CN110583856A - Method for producing protein feed by carrying out two-time different mixed fermentation on refined brewer grains and blueberry pomace - Google Patents
Method for producing protein feed by carrying out two-time different mixed fermentation on refined brewer grains and blueberry pomace Download PDFInfo
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- CN110583856A CN110583856A CN201911014407.9A CN201911014407A CN110583856A CN 110583856 A CN110583856 A CN 110583856A CN 201911014407 A CN201911014407 A CN 201911014407A CN 110583856 A CN110583856 A CN 110583856A
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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
- A23K10/10—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
- 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
-
- 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
-
- 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
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Polymers & Plastics (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Physiology (AREA)
- Animal Husbandry (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Health & Medical Sciences (AREA)
- Mycology (AREA)
- Botany (AREA)
- Sustainable Development (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for producing protein feed by carrying out two different mixed fermentations on refined brewer's grains and blueberry pomace, belonging to the technical field of production of protein feed. The method specifically comprises the following steps: respectively activating ordinary high-temperature actinomycetes, geotrichum candidum and lactobacillus plantarum by using a stock test tube, performing expanded culture in a triangular flask, performing seed tank culture, culturing and fermenting to obtain bacterial suspension, and mixing the cultured strains in proportion to obtain mixed bacteria; preparing fermentation raw materials for brewing beer grains, blueberry pomace and bran; preparing a solid mixed microbial inoculum by sterilizing bran from common high-temperature actinomycetes, geotrichum candidum and lactobacillus plantarum according to a certain inoculation amount; mixing the prepared solid mixed microbial inoculum with the refined brewer grains and the blueberry pomace, performing solid fermentation, inoculating common high-temperature actinomycetes at the constant temperature of 50 ℃ for primary culture and fermentation for 72h, inoculating the obtained geotrichum candidum and lactobacillus plantarum mixed bacteria at the temperature of 30 ℃ for secondary fermentation for 72h, drying and crushing the fermented product to obtain the protein feed.
Description
Technical Field
The invention belongs to the technical field of production of protein feed, and particularly relates to a method for producing protein feed by carrying out different mixed fermentation twice on refined brewer's grains and blueberry pomace.
Background
According to statistics of the research institute of the commercial industry, the feed yield is 28465.5 ten thousand tons in 2017 years in China, and the importation degree of feed raw materials, particularly protein feed raw materials is close to 80 percent, so that the feed is an important bottleneck for limiting the breeding industry and the feed processing industry in China.
Under the large background that the mass beer is becoming saturated, the China's refined beer market is being looked by more and more people. The Chinese beer market shows a high-end development trend, the intensive brewing beer is remarkably increased, and the market performance of attention in the future is expected to be obtained.
The fermented wine residues of the refined beer are directly transported to farms for feeding pigs, if the fermented wine residues are improper, pigs can be poisoned, even diseases are spread, and in addition, the water content and the organic matter content are high, so that the fermented wine residues are easy to rot and smell in a short time, mosquitoes and flies are bred, and the requirement of environmental sanitation cannot be met. Blueberry pulp is fine and smooth, the flavor is unique, the blueberry pulp contains rich nutrient substances such as saccharides, amino acids and proteins and functional substances such as anthocyanin, phenol, flavone and the like, the nutrition and health care value of the blueberry pulp is far higher than that of other fruits, the blueberry pulp is often known as 'the king of berries in the world' in recent years, the development of the blueberry industry of all countries in the world is rapid, blueberry products are layered endlessly, a large amount of fruit juice products are produced, and meanwhile, a large amount of waste blueberry pomace is produced, and the waste blueberry pomace can account for 20 percent of the quality of fresh fruits at most. It was shown that more than 15% polyphenols and 71.8% anthocyanins remained in the pomace. Therefore, the recycling of the blueberry pomace can not only reduce the environmental pollution, but also greatly improve the utilization rate of the nutrition and functional components in the blueberries.
The refined beer lees is rich in nutrition and mainly contains protein, amino acid, cellulose, calcium, phosphorus, manganese, iron, copper and the like. The blueberry residue is also very rich in nutrition, mainly contains protein, saccharides, cellulose, anthocyanin, flavone, polyphenol and the like, and has more antioxidant substances.
At present, only relevant reports of protein feed production by fermentation of industrial brewer's grains and no reports of protein feed production by fermentation of blueberry dregs exist in the prior art, the protein content of the refined brewer's grains is richer than that of the industrial brewer's grains, the fiber content is higher but the sugar content is lower, and the blueberry pomace can provide rich sugar and strong antioxidants such as polyphenol, anthocyanin and the like. Meanwhile, the blueberry pomace is also rich in cellulose. Some existing reports about industrial brewery mash fermentation adopt composite microorganisms consisting of yeast, mould and bacteria to perform primary fermentation to prepare protein feed. Because the cellulose content of the refined brewer's grains and the blueberry pomace is high, if the original process is adopted, the insufficient degradation degree of the cellulose can influence the generation rate of protein.
Therefore, the method has important significance for producing the protein feed by performing different mixed fermentation on the refined brewer's grains and the blueberry pomace twice.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provides a method for producing protein feed by carrying out two different mixed fermentations on refined brewer's grains and blueberry pomace.
