CN118581159A - Method for reutilizing brewery grains - Google Patents
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- CN118581159A CN118581159A CN202410942605.6A CN202410942605A CN118581159A CN 118581159 A CN118581159 A CN 118581159A CN 202410942605 A CN202410942605 A CN 202410942605A CN 118581159 A CN118581159 A CN 118581159A
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Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention belongs to the technical field of waste recycling, and particularly relates to a beer lees recycling method. The method comprises the steps of extruding and puffing brewer's grains, adding bacterial liquid of candida tropicalis (Candida tropicalis) SCYA4 for fermentation, and preserving the candida tropicalis in the China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: m20241049. According to the invention, after the brewer's grains are extruded and puffed, candida tropicalis (Candida tropicalis) SCYA4 is used for fermentation, so that the digestion efficiency of animals on nutrients in the brewer's grains can be remarkably improved.
Description
Technical Field
The invention belongs to the technical field of waste recycling, and particularly relates to a beer lees recycling method.
Background
Beer lees belongs to one of the lees, is a main byproduct waste in the beer brewing industry, and accounts for 80-85 percent. The beer industry has become an important field with the highest potential and development prospect in the Chinese beverage industry by the stimulation of economic development and consumption structure transformation, and the annual output is continuously increased. However, large amounts of brewery waste are produced during the brewing process, with annual yields exceeding 3,000 tens of thousands of tons, which are serious environmental hazards, since they require about 30-60% oxygen for complete oxidation. In addition, brewery waste is rich in essential nutrients, which is an ideal substrate for harmful microorganisms, which may cause groundwater pollution, greenhouse gas emissions, pathogen reproduction, etc. These adverse effects all stimulate the development of new sustainable animal feed resources. From a clean production perspective, it is possible to convert brewery waste into animal feed from linear economy to circular economy, thereby relieving the pressure of food supply and environmental protection. However, the use of nutrients in brewer's grains is inefficient due to the high content of insoluble fiber components in the brewer's grains.
In view of this, the present invention has been made.
Disclosure of Invention
In order to solve the problems in the background technology, the invention provides a method for recycling beer grains, which can obviously improve the digestion efficiency of nutrients in the beer grains.
In order to achieve the above purpose, the invention adopts the following technical scheme:
after extruding and puffing brewer's grains, adding bacterial liquid of candida tropicalis (Candida tropicalis) SCYA4 for fermentation, wherein the candida tropicalis is preserved in China Center for Type Culture Collection (CCTCC) NO: m20241049.
Preferably, the inoculation amount of the bacterial liquid is 10% -20%.
Preferably, the inoculation amount of the bacterial liquid is 15%.
The fermentation temperature and time are selected according to the proper fermentation temperature and time of the microorganism, and the fermentation time is 5-9d and the fermentation temperature is 31-37 ℃ without special limitation; more preferably, the fermentation time is 7d and the fermentation temperature is 34 ℃.
Preferably, the volume ratio of the brewery grain mass to the water in the fermentation process is (0.8-1.2): 1 g/mL.
Preferably, the volume ratio of the brewery grain mass to the water in the fermentation process is 1:1 g/mL. Preferably, the extrusion temperature of the extrusion expansion is 100-110 ℃, the screw speed is 15-17Hz, and the water content is 25-29%.
Preferably, the extrusion temperature of the extrusion expansion is 106 ℃, the screw speed is 16Hz, and the water content is 27%.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, after the brewer's grains are extruded and puffed, candida tropicalis (Candida tropicalis) SCYA4 is used for fermentation, so that the digestion efficiency of animals on nutrients in the brewer's grains can be remarkably improved.
Drawings
FIG. 1 shows the growth pattern of SCYA strain 24 h on wort agar medium;
FIG. 2 is gram stain of SCYA strain culture 24 h;
FIG. 3 is a scanning electron microscope observation of SCYA strain 4;
FIG. 4 shows the result of electrophoresis of the genomic 18S rDNA amplification product of SCYA strain 4;
FIG. 5 is a phylogenetic tree constructed by SCYA strain 4 based on the 18S rDNA sequence.
Detailed Description
The present invention will be further described in detail with reference to the following embodiments, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the description is only illustrative and is not intended to limit the scope of the invention. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The information on some reagents or instruments in the examples below is as follows:
Brewery grain: crushing dried brewery grain purchased from Sichuan Neotade biotechnology Co., ltd, and passing through 60 meshes;
Enrichment screening medium: extruding 10.0 g of brewer's grains, 15.0 g/L of yeast extract 4.5 g/L,NH4NO33.0 g/L,NaCl 5.0 g/L,K2HPO42.0 g/L、MgSO4 .7H2O 0.4 g/L, agar powder, 1L of distilled water and natural pH; the preparation method of the extruded brewer's grain comprises the following steps: adding water into brewer's grains, mixing uniformly to make the water content be 27%, extruding and puffing in DSE double-screw puffing tester to obtain extruded brewer's grains, the extrusion temperature is 106 deg.C, and screw speed is 16 Hz;
wort medium and wort agar medium were purchased from Qingdao sea Bo biotechnology Co., ltd;
Semi-solid fermentation medium: weighing 30g extruded brewer's grains in a fermentation bag, and adjusting the initial water content according to different screening conditions set by a test;
the culture media prepared in the following examples were sterilized by autoclaving at 121℃to 20 min.
