CN107509915B - Method for reducing cyanide content of cassava residue - Google Patents
Method for reducing cyanide content of cassava residue Download PDFInfo
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- CN107509915B CN107509915B CN201710743883.9A CN201710743883A CN107509915B CN 107509915 B CN107509915 B CN 107509915B CN 201710743883 A CN201710743883 A CN 201710743883A CN 107509915 B CN107509915 B CN 107509915B
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- 238000000034 method Methods 0.000 title claims abstract description 28
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Classifications
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- A—HUMAN NECESSITIES
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/28—Removal of unwanted matter, e.g. deodorisation or detoxification using microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
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- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
- A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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Abstract
The invention relates to the technical field of biology, in particular to a method for reducing cyanide content in cassava residue, which is characterized in that yeast and actinomucor elegans are mixed according to a volume ratio of 1-5:1-3 to obtain mixed bacterial liquid, then the mixed bacterial liquid of 10-20m L is mixed with 250g of cassava residue, and fermentation is carried out for 48-96h at 25-31 ℃.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to the technical field of biology, in particular to a method for reducing the cyanide content of cassava residue.
[ background of the invention ]
Guangxi is the main production area of cassava production in China, 250-300 million tons of fresh potatoes are produced in the whole area every year, and a large amount of waste residues are left after deep processing of the cassava, so that the serious pollution problem can be brought to the surrounding environment if the cassava is not properly treated. The cassava dregs are a cheap energy feed. Guangxi is a big province of buffalo breeding, the buffalo resources are rich, the total amount of buffalo breeding in the whole region reaches more than 400 thousands, and the common phenomenon in Guangxi is that cassava residues are used as one of the feed sources of the buffalo.
The cassava dregs contain high-content cyanogenic glycosides, including linarin and picroside, wherein the linarin accounts for 90-95% of the total amount. The toxicity of the linarin is low, but when the cells of cassava plants are damaged, such as crushing and pulping of fresh cassava, the hydrolase related to the linarin in the cells is released and contacts with the substrate of the linarin, and hydrocyanic acid is released after hydrolysis in the presence of water, so that the hydrocyanic acid is just used for causing the poisoning of cassava residues. After being absorbed by rumen, hydrocyanic acid is rapidly combined with ferric iron in oxidation type cytochrome oxidase to form cyaniding cytochrome oxidase, thereby inhibiting the activity of cytochrome oxidase, leading histiocyte not to utilize oxygen, causing 'intracellular asphyxia' to cause dysfunction of systems such as brain, cardiovascular and the like, particularly respiratory center and motor center, and finally influencing the improvement of animal production performance. Therefore, hydrocyanic acid in cassava limits the use of cassava dregs in a large amount in the livestock and poultry industry. Therefore, the finding of the cyanide content in the manioc waste for detoxification has important significance for expanding the development of the feed resources of the buffalo and promoting the healthy and sustainable development of the buffalo industry.
[ summary of the invention ]
The invention aims to: aiming at the problems, the invention provides a method for reducing the cyanide content of the cassava residue, which is simple and feasible, has the cyanide reduction rate of over 80 percent, improves the safety of the cassava residue as feed, can decompose hydrocyanic acid and convert the hydrocyanic acid into organic matters to become power elements of the feed, and increases the economic benefit of safe utilization of the cassava residue.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for reducing cyanide content in cassava residue comprises the steps of mixing saccharomycetes and actinomucor elegans according to a volume ratio of 1-5:1-3 to obtain a mixed bacterial liquid, then mixing the mixed bacterial liquid with the concentration of 10-20m L with 250g of cassava residue, and fermenting for 48-96 hours at the temperature of 25-31 ℃.
Further, the yeast and the actinomucor elegans are both domesticated and cultured before use.
The invention further provides a method for domesticating and culturing the actinomucor elegans, which comprises the following specific steps:
(1) inoculating actinomucor elegans to a first activated slant culture medium, and culturing for 8-16h at 40-50 ℃, wherein the first activated slant culture medium comprises peptone 12-18 g/L, beef extract powder 5-8 g/L, yeast extract powder 5-8 g/L0, aloe extract 5-8 g/L, astragalus extract 3-7 g/L, medlar extract 5-9 g/L, dipotassium hydrogen phosphate 0.5-0.8 g/L, diammonium hydrogen citrate 0.3-0.5 g/L, magnesium sulfate 0.3-0.5 g/L, manganese sulfate 0.05-0.1 g/L, agar 10-15 g/L, pH 6.0, and distilled water;
(2) domesticating strains: put the inclined plane 0.3cm above2The lawn is inoculated into a 250m L triangular flask filled with 15m L domestication culture medium, the rotating speed of a shaking table is 280 plus 300rpm, the shaking table is used for domestication culture at 29-31 ℃, each 3 days is a domestication period, domestication is carried out for 3 periods, and after the domestication of each period is finished, a culture solution with the highest degradation rate of a cassava residue washing solution is taken as an inoculation source for the next domestication, wherein the domestication culture medium comprises 10-20 g/L of the cassava residue washing solution, 3-5 g/L of dipotassium phosphate, 2-5 g/L of sodium chloride, 0.2-0.45 g/L of magnesium sulfate heptahydrate, 0.5-1.2 g/L of diamine citrate, 0.1-0.3 g/L of ferric sulfate, the pH value is 6.0, and distilled water is prepared;
(3) and (3) amplification culture of bacterial liquid: transferring the actinomucor elegans obtained by screening treatment after the final period acclimation into an amplification culture solution, and performing amplification culture in a constant-temperature shaking culture device at the rotation speed of 160-170rpm at 29-31 ℃ for 12-15h until the culture is finishedThe content of effective bacteria of actinomucor elegans is 3.6 × 108-9.2×108CFU/m L, wherein the enlarged culture solution comprises sweet potato powder 8-10 g/L, Hericium Erinaceus extract 3-5 g/L, Acanthopanax senticosus extract 1-3 g/L, Spirulina extract 1-3 g/L, yeast extract 1-3 g/L, sodium glutamate 1-3 g/L, dipotassium phosphate 2-5 g/L, vitamin B10.1-0.3 g/L, magnesium sulfate 0.3-0.8 g/L, pH 6.5, and distilled water.