The invention adopts the following technical scheme:
the invention provides a method for producing protein feed by carrying out two different mixed fermentations on refined brewer's grains and blueberry pomace, which comprises the following steps:
step one, activating and preparing strains:
respectively activating ordinary high-temperature actinomycetes, geotrichum candidum and lactobacillus plantarum by using a stock test tube, performing amplification culture in a triangular flask, performing seed tank culture, culturing and fermenting to obtain bacterial suspension, and mixing the cultured geotrichum candidum and lactobacillus plantarum strains in proportion to obtain mixed bacteria;
step two: preparing fermentation raw materials for brewing beer grains, blueberry pomace and bran;
step three: respectively preparing a solid mixed microbial inoculum from a mixed bacterium of common high-temperature actinomycetes, geotrichum candidum and lactobacillus plantarum by sterilized bran according to a certain inoculation amount;
step four: and (3) mixing the solid mixed microbial inoculum prepared in the third step with the refined brewer grains and the blueberry pomace, performing solid fermentation, performing primary culture fermentation at constant temperature, inoculating the mixed microbial inoculum obtained in the first step, performing secondary fermentation, drying and crushing the fermented product to prepare the protein feed.
In some embodiments, the mixing of the cultured strains in the first step according to the ratio is specifically: mixing the cultured Geotrichum candidum and the cultured Lactobacillus plantarum in a ratio of 4:1 to obtain a mixed strain.
In some embodiments, the mass ratio of the fermented brewer's grains and the blueberry pomace in the step two is 0.5-1.5:1, and the mass ratio of the mixture of the brewed brewer's grains and the blueberry pomace to the bran is 8-10: 1.
In some embodiments, in the third step, the common high-temperature actinomycetes are prepared into a solid mixed microbial inoculum by sterilized bran according to the inoculation amount of 10% of the weight of the fermentation raw material, and the mixed bacteria of geotrichum candidum and lactobacillus plantarum are prepared into a solid mixed microbial inoculum by sterilized bran according to the inoculation amount of 5% of the weight of the fermentation raw material; in the third step, the mass ratio of the solid mixed microbial inoculum to the material brewed beer lees and the blueberry pomace is 1: 9.
In some embodiments, the first culture fermentation in step four is performed at a temperature of 50 ℃ for a fermentation time of 72 hours.
In some embodiments, the temperature of the second fermentation in the fourth step is 30 ℃ and the fermentation time is 72 hours.
Compared with the common industrial brewer's grains, the refined brewer's grains are rich in more protein and crude fiber but low in sugar, and the blueberry pomace is rich in sugar, polyphenol, anthocyanin and other antioxidant substances, so that the combination of the sugar, the polyphenol, the anthocyanin and the like can make up for the deficiencies of the refined brewer's grains. The addition of a certain amount of bran not only can provide carbon source substances such as sugars and the like, but also can increase the air permeability of a culture substrate, and is beneficial to the growth of strains and the enzyme-producing fermentation. Because the content of crude fiber in the refined brewer's grains and the blueberry pomace is high and the cellulose can obstruct the absorption of nutrient components in the feed, the common high-temperature actinomycetes optimally cultured from the high-temperature compost has stronger cellulose degradation capability to be fermented for the first time, which is beneficial to decomposing the cellulose in the raw materials into utilizable saccharide substances. The optimum growth temperature of the common high-temperature actinomycetes is 50 ℃, the growth of mixed bacteria can be effectively inhibited, and the high-temperature actinomycetes has strong acid-base tolerance, so that the pH value does not need to be adjusted before fermentation.
The geotrichum candidum has strong reproduction capacity and high thallus protein content, lactobacillus plantarum can generate lactein in the growth process, the lactein is a biological preservative, the growth of mixed bacteria can be effectively inhibited, and the palatability of protein feed is improved through the generated lactic acid. Therefore, the cellulose is degraded through the first fermentation, then geotrichum candidum which is preferably selected by taking the protein content as an index is added, and the secondary fermentation is carried out after the compatibility of lactobacillus plantarum, and the key problems of optimization of mixed fermentation conditions and the like are solved, so that a two-time mixed fermentation system of the refined brewer's grains and the blueberry pomace is constructed, and the protein feed which is high in protein, rich in powerful antioxidant anthocyanin and has certain disease resistance and good palatability is produced. The growth of mixed bacteria can be inhibited no matter the first fermentation or the second fermentation, so the operation process can be finished without special sterilization.
Compared with the prior art, the invention has the beneficial effects that:
firstly, the method comprises the following steps: the Chinese beer market shows a high-end development trend, and the increase of the refined beer is obvious. The water content and the organic matter content of the wine residues after the fermentation of the fine brewing beer are high, and the wine residues are easy to rot and smell in a short time, thereby bringing environmental pollution. In recent years, the blueberry industry of all countries in the world develops rapidly, blueberry products emerge endlessly, a large amount of fruit juice products emerge, and meanwhile, a large amount of waste blueberry pomace is generated and can account for 20% of the quality of fresh fruits at most. According to the invention, the single-cell protein feed is produced by mixing and fermenting the two kinds of concentrated brewing brewer grains and blueberry residues with rich nutrition through a microorganism solid-state fermentation technology, so that the nutritional ingredients in the two kinds of waste residues can be fully utilized, the resource waste and the environmental pollution caused by the difficulty in utilizing the concentrated brewing brewer grains and the blueberry fruit residues are reduced, the disease resistance of livestock and poultry can be increased, and the productivity is improved.
