CN105994940B - Bioactive protein feed and preparation method thereof - Google Patents

Bioactive protein feed and preparation method thereof Download PDF

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CN105994940B
CN105994940B CN201610345696.0A CN201610345696A CN105994940B CN 105994940 B CN105994940 B CN 105994940B CN 201610345696 A CN201610345696 A CN 201610345696A CN 105994940 B CN105994940 B CN 105994940B
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microbial inoculum
mould
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CN105994940A (en
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来航线
李海洋
韦小敏
李肖
薛泉宏
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Northwest A&F University
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Abstract

The invention belongs to the field of feeds, and particularly discloses a bioactive protein feed which comprises the following raw material components: the biological fertilizer comprises pomace, oil residue, bran, urea and a fermentation microbial inoculum, wherein the fermentation microbial inoculum is a composite microbial inoculum of mould, saccharomycetes, bacillus and lactobacillus. The preparation method comprises the following steps: firstly, preparing a fermentation culture medium, and performing high-pressure steam sterilization on the fermentation culture medium; then sequentially inoculating a mould solid spore microbial inoculum, an activated yeast, a compound microbial inoculum of spore bacteria and lactic acid bacteria on the cooled sterilized fermentation medium, and fermenting to obtain an aerobic fermentation product; and finally, conveying the aerobic fermentation product to a closed anaerobic fermentation bin, carrying out anaerobic fermentation for 5 days, and then blowing air to heat and dry the obtained product to obtain the product. The invention can give full play to the metabolic capability of the zymophyte agent, has low production cost and simple and convenient operation, and effectively modifies the coarse feed into bioactive protein feed.

Description

Bioactive protein feed and preparation method thereof
Technical Field
The invention relates to the field of feeds, in particular to a bioactive protein feed and a preparation method thereof.
Background
With the increase of the demand of people for animal products, the animal husbandry is rapidly developed, however, food output in China is limited by the increase of population and the reduction of cultivated land, so that contradiction between people and livestock is increasingly prominent, particularly, the dependence on food is greatly determined by the animal husbandry production structure in China at the present stage, protein feed and energy raw materials face the situation of supply shortage, the annual demand in China reaches 8000 ten thousand tons and the gap reaches more than 4000 ten thousand tons, and the rise of the price of the protein feed raw materials in successive years seriously influences the development of the feed industry and the breeding industry in China, and the development of the animal husbandry cannot be far satisfied by only relying on conventional feed. Therefore, it has become a major topic in animal husbandry development to improve grain conversion rate and feed utilization rate, research new feed additives, develop and utilize unconventional raw material resources, and develop and establish a high-efficiency new sustainable feed industry.
At present, with the continuous development of fermentation technology, the microbial fermentation technology for producing protein feed and feed additives such as amino acid, vitamin, enzyme preparation, microbial preparation and the like is widely applied to the development of novel feed resources, so that the fermentation technology is more and more concerned and more emphasized in the feed industry. The definition of the expert committee of national engineering research center for biological feed development in 11 months in 2013 on the biological feed is as follows: the biological feed is a general name of a novel feed resource and a feed additive which are developed by taking feed and feed additive as objects and taking modern biotechnology such as genetic engineering, protein engineering, fermentation engineering and the like as means and utilizing fermentation of microbial engineering. The biological feed mainly comprises feed enzyme preparations, microbial feed additives, functional protein peptides, functional amino acids, organic trace elements, plant and microbial extracts, prebiotics, fermented and enzymolyzed feeds and other biotechnology related products. Common microorganisms used for biological fermentation feed are mold, yeast, lactobacillus and spore bacteria. The production process of the biological fermentation feed mainly comprises the following steps: the solid surface fermentation method utilizes a water-insoluble solid culture medium with certain humidity, has the unique advantages of water saving and energy saving, belongs to the hot point of research because the clean production technology gradually gains importance from all countries in the world; the large tank liquid fermentation method is mainly suitable for the production of aerobic microorganisms such as bacillus and the like, but the specific process for producing each strain is different, and the method is suitable for industrial production, is convenient for aseptic operation, and has high cost; and thirdly, anaerobic fermentation is mainly suitable for the production of absolute anaerobic bacteria such as bifidobacterium and the like.
