CN1304584C - Method for preparing lactic acid and feedstuff concurrent with crop straw fermentation - Google Patents
Method for preparing lactic acid and feedstuff concurrent with crop straw fermentation Download PDFInfo
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Abstract
The present invention provides a method for preparing lactic acid and feed while crop straws are fermented, which relates to the method for preparing the lactic acid and feed with good quality while the crop straws are fermented. The method uses synchronous diastatic fermentation, and water whose weigh is from 2 to 3 times of the weight of solid substrates is added to straws after pretreatment. After sterilization at the temperature of 121DEGC, cellulase prepared in a step (2) is added, and cellulase enzyme activity in a total substrate reaches 42 to 50 IU/g by the handiwork of cellulose filter paper enzymes. The inoculum concentration of high-temperature resistant lactic acid bacteria after inoculation activation is from 5 to 10% of the weight of the total substrate, fermentation is carried out for 72 to 108 hours at the temperature of 45 to 50DEGC, and the pH value of a fermentation substrate is adjusted to 5.0 to 6.0 during the fermentation. The method greatly shortens fermentation time, improves fermentation efficiency, synchronous fermentation is carried out for 72 to 108 hours, and each 100g of dry straw can produce 6 to 8g of the lactic acid and 60 to 70g of the feed with good quality. The method has the advantages of low production cost, little investment, no pollution, low energy consumption without waste residues or waste liquid and sufficiently used resource.
Description
Technical field:
The present invention relates to the method for a kind of fermenting crops straws while obtain lactic acid and high-quality feed.
Background technology:
Lactic acid is not only of many uses in foodstuffs industry, medicine industry, also can be used as industrial raw material and is used for leather, fiber industry.Particularly poly(lactic acid) has a lot of premium propertiess as biodegradable plastic in recent years, and the manufacturing of its material acid is more noticeable.In the past, being used for lactic acid fermented raw material, generally is the starch of farm crop such as corn, cassava, wheat, potato.But these farm crop are lactic acid fermented desirable feedstock, are again simultaneously valuable grain.From the angle of utilization of resources and reduction lactic acid-producing cost, utilizing other cheap carbon sources to ferment is one of research focus of lactic acid-producing.Agricultural crop straw is the main by product of grain-production, accounts for 50% of crop biomass, is the profuse Biological resources that can directly utilize of a class.Publication number is CN 1310949, and CN 1294861, and CN 1277813 discloses the method for being made protein fodder by stalk, and these methods can only obtain a kind of feed product from stalk.Open 2003-310243 of Japan's special permission and 1997-308494 disclose the method for utilizing waste paper and sludge of sewage treatment plant fermentation system lactic acid respectively, utilize Semen Maydis grit, potato slag, wheat bran, wheat bran in addition, and depleted beet leaves, bagasse, manufacture the research that waste after tankage, bread waste, paper mill sludge and the shellfish processing of residue behind the potato salad, cheese factory etc. is produced lactic acid.Above-mentioned these methods can only obtain lactic acid, but not only obtain lactic acid simultaneously but also produce feed was particularly produced feed with the stalk obtain lactic acid of farm crop simultaneously.