Further, the aloe extract in the step (2) is obtained by crushing aloe leaves into slurry, filtering to obtain precipitates, adding 2 times (v/v) of water, extracting with water, taking supernate, adding 75% by mass of ethanol solution, standing, taking precipitates, repeating the ethanol extraction for 3 times, combining the precipitates, dissolving the combined precipitates in 3-5 times (v/v) of water, introducing the solution into a percolation column for circular percolation, keeping the flow rate of the percolation solution at 0.2-0.5ml/s, extracting for 1-2 hours, taking filtrate, and intercepting the molecular weight of the percolation solution through an ultrafiltration membrane to be 6000-; wherein the content of polysaccharide in the aloe extract is more than 95%; the astragalus extract is prepared by soaking astragalus into an ethanol solution with the mass fraction of 15%, heating for 5 minutes, standing overnight, filtering to obtain a precipitate, pulverizing the precipitate, drying and sieving with a 100-mesh sieve, adding 10-50 times of mass water and 0.1-0.2 times of mass chitosan flocculant, heating to 35 ℃ while stirring at the speed of 100r/min for 10-20 minutes, stopping heating, cooling, adding an ethanol solution with the mass fraction of 65% which is 0.5-0.6 times of the volume (v/v), stirring, standing for 20-30 hours, and intercepting by an ultrafiltration membrane with the molecular weight of 3000 and 5000Da to obtain the astragalus extract; wherein the polysaccharide content in the astragalus extract reaches more than 95 percent; the wolfberry extract is prepared by adding 1-3 times (v/v) of water into wolfberry for pulping, oscillating in an ultrasonic oscillator for 3-4 hours at 20-25KHZ, filtering and collecting filtrate, adding sodium carbonate and ethanol solution for extraction, collecting extract, and concentrating the extract to 1.02 times of the original volume by a double-effect energy-saving concentrator. Wherein the content of polysaccharide in the fructus Lycii extract is above 95%.
Further, the hericium erinaceus extract in the step (3) is prepared by slicing hericium erinaceus, adding an ethanol solution with the mass fraction of 20%, soaking for 10 minutes under the assistance of ultrasonic waves, filtering to recover ethanol to obtain a precipitate, adding 25-30 times of water, heating to 40 ℃ for water bath boiling for 3-5 hours, filtering to obtain a filtrate, concentrating the filtrate to 2 times of the original volume, adding 75% ethanol to soak for 48 hours, taking the precipitate, and drying to obtain the hericium erinaceus extract; wherein the content of polysaccharide in the Hericium erinaceus extract reaches 95%; adding water of 1-3 times of volume of acanthopanax into acanthopanax, pulping, filtering to obtain a precipitate, adding 30-40 times of water, heating for water extraction, extracting for 20 minutes under the condition that the temperature of the water extraction is 75 ℃, performing auxiliary water extraction by using the frequency with the ultrasonic power of 1000W during the water extraction, performing water extraction for 3 times, filtering to remove the precipitate, concentrating the filtrate obtained by 3 times to obtain an extract, performing spray drying on the extract to obtain dry powder, sieving by using a 1000-mesh sieve, performing alcohol extraction for 2-5 hours by using 75% by mass of ethanol, and recovering the ethanol to obtain the precipitate so as to obtain the acanthopanax extract; wherein the content of polysaccharide in the acanthopanax senticosus extract reaches 95%; cleaning and crushing spirulina, performing water bath reflux extraction for 4-5 hours by using ethanol with the mass fraction of 85% and the weight of 3-5 times of that of the spirulina, filtering to obtain precipitate, adding water with the weight of 10-20 times of that of the precipitate to perform water bath extraction for 4-5 hours, filtering to obtain filtrate, concentrating the filtrate to 1.1 times of the original volume, adding ethanol with the mass fraction of 65% to perform precipitation for 28 hours, centrifuging to remove the precipitate, dissolving the precipitate by using distilled water, intercepting liquid with the molecular weight of 5000-6000Da by using a filter membrane with the surface of D20 resin, concentrating, and performing spray drying to obtain the spirulina extract; wherein the content of polysaccharide in Spirulina extract is above 95%.
In the invention, the yeasts comprise candida ethanolica and candida rugosa, and the volume ratio of the candida ethanolica to the candida rugosa is 1-3: 1-2.
In the invention, the Candida ethanolica is Candida ethanolica Y1(Candida ethamo L ica) separated from buffalo milk, the preservation number is CCTCC NO: M2016467, the Candida rugosa is Candida rugosa Y7(Candida rugosa) separated from buffalo milk, the preservation number is CCTCC NO: M2016468, and the Mucor elegans (Actinomucorolgans) is purchased from China general microbiological culture Collection center, and the preservation number is CMCC NO. 3.2468.
In the present invention, the method for acclimatizing and culturing candida ethanolica Y1 and candida rugosa Y7 comprises the following specific steps:
(1) the strain activation, namely culturing Candida ethanolica Y1(Candida ethamo L ica) and Candida rugosa Y7(Candida rugosa) which are separated from buffalo milk and are frozen and preserved in a glycerol tube form on YPD slant culture media respectively at the temperature of 34-36 ℃ for 22-24h, wherein the YPD slant culture media comprise 3-5 g/L of potato extract powder, 18-20 g/L of glucose, 12-15 g/L of agar, the pH value of 5.8 and distilled water preparation;
(2) domesticating strains:
a. mixing cassava residue, agricultural wastewater and distilled water according to a mass ratio of 1: 1: 2 stirring and mixing to obtain a mixture for later use;
b. 3-5 g/L of potato extract powder, 18-20 g/L of glucose, 3-5 g/L of agar, 5.5 of pH value and distilled water are prepared to obtain a domestication base liquid culture medium, then the mixture obtained in the step a is added to be compounded to obtain a compound liquid culture medium, 4 gradients are prepared in the compound liquid culture medium, the volume of the adding amount of the mixture in the first compound liquid culture medium is 10% of the volume of the domestication base liquid culture medium, the volume of the adding amount of the mixture in the second compound liquid culture medium is 20% of the volume of the domestication base liquid culture medium, the volume of the adding amount of the mixture in the third compound liquid culture medium is 40% of the volume of the domestication base liquid culture medium, and the volume of the adding amount of the mixture in the fourth compound liquid culture medium is 80% of the volume of the domestication base liquid culture medium for standby;
c. respectively inoculating the activated Candida ethanolica Y1(Candida ethhamo L ica) and Candida rugosa Y7(Candida rugosa) in the step (1) into a first compound liquid culture medium of 100m L according to the inoculation amount of 10%, culturing for 10h at 20-25 ℃ and 100r/min, respectively inoculating the cultured bacteria liquid into a second compound liquid culture medium of 100m L according to the inoculation amount of 10%, culturing for 10h at 25-30 ℃ and 200r/min, respectively inoculating the cultured bacteria liquid into a third compound liquid culture medium of 100m L according to the inoculation amount of 15%, culturing for 10h at 31-33 ℃ and 250r/min, respectively inoculating the cultured bacteria liquid into a fourth compound liquid culture medium of 100m L according to the inoculation amount of 15%, culturing for 10h at 33-37 ℃ and 300r/min, and then screening to obtain the domesticated Candida ethanolica Y1(Candida ethhama) and Candida rugosa 7;
d. expanding culture of the acclimatized strain, namely respectively transferring 1m L acclimatized Candida ethanolica Y1(Candida acethamo L ica) and the acclimatized Candida rugosa Y7(Candida rugosa) into a 250m L triangular flask filled with 30m L second expanding culture solution, and expanding culture for 10-13h in a constant-temperature shaking culture device at the rotating speed of 180-200rpm at the temperature of 30-35 ℃ until the effective bacterium content of the Candida ethanolica in each ml is 2.2 × 108-5.5×108CFU/m L, the effective bacteria content of the candida rugosa is 2.1 × 10 per milliliter8-4.8×108CFU/m L, wherein the second enlarged culture solution comprises herba Ephedrae extract 3-5 g/L, folium Bambusae extract 3-5 g/L, Tremella extract 3-5 g/L, ammonium sulfate 1-3 g/L, potassium dihydrogen phosphate 1-3 g/L, magnesium sulfate 0.5-1.0 g/L, pH 6.0, and distilled water.