Research shows that the use of antibiotics, hormones and other drugs in feed seriously affects the quality and safety of livestock products and aquatic products, and the development and popularization of a bioactive protein feed which can effectively replace antibiotics, hormones and preservatives and has multiple functions of health care, growth promotion and the like becomes a hotspot of current research. Therefore, more and more probiotics such as bacillus and lactic acid bacteria are applied to bio-fermented feed, and the fermentation of bacteria, mold and yeast becomes a new trend. However, the traditional solid-state surface fermentation process is based on an oxygen-consuming microorganism fermentation method, and cannot give full play to the characteristics of mould, yeast and bacteria and improve the commercial value of the feed at the same time; therefore, innovation and optimization of a fermentation process are urgently needed to realize solid-state fermentation production of high-quality bioactive protein feed and finally realize full development and utilization of unconventional feed resources and great improvement of grain conversion rate and feed utilization rate.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a bioactive protein feed and a preparation method thereof, the method has low production cost and simple and convenient operation, can fully exert the metabolic capability of mold, saccharomycetes, bacillus, lactobacillus and other microbial agents, and effectively modify a coarse feed into the bioactive protein feed.
In order to achieve the purpose, the invention is realized by adopting the following technical scheme.
The bioactive protein feed is characterized by comprising the following raw material components: pomace, oil residue, bran and urea; also comprises a zymophyte agent;
the fermentation microbial inoculum is a compound microbial inoculum of mould, microzyme, spore bacteria and lactic acid bacteria.
Further, the raw materials comprise the following components in percentage by mass: 75-78% of pomace, 13-15% of oil residue, 5-7% of bran and 2-3% of urea.
Further, the mould is inoculated into the raw material components in the form of a mould solid spore microbial inoculum, and the inoculation amount is 0.5-0.6% of the raw material components.
Further, the yeast is inoculated into the raw material component in the form of activated yeast, and the inoculation amount is 0.5-0.6% of the raw material component.
Further, the inoculation amount of the compound microbial inoculum of the bacillus and the lactic acid bacteria is 0.5 to 0.6 percent of the raw material components; wherein the mass ratio of the spore bacteria to the lactic acid bacteria is 2: 1-2: 1.5.
Further, the mould is aspergillus niger, and the yeast is Angel saccharomyces cerevisiae.
Further, the water content of the bioactive protein feed is less than or equal to 18 percent.
(II) a preparation method of bioactive protein feed, which is characterized by comprising the following steps:
step 1, preparing a fermentation medium: weighing the pomace, the oil residue, the bran and the urea of the raw material components according to the claim 2, adding water and inorganic salt, mixing and stirring, wherein the pH is natural, and the mass ratio of the raw material components to the water is 1: 1.2-1: 1.3;
step 2, sterilizing the fermentation medium by high-pressure steam;
step 3, cooling the sterilized fermentation medium, placing the cooled fermentation medium in a solid state fermentation bin, inoculating a mould solid state spore microbial inoculum, and fermenting for 34-38 hours under the conditions that the temperature is 30-35 ℃ and the humidity is 50-60%;
step 4, inoculating activated saccharomycetes after the mould fermentation is finished, continuously fermenting the mould and the saccharomycetes for 45-50 hours under the conditions that the temperature is 30-35 ℃ and the humidity is 50-60%, and stirring for 2-5 minutes by an air blower every 4-5 hours;
step 5, after the co-fermentation of the mould and the microzyme, inoculating a composite microbial inoculum of the spore bacteria and the lactic acid bacteria, fermenting for 24 to 30 hours under the conditions that the temperature is between 32 and 35 ℃ and the humidity is between 55 and 60 percent, and stirring for 2 to 5 minutes by using an air blower every 4 to 5 hours to obtain an aerobic fermentation product;
step 6, finally placing the aerobic fermentation product in a closed anaerobic fermentation chamber, and carrying out anaerobic fermentation for 4-5 days;
and 7, after the anaerobic fermentation is finished, blowing air to heat and dry the obtained product to obtain the bioactive protein feed.
