CN102172259B - Method for controlling solid state fermentation temperature of biological feed - Google Patents
Method for controlling solid state fermentation temperature of biological feed Download PDFInfo
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Abstract
The invention discloses a method for controlling solid state fermentation temperature of a biological feed. The method for controlling the fermentation temperature in solid state fermentation comprises the following steps: mixing a solid-state substrate, bacteria, a glucogen starting bacterium metabolism and a glucogen for providing energy required for the bacterium metabolism; and carrying out solid state fermentation. The method is characterized in that an product of enzyme hydrolysis is used as the glucogen for providing the energy required for bacterium metabolism in the process of solid state fermentation, so as to lower the central highest temperature of the fermentation in the fermentation process and/or inhibit the ascending speed of the fermentation temperature in the fermentation process; and the fermentations are all substances in a fermentation container. In the method provided by the invention, the starch is used as main glucogen in the solid state fermentation process; and gelatinized starch is decomposed step by step under the united action of amylase and glucoamylase, so as to generate yeast as well as monosaccharide, disaccharide and oligosaccharide which can be metabolized and digested by lactic acid bacteria, thus slowing down the fermentation speed of microorganisms, slowing down the heat production speed and heating-up speed of a fermentation system, further maintaining the bioactivities of the microorganisms in the fermentation system for a long time, prolonging the bacterial action time and improving the fermentation efficiency.
Description
Technical field
The present invention relates to a kind of method of controlling the solid state fermentation temperature, especially for the method for control temperature in the solid state fermentation of biological protein feedstuff.
Background technology
Solid state fermentation (solid-state fermentation) procedure definition is that microorganism is in the culture matrix growth that does not almost have free water and the course of reaction of metabolism.
Adopt the microbe in solid state anaerobic fermentation to produce the much-talked-about topic that biological feedstuff is present feed processing and herding production field.Microorganism solid fermentation is an ancient biotechnology, and the scope of application is very extensive.The research report that the using microbe solid state fermentation is produced biological protein feedstuff is a lot, but their overwhelming majority are confined to laboratory research, and the practical technique that really can apply in actual production also seldom.Its main cause is not that the screening of microorganism and metabolism research have technical difficulty, but exists great contradiction between the production capacity of round and the temperature control in the actual production.Round is larger, and temperature control is more difficult, and this contradiction has greatly limited applying of a lot of fermentation techniques.
The heat-transfer rate of solid fermented material is very slow.In the large tank of scale is produced, because the heat conductivility of material is poor, adopt common chuck cooling water temperature to be difficult to guarantee the harmony of fermentation temperature.When fermentation was carried out fast, although can obtain effective temperature-reducing near the material of container periphery, the material central temperature was difficult to lower, and forms a very large thermograde, causes product quality very poor.
Existing large tank solid state fermentation, the fermenting speed in early stage is slow, and heat production is seldom.Enter after the growth of microorganism peak period, fermenting speed is accelerated, and quantity of heat production also strengthens rapidly.If untimely heat radiation, temperature of charge meeting fast rise, activity and the metabolic capability of microorganism descend rapidly.A large amount of production practices prove that the charge of round surpasses 1m3, adopt conventional cooling measure, and in the peak period of microbial metabolism, the local temperature of material is easy to reach more than 60 ℃.Metabolism that not only can the grievous injury microorganism, but also can cause nutraceutical very big loss.More effective cool-down method is in the round inner coil pipe of installing, logical water at low temperature even chilled brine cooling at present.But device structure is complicated, and investment is large, and cleans difficulty, complex operation.The medicinal product that only production added value is very high generally seldom adopts.
The added value of animal feed product is generally not high, so round and treating capacity are all larger, it obviously is inappropriate adopting the inner coil pipe process for cooling.
Design a kind of method of easier control temperature of charge, the environment temperature of microbial metabolism is in the proper scope all the time, just can greatly alleviate the difficult problem of this long-term puzzlement actual production of solid state fermentation heat build-up, manufacturing requirement and operating procedure that not only can simplified apparatus, and can improve significantly the quality of fermented product.
Summary of the invention
An object of the present invention is to provide the method that a kind of solid state fermentation prepares biological feedstuff.
Solid state fermentation provided by the present invention prepares the method for biological feedstuff, comprises the steps: the sugared source of solid state substrate, bacterium, the metabolism of startup bacterium and provides the sugared source of bacterium metabolism institute energy requirement to mix, and the mixture that obtains is fermentation raw material; Described fermentation raw material is fermented, obtain biological feedstuff; The described enzymolysis product of sugared source for obtaining with amylase and carbohydrase enzymolysis starch that bacterium metabolism institute energy requirement is provided.
Above-mentioned solid state fermentation prepares in the method for biological feedstuff, and the proportioning of described starch, described amylase, described carbohydrase and described solid state substrate is (150kg-300kg): (1.0 * 10
6U-2 * 10
6U): (2.0 * 10
8U-3.5 * 10
8U): 4000kg is specially 150kg: 1.0 * 10
6U: 2.0 * 10
8U: 4000kg, 200kg: 1.3 * 10
6U: 2.5 * 10
8U: 4000kg, 250kg: 1.7 * 10
6U: 3.0 * 10
8U: 4000kg or 300kg: 2 * 10
6U: 3.5 * 10
8U: 4000kg.
Above-mentioned arbitrary solid state fermentation prepares in the method for biological feedstuff, and described enzymolysis product prepares according to the method that comprises the steps: with described starch gelatinization, obtain gelatinized starch; To wherein adding described amylase, mixing left standstill 120 minutes again; To wherein adding described carbohydrase, stir 3min again, obtain described enzymolysis product.
Above-mentioned arbitrary solid state fermentation prepares in the method for biological feedstuff, and the sugared source of described startup bacterium metabolism and the proportioning of described solid state substrate are 8kg: 4000kg.