EXAMPLE 1 separation screening, identification and characterization of candida tropicalis
1.1 Enrichment and screening of strains
Burying the extruded brewery grains in corrosive soil at 5-10 cm depth under the ground to enrich microorganisms, collecting surrounding soil samples after one month, taking soil sample 10 g, adding into 90mL sterile water, and shaking 30 min at 37deg.C and 150 r/min. Preparing enrichment screening culture medium by shaking, sterilizing, pouring into a flat plate, solidifying, sucking 100 mu L of soil suspension diluted to 10 -4, uniformly coating onto the enrichment screening culture medium, and culturing in a constant-temperature incubator at 30 ℃ in an inverted manner for 48h. And selecting a survival colony similar to the characteristic colony of the saccharomycete, repeatedly purifying the survival colony to be used as a rescreening object, then rescreening the protease activity, and selecting the saccharomycete with the highest protease activity.
1.2 Tolerance test
Temperature resistance: the selected saccharomycetes are inoculated into malt juice culture medium, and are respectively placed in 28, 31, 34, 37, 40 and 43 ℃ constant temperature culture 48 h, and the OD values of the saccharomycetes at different temperatures are measured by taking the initial culture medium as a reference.
Ethanol tolerance: inoculating the re-selected saccharomycetes into malt juice culture medium with alcohol concentration regulated to 0.5%, 1%, 2%, 4%, 6% and 8% separately, culturing at 30 deg.c for 48 h and measuring the OD value of saccharomycetes in different alcohol degrees.
Acid resistance test: inoculating the selected saccharomycetes into malt juice liquid culture mediums with the pH values of 2.5, 3.0, 3.5, 4.0, 4.5 and 5 respectively, culturing at 30 ℃ for 48 h, and measuring OD values of the saccharomycetes under different pH conditions.
A strain with high temperature resistance (37 ℃), ethanol resistance (4%) and acid resistance (pH value 3.5) and high protease activity (23.85U/mL) is obtained through multiple rounds of enrichment and purification, and is named SCYA.
1.3 Identifying and preserving strain
The attribution class of strain SCYA4 was initially identified by plating and routine visual inspection. Scanning electron microscopy was then used to observe colony morphology in wort agar medium: strain SCYA4 was plated on wort agar medium and incubated at 30 ℃ for 24: 24h, the colonies of which were nearly circular, slightly shiny, opaque, milky white (as shown in fig. 1), gram positive (as shown in fig. 2), and visible as isolated pseudohyphae (as shown in fig. 3). The specific physiological and biochemical characteristics are shown in Table 1, and as can be seen from Table 1, strain SCYA4 conforms to the characteristics of yeast.
TABLE 1 physiological and biochemical characteristics of strain SCYA4
。
Note that: "+" indicates positive and "-" indicates negative.
Further molecular biology identification: the method is characterized in that the method comprises the steps of performing a description operation by using a Baotou fungus genome DNA extraction kit, completing the extraction of strain DNA, and then performing PCR amplification on fungus 18S rDNA gene fragments to obtain an amplification product of about 500 bp, wherein the electrophoresis result of the amplification product is shown in figure 4. After the amplified product is detected by agarose gel electrophoresis, the amplified product is sent to the Koch (Orthobody) biological limited company for sequencing, and the base sequence of the 18S rDNA amplified product of the strain SCYA4 is shown as SEQ ID NO: 1. And submitting the sequencing result to NCBI database for Blast comparison analysis, so that the sequence similarity of the strain SCYA4 and candida tropicalis (Candida tropicalis) reaches 95%. The similar strain sequences in GenBank are used as reference sequences, the MEGA software is used for constructing a phylogenetic tree by an orthorhombic method, the phylogenetic tree is shown in figure 5, and the homology of the strain SCYA4 and Candida tropicalis is high.
The isolated strain is identified as candida tropicalis (Candida tropicalis) which is named candida tropicalis (Candida tropicalis) SCYA by combining with the morphological characteristics, physiological and biochemical characteristics identification results and molecular biological characteristics of the strain, and is preserved in China Center for Type Culture Collection (CCTCC) for 5 months and 23 days in 2024, wherein the preservation number is CCTCC NO: m20241049, the preservation address is Wuhan university (Wuhan in China).
Example 2 beer lees reuse method
Preparing fermentation seed liquid: inoculating the strain of candida tropicalis SCYA4 obtained in the example 1 into a malt juice liquid culture medium, carrying out constant-temperature shaking culture at 30 ℃ and 150 r/min for 14-16 hours to obtain a cultured bacterial liquid, and counting the number of viable bacteria to 10 6-107/mL by a flat plate. Inoculating the cultured bacterial liquid into a malt juice liquid culture medium, and culturing for a corresponding time to prepare fermentation seed liquid.