In the present invention, further, in the step d, the ephedra extract is obtained by pulverizing ephedra, adding 10-20 times by mass of water, adding 0.2-0.3 times by mass of potassium iodide solution by mass of water, shearing in a high speed shearing machine to obtain an ephedra soak solution, centrifuging, taking a filtrate, concentrating to 1.02 times of the original volume to obtain an extract, adding 3 times by volume of an ethanol solution with a mass concentration of 85% into the extract, stirring, standing for 48 hours, centrifuging at 1000r/min for 100S, centrifuging for 3 times in total, collecting precipitates, and freeze-drying to obtain the extract containing 99% of ephedra polysaccharide.
In the present invention, further, in the step d, the bamboo leaf extract is obtained by adding an ethanol solution with a mass concentration of 85% to fresh bamboo leaves, rapidly grinding the mixture into pulp, recovering ethanol, adding 10 times of mass of water to the precipitate for ultrasonic extraction, filtering the obtained leaching solution with an ultrafiltration membrane, concentrating the filtrate under reduced pressure to 1.1 times of the original volume, adding an ethanol solution with a mass fraction of 75% at 4 ℃, stirring, standing, filtering to obtain a precipitate, adding ethyl acetate to extract, collecting the supernatant, concentrating to 1.1 times of the original volume, and spray-drying to obtain an extract containing 99% of bamboo leaf polysaccharide; the tremella extract is prepared by drying and crushing tremella, leaching for 3 times by a hot bath alcohol extraction method, combining 3 times of leaching liquor, adding Sevage reagent to remove protein, concentrating filtrate to 1/3 of the original volume, adding ethanol solution with the mass concentration of 95% to enable the alcohol content of concentrated solution to reach 75%, standing for 24 hours, performing suction filtration, and finally performing spray drying to obtain the extract containing 99% of tremella polysaccharide.
The manioc waste, the agricultural wastewater, the chemicals and the traditional Chinese medicines used in the application can be purchased in the market.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
(1) the invention provides a method for reducing cyanide content in cassava residue, which is simple and easy to implement, adopts a microbial degradation mode, can greatly reduce the content of hydrocyanic acid, improves the safety of the cassava residue as feed, decomposes the hydrocyanic acid and converts the hydrocyanic acid into organic matters to become power elements of the feed, and increases the economic benefit of safe utilization of the cassava residue;
(2) the method for reducing the cyanide content in the cassava residue adopts a combined microbial degradation method, belongs to the field of biotechnology treatment, and has the advantages of good degradation effect, short degradation time, strong operation safety and simple required equipment; therefore, the detoxified cassava residues can be applied to the buffalo feed, and have important significance for expanding the development of buffalo feed resources and promoting the healthy and sustainable development of the buffalo industry.
[ detailed description ] embodiments
In order to make the technical means, the creation features, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the embodiment. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1:
test one:
1.2. design of experiments
1.2.1 preparation of fermented cassava dregs
The experiment takes the cassava dregs as the fermentation raw material. Sampling and measuring the hydrocyanic acid content before treatment.
1.2.2 optimal combinatorial screening of Mixed species
Activated saccharomycetes, lactobacillus plantarum (L Acobacter plantarum) SN12#, with the preservation number of CCTCC NO: M2015699) and Monascus ruber (Monascus ruber) strains purchased from China general microorganism strain preservation center with the preservation number of CMCC NO.3.4639) and actinomucor elegans culture solution (actinomucor elegans) purchased from China general microorganism strain preservation center with the preservation number of CMCCNO.3.2468 are combined according to different strains, 15 combinations are arranged in total, each combination has 4 repetitions, each repetition is subpackaged according to 180 and 250g cassava residue/bag (see Table 1), the cassava residue/bag is packaged into a polyethylene film bag to be uniformly mixed, the polyethylene film bag is sealed, the cassava residue/bag is stored at room temperature, the hydrocyanic acid content of the cassava residue/bag is measured, and the bacterial combination with the optimal fermentation effect is screened.
Control group: 5 percent of distilled water is added into the mixture,
treatment group 1: 5 percent of yeast, namely, yeast, wherein,
treatment group 2: 5 percent of the lactobacillus plantarum strain is selected,
treatment group 3: 5 percent of the monascus purpureus,
treatment group 4: 5 percent of the actinomucor elegans,
treatment group 5: 2.5% of yeast and 2.5% of lactobacillus plantarum,
treatment group 6: 2.5 percent of yeast and 2.5 percent of monascus,
treatment group 7: 2.5 percent of yeast and 2.5 percent of actinomucor elegans,
treatment group 8: 2.5 percent of lactobacillus plantarum and 2.5 percent of monascus,
treatment group 9: 2.5 percent of lactobacillus plantarum and 2.5 percent of actinomucor elegans,
treatment group 10: 2.5 percent of monascus and 2.5 percent of actinomucor elegans,
treatment group 11: 1.7 percent of yeast, 1.7 percent of lactobacillus plantarum and 1.7 percent of monascus,
treatment group 12: 1.7 percent of yeast, 1.7 percent of lactobacillus plantarum and 1.7 percent of actinomucor elegans,
the treatment group 13 was 1.7% lactobacillus plantarum + 1.7% monascus + 1.7% actinomucor elegans,
treatment group 14: 1.25% yeast, 1.25% lactobacillus plantarum, 1.25% monascus purpureus, and 1.25% actinomucor elegans.
TABLE 1 sample treatment settings and bacterial liquid additives per bag (calculated as 200g of cassava residues)
Note: "-" represents no addition of any bacteria liquid; calculating each bag according to 200g of fresh cassava residues; and subpackaging the bacterium liquid by using a pipette.
The raw potato dregs contain cyanide of 6.3 plus or minus 0.88 mg/kg; the above treatments were combined and subjected to hydrocyanic acid measurement (test center of research institute for tropical crops).
TABLE 2
The test and the test detection result can preliminarily judge that the fermentation by using the yeast and actinomucor elegans combined bacterial liquid in the test has better cyanide reducing capability.
And (2) test II:
1.1 test: fermentation test of cassava dregs (Box-Behnken test design, see Table 3)
1.2 method: bacterial liquids (yeast and actinomucor elegans are mixed in equal volumes) with different proportions are inoculated into each sample bag weighing 200g of cassava dregs according to the design in the table 4, the mixture is fully and uniformly mixed, and then the mixture is placed into a biochemical incubator to be cultured for different days at different temperatures, and then the cyanide content is detected.
TABLE 3 Bo-Behnken test design
Note: -1, 0 and +1 represent three levels of bacteria liquid, fermentation temperature and fermentation time, the bacteria liquid adding proportion is 5% (10ml), 7.5% (15ml) and 10% (20ml), and the fermentation temperature is: 25. 28 ℃ and 31 ℃, fermentation time: 48. 72 and 96 h.