Secondly, the method comprises the following steps: china is one of the biggest breeding and producing countries in the world, with the development of animal husbandry and breeding industry, the traditional feed resources (various grains, bran, soybean meal, potato residue and the like) cannot meet the rapidly-increased feed market, the shortage of protein feed, especially the continuous increase of the demand of the protein feed, becomes a prominent problem in China, and the healthy and rapid development of the animal husbandry and the breeding industry is seriously hindered. The protein feed produced by mixing and fermenting the refined brewer's grains and the blueberry pomace is beneficial to relieving the problem of shortage of protein feed resources in China, can save the feed input cost, changes waste into valuable and increases the income of manufacturers and farmers.
Thirdly, the method comprises the following steps: the strains used in the invention are all known beneficial strains, the cellulose content can be obviously reduced, the true protein content in the fermentation product can be improved, and the true protein content can reach 40.14%. After the fermentation of the microorganisms, the fermentation product is rich in polypeptides, amino acids and the like which are easy to be absorbed by livestock, and also rich in B vitamins, anthocyanin and the like, so that the nutritional value of the fermentation product is improved.
Drawings
FIG. 1 is a schematic diagram of the standard effect arrangement of the present invention.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be purely exemplary and are not intended to limit the scope of the invention, which is defined in the appended claims, as may be amended by those skilled in the art upon reading the present invention.
The percentage content related in the invention is mass percentage.
The invention provides a method for producing protein feed by carrying out two different mixed fermentations on refined brewer's grains and blueberry pomace, which comprises the following steps:
step one, activating and preparing strains:
respectively activating ordinary high-temperature actinomycetes, geotrichum candidum and lactobacillus plantarum by using a stock test tube, performing amplification culture in a triangular flask, performing seed tank culture, culturing and fermenting to obtain bacterial suspension, and mixing the cultured geotrichum candidum and lactobacillus plantarum strains in proportion to obtain mixed bacteria;
step two: preparing fermentation raw materials for brewing beer grains, blueberry pomace and bran;
step three: respectively preparing a solid mixed microbial inoculum from a mixed bacterium of common high-temperature actinomycetes, geotrichum candidum and lactobacillus plantarum by sterilized bran according to a certain inoculation amount;
step four: and (3) mixing the solid mixed microbial inoculum prepared in the third step with the refined brewer grains and the blueberry pomace, performing solid fermentation, performing primary culture fermentation at constant temperature, inoculating the mixed microbial inoculum obtained in the first step, performing secondary fermentation, drying and crushing the fermented product to prepare the protein feed.
In one embodiment, the mixing of the cultured strains in the first step according to the ratio is specifically as follows: mixing the cultured Geotrichum candidum and the cultured Lactobacillus plantarum in a ratio of 4:1 to obtain a mixed strain.
In one embodiment, the mass ratio of the fermented brewer grains to the blueberry pomace in the second step is 0.5-1.5:1, and the mass ratio of the mixture of the brewed brewer grains and the blueberry pomace to the bran is 8-10: 1.
In one embodiment, in the third step, respectively preparing a solid mixed microbial inoculum from ordinary high-temperature actinomycetes and sterilized bran according to the inoculation amount of 10% by weight of the fermentation raw material, and preparing a solid mixed microbial inoculum from a mixed microbial inoculum of geotrichum candidum and lactobacillus plantarum according to the inoculation amount of 5% by weight of the fermentation raw material; in the third step, the mass ratio of the solid mixed microbial inoculum to the material brewed beer lees and the blueberry pomace is 1: 9.
In one embodiment, the temperature of the first cultivation fermentation in step four is 50 ℃ and the fermentation time is 72 hours.
In one embodiment, the temperature of the secondary fermentation in the fourth step is 30 ℃ and the fermentation time is 72 hours.
The present invention will be described in detail with reference to specific examples
Example 1
Firstly, testing the crude fiber content of the mixture of the refined brewer's grain, the blueberry pomace and the bran (the proportion of the refined brewer's grain to the blueberry pomace is 1:1, and the proportion of the mixture of the refined brewer's grain and the blueberry pomace to the bran is 4:1)
The test method comprises the following steps: weighing 2g of sample, degreasing with diethyl ether, putting into a digester, adding 200ml of boiled sulfuric acid solution with the accurate concentration of 0.255N and 1 drop of N-octanol, immediately heating to boil within 2min, continuously slightly boiling for 30 +/-1 min, keeping the sulfuric acid concentration unchanged, filtering, washing with boiling distilled water until the solution does not contain acid, taking off insoluble substances, putting into an original container, adding 200ml of boiled sodium hydroxide solution with the accurate concentration, and slightly boiling for 30 min. Immediately performing suction filtration on a Gu's crucible paved with asbestos, washing with 25ml of sulfuric acid solution, then washing with boiling distilled water until the washing liquid is neutral, washing residues with 15ml of ethanol, then putting the Gu's crucible and the residues into an oven, drying for 2 hours at the temperature of 130 +/-2 ℃, cooling to room temperature in a dryer, and weighing. Then burning the mixture in a high-temperature furnace at 550 +/-25 ℃ for 30min, cooling the mixture to room temperature in a drier and weighing the mixture.