Above-mentioned arbitrary solid state fermentation prepares in the method for biological feedstuff, and described bacterium is saccharomycete and/or lactic acid bacteria;
Above-mentioned arbitrary solid state fermentation prepares in the method for biological feedstuff, and the proportioning of described saccharomycete and described solid state substrate is 2 * 10
5Hundred million cfu: 4000kg, the proportioning of described lactic acid bacteria and described solid state substrate is 5 * 10
5Hundred million cfu: 4000kg.
Above-mentioned arbitrary solid state fermentation prepares in the method for biological feedstuff, and the method for described mixing comprises the steps:
1) described bacterium is mixed with the sugared source of described startup bacterium metabolism, the mixture that obtains is denoted as strain liquid B;
2) described enzymolysis product is mixed with described solid state substrate, the mixture that obtains is denoted as mixture C;
3) described mixture C is mixed with described strain liquid B, the mixture that obtains is fermentation raw material;
The step that comprises logical cooling water in the described fermentation;
Above-mentioned arbitrary solid state fermentation prepares in the method for biological feedstuff, described step 3) in, the temperature of described mixture C is 27 ℃-29 ℃ or 28 ℃ during described the mixing; The temperature of described cooling water is 13 ℃-22 ℃ or 13 ℃-15 ℃ or 20 ℃-22 ℃)
Ordinary material is packed into after the fermentation tank, and sweat and environment temperature are irrelevant, and is only relevant with cooling water temperature.
Above-mentioned arbitrary solid state fermentation prepares in the method for biological feedstuff, and the water content of described fermentation raw material is 25～85% or 33%-36%, is specially 34%, 33.78%, 34.93% or 35.40%.
Above-mentioned arbitrary solid state fermentation prepares in the method for biological feedstuff, the method of mixing described step 1) comprises the steps: the sugared source of described startup bacterium metabolism water-soluble, obtain the aqueous solution in the sugared source of described startup bacterium metabolism, when the temperature of the aqueous solution for the treatment of the sugared source of described startup bacterium metabolism is 31 ℃-33 ℃ or 32 ℃, add dusty yeast and lactic acid bacteria culture solution, left standstill 30 minutes, and obtained described strain liquid B;
Above-mentioned arbitrary solid state fermentation prepares in the method for biological feedstuff, described dusty yeast prepares according to the method that comprises the steps: saccharomycete is accessed microzyme culture medium, be that 30C, ventilation are to cultivate 8 hours-9 hours under the condition of 0.4vvm-0.6vvm and stirring in temperature, obtain saccharomycetes to make fermentation liquid; Saccharomycetes to make fermentation liquid is carried out concentrate drying, and the material that obtains is dusty yeast; All substances in the culture vessel are denoted as saccharomycetes to make fermentation liquid;
Above-mentioned arbitrary solid state fermentation prepares in the method for biological feedstuff, described lactic acid bacteria culture solution prepares according to the method that comprises the steps: white granulated sugar is dissolved in the plain chocolate of antibiotic-free pollution, the proportioning of the plain chocolate that white granulated sugar and antibiotic-free pollute is 300g: 5000g, obtains the lactic acid bacteria culture medium; Access lactic acid bacteria in the lactic acid bacteria culture medium, constant temperature culture is 20 hours-24 hours between 36 ℃-40 ℃, and the culture that obtains is lactic acid bacteria culture solution; All substances in the culture vessel are denoted as lactic acid bacteria culture solution.
Above-mentioned arbitrary solid state fermentation prepares in the method for biological feedstuff, and the sugared source of described startup bacterium metabolism is brown sugar, white granulated sugar and/or sucrose.
Above-mentioned arbitrary solid state fermentation prepares in the method for biological feedstuff, and described starch is rice, cornstarch or farina;
Above-mentioned arbitrary solid state fermentation prepares in the method for biological feedstuff, and described solid state substrate is dregs of beans, cotton dregs and/or the dish dregs of rice;
Above-mentioned arbitrary solid state fermentation prepares in the method for biological feedstuff, and described saccharomycete is saccharomyces cerevisiae (Sacharomycescerevisiae) CGMCC No.2.399;
Above-mentioned arbitrary solid state fermentation prepares in the method for biological feedstuff, and described lactic acid bacteria is saliva chain coccus thermophilous subspecies (Streptococcus thermophilus) CGMCC 1.1864;
Above-mentioned arbitrary solid state fermentation prepares in the method for biological feedstuff, and described amylase is Isosorbide-5-Nitrae-α-D-glucan hydrolase; Described carbohydrase is α-Isosorbide-5-Nitrae-glucose hydrolysis enzyme.
The biological feedstuff that is obtained by above-mentioned arbitrary described method also belongs to protection scope of the present invention.
Another object of the present invention provides a kind of method of controlling fermentate temperature in the solid state fermentation.
The method of fermentate temperature comprises the steps: the sugared source of solid state substrate, bacterium, the metabolism of startup bacterium and provides the sugared source of bacterium metabolism institute energy requirement to mix in the control solid state fermentation provided by the present invention, carries out solid state fermentation; In the process of described solid state fermentation with enzymolysis product as the described sugared source that bacterium metabolism institute energy requirement is provided, with the center maximum temperature that reduces fermentate in the sweat and/or with the rate of climb of fermentate temperature in the stopping fermentation process; Described fermentate is all substances in the round.The enzymolysis product of described enzymolysis product for obtaining with amylase and carbohydrase enzymolysis starch.
In the method for above-mentioned control temperature, the proportioning of described starch, described amylase, described carbohydrase and described solid state substrate is (150kg-300kg): (1.0 * 10
6U-2 * 10
6U): (2.0 * 10
8U-3.5 * 10
8U): 4000kg is specially 150kg: 1.0 * 10
6U: 2.0 * 10
8U: 4000kg, 200kg: 1.3 * 10
6U: 2.5 * 10
8U: 4000kg, 250kg: 1.7 * 10
6U: 3.0 * 10
8U: 4000kg or 300kg: 2 * 10
6U: 3.5 * 10
8U: 4000kg.
In the method for above-mentioned control temperature, described enzymolysis product prepares according to the method that comprises the steps: with described starch gelatinization, obtain gelatinized starch; To wherein adding described amylase, mixing left standstill 120 minutes again; To wherein adding described carbohydrase, stir 3min again, obtain described enzymolysis product.