TABLE 4 sample treatment settings per bag (calculated as 200g of cassava residues)
Note: each group is repeated for 3 times, each time the cassava residue is calculated by 200g, bacterial liquid is added, well mixed, compacted and sealed, and the mixture is placed into a corresponding temperature condition for cultivation (an incubator).
1.3 the results are shown in Table 5 (6.3. + -. 0.88mg/kg cyanide is present in the raw potato residue).
TABLE 5
| Group of | Cyanide content mg/kg |
| 1 | 1.73±0.34 |
| 2 | 2.37±0.28 |
| 3 | 2.83±0.31 |
| 4 | 1.90±0.23 |
| 5 | 1.53±0.12 |
| 6 | 1.67±0.38 |
| 7 | 1.85±0.32 |
| 8 | 1.51±0.30 |
| 9 | 1.90±0.26 |
| 10 | 1.85±0.34 |
| 11 | 1.85±0.23 |
| 12 | 3.3±0.42 |
| 13 | 1.67±0.28 |
| 14 | 1.63±0.41 |
| 15 | 0.89±0.33 |
As can be seen from the above table, the fermentation is carried out by using the yeast and actinomucor elegans combined bacterial liquid, the volume of the bacterial liquid is 10-20m L, the fermentation temperature is 25-31 ℃, and the fermentation time is 48-96h, so that the cyanide reducing capability is better.
Example 2:
a method for reducing cyanide content in cassava residue comprises mixing yeast and actinomucor elegans at a volume ratio of 5:3 to obtain a mixed bacterial liquid, mixing the mixed bacterial liquid with 10m L and 180g of cassava residue, and fermenting at 25 deg.C for 48 h.
The yeasts comprise Candida ethanolica and Candida rugosa in a volume ratio of 3:2, the Candida ethanolica is Candida ethanolica Y1(Candida ethamo L ica) separated from buffalo milk and has a preservation number of CCTCCNO: M2016467, the Candida rugosa is Candida rugosa Y7(Candida rugosa) separated from the buffalo milk and has a preservation number of CCTCC NO: M2016468, and the Mucor elegans (Actinomucorens) is purchased from the China general microbiological culture Collection center and has a preservation number of CMCC NO. 3.2468.
The pre-yeast and actinomucor elegans are used for acclimatization culture in advance.
The method for domesticating and culturing the actinomucor elegans comprises the following specific steps:
(1) the strain activation comprises the steps of inoculating actinomucor elegans on a first activation slant culture medium, and culturing for 8 hours at 40 ℃, wherein the first activation slant culture medium comprises 12 g/L of peptone, 5 g/L of beef extract powder, 5 g/L0 of yeast extract powder, 5 g/L of aloe extract, 3 g/L of astragalus extract, 5 g/L of medlar extract, 0.5 g/L of dipotassium hydrogen phosphate, 0.3 g/L of diammonium hydrogen citrate, 0.3 g/L of magnesium sulfate, 0.05 g/L of manganese sulfate, 10 g/L of agar, 6.0 of pH value and distilled water;
(2) domesticating strains: put the inclined plane 0.3cm above2Inoculating the lawn into 250m L triangular flask containing 15m L acclimatization culture medium, and rotating the table at a certain speedCarrying out shake flask domestication culture at 280rpm and 29 ℃, wherein each 3 days is a domestication period, carrying out domestication for 3 periods, and after the domestication of each period is finished, taking a culture solution with the highest degradation rate of a cassava residue washing solution as an inoculation source for next domestication, wherein the domestication culture medium comprises 10 g/L of the cassava residue washing solution, 3 g/L of dipotassium phosphate, 2 g/L of sodium chloride, 0.2 g/L of magnesium sulfate heptahydrate, 0.5 g/L of diamine citrate, 0.1 g/L of ferric sulfate, the pH value of the culture medium is 6.0, and the culture medium is prepared from distilled water;
(3) expanding culture of bacterial liquid, transferring the actinomucor elegans obtained by screening after the last period of acclimation into the expanding culture liquid, expanding culture for 12h in a constant temperature shaking culture device at the rotating speed of 160rpm and 29 ℃ until the effective bacterial content of the actinomucor elegans is 3.6 × 108-9.2×108CFU/m L, wherein the enlarged culture solution comprises sweet potato powder 8 g/L, Hericium erinaceus extract 3 g/L, acanthopanax senticosus extract 1 g/L, spirulina extract 1 g/L, yeast extract 1 g/L, sodium glutamate 1 g/L, dipotassium chloride phosphate 2 g/L, vitamin B10.1g/L, magnesium sulfate 0.3 g/L, pH value 6.5, and distilled water.
The aloe extract in the step (2) is prepared by crushing aloe leaves into pulp, filtering to obtain precipitate, adding 2 times (v/v) of water, extracting with water, taking supernatant, adding 75% by mass of ethanol solution, standing, taking precipitate, repeating ethanol extraction for 3 times, combining the precipitate, dissolving the combined precipitate in 3 times (v/v) of water, circulating and percolating in a percolation column, keeping the flow rate of the percolation liquid at 0.2ml/s, extracting for 1 hour, taking filtrate, and intercepting the molecular weight of the percolation liquid by an ultrafiltration membrane to be 6000Da to obtain the aloe extract; wherein the aloe extract contains 95% of polysaccharides;
soaking astragalus into an ethanol solution with the mass fraction of 15%, heating for 5 minutes, standing overnight, filtering to obtain a precipitate, pulverizing the precipitate, drying and sieving with a 100-mesh sieve, adding 10 times of mass water and 0.1 time of mass chitosan flocculant, heating to 35 ℃ while stirring at the speed of 100r/min for 10 minutes, stopping heating, cooling, adding an ethanol solution with the mass fraction of 65% which is 0.5 times of the volume (v/v), stirring, standing for 20 hours, and performing ultrafiltration membrane to cut off the molecular weight of 3000Da to obtain the astragalus extract; wherein the content of polysaccharide in the radix astragali extract reaches 95%;
the wolfberry extract is prepared by adding 1 time (v/v) of water into wolfberry, pulping, oscillating in an ultrasonic oscillator for 3 hours at 20KHZ, filtering, collecting filtrate, adding sodium carbonate and ethanol solution for extraction, collecting extract, and concentrating the extract to 1.02 times of the original volume by a double-effect energy-saving concentrator. Wherein the content of polysaccharide in the fructus Lycii extract is 95%.