Summary of the invention:
The purpose of this invention is to provide a kind of fermenting crops straws method of obtain lactic acid and feed simultaneously, it has, and production cost is low, less investment, pollution-free, energy consumption is low, no waste residue, no waste liquid, makes full use of characteristics of resources.The step that the present invention taked is: (1) crop straw pre-treatment: behind crushed stalk to 80～140 orders, soaked 12～36 hours with sig water, filter, it is neutral washing to washings.The waste water utilization bipolar membrane electrodialysis method that pre-treatment produces reclaims xylogen and alkali lye.(2) solid state fermentation prepares CELLULASE: wooden mould, the fungi of selecting cellulase-producing for use is as starting strain, and solid state fermentation is 60～108 hours under 25～30 ℃ condition, and the CELLULASE that fermentation produces directly uses in the follow-up synchronous fermentation.The concrete steps of preparation CELLULASE are: a, with the starting strain of cellulase-producing, and be inoculated into slant medium and cultivate, scrape the sterilized water that a few ring spores insert the band granulated glass spherees from the activatory inclined-plane, vibration, dispersal spore is measured spore concentration, makes spore suspension concentration reach 10
6～10
7Individual/mL; B, in container, add straw powder, wheat bran (molasses, residue from beans after making), the buffering nutritive medium is mixed with substratum, straw powder and wheat bran weight ratio are 1: 1～1.5: 1, the solid-liquid weight ratio is 1: 2～1: 3, buffering nutritive medium pH value is 4.0～6.0, wherein add ammonium sulfate, potassium primary phosphate, sal epsom, its concentration is respectively 2%, 0.1%, 0.05%; C, with the medium sterilization in the b step, 5～8% spore suspension of cooling back inoculation gross weight obtains CELLULASE after cultivating 60～108 hours under 25 ℃～30 ℃ conditions.(3) milk-acid bacteria activation: select for use and can under 45～50 ℃ of conditions, produce the good high temperature resistant milk-acid bacteria of acid, with MRS substratum activation 12～24 hours.(4) simultaneous saccharification and fermentation: adding weight in the stalk of (not oven dry) after pre-treatment is the water of 2～3 times of solid substrates, after 121 ℃ of sterilizations, add the CELLULASE that makes in the step (2), make the cellulase inulinase work in total substrate reach 42～50IU/g in the Mierocrystalline cellulose filter paper enzyme activity, high temperature resistant milk-acid bacteria after the inoculation activation, inoculum size is 5～10% of total substrate weight, and in 45～50 ℃ of condition bottom fermentations 72～108 hours, the pH value of regulating fermentation substrate between yeast phase was 5.0～6.0.(5) aftertreatment of solid-liquid separation and product: solid-liquid separation, collect the lactic acid in the supernatant liquor, methods such as employing electrodialysis are further purified.Raffinate behind the purification of lactic acid can be back to synchronous fermenting process.The fermentation residue that obtains after the solid-liquid separation is directly made feed, or further in fermentation residue, adds the integration engineering flora of 5～40% (wt%) and 10～30% auxiliary material; Solid state fermentation is 48～240 hours under 10～35 ℃ of conditions, and tunning is dried under field conditions (factors) or under 45 ℃ of conditions, makes protein content at 10～40% high protein feed.The present invention has realized that stalk ferments synchronously and has transformed and obtained simultaneously lactic acid and two kinds of products of feed, it has following advantage: the present invention utilizes and produces that cellulose wood is mould, fungi etc. is as starting strain, the solid state fermentation production of cellulose, the yield of enzyme height, and saved the process of purifying, preparation cellulase preparation, the substratum that while cellulase-producing song is utilized is based on stalk, only add on a small quantity wheat bran and ammonium sulfate etc. cheaply, not only utilize stalk, and greatly reduced the cost of preparation enzyme; Select resistant to elevated temperatures yeast for use, solved the inconsistent technical barrier of optimum temperuture of yeast optimum growth temperature and cellulase hydrolysis, satisfied the requirement of synchronous fermentation; By research to synchronous fermentation condition, obtain the zymotechnique route of optimizing, shortened fermentation time greatly, improved fermentation efficiency, fermentation synchronously 72～108 hours can obtain every 100g dry straw and produce the lactic acid of 6～8g and the high-quality feed (dry weight) of 60～70g.Utilize present method not only agricultural wastes such as stalk can be converted into lactic acid and high-quality feed simultaneously, low, the less investment of production cost, and low, the non-secondary pollution of whole technological process energy consumption are that the agricultural crop straw of environmental protection utilizes method.Contain abundant cellulose substances in the stalk, produce lactic acid with stalk as fermenting raw materials, not only can fundamentally change present lactic acid is the production status of raw material with the starchy material, and can obtain straw feed of high quality and at a reasonable price simultaneously, wherein contain more rich protein and partial reduction carbohydrate, also be rich in living lactic acid bacteria (but feed need be sterilized after using the non-food product milk-acid bacteria) and various digestive ferment and cellulase, have bigger economic benefit and application prospect.