In the method for above-mentioned control temperature, the sugared source of described startup bacterium metabolism and the proportioning of described solid state substrate are 8kg: 4000kg.
In the method for above-mentioned control temperature, described bacterium is saccharomycete and/or lactic acid bacteria;
In the method for above-mentioned control temperature, the proportioning of described saccharomycete and described solid state substrate is 2 * 10
5Hundred million cfu: 4000kg, the proportioning of described saliva chain coccus thermophilous subspecies and described solid state substrate is 5 * 10
5Hundred million cfu: 4000kg.
In the method for above-mentioned control temperature, the method for described mixing comprises the steps:
1) described bacterium is mixed with the sugared source of described startup bacterium metabolism, the mixture that obtains is denoted as strain liquid B;
2) described enzymolysis product is mixed with described solid state substrate, the mixture that obtains is denoted as mixture C;
3) described mixture C is mixed with described strain liquid B, the mixture that obtains is fermentation raw material;
In the method for above-mentioned control temperature, comprise the step of logical cooling water in the described fermentation;
In the method for above-mentioned control temperature, described step 3) in, the temperature of described mixture C is 27 ℃-29 ℃ or 28 ℃ during described the mixing;
The temperature of described cooling water is 13 ℃-22 ℃ or 13 ℃-15 ℃ or 20 ℃-22 ℃.
In the method for above-mentioned control temperature, the water content of described fermentation raw material is 25～85% or 33%-36%, is specially 34%, 33.78%, 34.93% or 35.40%.
In the method for above-mentioned control temperature, the method of mixing described step 1) comprises the steps: the sugared source of described startup bacterium metabolism water-soluble, obtain the aqueous solution in the sugared source of described startup bacterium metabolism, when the temperature of the aqueous solution for the treatment of the sugared source of described startup bacterium metabolism is 31 ℃-33 ℃ or 32 ℃, add dusty yeast and lactic acid bacteria culture solution, left standstill 30 minutes, and obtained described strain liquid B;
In the method for above-mentioned control temperature, described dusty yeast prepares according to the method that comprises the steps: saccharomycete is accessed microzyme culture medium, be that 30 ℃, ventilation are to cultivate 8 hours-9 hours under the condition of 0.4vvm-0.6vvm and stirring in temperature, obtain saccharomycetes to make fermentation liquid; Saccharomycetes to make fermentation liquid is carried out concentrate drying, and the material that obtains is dusty yeast; All substances in the culture vessel are denoted as saccharomycetes to make fermentation liquid;
In the method for above-mentioned control temperature, described lactic acid bacteria culture solution prepares according to the method that comprises the steps: white granulated sugar is dissolved in the plain chocolate of antibiotic-free pollution, the proportioning of the plain chocolate that white granulated sugar and antibiotic-free pollute is 300g: 5000g, obtains the lactic acid bacteria culture medium; Access lactic acid bacteria in the lactic acid bacteria culture medium, constant temperature culture is 20 hours-24 hours between 36 ℃-40 ℃, and the culture that obtains is lactic acid bacteria culture solution; All substances in the culture vessel are denoted as lactic acid bacteria culture solution.
In the method for above-mentioned control temperature, the sugared source of described startup bacterium metabolism is brown sugar, white granulated sugar and/or sucrose;
In the method for above-mentioned control temperature, described starch is rice, cornstarch or farina;
In the method for above-mentioned control temperature, described solid state substrate is dregs of beans, cotton dregs and/or the dish dregs of rice;
In the method for above-mentioned control temperature, described saccharomycete is saccharomyces cerevisiae (Sacharomyces cerevisiae) CGMCC No.2.399;
In the method for above-mentioned control temperature, described lactic acid bacteria is saliva chain coccus thermophilous subspecies (Streptococcusthermophilus) CGMCC 1.1864;
In the method for above-mentioned control temperature, described amylase is Isosorbide-5-Nitrae-α-D-glucan hydrolase;
In the method for above-mentioned control temperature, described carbohydrase is α-Isosorbide-5-Nitrae-glucose hydrolysis enzyme.
According to technological requirement, the technology of the present invention is mainly used in the anaerobic solid-state fermentation (including the closed container of air bleeding valve) of closed container, also can be applied to the aerobic solid ferment process.
The inventive method under the synergy of amylase and carbohydrase, is progressively decomposed the starch through gelatinization, liquefaction with the main sugared source of liquefying starch as microorganism solid fermentation, but produces monosaccharide and disaccharide and the oligosaccharides of saccharomycete and lactic acid bacteria digestion and metabolism.By the mode of this progressively releasing microbe metabolizable sugar, can control preferably the fermenting speed of microorganism, slow down the heat production speed of fermentation system.The degree of slowing down depends primarily on temperature and the flow of volume, thermal conductivity factor and the cooling water of fermentation materials.The volume of material is larger, the capacity of heat transmission is poorer, the corresponding metabolizable sugar rate of release that requires is also lower, make conventional chuck cooling can eliminate timely and effectively the metabolic heat of microorganism, thereby make the microorganism in the fermentation system can keep for a long time preferably metabolic activity, sweat can be stablized and carry out equably, although fermentation time has prolonged, the quality of fermented product is greatly improved, and can obtain the good finished product of quality.
The present invention has solved the difficulties such as fast because of fermenting speed in the solid ferment process, that heat radiation is difficult, the microbial activity loss is large effectively.The present invention is simple to operate, device fabrication cost low (common chuck cooling just can meet the demands fully), and end product quality is fine, all has broad application prospects at a lot of production fields such as food brewing, biological feedstuff and biology enzyme solid state fermentation.
Except metabolizable sugar, also have some other factor also can regulate the fermenting speed of microorganism.Such as: the water content of the production bacterial classification of inoculation and the ratio of inoculation thereof, material, the initial temperature of material, initial metabolizable sugar content etc.