Further, in step 1, the inorganic salt comprises: KH (Perkin Elmer)2PO42.8~3.2g/kg、CaCl20.45~0.55g/kg、MgSO4·7H2O 0.45~0.55g/kg、FeSO4·7H2O 7.4~7.6mg/kg、MnSO4·H2O 2.4~2.6mg/kg、ZnSO4·7H2O 3.5~3.7mg/kg。
Further, in the step 2, the inoculation amount of the solid mold spore fungicide is 0.5-0.6% of the ingredient components.
Further, in the step 2, the mould solid spore microbial inoculum is obtained by inoculating mould spore suspension into a sterilized bran culture medium with the water content of 50%, and culturing for 4-6 days at the temperature of 28-31 ℃.
Further, in step 3, the inoculation amount of the activated yeast is 0.5% -0.6% of the ingredient components.
Further, in step 3, the activation of Angel Saccharomyces cerevisiae is carried out by inoculating Saccharomyces cerevisiae into 4% brown sugar sterile water solution, and culturing at 30 deg.C and 1000rpm for 24 hr.
Further, in the step 4, the inoculation amount of the compound microbial inoculum of the spore bacteria and the lactic acid bacteria is 0.5-0.6% of the ingredient components, wherein the mass ratio of the spore bacteria to the lactic acid bacteria is 2: 1-2: 1.5.
Further, in the step 6, the ventilation temperature of the air blowing heating drying is 75-80 ℃, the material temperature is 28-30 ℃, and the time is 4-4.5 hours.
The fermentation medium selects the pomace, reasonably combines the oil pomace, the bran, the urea and the inorganic salt, reasonably supplements organic and inorganic quick-acting nitrogen sources for the growth of microorganisms, increases the content of basic protein in the raw materials, and can promote the production of extracellular enzymes of fungi by adding the inorganic salt.
According to the invention, mould and yeast are inoculated in turn creatively, the mould grows in a fermentation culture medium, and the produced enzyme activities such as cellulase and pectinase are utilized to degrade macromolecular nutrients such as cellulose and pectin into easily utilized reducing sugar and galacturonic acid, so that nutrient substances are provided for the rapid propagation of the yeast, the conversion of inorganic nitrogen into organic nitrogen is facilitated, and the accumulation of protein is promoted; meanwhile, the saccharomycetes utilize enzymolysis products such as glucose and the like, so that the inhibition of a feedback mechanism on the secretion of extracellular enzymes of the fungi is slowed down to a certain extent, and the fermentation product has high enzyme activity of spectrum.
Meanwhile, the invention combines aerobic fermentation and anaerobic fermentation, so that the fermentation benefits of the fungus agent and the bacterial agent are maximized, and the bacterial agent enters an anaerobic stage after aerobic fermentation for 24 hours, at the moment, the bacteria adapt to a fermentation substrate and accumulate the quantity. In the anaerobic stage, although the activity of the mould and the microzyme is inhibited, the produced extracellular enzyme continues to perform enzymolysis at a proper temperature, so that the modification effect on the raw materials is prolonged, and the nutrient proportion easily absorbed and utilized by animals in the feed is increased; meanwhile, the anaerobic degree is gradually strengthened along with the progress of anaerobic fermentation, so that conditions and time are provided for spore production of spore bacteria, and the existence of high activity of the spore bacteria is ensured; the lactic acid accelerates metabolism in the anaerobic stage, is beneficial to accumulation of metabolites, and increases the palatability and the probiotic effect of the feed.
In addition, the invention adopts low-temperature blast drying, can realize the quick air drying of the feed by controlling the blast temperature and the material temperature, is beneficial to the storage and transportation of the biological feed, avoids the pollution and the deterioration of the feed, and protects the enzyme activity of the biological active feed and the activity and the content of probiotics such as bacillus, lactobacillus and the like to the maximum extent.
The invention adopts a solid state fermentation method, reduces the fermentation cost, improves the operability, reduces the environmental pollution and is beneficial to the development and utilization of solid feed raw materials.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention.