Description of drawings:
Fig. 1 is preparation technology's schema of CELLULASE among the present invention, and Fig. 2 is the process flow sheet of the present invention with synchronous fermenting lactic acid of stalk and feed.
Embodiment:
Embodiment one: (referring to Fig. 1, Fig. 2) present embodiment realizes as follows: (1) crop straw pre-treatment: behind crushed stalk to 80～140 orders, soaked 12～36 hours with sig water, filter, it is neutral washing to washings.The waste water utilization bipolar membrane electrodialysis method that pre-treatment produces reclaims xylogen and alkali lye.(2) solid state fermentation prepares CELLULASE: wooden mould, the fungi of selecting cellulase-producing for use is as starting strain, and solid state fermentation is 60～108 hours under 25～30 ℃ condition, and the CELLULASE that fermentation produces directly uses in the follow-up synchronous fermentation.The concrete steps of preparation CELLULASE are: a, with the starting strain of cellulase-producing, and be inoculated into slant medium and cultivate, scrape the sterilized water that a few ring spores insert the band granulated glass spherees from the activatory inclined-plane, vibration, dispersal spore is measured spore concentration, makes spore suspension concentration reach 10
6～10
7Individual/mL; B, in container, add straw powder, wheat bran (molasses, residue from beans after making), the buffering nutritive medium is mixed with substratum, straw powder and wheat bran weight ratio are 1: 1～1.5: 1, the solid-liquid weight ratio is 1: 2～1: 3, buffering nutritive medium pH value is 4.0～5.0, wherein add ammonium sulfate, potassium primary phosphate, sal epsom, its concentration is respectively 2%, 0.1%, 0.05%; C, with the medium sterilization in the b step, 5～8% spore suspension of cooling back inoculation gross weight obtains CELLULASE after cultivating 60～108 hours under 25 ℃～30 ℃ conditions.(3) milk-acid bacteria activation: select for use and can under 45～50 ℃ of conditions, produce the good high temperature resistant milk-acid bacteria of acid, with MRS substratum activation 12～24 hours.(4) simultaneous saccharification and fermentation: adding weight in the stalk of (not oven dry) after pre-treatment is the water of 2～3 times of solid substrates, after 121 ℃ of sterilizations, add the CELLULASE that makes in the step (2), make the cellulase inulinase work in total substrate reach 42～50IU/g in the Mierocrystalline cellulose filter paper enzyme activity, high temperature resistant milk-acid bacteria after the inoculation activation, inoculum size is 5～10% of total substrate weight, and in 45～50 ℃ of condition bottom fermentations 72～108 hours, the pH value of regulating fermentation substrate between yeast phase was 5.0～6.0.(5) aftertreatment of solid-liquid separation and product: solid-liquid separation, collect the lactic acid in the supernatant liquor, methods such as employing electrodialysis are further purified.Raffinate behind the purification of lactic acid can be back to synchronous fermenting process.The fermentation residue that obtains after the solid-liquid separation is directly made feed, or further in fermentation residue, adds the integration engineering flora of 5～40% (wt%) and 10～30% auxiliary material; Solid state fermentation is 48～240 hours under 10～35 ℃ of conditions, and tunning is dried under field conditions (factors) or under 45 ℃ of conditions, makes protein content at 10～40% high protein feed.Described agricultural crop straw is maize straw, soybean stalk, rice straw, broomcorn straw or wheat stalk.In the described CELLULASE preparation process, the bacterial classification of used cellulase-producing is any in koning trichoderma, viride, terreus, the thermophilic fiber clostridium or their mixed bacterial.Regulate the used alkali of fermentation substrate pH value in the described step (4) between yeast phase and be sodium hydroxide, calcium hydroxide, potassium hydroxide or ammoniacal liquor one or more mixture wherein.For further producing in the solid state fermentation that high protein feed carries out, the auxiliary material that is added is at least a in beet pulp, bagasse, bean dregs, wheat bran or the cavings.Described integration engineering flora is the mixture of candida tropicalis bacterium, geotrichum candidum bacterium, koning trichoderma bacterium and plant lactobacillus.