In biological feedstuff fermenting and producing bacterium commonly used at present, saccharomyces cerevisiae is the most frequently used yeast, and its heat production speed and metabolism intensity will be far above lactic acid bacterias.In the lactic acid bacteria that actual production is used, metabolism intensity and the heat production speed of heterolactic fermentation will be higher than facultative lactic fermentation, and facultative lactic acid fermented metabolism intensity and heat production speed will be higher than the homotype lactic fermentation.So in bulk container fermentation to produce biological feed, the lactic acid bacteria of use is the lactic acid fermented streptococcus thermophilus of homotype or lactobacillus acidophilus preferably.
If only consider the utilization ratio of equipment, the ratio of inoculation is the bigger the better.But the preparation of bacterial classification needs higher cost, so should consider a proper inoculative proportion, this ratio can make initial period of delay short as far as possible, and the consumption of bacterial classification is also proper.
The water content of material has material impact to fermenting speed.Generally speaking, moisture is higher, and fermenting speed is faster.For the heat production speed of alleviating fermentation and the contradiction between the radiating capacity of equipment, comprise the drying process that the later stage may need, in the bulk container sweat, the moisture of material should be low as far as possible.But in order to guarantee that fermentation energy carries out smoothly and suitable production capacity is arranged, the content of material moisture can not be too small.When material moisture less than 25% the time, fermenting speed is extremely slow, production capacity is extremely low.Experimental study according to early stage proves that proper water content is between 28% to 32%.
Initial fermentation temperature preferably is no more than 28 ℃, (optimum growth temperature of saccharomyces cerevisiae is about 32 ℃ although this temperature growth temperature suitableeer than yeast wine brewing and lactic acid bacteria is all low, the optimum growth temperature of lactic acid bacteria is about 38 ℃), but consider the difficulty of fermentation heat production and cooling, it is still more rational suitably to reduce the cultivation temperature that material begins.In actual production, the treating capacity of material is larger, and initial temperature is corresponding lower.
In order to accelerate equipment efficiency of usage, reduce simultaneously the probability that miscellaneous bacteria infects, need in raw material, add an amount of direct metabolizable sugar to shorten the stand-down of growth of microorganism.But directly the concentration of metabolizable sugar can not be too high, otherwise can cause microbial fermentation intensity excessive, the post drop difficulty.More satisfactory addition is after microorganism has just run out of these metabolizable sugars, and enzymolysis starch has discharged the metabolizable sugar that satisfies the follow-up metabolism of microorganism within the corresponding time.According to the research in early stage, find that the direct metabolizable sugar in the raw material just can significantly shorten greatly the stop time of sweat about about 0.2%.
The present invention is with the main energy sources of enzymolysis gelatinized starch as microbial metabolism, make the speed of microbial metabolism be subject to the restriction of enzymolysis starch speed, fermentation heat production speed is effectively controlled, recirculated cooling water can be eliminated the used heat that fermentation system produces more in time, effectively alleviates the difficult problem of this long-term puzzlement actual production of solid state fermentation heat build-up.
Description of drawings
Fig. 1 is that the sugar fermentation type is on the impact of fermentation central temperature.
Fig. 2 is the solid state fermentation of cotton dregs and the dish dregs of rice.
Fig. 3 is the solid state fermentation of cornstarch and farina enzymolysis.
Fig. 4 is that cooling water temperature improves the impact on the fermentation central temperature.
The specific embodiment
Employed experimental technique is conventional method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The old rice powder is the rice of depositing more than 3 years, and old rice is cheap, water content 5%;
Amylase is Isosorbide-5-Nitrae-α-D-glucan hydrolase (zymetology numbering EC3.2.1.1.)
Carbohydrase is α-Isosorbide-5-Nitrae-glucose hydrolysis enzyme.
Saccharomycete is saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC 2.399, available from Chinese common micro-organisms culture presevation administrative center (CGMCC).
Lactic acid bacteria is saliva chain coccus thermophilous subspecies (Streptococcus thermophilus) CGMCC 1.1864, available from Chinese common micro-organisms culture presevation administrative center (CGMCC).
Streptococcus thermophilus is the homotype lactic acid fermenting bacteria, and the lactic acid that produces all is L-type, has BA.
Every bacterium that can produce lactic acid from the sweat of glucose or lactose is referred to as lactic acid bacteria.
1, the saliva chain coccus thermophilous subspecies nutrient solution is prepared as follows: the plain chocolate 5000g that antibiotic-free pollutes, add the 300g white granulated sugar, and be heated to 70 ℃, until white granulated sugar dissolves fully.Then the milk that is dissolved with white granulated sugar is divided in the 1000ml triangular flask, uses the cotton plug jam-pack, outsourcing brown paper.110 ℃ of left and right sides sterilizations 20 minutes, inoculation saliva chain coccus thermophilous subspecies (Streptococcus thermophilus) CGMCC NO.1.1864 after the cooling (bacterial classification with the freeze-dried vaccine powder of Refrigerator store).40 ℃ of constant temperature culture 24 hours, obtain the saliva chain coccus thermophilous subspecies nutrient solution.
2, the preparation method of the active dry yeast powder of saccharomyces cerevisiae (Sacharomyces cerevisiae) CGMCC 2.399:
Primary seed solution is cultivated: cultivate with the triangular flask shaking flask, inoculation is than being 0.5-2.0%, and 500mL triangular flask liquid amount is 150mL, and rotating speed is controlled between the 200-250rpm.At 30 ℃ of constant temperature culture 8-10 hours, access secondary seed tank.
Secondary seed solution is cultivated: rise canister with 80-100, inoculation is than being 2.0-5.0%, and ventilating ratio is 1.0-1.2vvm.At 30 ℃ of aerlbic culture 7-8 hours, access was produced and is used large tank.