Calculation of the results of the measurement: crude fiber (%) ═ W2-W1/W × 100 formula: w1 shows the weight of the residue of the crucible and the sample after drying at 130 ℃, and W2 shows the weight of the residue of the crucible and the sample after burning at 550 ℃. W is the weight of the sample (when not degreased).
And (3) calculating the result: the crude fiber content of the mixture of the refined brewer grains, the blueberry pomace and the bran is 23.7 percent.
Secondly, the degradation capability of common high-temperature actinomycetes, aspergillus niger and trichoderma cellulose is compared
1. Materials: thermoactinomyces vulgaris (CICC 10672, Thermoactinomyces vulgari), Aspergillus niger (A. niger)2103 Aspergillus niger) and Trichoderma viride (CICC 13002, Trichoderma viride) were purchased from China center for Industrial culture Collection of microorganisms
2. Culture medium and culture method
Slant culture medium: the common actinomycete culture medium comprises glucose 4.0g, yeast extract 4.0g, malt extract 10.0g, CaCO32.0g, agar 12.0g, distilled water 1.0L, pH7.2, and the prepared slant culture medium is sterilized at 121 deg.C for 20min before use.
The culture medium of trichoderma viride and aspergillus niger is a 5 DEG Bee wort agar culture medium, and the preparation steps are as follows: weighing 50g of dry malt, grinding, adding 400ml of distilled water, saccharifying in a water bath at 50-60 deg.C for 3-4 hr, and boiling completely. Filtering the boiled saccharified liquid with 4-6 layers of gauze, putting the filtrate into a plastic centrifugal tank, centrifuging by a centrifugal machine at 4000r/min for 10min, and taking the supernatant. Diluting the wort supernatant to 5-6 Baume degree, pH about 6.4, and adding 2% agar to obtain solid culture medium. Sterilizing at 121 deg.C for 20 min;
seed culture medium: transferring the common high-temperature actinomycetes cultured in the slant test tube into a 1000ml shake flask, and putting the shake flask into a constant-temperature shaking table for culturing at the temperature of 50 ℃, the rotating speed of 180r/min and the culturing time of 72 h; inoculating the general actinomycetes cultured by the shake flask into a seeding tank according to the inoculation amount of 5% for culturing, wherein the culture temperature is 50 ℃, and the culture time is 48 h; and respectively transferring the trichoderma viride and the aspergillus niger which are cultured by the slant test tube into 1000ml shake flasks, putting the shake flasks into a constant-temperature shaking table for culturing at the temperature of 30 ℃, the rotating speed of 180r/min and the culturing time of 72h, respectively inoculating the trichoderma viride and the aspergillus niger which are cultured by the shake flasks into a seeding tank for culturing according to the inoculum size of 5 percent, wherein the culturing temperature is 30 ℃ and the culturing time is 48 h.
Fermentation medium: 30g of the processed refined brewer grains, blueberry pomace and bran are filled into a 250mL triangular flask (the proportion of the refined brewer grains to the blueberry pomace is 1:1, and the proportion of the mixture of the refined brewer grains to the blueberry pomace to the bran is 4: 1).
Fermentation culture: adding 30g of fermentation medium into a 250mL triangular flask, adding 0.8 time of distilled water, sterilizing at 121 ℃ for 20min, inoculating 20% of common high-temperature actinomycetes, trichoderma viride and aspergillus niger seed liquid respectively, and culturing at 50 ℃ and 30 ℃ for 72h respectively.
The results show that: after 72h fermentation culture, the crude fiber of the raw materials can be reduced to 7.9% by common high-temperature actinomycetes, and the crude fiber of the raw materials can be reduced to 9.8% and 8.4% by trichoderma viride and aspergillus niger respectively. Therefore, the common thermoactinomycetes have strong capability of decomposing crude fibers.
Optimization of mixed bacteria condition of three-time and two-time fermentation
The Geotrichum candidum (strain preservation number: CICC 1443 latin name: Geotrichum candidum) and the Lactobacillus plantarum (strain preservation number: CICC 21794 latin name: Lactobacillus plantarum) are purchased from China center for Industrial culture Collection of microorganisms.
(II) culture medium and culture method
The geotrichum candidum culture medium is a 5 DEG Bee wort agar culture medium and is prepared by the following steps: weighing 50g of dry malt, grinding, adding 400ml of distilled water, saccharifying in a water bath at 50-60 deg.C for 3-4 hr, and boiling completely. Filtering the boiled saccharified liquid with 4-6 layers of gauze, putting the filtrate into a plastic centrifugal tank, centrifuging by a centrifugal machine at 4000r/min for 10min, and taking the supernatant. Diluting the wort supernatant to 5-6 Baume degree, pH about 6.4, and adding 2% agar to obtain solid culture medium. Sterilizing at 121 deg.C for 20 min; the agar is removed when preparing liquid culture medium.