Example 1
(1) Bacterial species and sources
The mold of the bioactive protein feed of the embodiment is Aspergillus niger MHQ1, which is preserved by the microbiological laboratory of resource environmental institute of northwest university of agriculture and forestry; the yeast is Angel Saccharomyces cerevisiae, and is available from Angel Yeast GmbH; the complex bacteria of lactobacillus and spore bacteria are purchased from Shandong blue Biotech limited.
(2) Preparation and activation of microbial inoculum
Preparation of solid spore preparation of aspergillus niger MHQ 1: activating Aspergillus niger MHQ1 preserved in a test tube by using a PDA solid inclined plane, eluting activated inclined plane spores by using sterile water to prepare a small amount of Aspergillus niger MHQ1 spore suspension, inoculating the Aspergillus niger MHQ1 spore suspension into a PDA triangular flask solid culture medium, culturing until a large amount of spores grow out, and eluting by using sterile water to obtain a large amount of Aspergillus niger MHQ1 spore suspension. Then adopting a tissue culture bottle for solid state fermentation, accurately weighing 50g of bran, adding 60mL of tap water, uniformly stirring, sterilizing at 121 ℃ for 30 minutes, cooling, then inoculating 5mL of Aspergillus niger MHQ1 spore suspension, and then culturing in a culture chamber at the constant temperature of 30 ℃ for 5 days.
Activating Angel saccharomyces cerevisiae: inoculating Angel Saccharomyces cerevisiae into sterile water solution containing 7.5kg of 4% brown sugar, and culturing at 30 deg.C under stirring at 100rpm for 24 hr.
Activation of the composite microbial inoculum of the spore bacteria and the lactic acid bacteria: the compound microbial inoculum is inoculated in 4 percent of brown sugar sterile water solution, and stirred and cultured for 24 hours at the temperature of 35 ℃ and the rpm of 100.
(3) Preparation of fermentation Medium
The mass ratio of the total mass of the pomace, the oil pomace, the bran and the urea to the water is 1:1.2, wherein the mass ratio of the pomace to the oil pomace to the urea is 77%, the mass ratio of the bran to the water pomace to the oil pomace is 15%, and the mass ratio of the bran to the urea is 3%; the total mass of the pomace, the oil residue, the bran and the urea is 1kg as a reference, and the composition of the inorganic salt is KH2PO43g/kg、CaCl20.5g/kg、MgSO4·7H2O 0.5g/kg、FeSO4·7H2O 7.5mg/kg、MnSO4·H2O2.5mg/kg、ZnSO4·7H2O3.6 mg/kg. Urea and inorganic salts are added to the fermentation medium in the form of aqueous solutions, respectively.
(4) And (3) fermentation process:
step 1, preparing a fermentation medium, and performing high-pressure steam sterilization on the fermentation medium;
step 2, cooling the sterilized fermentation medium on a fluidized bed, conveying the cooled fermentation medium to a solid state fermentation bin, inoculating an Aspergillus niger MHQ1 solid state spore microbial inoculum, and fermenting for 34-38 hours at the temperature of 30-35 ℃ and the humidity of 50-60%;
step 3, inoculating activated Angel saccharomyces cerevisiae after the fermentation of the Aspergillus niger MHQ1 is finished, and co-fermenting the Aspergillus niger MHQ1 and the Angel saccharomyces cerevisiae for 48 hours under the conditions that the temperature is 30-35 ℃ and the humidity is 50-60%, wherein the blower is used for stirring for 2 minutes every 4 hours;
step 4, after co-fermentation of Aspergillus niger MHQ1 and Angel saccharomyces cerevisiae is finished, inoculating a composite microbial inoculum of bacillus and lactic acid bacteria, fermenting for 24 hours under the conditions that the temperature is 32-35 ℃ and the humidity is 55-60%, and stirring for 5 minutes by a blower every 4 hours to obtain an aerobic fermentation product;
step 5, finally conveying the aerobic fermentation product to a closed anaerobic fermentation chamber for anaerobic fermentation for 5 days;
and 6, after the anaerobic fermentation is finished, blowing air to heat and dry the obtained product, and drying for 4 hours at the ventilation temperature of 78 ℃ and the material temperature of 27 ℃ to obtain the bioactive protein feed.