Fermented and cultured: the volume of large tank is 3 to 5 tons, can be non-pressure vessel.The inoculation of large tank is than being 1.0-8.0%, and ventilation is controlled at 0.4vvm, and temperature is 30 ℃, and the stirring motor power setting is at 1.0kw/m
3About (nutrient solution), whole incubation time is 8 hours.Its leading indicator is the remaining sugar concentration in the solution.When the sugared concentration in the nutrient solution is lower than 1.0%, just finish to cultivate, obtain zymotic fluid (all substances in the large tank are denoted as zymotic fluid).Cultivate the zymotic fluid centrifuge centrifugal concentrating after finishing, isolate concentrated broth, then carry out spray-drying at 60 ℃, the dry thing of acquisition is the saccharomycete powder.
The culture medium of culture yeasts bacterium is existing culture medium, and specifically consist of: 1 liter of culture medium is comprised of 10mL corn steep liquor, 5.0g peptone, 0.3g phosphoric acid, 5.0g ammonium sulfate, 40.0g brown sugar and water, and water is supplied volume.
3, the assay method of lactic acid content---colorimetric method
Principle: rare lactic acid heats under concentrated sulfuric acid effect, can become acetaldehyde.Acetaldehyde and xenol effect can generate red compound, and colorimetric estimation under the 570nm condition can be determined the content of lactic acid.
One, reagent:
1.4.0% copper-bath: 4 gram cupric sulfate pentahydrates are dissolved in the aqueous solution, are settled to 100ml.
2. xenol (C
6H
5C
6H
4OH) solution: 1 gram xenol is dissolved in the NaOH solution that 100ml concentration is 0.08mol/L, is stored in the brown reagent bottle.
3. lactic acid titer: the pure lithium lactate of 280mg is soluble in water, be made into 250ml, its lactic acid concn is 1mg/ml, leaves in 4 ℃ of refrigerators.With 10 times of solution dilutions, making lactic acid content is 100 μ g/ml when doing calibration curve.Get respectively again 1,2., 3,4,5, the 6ml dilution, dilution is mixed with 1 μ g/ml, 2 μ g/ml, 3 μ g/ml, 4 μ g/ml, 5 μ g/ml, 6 μ g/ml lactic acid titers.
4. concentrated sulfuric acid solution: in colorimetric cylinder, add respectively 1ml lactic acid titer and 0.05ml copper-bath, then add the 6ml concentrated sulfuric acid, placed 5 minutes, be cooled to below 20 ℃.
Two, calibration curve
In above-mentioned colorimetric cylinder, add 0.05ml xenol solution, mix, at room temperature placed 6-8 hour, spend the night.
Under the 570nm wavelength, carry out colorimetric estimation, the drawing standard curve.
Three, lactic acid content is measured in the sample
The making of reference standard curve, the absorbance of working sample reactant liquor, the reference standard curve calculates lactic acid content.
Embodiment 1, solid fermentation prepare biological feedstuff
One, fermentation process
(1) experimental group
1, raw material preparation
(1) get dregs of beans 4000kg, water content is 13% (quality percentage composition), pulverizes, and crosses 20 mesh sieves.
(2) preparation in bacterium metabolism sugar source: get old rice 150kg, pulverize, cross 40 mesh sieves.Be 2.0m at volume
3Cooker in add successively 1000kg clear water and 150kg old rice powder, progressively pass into high-temperature steam 250kg, be heated to 100 ℃, and then be incubated 20 minutes, until the complete gelatinization of starch.Be cooled to 70 ℃, (enzyme is lived: 2000u/g, the enzyme definition of living: under 60 ℃, pH6.0 condition, the needed enzyme amount of 1 gram soluble starch that per hour liquefies is the enzyme u of unit that lives to add 500g amylase.), abundant mixing, left standstill 120 minutes, (enzyme is lived: 100,000 u/g to add 2000 gram carbohydrase again, under 40 ℃, pH4.6 condition, per hour 1 milligram of needed enzyme amount of glucose of decomposing soluble starch generation is the enzyme u of unit alive), stirred 3 minutes, obtain liquefied starch A (this moment, the temperature of solution was 58 ℃).
(3) add successively 75kg clear water and 8kg brown sugar in volume is 300 liters constant temperature water pot, then pass into 5kg steam, appropriateness stirs.After brown sugar dissolves fully, continue to stir cooling.When about the drop in temperature to 32 of solution ℃, add again 2000g active dry yeast powder (saccharomyces cerevisiae, water content is about 8%, the number of active yeast is 10,000,000,000 cfu in the 1g active dry yeast powder) and 5000g saliva chain coccus thermophilous subspecies nutrient solution (number of active bacterium is 10,000,000,000 cfu in 1ml (the being 1g) nutrient solution), static maintenance 30 minutes obtains strain liquid B.
(4) liquefying starch A is progressively joined in the dregs of beans, mix, keep preferably uniformity and free-running property.Obtain the mixture C of liquefying starch and dregs of beans, temperature is waited for inoculation at 28 ℃.
(5) strain liquid B is evenly mixed again with mixture C, obtain complete fermentation raw material D.Again fermentation raw material D is contained in the fermentation tank, fills up as far as possible container.Fermentation tank seals, and an one-way exhaust valve (can only give vent to anger, can not air inlet) is arranged at top, and the pressure at expulsion of setting is 2000Pa (about 20cm H
2O).At the material center of fermentation tank a temperature sensor probe is arranged.
(6) concentration of each composition is as follows in the fermentation raw material:
Table 1: experimental group weight of material, water content (kg)
Moisture in the brown sugar is ignored.
The gross weight of fermentation materials (kg): 4000+150+1250+80+8.0+2+5+2.5=5497.5; (80 kilograms of clear water have dissolved brown sugar and enzyme preparation, have also all entered fermentation system.)
The water content of fermentation materials (kg): 520+7.5+1250+80+0.16+5.0+0.25=1862.91;
The moisture content of fermentation materials: 1862.73/5497.5=34.0%;
The proportioning of old rice starch, amylase, carbohydrase is 150kg: 1.0 * 10
6U: 2.0 * 10
8U;
The proportioning of old rice starch, amylase, carbohydrase, dregs of beans is 150kg: 1.0 * 10
6U: 2.0 * 10
8U: 4000kg;
The proportioning of brown sugar and dregs of beans is 8kg: 4000kg;
The proportioning of saccharomycete and dregs of beans is 2 * 10
5Hundred million cfu: 4000kg;
The proportioning of saliva chain coccus thermophilous subspecies and dregs of beans is 5 * 10
5Hundred million cfu: 4000kg.