The preparation method of the lactobacillus plantarum slant culture medium comprises the following steps: weighing 10.0g of casein peptone, 10.0g of beef extract, 5.0g of yeast powder, 5.0g of glucose, 5.0g of sodium acetate, 2.0g of diammonium citrate, 801.0 g of Tween and K2HPO4 2.0g，MgSO4.7H2O 0.2g，MnSO4.H2O 0.05g，CaCO320.0g of agar and 15.0g of agar are added with 1.0L of distilled water, boiled and melted, and sterilized at 121 ℃ for 20min to obtain the product, wherein the pH value is adjusted to 5.0. The agar is removed when preparing liquid culture medium.
The geotrichum candidum and the lactobacillus plantarum cultured by the inclined tube are respectively transferred into 1000ml shake flasks and put into a constant temperature shaking table for culturing at the temperature of 30 ℃, the rotating speed of 180r/min and the culturing time of 72 h.
The geotrichum candidum and the lactobacillus plantarum which are cultured in a shake flask are respectively inoculated into a seeding tank for culture according to the inoculation amount of 5 percent, the culture temperature is 30 ℃, and the culture time is 48 hours.
The geotrichum candidum and the lactobacillus plantarum obtained after the culture in the seeding tank are also inoculated into a fermentation tank according to the inoculation amount of 10 percent for culture at the culture temperature of 30 ℃ for 48-72h, and the bacterial suspension is obtained.
Fermentation medium: 30g of the processed refined brewer grains, blueberry pomace and bran are filled into a 250mL triangular flask (the proportion of the refined brewer grains to the blueberry pomace is 1:1, and the proportion of the mixture of the refined brewer grains to the blueberry pomace to the bran is 9: 1).
Fermentation culture: adding 30g of fermentation medium into a 250mL triangular flask, adding 0.8 time of distilled water, sterilizing at 121 ℃ for 20min, inoculating 10% of common high-temperature actinomycetes respectively, culturing at 50 ℃ for 72h, adjusting the temperature to 30 ℃, inoculating mixed bacteria of geotrichum candidum and lactobacillus plantarum with the volume ratio of 2:1 and 4:1 respectively, fermenting for 72h, and determining the true protein content.
The method for measuring the true protein content comprises the following steps:
1. weighing 0.5-1g (accurate to 0.1mg) of a sample to be detected in a 200ml beaker, adding 50ml of distilled water, stirring uniformly by using a glass rod, heating and boiling on an electric furnace, adding 20ml of 10% copper sulfate solution, adding 20ml of 2.5% sodium hydroxide solution, stirring uniformly, taking down from the electric furnace, standing at room temperature for 2h, filtering the sample on filter paper by using a pouring method, washing the beaker and precipitate for multiple times by using a small amount of warm water until a washing solution is free of sulfate ions (the filtrate is free of precipitate by using a 10% barium chloride solution), putting the beaker and the precipitate together into a 80 ℃ drying box for drying, carefully putting the filtrate and the precipitate into a 250ml Kjeldahl flask, adding 6.4g of catalyst and 20ml of concentrated sulfuric acid, digesting on the electric furnace, clarifying the sample solution, then boiling for 2h, taking down, cooling, then adding 60-100ml of distilled water, shaking uniformly and cooling. And simultaneously, carrying out a blank experiment.
2. The end of the condenser tube of the distillation apparatus was immersed in a conical flask containing 25ml of boric acid absorbent and 2 drops of mixing indicator. Carefully adding 50ml of sodium hydroxide solution into the Kjeldahl flask, slightly shaking the Kjeldahl flask to uniformly mix the solution, then heating for distillation until the effluent liquid is 100ml, lowering the conical flask to enable the tail end of the condenser tube to be away from the liquid level, continuing to distill for 1-2min, washing the tail end of the condenser tube with distilled water, enabling the washing liquids to flow into the conical flask, and then stopping the distillation. The titrated and distilled absorption solution is immediately titrated by 0.1mol/L hydrochloric acid standard solution, and the end point is that the solution is changed from blue-green color to grey-red color.
3. Examination of distillation step: accurately weighing 0.2g of ammonium sulfate instead of the sample, and operating according to the steps to obtain the nitrogen content of the ammonium sulfate to be 21.19 +/-0.2%, otherwise, checking whether the steps of adding alkali, distilling and titrating are correct.
4. Blank determination: 0.5g of sucrose is weighed to replace a sample, blank measurement is carried out according to the previous steps, and the volume of 0.1mol/L hydrochloric acid standard solution consumed cannot exceed 0.2 ml. The volume of the standard solution consuming 0.02mol/L hydrochloric acid should not exceed 0.3 ml.