Embodiment two: (referring to Fig. 1, Fig. 2) present embodiment realizes as follows: after (1) is crushed to 140 orders with soybean stalk,, filter with the ammoniacal liquor soaking at room temperature of 10% concentration 24 hours, wash to washings for neutral.Waste water utilization bipolar membrane electrodialysis method separating lignin and alkali lye that pre-treatment produces.(2) koning trichoderma being inoculated into slant medium, to be cultured to mycelial growth vigorous, scrape the sterilized water that a few ring spores insert the band granulated glass spherees from the inclined-plane, and vibration, dispersal spore is measured spore concentration, makes spore suspension concentration reach 10
7Individual/mL.(3) in container, add straw powder, wheat bran, acetic acid-sodium acetate buffer solution etc. and be mixed with substratum, making straw powder and wheat bran weight ratio is 1: 1, the solid-liquid weight ratio is 2: 5, acetic acid-sodium acetate buffer pH value of solution value is 4.5, add ammonium sulfate, potassium primary phosphate, sal epsom, its concentration is respectively 2%, 0.1%, 0.05%, mix, sterilization, 5% of the inoculation gross weight spore suspension after the cooling, solid state fermentation obtained CELLULASE after 96 hours under 30 ℃ of conditions, and its cellulase activity is 798.84FPU/ml.(4) the high temperature resistant lactobacillus strain 1 preserved of inclined-plane encircles in the MRS liquid nutrient medium, and 120r/min cultivated 16 hours under 50 ℃ of conditions, then by 5% inoculum size enlarged culturing successively.(5) adding weight in pretreated wet stalk is the water of 2.5 times of solid substrates, after 121 ℃ of sterilizations, add the CELLULASE that makes in the step (2), make the cellulase inulinase work in total substrate reach 42IU/g in the Mierocrystalline cellulose filter paper enzyme activity, high temperature resistant milk-acid bacteria after the inoculation activation, inoculum size is 5% of total substrate weight, and in 50 ℃ of condition bottom fermentations 96 hours, the pH value of regulating fermentation substrate between yeast phase was 5.0～6.0.(6) carry out solid-liquid separation with supercentrifuge, analyze the lactic acid content in the fermented liquid, can obtain the lactic acid 6.2g of every 100g dry straw output.Can adopt purification that method such as electrodialysis carries out and refining.(7) crude protein, Mierocrystalline cellulose, hemicellulose, the xylogen equal size of analyzing in the solid-liquid separation gained fermentation residue sees Table 1.Can in the gained fermentation residue, add the integration engineering flora of 5～40% (wt%) and 10～30% auxiliary material if need further increase percent protein; Solid state fermentation is 48～240 hours under 10～35 ℃ of conditions, and tunning is dried under field conditions (factors) or under 45 ℃ of conditions, makes protein content at 1540% high protein feed.