2, fermentation process
After charging finished, the temperature of charge that temp probe shows was 26 ℃.Finish from charging and just to begin logical cooling water, flow is 2.0m
3/ h (volume of fermentation tank is 5 cubic metres).Environment temperature is 13-20 ℃, and cooling water temperature is 13-15 ℃.Measure 1 (the sugar fermentation type is on the impact of fermentation materials central temperature) that the results are shown in Figure of fermentate central temperature.
Phase I (0-24 hour): the metabolic activity of microorganism is more weak, substantially is in to treat germinating, and available direct fermentable sugars content is very low, and temperature rises mild.
Second stage (24-72 hour): the growth of microorganism metabolism is vigorous, and quantity of heat production is larger, and the temperature-rise ratio of material is very fast, and the center maximum temperature reaches 45 ℃, but fluctuation is mild.
Phase III (72-96 hour): the metabolic activity of microorganism obviously weakens, and quantity of heat production is very little, and the temperature of material progressively descends.
(2) control group
1, raw material preparation
Basic identical with experimental group, different is: source (red sugar) preparation method is different, specific as follows for bacterium metabolism sugar:
Bacterium metabolism sugar source (red sugar) preparation: be 2.0m at volume
3Cooker in add successively 1200kg clear water and 150kg brown sugar, pass into again 70kg heating steam, appropriateness stirs to promote the brown sugar dissolving.Deng brown sugar all after the dissolving (this moment, the temperature of solution was 43 ℃) again with mixing of materials, the temperature of material is about 28 ℃ after mixing.
Table 2: the weight of control group material and water content (kg)
Moisture in the brown sugar is ignored.
The total amount of fermentation materials (kg): 4000+1270+80+158+2+5=5515
The Total Water of fermentation materials (kg): 520+1270+80+0.16+5.0=1875.16
The moisture content of fermentation materials: 1875.16/5515=34.0%.
The proportioning of brown sugar and dregs of beans is 158kg: 4000kg.
2, sweat
After the charging, the initial temperature of material is 28 ℃, the very fast rising of temperature of charge, and the process of this sweat is faster than test group.
Phase I (0-24 hour): temperature of charge steadily rises to 41 ℃ from 28 ℃, and the metabolic activity of microorganism is slow.
Second stage (24-48 hour): temperature of charge begins fast rise, to 36 hours, has risen to 56 ℃.Up to 48 hours, maintain all the time hot stage later on.This, metabolic activity of microorganism was very vigorous in period.
Phase III (48-96 hour): the temperature of material begins to descend, and the metabolic activity of microorganism is more weak, and to 72 hours, the material central temperature was reduced to 40 ℃, and to 96 hours, the material central temperature dropped to 35 ℃.
Begin just to open cooling water from the material fermentation tank of packing into, until fermentation ends.The sensor measurement central temperature.
3 repetitions are all established in above-mentioned experiment, and the result takes the mean.
Two, result
Table 3: the raw material of experimental group and control group forms
| Experimental group | Control group | |
| Dregs of beans (kg) | 4000 | 4000 |
| Brown sugar (kg) | 8 | 158 |
| Old rice (kg) | 150 | 0 |
| Amylase (kg) | 0.5 | 0 |
| Carbohydrase (kg) | 2.0 | 0 |
| Dusty yeast (kg) | 2 | 2 |
| Lactobacillus suspension (liter) | 5 | 5 |
| Water content (%) | 34 | 34 |
The price of brown sugar is far above old rice.Brown sugar is 7500 yuan per ton at present, 1000 yuan of old rice less thaies per ton.The price per kilogram of amylase and carbohydrase (kind that this test is adopted) is about 12 yuan.Obviously, aspect cost of material, experimental group contrast group has apparent in view advantage.
Table 4: the result of experimental group and control group relatively
| Experimental group | Control group | |
| Lactic acid production (%) | 1.12 | 0.96 |
| Maximum temperature (℃) | 45 | 56 |
| The fermenting-ripening time (hour) | 96 | 72 |
Lactic acid production refers to " quality of generation lactic acid accounts for the percentage composition of dregs of beans gross mass " in the table.The lactic acid production contrast group of test group is high by about 15%, low 11 ℃ of maximum temperature contrast group, and cooling-down effect is very remarkable.Although fermenting-ripening time contrast group has prolonged 24 hours, the benefit that obtains still is worth fully.
The definition of fermenting-ripening time: the output of lactic acid substantially no longer increases, and the time that at this moment fermentation is carried out is called the fermenting-ripening time.
Cooling water is recirculated water, can be recycling after processing through cooling tower.The power consumption that increases is no more than 20 yuan within a production cycle.
The experimental group method is basic identical among method and the embodiment 1, different is: respectively the consumption of old rice is set as 3 gradients (200kg, 250kg, 300kg), the consumption of initial clear water amount, heating steam consumption, amylase and carbohydrase in cooker also adjusts accordingly respectively, and is concrete such as table 5 and table 6.3 repetitions are established in experiment, and the result takes the mean.Result such as table 7.
Table 5: old rice pre-treatment gradient experimental data
Table 6: the water content of material after the inoculation
Table 7: old rice gradient experimental result
The experimental result explanation, adopt new technology (metabolizable sugar slow release method) can bring into play the fermentation potentiality of microorganism (particularly lactic acid bacteria) to a greater degree.
The 100kg old rice can produce the 85kg metabolizable sugar through boiling, gelatinization, liquefaction and fully saccharification.The metabolizable sugar total content of experimental group A is approximately 178kg (contain activation saccharomycete used 8kg brown sugar), with the relative scale of dregs of beans raw material 4000kg be 4.45%.