5. Presentation of analytical results
The calculation is shown in the following formula:
true protein (%) - (V1-V2) × C × 0.0140 × 6.25 × 100m × V'/V
In the formula: v1-volume of standard solution required for titration of sample, ml;
v2-volume of standard solution required for titration of blank, ml;
c, the concentration of the hydrochloric acid standard solution, mol/L;
v is the total volume of the sample decomposition liquid, ml;
v' -volume for sample decomposition distillation, ml;
the results show that the true protein content of the mixed bacterial liquid of geotrichum candidum and lactobacillus plantarum with the volume ratio of 4:1 is 36.79 percent after being fermented for 72 hours, and the true protein content of the mixed bacterial liquid of geotrichum candidum and lactobacillus plantarum with the volume ratio of 2:1 is 31.06 percent after being fermented for 72 hours. Therefore, the geotrichum candidum and the lactobacillus plantarum are preferably mixed in a volume ratio of 4: 1.
Example 2
(1) Bacterial species and sources
Common Thermoactinomyces (strain accession number: CICC 10672, latin name: Thermoactinomyces vulgaris), Geotrichum candidum (strain accession number: CICC 1443, latin name: Geotrichum candidum) and Lactobacillus plantarum (strain accession number: CICC 21794, latin name: Lactobacillus plantarum) were purchased from China center for Industrial culture Collection of microorganisms.
(2) Activation and preparation of bacterial species
Activation of stock test tube, triangular flask expanding culture, seeding tank culture and fermentation tank culture
The culture medium of common actinomycetes comprises glucose 4.0g, yeast extract 4.0g, malt extract 10.0g, CaCO32.0g of agar, 12.0g of agar, 1.0L of distilled water, pH7.2, and sterilizing the prepared slant culture medium at 121 ℃ for 20min before use; the agar is removed when preparing liquid culture medium.
The geotrichum candidum culture medium is a 5 DEG Bee wort agar culture medium and is prepared by the following steps: weighing 50g of dry malt, grinding, adding 400ml of distilled water, saccharifying in a water bath at 50-60 deg.C for 3-4 hr, and boiling completely. Filtering the boiled saccharified liquid with 4-6 layers of gauze, putting the filtrate into a plastic centrifugal tank, centrifuging by a centrifugal machine at 4000r/min for 10min, and taking the supernatant. Diluting the wort supernatant to 5-6 Baume degree, pH about 6.4, and adding 2% agar to obtain solid culture medium. Sterilizing at 121 deg.C for 20 min; the agar is removed when preparing liquid culture medium.
The preparation method of the lactobacillus plantarum slant culture medium comprises the following steps: weighing 10.0g of casein peptone, 10.0g of beef extract, 5.0g of yeast powder, 5.0g of glucose, 5.0g of sodium acetate, 2.0g of diammonium citrate, 801.0 g of Tween and K2HPO4 2.0g，MgSO4.7H2O 0.2g，MnSO4.H2O 0.05g，CaCO3Adding agar 20.0g and agar 15.0g into distilled water 1.0L, boiling to melt, adjusting pH to 5.0, and sterilizing at 121 deg.C for 20 min. The agar is removed when preparing liquid culture medium.
Transferring the common high-temperature actinomycetes cultured in the slant test tube into a 1000ml shake flask, and putting the shake flask into a constant-temperature shaking table for culturing at the temperature of 50 ℃, the rotating speed of 180r/min and the culturing time of 72 h; separately transferring geotrichum candidum and lactobacillus plantarum into 1000ml shake flasks, and placing the shake flasks into a constant-temperature shaking table for cultivation at the temperature of 30 ℃ and the rotation speed of 180r/min for 72 h.
Inoculating the general actinomycetes cultured by the shake flask into a seeding tank according to the inoculation amount of 5% for culturing, wherein the culture temperature is 50 ℃, and the culture time is 48 h; the geotrichum candidum and the lactobacillus plantarum are respectively inoculated into a seed tank according to the inoculation amount of 5 percent for culture, the culture temperature is 30 ℃, and the culture time is 48 hours.
Inoculating the obtained common actinomycetes cultured in the seeding tank into a fermentation tank according to the inoculation amount of 10% for culturing, wherein the culture temperature is 50 ℃, and the culture time is 48-72h, so as to obtain a bacterial suspension; the geotrichum candidum and the lactobacillus plantarum are also inoculated into a fermentation tank according to the inoculation amount of 10 percent for culture at the culture temperature of 30 ℃ for 48-72h, and the bacterial suspension is obtained.
And mixing the geotrichum candidum and the lactobacillus plantarum obtained by three-stage culture according to the volume ratio of 2:1 to obtain the mixed strain I.
(3) Preparation of fermentation feedstock
28% of refined beer lees, 56% of blueberry pomace and 8% of bran, wherein the inoculation amount of the mixed strain I is 3%.
(4) Bacterial inoculation and preparation process flow
Firstly, carrying ordinary high-temperature actinomycetes by sterilized bran according to the inoculation amount of 10% of the weight of a fermentation raw material through a conventional carrying technology to prepare a solid mixed microbial inoculum II, and carrying the geotrichum candidum and lactobacillus plantarum with the volume ratio of 2:1 by sterilized bran according to the inoculation amount of 3% of the weight of the fermentation raw material through the conventional carrying technology to prepare the solid mixed microbial inoculum I.