Table 1 pre-treatment and lactic fermentation process are to the influence of stalk composition
| Crude protein (%) | Mierocrystalline cellulose (%) | Hemicellulose (%) | Xylogen (%) | Cellulase activity (IU/g) | |
| Before the pre-treatment | 5.83 | 24.99 | 11.91 | 17.64 | |
| After the pre-treatment (before the lactic fermentation) | 6.22 | 42.55 | 6.97 | 12.32 | |
| After the lactic fermentation | 12.77 | 17.62 | 5.31 | 20.32 | 24.39 |
Embodiment three: (referring to Fig. 1, Fig. 2) present embodiment realizes as follows: (1) pulverizes maize straw, cross 80 purposes sieve, the ammoniacal liquor with 8% concentration under the room temperature soaked 24 hours, and solid-to-liquid ratio is 1: 10, filter afterwards, it is neutral washing to washings.Waste water utilization bipolar membrane electrodialysis method separating lignin and alkali lye that pre-treatment produces.(2) koning trichoderma being inoculated into slant medium, to be cultured to mycelial growth vigorous, scrape the sterilized water that a few ring spores insert the band granulated glass spherees from the inclined-plane, and vibration, dispersal spore is measured spore concentration, makes spore suspension concentration reach 10
7Individual/mL.(3) in container, add pretreated straw powder, wheat bran, water, ammonium sulfate, the weight ratio that makes straw powder and wheat bran is 1: 1, the solid-liquid weight ratio is 1: 3, ammonium sulfate concentrations is 2% in the nutritive medium, mix sterilization, 5% of the inoculation gross weight spore suspension after the cooling, solid state fermentation obtained CELLULASE after 96 hours under 27 ℃ of conditions, and the cellulase activity of its generation can reach 1392.93FPU/ (g. stalk).(4) the high temperature resistant lactobacillus strain 1 preserved of inclined-plane encircles in liquid nutrient medium, and 120r/min cultivated 16 hours under 50 ℃ of conditions, enlarges successively by 5% inoculum size then.(5) in pretreated straw powder, add CELLULASE, make the cellulase inulinase work in total substrate reach 50IU/g, add the water of 2.5 times of weight of solid substrate in the Mierocrystalline cellulose filter paper enzyme activity; High temperature resistant milk-acid bacteria after the inoculation activation, inoculum size is 5% of total substrate weight.In 50 ℃ of condition bottom fermentations 84 hours.The pH value of regulating fermentation substrate is 5.5～6.0.(6) carry out solid-liquid separation with supercentrifuge, analyze the lactic acid content in the fermented liquid, can obtain the lactic acid 7.35g of the pretreated dry straw output of every 100g.Can adopt purification that method such as electrodialysis carries out and refining.(7) analyze the content of various compositions in the solid-liquid separation gained fermentation residue, learn lactic fermentation after the residue crude protein content improve, Mierocrystalline cellulose reduces, and has high enzyme and live.The results are shown in Table 2.Can in the gained fermentation residue, add the integration engineering flora of 5～40% (wt%) and 10～30% auxiliary material if need further increase percent protein; Solid state fermentation is 48～240 hours under 10～35 ℃ of conditions, and tunning is dried under field conditions (factors) or under 45 ℃ of conditions, makes protein content at 15～40% high protein feed.