Along with the increase of starch consumption, although fermentation energy is proceeded, the relative efficiency that lactic acid transforms is more and more lower, and (output of lactic acid is an absolute magnitude, and transformation efficiency is a relative quantity; Lactic acid mainly is transformed by starch or metabolizable sugar, and the lactic acid production height is might not transformation efficiency high; Only under the condition that content of starch equates or metabolizable sugar content is equal to, lactic acid production is high to represent that just transformation efficiency is also high), the temperature of material is more and more higher, and the local flavor of product is worse and worse.The ratio of more rational metabolizable sugar total content and substrate raw material is greatly between 3.0-5.0% in the raw material.
Embodiment 3: the solid state fermentation of biological feedstuff
Experimental group is described basic identical among method and the embodiment 1, and different is: substitute the 4000kg dregs of beans with 4000kg cotton dregs and the 4000kg dish dregs of rice respectively.3 repetitions are established in experiment, and the result takes the mean.Result such as Fig. 2 and table 8.
Table 8: the solid state fermentation of cotton dregs and the dish dregs of rice
Compare with bean pulp fermentation, it is slow that the fermenting speed of cotton dregs is wanted, and central temperature is also slightly low.The output of lactic acid also only has about 77% of bean pulp fermentation output.The lactic acid production of the dish dregs of rice is lower, and fermenting speed is slower (the available albumen of microorganism seldom) also.But be that the output of temperature controlled effect and lactic acid or more satisfactory is compared in the fermentation of main body with adopting direct fermentable sugars.
The enzymatic hydrolysis and fermentation of embodiment 4, other starch material
Experimental group is described basic identical among method and the embodiment 1, and different is: 1) substitute 150kg old rice starch with 150kg cornstarch and 150kg farina respectively; 2) environment temperature is 13-20 ℃, and cooling water temperature is 13-15 ℃, and (this situation often occurs in the north, and in the environment that especially relative air humidity is lower, the temperature of ambient air temperature and water is also inconsistent.)。3 repetitions are established in experiment, and the result takes the mean.Result such as Fig. 3 and table 9.
Table 9: the solid state fermentation of cotton dregs and the dish dregs of rice
Cornstarch starts fast, and farina starts slow.But final result is very approaching.
Bean pulp solid-state fermentation under embodiment 5, the high ambient temperature
When environment temperature is higher, need suitably to reduce inoculation temperature.Starch needs to be cooled to after through liquefaction after the lower temperature and could mix with the raw material such as dregs of beans.If under hot conditions, mix with dregs of beans, then need to inoculate after the cooling.
In addition, suitably reduce the consumption of carbohydrase, appropriateness reduces the rate of release of metabolizable sugar, prolongs the fermentation heat production time, reduces the heat radiation load of equipment.
Environment temperature in the present embodiment is 20-26 ℃, and the temperature of cooling water is 20-22 ℃.
(1) get dregs of beans 4000kg, water content is 13.0% (quality percentage composition), pulverizes, and crosses 20 mesh sieves.
(2) get old rice 150kg, pulverize, cross 40 mesh sieves.Be 2.0m at volume
3Cooker in add successively 1000kg clear water and 150kg old rice powder, progressively pass into high-temperature steam 250kg, be heated to 100 ℃, be incubated 20 minutes, until the complete gelatinization of starch.Be cooled to 70 ℃, add 500g amylase, fully mixing left standstill 120 minutes, added 1200 gram carbohydrase again and obtained liquefied starch (this moment, the temperature of solution was 52 ℃).
(3) add successively 75kg clear water and 8kg brown sugar in volume is 300 liters constant temperature water pot, then pass into 5kg steam, appropriateness stirs.After brown sugar dissolves fully, continue to stir cooling.When about the drop in temperature to 32 of solution ℃, add again 2000g active dry yeast powder (saccharomyces cerevisiae, water content is 8.0%, the number of active yeast is about 1,000,000,000 cfu in the 1g active dry yeast powder) and 5000g saliva chain coccus thermophilous subspecies nutrient solution 5kg (number of active bacterium is about 10,000,000,000 cfu in the 1ml nutrient solution), static maintenance 40 minutes obtains strain liquid.
(4) liquefying starch is progressively joined in the dregs of beans, mix, the temperature of mixture is about 32 ℃.Turn heat radiation, temperature is reduced to 28 ℃, wait for inoculation.
(5) strain liquid is evenly mixed again with mixture, obtain complete fermentation raw material, in the fermentation tank of packing into, fill up as far as possible container.Then seal fermentation tank, open cooling water.
Through fermentation in 108 hours, the result was referring to Fig. 4 and table 10.
Table 10: the solid state fermentation result (raw material is dregs of beans and rice starch) who improves cooling water temperature
| Index | Parameter |
| Lactic acid production (%) | 1.08 |
| Maximum temperature (℃) | 46 |
| The fermenting-ripening time (hour) | 108 |
Environment temperature rises and causes the temperature of recirculated cooling water also to rise, and needs suitably to reduce the rate of release of metabolizable sugar during operation, prolongs fermentation time.
Claims (8)
1. a solid state fermentation prepares the method for biological feedstuff, comprises the steps: the sugared source of solid state substrate, bacterium, the metabolism of startup bacterium and provides the sugared source of bacterium metabolism institute energy requirement to mix, and the mixture that obtains is fermentation raw material; Described fermentation raw material is fermented, obtain biological feedstuff; The described enzymolysis product of sugared source for obtaining with amylase and carbohydrase enzymolysis starch that bacterium metabolism institute energy requirement is provided;
The proportioning of described starch, described amylase, described carbohydrase and described solid state substrate is (150kg-300kg): (1.0 * 10
6U-2 * 10
6U): (2.0 * 10
8U-3.5 * 10
8U): 4000kg;
The sugared source of described startup bacterium metabolism and the proportioning of described solid state substrate are 8kg: 4000kg;
Described bacterium is saccharomycete and/or lactic acid bacteria;
The proportioning of described saccharomycete and described solid state substrate is 2 * 10
5Hundred million cfu: 4000kg, the proportioning of described lactic acid bacteria and described solid state substrate is 5 * 10
5Hundred million cfu: 4000kg;
The water content of described fermentation raw material is 25～85%;
Described enzymolysis product prepares according to the method that comprises the steps: with described starch gelatinization, obtain gelatinized starch; To wherein adding described amylase, mixing left standstill 120 minutes again; To wherein adding described carbohydrase, stir 3min again, obtain described enzymolysis product.