Mixing a common high-temperature actinomycete solid mixed microbial inoculum II with the refined brewer grains and the blueberry pomace, carrying out solid fermentation, carrying out primary culture fermentation at the constant temperature of 50 ℃ for 24 hours, then inoculating a solid mixed microbial inoculum I (the inoculation amount is 3%) prepared by mixing geotrichum candidum and lactobacillus plantarum (the volume ratio is 2:1), adjusting the temperature to 30 ℃, carrying out secondary fermentation for 72 hours, drying and crushing the fermentation product, and thus obtaining the protein feed.
Example 3
(1) Strains and sources: same as in example 2
(2) Activation and preparation of bacterial species
Geotrichum candidum and Lactobacillus plantarum were mixed in a ratio of 4:1, otherwise the same as in example 2.
(3) Preparation of fermentation raw materials: 51.4% of refined brewer's grains, 36.2% of blueberry pomace, 9.9% of bran and 5% of mixed strain of geotrichum candidum and lactobacillus plantarum.
(4) Bacterial inoculation and preparation process flow
Firstly, carrying out vectorization on common high-temperature actinomycetes by sterilized bran according to the inoculum size of 10% of the weight of a fermentation raw material through a conventional vectorization technology to prepare a solid mixed microbial inoculum II, and carrying out vectorization on geotrichum candidum and lactobacillus plantarum according to the inoculum size of 5% of the weight of the fermentation raw material by sterilized bran according to the conventional vectorization technology to prepare the solid mixed microbial inoculum I.
Mixing a common high-temperature actinomycete solid mixed microbial inoculum II with the refined brewer grains and the blueberry pomace, carrying out solid fermentation, carrying out primary culture fermentation at the constant temperature of 50 ℃ for 72 hours, then inoculating a solid mixed microbial inoculum I (the inoculation amount is 5%) prepared by mixing geotrichum candidum and lactobacillus plantarum (the volume ratio is 4:1), adjusting the temperature to 30 ℃, carrying out secondary fermentation for 72 hours, drying and crushing the fermentation product, and thus obtaining the protein feed.
The true protein content of the protein feeds obtained in examples 2 and 3 was determined as follows:
1. weighing 0.5-1g (accurate to 0.1mg) of sample to be tested in a 200ml beaker, adding 50ml of distilled water, stirring uniformly by using a glass rod, heating and boiling on an electric furnace, adding 20ml of copper sulfate solution with the mass concentration of 10%, adding 20ml of sodium hydroxide solution with the mass concentration of 2.5%, stirring uniformly, taking down from the electric furnace, standing at room temperature for 2h, filtering the sample on filter paper by using a dumping method, washing the beaker and precipitate for multiple times by using a small amount of warm water until eluate has no sulfate ions (the eluate has no precipitate by using barium chloride solution with the mass concentration of 10%), putting the beaker and the precipitate together with a funnel into a drying box at 80 ℃ for drying, carefully putting the filtrate and the precipitate into a 250ml Kjeldahl flask, adding 6.4g of catalyst and 20ml of concentrated sulfuric acid, digesting on the electric furnace, then boiling for 2h after the sample is clarified, taking down, cooling, adding 60-100ml of distilled water, shaking up and cooling. And simultaneously, carrying out a blank experiment.
2. The end of the condenser tube of the distillation apparatus was immersed in a conical flask containing 25ml of boric acid absorbent and 2 drops of mixing indicator. Carefully adding 50ml of 40% sodium hydroxide solution into the Kjeldahl flask, slightly shaking the Kjeldahl flask to mix the solution uniformly, then heating for distillation until the effluent is 100ml, lowering the conical flask to enable the tail end of the condenser tube to be away from the liquid level, continuing to distill for 1-2min, washing the tail end of the condenser tube with distilled water, enabling the washing liquids to flow into the conical flask, and then stopping distillation. The distilled absorption solution was titrated and immediately titrated with a 0.1mol/L hydrochloric acid standard solution, and the end point was the solution changed from blue-green to gray-red.
3. Examination of distillation step: accurately weighing 0.2g of ammonium sulfate instead of the sample, and operating according to the steps to obtain the nitrogen content of the ammonium sulfate to be 21.19 +/-0.2%, otherwise, checking whether the steps of adding alkali, distilling and titrating are correct.
4. Blank determination: 0.5g of sucrose is weighed to replace a sample, blank measurement is carried out according to the previous steps, and the volume of 0.1mol/L hydrochloric acid standard solution consumed cannot exceed 0.2 ml. The volume of the standard solution consuming 0.02mol/L hydrochloric acid should not exceed 0.3 ml.
The calculation is shown in the following formula:
true protein (%) - (V1-V2) × C × 0.0140 × 6.25 × 100m × V'/V,
in the formula: v1-volume of standard solution required for titration of sample, ml;
v2-volume of standard solution required for titration of blank, ml;
c, the concentration of the hydrochloric acid standard solution, mol/L;
v is the total volume of the sample decomposition liquid, ml;
v' -volume for sample decomposition distillation, ml;
the authentic protein content was found to be 29.15% in the product of example 2 and 40.14% in the product of example 3.