The hericium erinaceus extract in the step (3) is prepared by slicing hericium erinaceus, adding an ethanol solution with the mass fraction of 20%, soaking for 10 minutes under the assistance of ultrasonic waves, filtering to recover ethanol to obtain a precipitate, adding 25 times of water, heating to 40 ℃ for water bath boiling for 3 hours, filtering to obtain a filtrate, concentrating the filtrate to 2 times of the original volume, adding 75% ethanol, soaking for 48 hours, taking the precipitate, and drying to obtain the hericium erinaceus extract; wherein the content of polysaccharide in the Hericium erinaceus extract reaches 95%;
adding 1-fold water into acanthopanax senticosus, pulping, filtering to obtain a precipitate, adding 30 times of water, heating for water extraction, extracting for 20 minutes under the condition that the temperature of the water extraction is 75 ℃, performing water extraction assisted by the frequency with the ultrasonic power of 1000W during the water extraction, performing water extraction for 3 times, filtering to remove the precipitate, concentrating the filtrate obtained by 3 times to obtain an extract, performing spray drying on the extract to obtain dry powder, sieving by a 1000-mesh sieve, performing alcohol extraction for 2 hours by using 75% by mass of ethanol, and recovering the ethanol to obtain the precipitate, thereby obtaining the acanthopanax senticosus extract; wherein the content of polysaccharide in the acanthopanax senticosus extract reaches 95%;
cleaning and crushing spirulina, performing water bath reflux extraction for 4 hours by using ethanol with the mass fraction of 85% and the weight of 3 times of that of the spirulina, filtering to obtain precipitate, adding water with the weight of 10 times of that of the precipitate to perform water bath extraction for 4 hours, filtering to obtain filtrate, concentrating the filtrate to 1.1 times of the original volume, adding ethanol with the mass fraction of 65% to perform precipitation for 28 hours, centrifuging to remove the precipitate, dissolving the precipitate by using distilled water, intercepting liquid with the molecular weight of 5000Da by using a filter membrane with the surface of D20 resin, concentrating, and performing spray drying to obtain the spirulina extract; wherein the content of polysaccharide in Spirulina extract is 97%.
Furthermore, the domestication and culture method of the candida ethanolica Y1 and the candida rugosa Y7 comprises the following specific steps:
(1) activating strains, namely respectively culturing Candida ethanolica Y1(Candida ethamo L ica) and Candida rugosa Y7(Candida rugosa) which are separated from buffalo milk and are frozen and preserved in a glycerol tube form on YPD slant culture media at 34 ℃ for 22 hours, wherein the YPD slant culture media comprise 3 g/L of potato extract powder, 18 g/L of glucose, 12 g/L of agar, 5.8 of pH value and distilled water preparation;
(2) domesticating strains:
a. mixing cassava residue, agricultural wastewater and distilled water according to a mass ratio of 1: 1: 2 stirring and mixing to obtain a mixture for later use;
b. 3 g/L g of potato extract powder, 18 g/L g of glucose, 3 g/L g of agar, 5.5 of pH value and distilled water, preparing a domesticated basic liquid culture medium, adding the mixture obtained in the step a, and compounding to obtain a compound liquid culture medium, wherein 4 gradients are prepared in the compound liquid culture medium, the volume of the added amount of the mixture in the first compound liquid culture medium is 10% of the volume of the domesticated basic liquid culture medium, the volume of the added amount of the mixture in the second compound liquid culture medium is 20% of the volume of the domesticated basic liquid culture medium, the volume of the added amount of the mixture in the third compound liquid culture medium is 40% of the volume of the domesticated basic liquid culture medium, and the volume of the added amount of the mixture in the fourth compound liquid culture medium is 80% of the volume of the domesticated basic liquid culture medium for standby;
c. respectively taking the activated Candida ethanolica Y1(Candida ethanolica L ica) and Candida rugosa Y7(Candida rugosa) in the step (1) according to the inoculum size of 10%, respectively inoculating the activated Candida ethanolica Y1 and Candida rugosa Y7 into a first compound liquid culture medium of 100m L, culturing for 10h at 20 ℃ and 100r/min, respectively inoculating the cultured bacteria liquid into a second compound liquid culture medium of 100m L according to the inoculum size of 10%, culturing for 10h at 25 ℃ and 200r/min, respectively inoculating the cultured bacteria liquid into a third compound liquid culture medium of 100m L according to the inoculum size of 15%, culturing for 10h at 31 ℃ and 250r/min, respectively inoculating the cultured bacteria liquid into a fourth compound liquid culture medium of 100m L according to the inoculum size of 15%, culturing for 10h at 33 ℃ and 300r/min, and then screening and obtaining the domesticated Candida ethanolica Y1(Candida rugosa L Candida rugosa 7) and Candida rugosa 7 after domestication;
d. expanding culture of acclimatized strain, namely taking 1m L acclimatized Candida ethanolica Y1(Candida acethamo L ica) and acclimatized Candida rugosa Y7(Candida rugosa) to respectively transfer into a 250m L triangular flask filled with 30m L second expanding culture solution, expanding culture for 10 hours in a constant-temperature shaking culture apparatus at the rotating speed of 180rpm at 30 ℃ until the effective bacteria content of the Candida ethanolica per ml is 2.2 × 108-5.5×108CFU/m L, the effective bacteria content of the candida rugosa is 2.1 × 10 per milliliter8-4.8×108CFU/m L, wherein the second enlarged culture solution comprises herba Ephedrae extract 3 g/L, folium Bambusae extract 3 g/L, Tremella extract 3 g/L, ammonium sulfate 1 g/L, potassium dihydrogen phosphate 1 g/L, magnesium sulfate 0.5 g/L, pH 6.0, and distilled water.
In the step d, the ephedra extract is prepared by crushing ephedra, adding 10 times of water by mass, adding a potassium iodide solution with the volume 0.2 times of that of the water by mass, shearing in a high-speed shearing machine to obtain an ephedra soak solution, centrifuging, taking a filtrate, concentrating to 1.02 times of the original volume to obtain an extract, adding a 3 times of ethanol solution with the mass concentration of 85% into the extract, stirring, standing for 48 hours, centrifuging at 1000r/min for 100S for 3 times, collecting precipitates, and freeze-drying to obtain the extract containing the ephedra polysaccharide of 99%. Adding fresh bamboo leaves into an ethanol solution with the mass concentration of 85%, quickly grinding the mixture into pulp, recovering ethanol, adding 10 times of mass water into the precipitate for ultrasonic extraction, filtering the obtained leaching liquor by an ultrafiltration membrane, concentrating the filtrate under reduced pressure to 1.1 times of the original volume, adding the ethanol solution with the mass fraction of 75% at 4 ℃, stirring, standing, filtering, taking the precipitate, adding ethyl acetate for extraction, taking the supernatant, concentrating to 1.1 times of the original volume, spray-drying to obtain an extract containing 99% of bamboo leaf polysaccharide, drying and crushing tremella, leaching 3 times by a hot bath alcohol extraction method, combining the leaching liquor for 3 times, adding a Sevage reagent to remove protein, concentrating the filtrate to 1/3 of the original volume, adding an ethanol solution with the mass concentration of 95% to ensure that the alcohol content of the concentrated solution reaches 75%, standing for 24 hr, vacuum filtering, and spray drying to obtain extract containing 99% of Tremella polysaccharide.
Example 3:
a method for reducing cyanide content in cassava residue comprises mixing yeast and actinomucor elegans at a volume ratio of 5:1 to obtain a mixed bacterial liquid, mixing the mixed bacterial liquid with 20m L and 250g of cassava residue, and fermenting at 31 deg.C for 96 h.
The yeasts comprise Candida ethanolica and Candida rugosa in a volume ratio of 3:1, the Candida ethanolica is Candida ethanolica Y1(Candida ethamo L ica) separated from buffalo milk and has a preservation number of CCTCCNO: M2016467, the Candida rugosa is Candida rugosa Y7(Candida rugosa) separated from the buffalo milk and has a preservation number of CCTCC NO: M2016468, and the Mucor elegans (Actinomucorens) is purchased from the China general microbiological culture Collection center and has a preservation number of CMCC NO. 3.2468.