2. method according to claim 1, it is characterized in that: the proportioning of described starch, described amylase, described carbohydrase and described solid state substrate is 150kg: 1.0 * 10
6U: 2.0 * 10
8U: 4000kg, 200kg: 1.3 * 10
6U: 2.5 * 10
8U: 4000kg, 250kg: 1.7 * 10
6U: 3.0 * 10
8U: 4000kg or 300kg: 2 * 10
6U: 3.5 * 10
8U: 4000kg.
3. method according to claim 1 and 2, it is characterized in that: the method for described mixing comprises the steps:
1) described bacterium is mixed with the sugared source of described startup bacterium metabolism, the mixture that obtains is denoted as strain liquid B;
2) described enzymolysis product is mixed with described solid state substrate, the mixture that obtains is denoted as mixture C;
3) described mixture C is mixed with described strain liquid B, the mixture that obtains is fermentation raw material;
The step that comprises logical cooling water in the described fermentation.
4. method according to claim 1, it is characterized in that: the water content of described fermentation raw material is 33%-36%.
5. method according to claim 4, it is characterized in that: the water content of described fermentation raw material is 34%, 33.78%, 34.93% or 35.40%.
6. method according to claim 3, it is characterized in that: the method for mixing described step 1) comprises the steps: the sugared source of described startup bacterium metabolism water-soluble, obtain the aqueous solution in the sugared source of described startup bacterium metabolism, when the temperature of the aqueous solution for the treatment of the sugared source of described startup bacterium metabolism is 31 ℃-33 ℃, add dusty yeast and lactic acid bacteria culture solution, left standstill 30 minutes, and obtained described strain liquid B;
Described dusty yeast prepares according to the method that comprises the steps: with described saccharomycete access microzyme culture medium, be that 30C, ventilation are to cultivate 8 hours-9 hours under the condition of 0.4vvm-0.6vvm and stirring in temperature, obtain saccharomycetes to make fermentation liquid; Saccharomycetes to make fermentation liquid is carried out concentrate drying, and the material that obtains is dusty yeast; All substances in the culture vessel are denoted as saccharomycetes to make fermentation liquid;
Described lactic acid bacteria culture solution prepares according to the method that comprises the steps: white granulated sugar is dissolved in the plain chocolate of antibiotic-free pollution, the proportioning of the plain chocolate that white granulated sugar and antibiotic-free pollute is 300g: 5000g, obtains the lactic acid bacteria culture medium; Access described lactic acid bacteria in the lactic acid bacteria culture medium, constant temperature culture is 20 hours-24 hours between 36 ℃-40 ℃, and the culture that obtains is lactic acid bacteria culture solution; All substances in the culture vessel are denoted as lactic acid bacteria culture solution.
7. method according to claim 6 is characterized in that:
The sugared source of described startup bacterium metabolism is brown sugar, white granulated sugar and/or sucrose;
Described starch is rice, cornstarch or farina;
Described solid state substrate is dregs of beans, cotton dregs and/or the dish dregs of rice;
Described saccharomycete is saccharomyces cerevisiae (Sacharomyces cerevisiae) CGMCC No.2.399;
Described lactic acid bacteria is saliva chain coccus thermophilous subspecies (Streptococcus salivarius subsp.thermophilus) CGMCC 1.1864;
Described amylase is Isosorbide-5-Nitrae-α-D-glucan hydrolase;
Described carbohydrase is α-Isosorbide-5-Nitrae-glucose hydrolysis enzyme.
8. method of controlling fermentate temperature in the solid state fermentation comprises the steps: the sugared source of solid state substrate, bacterium, the metabolism of startup bacterium and provides the sugared source of bacterium metabolism institute energy requirement to mix, and carries out solid state fermentation; It is characterized in that: in the process of described solid state fermentation with the enzymolysis product in arbitrary described method among the claim 1-7 as the described sugared source that bacterium metabolism institute energy requirement is provided, with the center maximum temperature that reduces fermentate in the sweat and/or with the rate of climb of fermentate temperature in the stopping fermentation process; Described fermentate is all substances in the round.
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| CN104365993A (en) * | 2014-11-13 | 2015-02-25 | 中国农业大学 | Method for reducing viscosity of solid-state fermented soybean meal |
| CN106333060A (en) * | 2016-08-26 | 2017-01-18 | 曹蕊 | Microbial fermentation feed and preparation method and application thereof |
| CN106480100B (en) * | 2016-10-12 | 2023-03-31 | 武汉贵言机械制造有限公司 | Method and device for efficient solid state fermentation microorganism inoculation metering |
| CN106666161A (en) * | 2017-01-06 | 2017-05-17 | 北京中农弘科生物技术有限公司 | Application of yeast culture in preparation of feed capable of improving reproductive performance and relieving constipation of sows |
| CN106689819A (en) * | 2017-01-06 | 2017-05-24 | 北京中农弘科生物技术有限公司 | Application of yeast culture in preparation of feed for improving fish growth and improving non-specific immunity of fish |
| CN106666129A (en) * | 2017-01-06 | 2017-05-17 | 北京中农弘科生物技术有限公司 | Application of yeast culture in preparing feed for improving production performance of lactating dairy cow |
| CN111869797A (en) * | 2020-06-23 | 2020-11-03 | 中国农业大学 | Laying hen fermented feed and preparation method and application thereof |
| CN115226810B (en) * | 2022-07-21 | 2023-10-31 | 中国农业大学 | Preparation method of solid state fermentation feed |
| CN115624086B (en) * | 2022-09-23 | 2024-07-26 | 亚太星原农牧科技海安有限公司 | Method for avoiding overhigh temperature of material center in fermentation process |
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