Table 2 pre-treatment and lactic fermentation process are to the influence of stalk composition
| Crude protein (%) | Mierocrystalline cellulose (%) | Hemicellulose (%) | Xylogen (%) | Cellulase activity (IU/g) | |
| Before the pre-treatment | 3.92 | 28.56 | 26.79 | 22.08 | |
| After the pre-treatment (before the lactic fermentation) | 4.82 | 40.96 | 28.57 | 18.83 | |
| After the lactic fermentation | 12.56 | 15.82 | 20.38 | 25.88 | 25.42 |
Claims (5)
1, the method for fermenting crops straws while obtain lactic acid and feed, the step that it is characterized in that it is: (1) soybean stalk pre-treatment: after soybean stalk is crushed to 80～140 orders, soaked 12～36 hours with sig water, filter, it is neutral washing to washings, and the waste water utilization bipolar membrane electrodialysis method that pre-treatment produces reclaims xylogen and alkali lye; (2) solid state fermentation prepares CELLULASE: select the wooden mould as starting strain of cellulase-producing for use, solid state fermentation is 60～108 hours under 25～30 ℃ condition, and the CELLULASE that fermentation produces directly uses in the follow-up synchronous fermentation; (3) milk-acid bacteria activation: select for use and can under 45～50 ℃ of conditions, produce the good high temperature resistant milk-acid bacteria of acid, with MRS substratum activation 12～24 hours; (4) simultaneous saccharification and fermentation: adding weight in pretreated soybean stalk is the water of 2～3 times of solid substrates, after 121 ℃ of sterilizations, add the CELLULASE that makes in the step (2), make the cellulase inulinase work in total substrate reach 42～50IU/g in the Mierocrystalline cellulose filter paper enzyme activity, high temperature resistant milk-acid bacteria after the inoculation activation, inoculum size is 5～10% of total substrate weight, and in 45～50 ℃ of condition bottom fermentations 72～108 hours, the pH value of regulating fermentation substrate between yeast phase was 5.0～6.0; (5) aftertreatment of solid-liquid separation and product: solid-liquid separation, collect the lactic acid in the supernatant liquor, adopt electrodialytic method further to purify, the raffinate behind the purification of lactic acid can be back to synchronous fermenting process, and the fermentation residue that obtains after the solid-liquid separation is directly made feed.
2, the method for fermenting crops straws while obtain lactic acid according to claim 1 and feed is characterized in that in the described CELLULASE preparation process, the bacterial classification of used cellulase-producing is koning trichoderma or viride.
3, the fermenting crops straws according to claim 1 method of obtain lactic acid and feed simultaneously is characterized in that regulating between yeast phase in the described step (4) the used alkali of fermentation substrate pH value and is sodium hydroxide, calcium hydroxide, potassium hydroxide or ammoniacal liquor one or more mixture wherein.
4, the method for fermenting crops straws while obtain lactic acid according to claim 1 and feed is characterized in that in the gained fermentation residue, adds the integration engineering flora of wt5～40% and 10～30% auxiliary material; Solid state fermentation is 48～240 hours under 10～35 ℃ of conditions, tunning is dried under field conditions (factors) or under 45 ℃ of conditions, make protein content at 15～40% high protein feed, wherein said integration engineering flora is the mixture of candida tropicalis bacterium, geotrichum candidum bacterium, koning trichoderma bacterium and plant lactobacillus, and auxiliary material is at least a in beet pulp, bagasse, bean dregs, wheat bran or the cavings.
5, the method for fermenting crops straws while obtain lactic acid according to claim 1 and feed, it is characterized in that the concrete steps that prepare CELLULASE are: a, with the starting strain of cellulase-producing, being inoculated into slant medium cultivates, scrape a few ring spores inserts the sterilized water of being with granulated glass sphere from the activatory inclined-plane, vibration, dispersal spore is measured spore concentration, makes spore suspension concentration reach 10
6～10
7Individual/mL; B, in container, add straw powder, wheat bran, buffering nutritive medium and be mixed with substratum, straw powder and wheat bran weight ratio are 1: 1～1.5: 1, the solid-liquid weight ratio is 1: 2～1: 3, buffering nutritive medium pH value is 4.0～5.0, wherein add ammonium sulfate, potassium primary phosphate, sal epsom, its concentration is respectively 2%, 0.1%, 0.05%; C, with the medium sterilization in the b step, 5～8% spore suspension of cooling back inoculation gross weight obtains CELLULASE after cultivating 60～108 hours under 25 ℃～30 ℃ conditions.
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| CN110037176A (en) * | 2018-01-15 | 2019-07-23 | 华东理工大学 | A kind of steam pre-treatment straw feed processing method of endogenous lactate catalysis |
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| JPS53113085A (en) * | 1977-03-15 | 1978-10-03 | Bio Research Center Co | Production of lactic acid from cellulosic substance |
| CN1310949A (en) * | 2001-01-17 | 2001-09-05 | 中国科学院微生物研究所 | Method for producing straw protein fodder by using microbiological straw fermentation |
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