The pre-yeast and actinomucor elegans are used for acclimatization culture in advance.
The method for domesticating and culturing the actinomucor elegans comprises the following specific steps:
(1) the strain activation comprises the steps of inoculating actinomucor elegans on a first activation slant culture medium, and culturing for 16h at 50 ℃, wherein the first activation slant culture medium comprises 18 g/L of peptone, 8 g/L of beef extract powder, 8 g/L0 of yeast extract powder, 8 g/L of aloe extract, 7 g/L of astragalus extract, 9 g/L of medlar extract, 0.8 g/L of dipotassium hydrogen phosphate, 0.5 g/L of diammonium hydrogen citrate, 0.5 g/L of magnesium sulfate, 0.1 g/L of manganese sulfate, 15 g/L of agar, 6.0 of pH value and distilled water;
(2) domesticating strains: put the inclined plane 0.3cm above2The bacterial lawnThe method comprises the following steps of inoculating the cassava residues into a 250m L triangular flask filled with a 15m L domestication culture medium, carrying out domestication for 3 periods by shaking and domesticating the culture medium at the rotating speed of 300rpm at 31 ℃ in a shaking way, taking a culture solution with the highest degradation rate of a cassava residue washing solution as an inoculation source for the next domestication after domestication for 3 days, wherein the domestication culture medium comprises the components of 20 g/L of the cassava residue washing solution, 5 g/L of dipotassium phosphate, 5 g/L of sodium chloride, 0.45 g/L of magnesium sulfate heptahydrate, 1.2 g/L of diamine citrate, 0.3 g/L of ferric sulfate, the pH value of 6.0 and distilled water;
(3) expanding culture of bacterial liquid, transferring the actinomucor elegans obtained by screening after the last period of acclimation into the expanding culture liquid, expanding culture for 15h in a constant temperature shaking culture device at the rotating speed of 170rpm and the temperature of 31 ℃ until the effective bacterial content of the actinomucor elegans is 3.6 × 108-9.2×108CFU/m L, wherein the enlarged culture solution comprises sweet potato powder 10 g/L, Hericium erinaceus extract 5 g/L, acanthopanax senticosus extract 3 g/L, spirulina extract 3 g/L, yeast extract 3 g/L, sodium glutamate 3 g/L, dipotassium chloride phosphate 5 g/L, vitamin B10.3 g/L, magnesium sulfate 0.8 g/L, pH value 6.5, and distilled water.
The aloe extract in the step (2) is prepared by crushing aloe leaves into pulp, filtering to obtain precipitate, adding 2 times (v/v) of water, extracting with water, taking supernatant, adding 75% by mass of ethanol solution, standing, taking precipitate, repeating ethanol extraction for 3 times, combining the precipitate, dissolving the combined precipitate in 5 times (v/v) of water, circulating and percolating in a percolation column, keeping the flow rate of the percolation liquid at 0.5ml/s, extracting for 2 hours, taking filtrate, and intercepting the molecular weight of the percolation liquid by an ultrafiltration membrane to 8000Da to obtain the aloe extract; wherein the aloe extract contains 95% of polysaccharides;
soaking astragalus into an ethanol solution with the mass fraction of 15%, heating for 5 minutes, standing overnight, filtering to obtain a precipitate, pulverizing the precipitate, drying and sieving with a 100-mesh sieve, adding 50 times of mass water and 0.2 times of mass chitosan flocculant, heating to 35 ℃ while stirring at the speed of 100r/min for 20 minutes, stopping heating, cooling, adding an ethanol solution with the volume of 0.6 times (v/v) and the mass fraction of 65%, stirring, standing for 30 hours, and performing ultrafiltration membrane to cut off the molecular weight of 5000Da to obtain the astragalus extract; wherein the content of polysaccharide in the radix astragali extract reaches 95%;
the wolfberry extract is prepared by adding 3 times (v/v) of water into wolfberry, pulping, oscillating for 4 hours in an ultrasonic oscillator at 25KHZ, filtering, collecting filtrate, adding sodium carbonate and ethanol solution for extraction, collecting extract, and concentrating the extract to 1.02 times of the original volume by a double-effect energy-saving concentrator. Wherein the content of polysaccharide in the fructus Lycii extract is 95%.
The hericium erinaceus extract in the step (3) is prepared by slicing hericium erinaceus, adding an ethanol solution with the mass fraction of 20%, soaking for 10 minutes under the assistance of ultrasonic waves, filtering to recover ethanol to obtain a precipitate, adding 30 times of water, heating to 40 ℃ for water bath boiling for 5 hours, filtering to obtain a filtrate, concentrating the filtrate to 2 times of the original volume, adding 75% ethanol, soaking for 48 hours, taking the precipitate, and drying to obtain the hericium erinaceus extract; wherein the content of polysaccharide in the Hericium erinaceus extract reaches 95%;
adding 3-fold water into acanthopanax senticosus, pulping, filtering to obtain a precipitate, adding 40 times of water, heating for water extraction, extracting for 20 minutes under the condition that the temperature of the water extraction is 75 ℃, performing water extraction with the frequency of 1000W of ultrasonic power during the water extraction, performing water extraction for 3 times, filtering to remove the precipitate, concentrating the filtrate obtained by 3 times to obtain an extract, performing spray drying on the extract to obtain dry powder, sieving with a 1000-mesh sieve, performing alcohol extraction for 5 hours by using 75% by mass of ethanol, and recovering the ethanol to obtain the precipitate, thereby obtaining the acanthopanax senticosus extract; wherein the content of polysaccharide in the acanthopanax senticosus extract reaches 95%;
cleaning and crushing spirulina, performing water bath reflux extraction for 5 hours by using ethanol with the mass fraction of 85% in an amount which is 5 times that of the spirulina, filtering to obtain precipitate, adding water with the weight of 20 times that of the precipitate to perform water bath extraction for 5 hours, filtering to obtain filtrate, concentrating the filtrate to be 1.1 times that of the original volume, adding ethanol with the mass fraction of 65% to perform precipitation for 28 hours, centrifuging to remove the precipitate, dissolving the precipitate by using distilled water, intercepting liquid with the molecular weight of 6000Da by using a filter membrane with the surface of D20 resin, concentrating, and performing spray drying to obtain the spirulina extract; wherein the content of polysaccharide in Spirulina extract is up to 99%.