Preparing extruded brewer's grains: adding water into brewer's grains, mixing uniformly to make its water content be 27%, extruding and puffing in DSE double-screw puffing tester to obtain extruded brewer's grains, its extrusion temp. is 106 deg.C, and screw speed is 16 Hz.
Fermentation: weighing extruded brewer's grain 30 g, placing in a solid state fermentation breathing bag, and adding the prepared fermentation seed liquid. Different solid state fermentation conditions are selected for testing, the stirring is uniform, the static culture is carried out, the sampling is carried out at fixed time in the fermentation process, and the drying and the crushing are carried out at 65 ℃ after the fermentation. The setting conditions are as follows: the fermentation temperature is 34 ℃, the fermentation time is 7d, and the volume ratio of the brewery grain mass to the water (namely the feed water ratio) and the bacterial liquid inoculation amount are shown in Table 2.
TABLE 2 setting of feed to Water ratio and inoculum size
。
Comparative example 1
The difference compared with sample 3 in example 2 is that fermentation seed liquid is not used, that is, only the brewer's grains are extruded and puffed, and the rest of the method and parameters are the same as those of sample 3 in example 2.
Comparative example 2
The difference compared to sample 3 of example 2 is that no brewer's grain is extruded and puffed, and the rest of the method and parameters are the same as sample 3 of example 2.
Test example dry matter, crude protein and total in vitro digestibility assay
The dry matter and crude protein digestibility in vitro of each of the untreated brewery, example 2, comparative examples 1-3 were determined by reference to the in vitro simulated digestion method of Boisen et al, and were performed as follows: 1.0 g (accurate to 0.001 g) of the sample is weighed into a 50 mL centrifuge tube, 10 mL of 1 mg/mL pig pepsin solution (pH 2.0) is added, meanwhile, 0.5 mL chloramphenicol solution is added to prevent microorganism growth, and after sealing, the mixture is incubated for 4h at 37 ℃ on a constant temperature water bath shaking table; after the gastric digestion period has ended, the mixture is neutralized with 0.2mo1/LNaOH, 10 mL phosphate buffer (0.2 mol/L, pH 6.8) is added, and the pH is adjusted to 6.8 with 1mol/L HCl or 1mol/L NaOH; then 1mL of 50 mg/mL porcine pancreatin solution is added, sealed and put into a constant temperature water bath oscillator at 39 ℃ to be digested for 24h respectively; after the digestion period of the small intestine is finished, 5mL of 20% sulfosalicylic acid is added to stop the digestion reaction, 15min is centrifuged at 15000 r/min, the supernatant is discarded, and the precipitate is placed in a drying oven at 80 ℃ for overnight and is to be tested.
The results are shown in Table 3. The in vitro digestibility of dry matter and crude protein of comparative example 1 (extruded brewer's grains) was significantly improved (P < 0.05) compared to untreated brewer's grains, by 4.49 and 6.27 percentage points respectively; the in vitro digestibility of dry matter and crude protein of sample 3 of example 2 (fermented extruded brewer under optimal fermentation conditions) was also significantly increased (P < 0.05) compared to comparative example 1 (extruded brewer's grain), by 5.21 percentage points and 12.98 percentage points, respectively, indicating that extrusion pretreatment combined with microbial fermentation can increase the nutrient digestibility of brewer's grain.
TABLE 3 in vitro digestibility of dry matter and crude protein in each sample
。
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. The beer lees reutilization method is characterized in that after beer lees are extruded and puffed, bacterial liquid of candida tropicalis (Candida tropicalis) SCYA is added for fermentation, and the candida tropicalis is preserved in China center for type culture collection (CCTCC NO) in 5 months of 2024: m20241049.
2. The method of claim 1, wherein the bacterial fluid is inoculated in an amount of 10% to 20%.
3. The method of claim 2, wherein the bacterial fluid is inoculated at 15%.
4. The method of claim 1, wherein the fermentation time is 5-9d and the fermentation temperature is 31-37 ℃.
5. The method of claim 4, wherein the fermentation time is 7d and the fermentation temperature is 34 ℃.
6. The method of claim 1, wherein the volume ratio of brewer's grain mass to water in the fermentation process is (0.8-1.2): 1 g/mL.
7. The method of claim 6, wherein the volume ratio of brewer's grain mass to water in the fermentation process is 1:1 g/mL.
8. The method of claim 1, wherein the extrusion temperature of the extrusion is 100-110 ℃, the screw speed is 15-17Hz, and the moisture content is 25-29%.
9. The method of claim 8 wherein the extrusion temperature of the extrusion is 106 ℃, the screw speed is 16 Hz, and the moisture content is 27%.
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| CN202410942605.6A CN118581159A (en) | 2024-07-15 | 2024-07-15 | Method for reutilizing brewery grains |
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