The response surface experimental method adopted in the invention is as follows:
1. Plackett-Burman design: selecting possible factors influencing the true protein content of a fermentation product, comprehensively inspecting 6 factors of fermentation time, strain ratio, ratio of refined brewer's grains to blueberry residues, mixing amount of the refined brewer's grains to blueberry residues, bran amount and inoculation amount, selecting a Plackett-Burman design, taking 2 levels of each factor and taking the true protein content as a response value, inspecting the main effect and interaction of each factor, and determining important influencing factors.
TABLE 1 Plackett-Burman design factors and levels
The results of the experiment are shown in table 2 and fig. 1:
TABLE 2 results of the experiment
The result shows the mixing amount of the refined brewer grains and the blueberry residues, the refined brewer grains: the ratio of blueberry pomace to bran is the three most important influencing factors.
2. Response surface analysis
According to the center combination design principle of Box-Behnken, the Box-Behnken test design is carried out on 3 main influence factors screened by Plackett-Burman design, and the value levels of all the factors are as follows:
TABLE 3 Main influencing factors and levels
The experimental results are as follows:
TABLE 4 results of the experiment
The optimal fermentation optimization conditions determined by the response surface method are that the ratio of the refined brewer grains to the blueberry residues is 1.42:1, the mixing amount of the refined brewer grains to the blueberry residues is 87.66%, the bran amount is 9.9%, and the true protein content is 40.15%.
In order to test the feasibility of the response surface method, the obtained optimal fermentation optimization conditions are adopted for testing, the true protein content obtained by three parallel experiments is 40.14 percent and is 1 percent less than the theoretical value, and the correctness of the experimental scheme is explained. Therefore, the best fermentation scheme is that 51.4 percent of the refined beer lees, 36.2 percent of the blueberry pomace and 9.9 percent of the bran (the balance being water) are mixed according to the mass fraction to prepare a fermentation solid culture medium, common high-temperature actinomycetes are inoculated firstly and cultured for 72 hours at the constant temperature of 50 ℃, 5 percent of mixed strain of geotrichum candidum and lactobacillus plantarum with the ratio of 4:1 is inoculated, the temperature is adjusted to 30 ℃ and fermented for 72 hours, and the fermented product is dried and crushed to prepare the protein feed.
The embodiments of the present invention have been described in detail with reference to the above examples, but the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art.
Claims (6)
1. The method for producing the protein feed by carrying out two different mixed fermentations on the refined brewer's grains and the blueberry pomace is characterized by comprising the following steps:
step one, activating and preparing strains:
respectively activating ordinary high-temperature actinomycetes, geotrichum candidum and lactobacillus plantarum by using a stock test tube, performing amplification culture in a triangular flask, performing seed tank culture, culturing and fermenting to obtain bacterial suspension, and mixing the cultured geotrichum candidum and lactobacillus plantarum strains in proportion to obtain mixed bacteria;
step two: preparing fermentation raw materials for brewing beer grains, blueberry pomace and bran;
step three: respectively preparing a solid mixed microbial inoculum from a mixed bacterium of common high-temperature actinomycetes, geotrichum candidum and lactobacillus plantarum by sterilized bran according to a certain inoculation amount;
step four: and (3) mixing the solid mixed microbial inoculum prepared in the third step with the refined brewer grains and the blueberry pomace, performing solid fermentation, performing primary culture fermentation at constant temperature, inoculating the mixed microbial inoculum obtained in the first step, performing secondary fermentation, drying and crushing the fermented product to prepare the protein feed.
2. The method for producing the protein feed by using the brewer's grains and the blueberry pomace for two different mixed fermentations according to claim 1, wherein the step one of mixing the cultured strains according to the proportion specifically comprises the following steps: mixing the cultured Geotrichum candidum and the cultured Lactobacillus plantarum in a ratio of 4:1 to obtain a mixed strain.
3. The method for producing the protein feed by using the refined brewer's grains and the blueberry pomace through two different mixed fermentations according to claim 1, wherein the mass ratio of the fermentation raw materials of the refined brewer's grains and the blueberry pomace in the step two is 0.5-1.5:1, and the mass ratio of the mixture of the refined brewer's grains and the blueberry pomace to the bran is 8-10: 1.
4. The method for producing protein feed by using the two different mixed fermentations of the brewer's grains and the blueberry pomaces according to the claim 1, characterized in that, in the third step, the ordinary high temperature actinomycetes is prepared into the solid mixed microbial inoculum by sterilized bran according to the inoculation amount of 10 percent of the weight of the fermentation raw materials, and the mixed microbial inoculum of geotrichum candidum and lactobacillus plantarum is prepared into the solid mixed microbial inoculum by sterilized bran according to the inoculation amount of 5 percent of the weight of the fermentation raw materials; in the third step, the mass ratio of the solid mixed microbial inoculum to the material brewed beer lees and the blueberry pomace is 1: 9.
5. The method for producing protein feed by using the two different mixed fermentations of the brewer's grains and the blueberry pomace according to the claim 1, wherein the temperature of the first culture fermentation in the fourth step is 50 ℃ and the fermentation time is 72 hours.
6. The method for producing the protein feed by using the two different mixed fermentations of the brewer's grains and the blueberry pomace according to the claim 1, wherein the temperature of the second fermentation in the fourth step is 30 ℃, and the fermentation time is 72 hours.
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