Furthermore, the domestication and culture method of the candida ethanolica Y1 and the candida rugosa Y7 comprises the following specific steps:
(1) activating strains, namely respectively culturing Candida ethanolica Y1(Candida ethamo L ica) and Candida rugosa Y7(Candida rugosa) which are separated from buffalo milk and are frozen and preserved in a glycerol tube form on a YPD slant culture medium at 36 ℃ for 24 hours, wherein the YPD slant culture medium comprises 5 g/L of potato extract powder, 20 g/L of glucose, 15 g/L of agar, 5.8 of pH value and distilled water;
(2) domesticating strains:
a. mixing cassava residue, agricultural wastewater and distilled water according to a mass ratio of 1: 1: 2 stirring and mixing to obtain a mixture for later use;
b. 5 g/L of potato extract powder, 20 g/L of glucose, 5 g/L of agar, 5.5 of pH value and distilled water are prepared to obtain a domesticated basic liquid culture medium, then the mixture obtained in the step a is added to be compounded to obtain a compound liquid culture medium, 4 gradients are prepared in the compound liquid culture medium, the volume of the added amount of the mixture in the first compound liquid culture medium is 10% of the volume of the domesticated basic liquid culture medium, the volume of the added amount of the mixture in the second compound liquid culture medium is 20% of the volume of the domesticated basic liquid culture medium, the volume of the added amount of the mixture in the third compound liquid culture medium is 40% of the volume of the domesticated basic liquid culture medium, and the volume of the added amount of the mixture in the fourth compound liquid culture medium is 80% of the volume of the domesticated basic liquid culture medium for standby;
c. respectively taking the activated Candida ethanolica Y1(Candida ethanolica L ica) and Candida rugosa Y7(Candida rugosa) in the step (1) and respectively inoculating the activated Candida ethanolica Y1(Candida ethanolica L ica) and Candida rugosa Y7(Candida rugosa) into a first compound liquid culture medium of 100m L according to the inoculation amount of 10%, culturing for 10 hours at 25 ℃ and 100r/min, respectively inoculating the cultured bacteria liquid into a second compound liquid culture medium of 100m L according to the inoculation amount of 10%, culturing for 10 hours at 30 ℃ and 200r/min, respectively inoculating the cultured bacteria liquid into a third compound liquid culture medium of 100m L according to the inoculation amount of 15%, culturing for 10 hours at 33 ℃ and 250r/min, respectively inoculating the cultured bacteria liquid into a fourth compound liquid culture medium of 100m L according to the inoculation amount of 15%, culturing for 10 hours at 37 ℃ and 300r/min, and then screening for obtaining the domesticated Candida ethanolica Y1(Candida rugosa L Candida rugosa) and Candida rugosa 7 after domestication;
d. expanding culture of acclimatized strain, namely taking 1m L acclimatized Candida ethanolica Y1(Candida acethamo L ica) and acclimatized Candida rugosa Y7(Candida rugosa) to respectively transfer into a 250m L triangular flask filled with 30m L second expanding culture solution, expanding culture for 13h in a constant-temperature shaking culture apparatus at the rotating speed of 200rpm at 35 ℃ until the effective bacteria content of the Candida ethanolica per ml is 2.2 × 108-5.5×108CFU/m L, the effective bacteria content of the candida rugosa is 2.1 × 10 per milliliter8-4.8×108CFU/m L, wherein the second enlarged culture solution comprises herba Ephedrae extract 5 g/L, folium Bambusae extract 5 g/L, Tremella extract 5 g/L, ammonium sulfate 3 g/L, potassium dihydrogen phosphate 3 g/L, magnesium sulfate 1.0 g/L, pH 6.0, and distilled water.
In the step d, the ephedra extract is prepared by crushing ephedra, adding 20 times of water by mass, adding a potassium iodide solution with the volume 0.3 times of that of the water by mass, shearing in a high-speed shearing machine to obtain an ephedra soak solution, centrifuging, taking a filtrate, concentrating to 1.02 times of the original volume to obtain an extract, adding an ethanol solution with the mass concentration of 85% with the volume 3 times of the extract, stirring, standing for 48 hours, centrifuging at 1000r/min for 100S for 3 times, collecting precipitates, and freeze-drying to obtain the extract containing the ephedra polysaccharide with the content of 99%. Adding fresh bamboo leaves into an ethanol solution with the mass concentration of 85%, quickly grinding the mixture into pulp, recovering ethanol, adding 10 times of mass water into the precipitate for ultrasonic extraction, filtering the obtained leaching liquor by an ultrafiltration membrane, concentrating the filtrate under reduced pressure to 1.1 times of the original volume, adding the ethanol solution with the mass fraction of 75% at 4 ℃, stirring, standing, filtering, taking the precipitate, adding ethyl acetate for extraction, taking the supernatant, concentrating to 1.1 times of the original volume, spray-drying to obtain an extract containing 99% of bamboo leaf polysaccharide, drying and crushing tremella, leaching 3 times by a hot bath alcohol extraction method, combining the leaching liquor for 3 times, adding a Sevage reagent to remove protein, concentrating the filtrate to 1/3 of the original volume, adding an ethanol solution with the mass concentration of 95% to ensure that the alcohol content of the concentrated solution reaches 75%, standing for 24 hr, vacuum filtering, and spray drying to obtain extract containing 99% of Tremella polysaccharide.
Example 4:
a method for reducing cyanide content in cassava residue comprises mixing yeast and actinomucor elegans at a volume ratio of 1:1 to obtain a mixed bacterial liquid, mixing the mixed bacterial liquid 15m L with 200g of cassava residue, and fermenting at 27 deg.C for 60 h.
The yeasts comprise Candida ethanolica and Candida rugosa in a volume ratio of 1:1, the Candida ethanolica is Candida ethanolica Y1(Candida ethamo L ica) separated from buffalo milk and has a preservation number of CCTCCNO: M2016467, the Candida rugosa is Candida rugosa Y7(Candida rugosa) separated from the buffalo milk and has a preservation number of CCTCC NO: M2016468, and the Mucor elegans (Actinomucorens) is purchased from the China general microbiological culture Collection center and has a preservation number of CMCC NO. 3.2468.
The pre-yeast and actinomucor elegans are used for acclimatization culture in advance.
The method for domesticating and culturing the actinomucor elegans comprises the following specific steps:
(1) the strain activation comprises the steps of inoculating actinomucor elegans to a first activation slant culture medium, and culturing for 10 hours at 45 ℃, wherein the first activation slant culture medium comprises 14 g/L of peptone, 6 g/L of beef extract powder, 6 g/L0 of yeast extract powder, 6 g/L of aloe extract, 5 g/L of astragalus extract, 6 g/L of medlar extract, 0.6 g/L of dipotassium hydrogen phosphate, 0.4 g/L of diammonium hydrogen citrate, 0.35 g/L of magnesium sulfate, 0.07 g/L of manganese sulfate, 12 g/L of agar, 6.0 of pH value and distilled water;
(2) domesticating strains: put the inclined plane 0.3cm above2The lawn is inoculated into a 250m L triangular flask filled with 15m L domestication culture medium, the rotation speed of a shaking table is 285rpm, the domestication culture is carried out in a shaking bottle at 30 ℃, each 3 days is a domestication period, 3 periods of domestication are carried out, after the domestication of each period is finished, a culture solution with the highest degradation rate of a cassava residue washing solution is taken as an inoculation source for the next domestication, and the domestication culture medium comprises 15 g/L of the cassava residue washing solution, 3.5 g/L of dipotassium phosphate, 3 g/L of sodium chloride, 0.3 g/L of magnesium sulfate heptahydrate, 0.8 g/L of diamine citrate, 0.15 g/L of ferric sulfate, the pH value is 6.0, and distilled water is prepared;
(3) expanding culture of bacterial liquid, transferring the actinomucor elegans obtained by screening after the last period of acclimation into the expanding culture liquid, expanding culture for 13h in a constant temperature shaking culture device at the rotating speed of 165rpm and the temperature of 30 ℃ until the effective bacterial content of the actinomucor elegans is 3.6 × 108-9.2×108CFU/m L, wherein the enlarged culture solution comprises 8.5 g/L of sweet potato powder, 3.5 g/L of hericium erinaceus extract, 1.5 g/L of acanthopanax senticosus extract, 1.5 g/L of spirulina extract, 1.5 g/L of yeast extract, 1.5 g/L of sodium glutamate, 3 g/L of dipotassium phosphate, 10.15g/L of vitamin B, 0.5 g/L of magnesium sulfate, 6.5 of pH value and distilled water.
