent of DDGS, 10 to 35 percent of Aspergillus oryzae fermentation products and 1 to 5 percent of fermentation bags in percentage by weight;
the fermentation bag comprises 50 to 70 percent of calcium carbonate, 10 to 25 percent of enterococcus faecalis and 10 to 25 percent of pichia yeast;
the addition amount of the bacillus subtilis zymocyte liquid is 18 to 25 percent of the fermentation substrate.
2. The method of claim 1, wherein the effective viable count of the bacillus subtilis zymocyte solution is 1 in10 9 ~3×10 9 cfu/mL, protease activity 1200-1500U/mL, and lipase activity 40-60U/mL.
3. The method of claim 1, wherein the number of Aspergillus oryzae spores in the Aspergillus oryzae fermentation product is 1X 10 9 ~3×10 9 cfu/g, the activity of the protease is 800 to 1200U/g, and the activity of the xylanase is 1000 to 1500U/g.
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