The obtained bioactive protein feed has water content of 16.3%, and dried feed contains crude protein 27.8%, carboxymethyl cellulase activity 5389U/g, pectase activity 1176U/g, and lactobacillus content 1.7 × 108cfu/g, the content of spore bacteria is 6.7 × 107cfu/g, yeast content 4.5 × 107cfu/g。
Example 2
(1) Bacterial species and sources
The mold of the bioactive protein feed is aspergillus niger A8 which is preserved by a microbiological laboratory of resource environmental institute of northwest university of agriculture and forestry; the yeast is Angel Saccharomyces cerevisiae, and is available from Angel Yeast GmbH; the complex bacteria of lactobacillus and spore bacteria are purchased from Shandong blue Biotech limited.
(2) Preparation and activation of microbial inoculum
Preparation of aspergillus niger A8 solid spore preparation: activating Aspergillus niger A8 preserved in a test tube by using a PDA solid inclined plane, eluting spores on the activated inclined plane by using sterile water to prepare a small amount of Aspergillus niger A8 spore suspension, inoculating the Aspergillus niger A8 spore suspension into a PDA triangular flask solid culture medium, culturing until a large amount of spores grow out, and eluting by using sterile water to obtain a large amount of Aspergillus niger MHQ1 spore suspension. Then adopting a tissue culture bottle for solid state fermentation, accurately weighing 50g of bran, adding 60mL of tap water, uniformly stirring, sterilizing at 121 ℃ for 30 minutes, cooling, then inoculating 5mL of Aspergillus niger A8 spore suspension, and then culturing in a culture chamber at the constant temperature of 30 ℃ for 5 days.
Activating Angel saccharomyces cerevisiae: inoculating Angel Saccharomyces cerevisiae into sterile water solution containing 7.5kg of 4% brown sugar, and culturing at 30 deg.C under stirring at 100rpm for 24 hr.
Activation of the composite microbial inoculum of the spore bacteria and the lactic acid bacteria: the compound microbial inoculum is inoculated in 4 percent of brown sugar sterile water solution, and stirred and cultured for 24 hours at the temperature of 35 ℃ and the rpm of 100.
(3) Preparation of fermentation Medium
The mass ratio of the total mass of the pomace, the oil pomace, the bran and the urea to the water is 1:1.2, wherein the mass ratio of the pomace to the oil pomace to the urea is 77%, the mass ratio of the bran to the water pomace to the oil pomace is 15%, and the mass ratio of the bran to the urea is 3%; the total mass of the pomace, the oil residue, the bran and the urea is 1kg as a reference, and the composition of the inorganic salt is KH2PO43g/kg、Cacl20.5g/kg、MgSO4·7H2O 0.5g/kg、FeSO4·7H2O 7.5mg/kg、MnSO4·H2O2.5mg/kg、ZnSO4·7H2O3.6 mg/kg. Urea and inorganic salts are added to the fermentation medium in the form of an aqueous solution.
(4) And (3) fermentation process:
step 1, preparing a fermentation medium, and performing high-pressure steam sterilization on the fermentation medium;
step 2, cooling the sterilized fermentation medium on a fluidized bed, conveying the cooled fermentation medium to a solid state fermentation bin, inoculating an Aspergillus niger A8 solid spore microbial inoculum, and fermenting for 36 hours under the conditions that the temperature is 30-35 ℃ and the humidity is 50-60%;
step 3, inoculating activated Angel saccharomyces cerevisiae after the fermentation of the Aspergillus niger A8 is finished, co-fermenting the Aspergillus niger A8 and the Angel saccharomyces cerevisiae for 48 hours under the conditions that the temperature is 30-35 ℃ and the humidity is 50-60%, and stirring for 5 minutes by a blower every 4 hours;
step 4, after the co-fermentation of the Aspergillus niger A8 and the Angel saccharomyces cerevisiae is finished, inoculating a composite microbial inoculum of bacillus and lactic acid bacteria, fermenting for 24 hours under the conditions that the temperature is 32-35 ℃ and the humidity is 55-60%, and stirring for 5 minutes by a blower every 4 hours to obtain an aerobic fermentation product;
step 5, finally conveying the aerobic fermentation product to a closed anaerobic fermentation chamber for anaerobic fermentation for 5 days;
and 6, after the anaerobic fermentation is finished, blowing air to heat and dry the obtained product, and drying for 4 hours at the ventilation temperature of 80 ℃ and the material temperature of 27 ℃ to obtain the bioactive protein feed.
The obtained bioactive protein feed has water content of 14.7%, and dried feed contains crude protein 24.6%, carboxymethyl cellulose 4957U/g, pectase 973U/g, and lactobacillus 1.2 × 108cfu/g, the content of spore bacteria is 4.6 × 107cfu/g, yeast content 2.9 × 107cfu/g。
Although the present invention has been described in detail in this specification with reference to specific embodiments and illustrative embodiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto based on the present invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (1)

1. The bioactive protein feed is characterized by comprising the following raw material components: pomace, oil residue, bran and urea; the raw material components comprise the following components in percentage by mass: 75-78% of pomace, 13-15% of oil residue, 5-7% of bran and 2-3% of urea; also comprises a zymophyte agent;
the fermentation microbial inoculum is a compound microbial inoculum of mould, microzyme, spore bacteria and lactic acid bacteria;
the preparation method of the bioactive protein feed is characterized by comprising the following steps:
step 1, preparing a fermentation medium: weighing the pomace, the oil residue, the bran and the urea of the raw material components, adding water and inorganic salt, mixing and stirring, wherein the pH is natural, and the mass ratio of the raw material components to the water is 1: 1.2-1: 1.3; the mould is inoculated into the raw material components in the form of a mould solid spore microbial inoculum, and the inoculation amount is 0.5-0.6% of the raw material components; the yeast is inoculated into the raw material component in the form of activated yeast, and the inoculation amount is 0.5-0.6% of the raw material component; the inoculation amount of the compound microbial inoculum of the spore bacteria and the lactic acid bacteria is 0.5 to 0.6 percent of the raw material components; wherein the spore bacteria and lactic acid bacteriaThe mass ratio is 2: 1-2: 1.5; the water content of the bioactive protein feed is less than or equal to 18 percent; the inorganic salt comprises: KH (Perkin Elmer)2PO42.8~3.2g/kg、CaCl20.45~0.55g/kg、MgSO4·7H2O 0.45~0.55g/kg、FeSO4·7H2O 7.4~7.6mg/kg、MnSO4·H2O 2.4~2.6mg/kg、ZnSO4·7H2O 3.5~3.7mg/kg;
Step 2, sterilizing the fermentation medium by high-pressure steam;
step 3, cooling the sterilized fermentation medium, placing the cooled fermentation medium in a solid state fermentation bin, inoculating a mould solid state spore microbial inoculum, and fermenting for 34-38 hours under the conditions that the temperature is 30-35 ℃ and the humidity is 50-60%; the mould solid spore microbial inoculum is obtained by inoculating mould spore suspension into a sterilized bran culture medium with the water content of 50 percent and culturing for 4 to 6 days at the temperature of between 28 and 31 ℃;
step 4, inoculating activated saccharomycetes after the mould fermentation is finished, continuously fermenting the mould and the saccharomycetes for 45-50 hours under the conditions that the temperature is 30-35 ℃ and the humidity is 50-60%, and stirring for 2-5 minutes by an air blower every 4-5 hours;
step 5, after the co-fermentation of the mould and the microzyme, inoculating a composite microbial inoculum of the spore bacteria and the lactic acid bacteria, fermenting for 24 to 30 hours under the conditions that the temperature is between 32 and 35 ℃ and the humidity is between 55 and 60 percent, and stirring for 2 to 5 minutes by using an air blower every 4 to 5 hours to obtain an aerobic fermentation product;
step 6, finally placing the aerobic fermentation product in a closed anaerobic fermentation chamber, and carrying out anaerobic fermentation for 4-5 days;
step 7, after the anaerobic fermentation is finished, blowing air to heat and dry the obtained product to obtain the bioactive protein feed; the ventilation temperature of the blast heating drying is 75-80 ℃, the temperature of the materials is 28-30 ℃, and the time is 4-4.5 hours.
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