The aloe extract in the step (2) is prepared by crushing aloe leaves into pulp, filtering to obtain precipitate, adding 2 times (v/v) of water, extracting with water, taking supernatant, adding 75% ethanol solution, standing, taking precipitate, repeatedly extracting with alcohol for 3 times, combining the precipitate, dissolving the combined precipitate in 4 times (v/v) of water, circularly percolating through a percolation column, keeping the percolation flow rate at 0.3ml/s, extracting for 1.5 hours, taking filtrate, and intercepting the molecular weight with an ultrafiltration membrane at 7000Da to obtain the aloe extract; wherein the aloe extract contains 95% of polysaccharides;
soaking astragalus into an ethanol solution with the mass fraction of 15%, heating for 5 minutes, standing overnight, filtering to obtain a precipitate, pulverizing the precipitate, drying and sieving with a 100-mesh sieve, adding 20 times of mass water and 0.1 time of mass chitosan flocculant, heating to 35 ℃ while stirring at the speed of 100r/min for 15 minutes, stopping heating, cooling, adding an ethanol solution with the mass fraction of 65% which is 0.5 times of the volume (v/v), stirring, standing for 25 hours, and performing ultrafiltration membrane to intercept the molecular weight of 4000Da to obtain the astragalus extract; wherein the content of polysaccharide in the radix astragali extract reaches 95%;
the wolfberry extract is prepared by adding 2 times (v/v) of water into wolfberry for pulping, then oscillating in an ultrasonic oscillator for 3.5 hours at 22KHZ, then filtering and collecting filtrate, then adding sodium carbonate and ethanol solution for extraction, collecting extract, and then concentrating the extract to 1.02 times of the original volume through a double-effect energy-saving concentrator. Wherein the content of polysaccharide in the fructus Lycii extract is 95%.
The hericium erinaceus extract in the step (3) is prepared by slicing hericium erinaceus, adding an ethanol solution with the mass fraction of 20%, soaking for 10 minutes under the assistance of ultrasonic waves, filtering to recover ethanol to obtain a precipitate, adding 27 times of water, heating to 40 ℃ for water bath boiling for 4 hours, filtering to obtain a filtrate, concentrating the filtrate to 2 times of the original volume, adding 75% ethanol, soaking for 48 hours, taking the precipitate, and drying to obtain the hericium erinaceus extract; wherein the content of polysaccharide in the Hericium erinaceus extract reaches 95%;
adding 2-fold water into acanthopanax senticosus, pulping, filtering to obtain a precipitate, adding 35 times of water, heating for water extraction, extracting for 20 minutes under the condition that the temperature of the water extraction is 75 ℃, performing water extraction assisted by the frequency with the ultrasonic power of 1000W during the water extraction, performing water extraction for 3 times, filtering to remove the precipitate, concentrating the filtrate obtained by 3 times to obtain an extract, performing spray drying on the extract to obtain dry powder, sieving by a 1000-mesh sieve, performing alcohol extraction for 3 hours by using 75% by mass of ethanol, and recovering the ethanol to obtain the precipitate, thereby obtaining the acanthopanax senticosus extract; wherein the content of polysaccharide in the acanthopanax senticosus extract reaches 95%;
cleaning and crushing spirulina, performing water bath reflux extraction for 4.5 hours by using ethanol with the mass fraction of 85% in an amount which is 4 times that of the spirulina, filtering to obtain precipitate, adding water with the weight of 15 times that of the precipitate to perform water bath extraction for 4.5 hours, filtering to obtain filtrate, concentrating the filtrate to be 1.1 times that of the original volume, adding ethanol with the mass fraction of 65% to perform precipitation for 28 hours, centrifuging to remove the precipitate, dissolving the precipitate by using distilled water, intercepting liquid with the molecular weight of 5500Da through a filter membrane with the surface being D20 resin, concentrating, and performing spray drying to obtain the spirulina extract; wherein the content of polysaccharide in the spirulina extract reaches 98%.
Furthermore, the domestication and culture method of the candida ethanolica Y1 and the candida rugosa Y7 comprises the following specific steps:
(1) activating strains, namely respectively culturing Candida ethanolica Y1(Candida ethamo L ica) and Candida rugosa Y7(Candida rugosa) which are separated from buffalo milk and are frozen and preserved in a glycerol tube form on a YPD slant culture medium at 35 ℃ for 23 hours, wherein the YPD slant culture medium comprises 3.5 g/L of potato extract powder, 19 g/L of glucose, 13 g/L of agar, 5.8 of pH value and distilled water;
(2) domesticating strains:
a. mixing cassava residue, agricultural wastewater and distilled water according to a mass ratio of 1: 1: 2 stirring and mixing to obtain a mixture for later use;
b. 4 g/L of potato extract powder, 19 g/L of glucose, 3.5 g/L of agar, 5.5 of pH value and distilled water are prepared into a domesticated basic liquid culture medium, then the mixture obtained in the step a is added to be compounded to obtain a compound liquid culture medium, 4 gradients are prepared in the compound liquid culture medium, the volume of the added amount of the mixture in the first compound liquid culture medium is 10% of the volume of the domesticated basic liquid culture medium, the volume of the added amount of the mixture in the second compound liquid culture medium is 20% of the volume of the domesticated basic liquid culture medium, the volume of the added amount of the mixture in the third compound liquid culture medium is 40% of the volume of the domesticated basic liquid culture medium, and the volume of the added amount of the mixture in the fourth compound liquid culture medium is 80% of the volume of the domesticated basic liquid culture medium for standby application;
c. respectively taking the activated Candida ethanolica Y1(Candida ethanolica L ica) and Candida rugosa Y7(Candida rugosa) in the step (1) according to the inoculum size of 10%, respectively inoculating the activated Candida ethanolica Y1 and Candida rugosa Y7 into a first compound liquid culture medium of 100m L, culturing for 10h at 22 ℃ and 100r/min, respectively inoculating the cultured bacteria liquid into a second compound liquid culture medium of 100m L according to the inoculum size of 10%, culturing for 10h at 27 ℃ and 200r/min, respectively inoculating the cultured bacteria liquid into a third compound liquid culture medium of 100m L according to the inoculum size of 15%, culturing for 10h at 32 ℃ and 250r/min, respectively inoculating the cultured bacteria liquid into a fourth compound liquid culture medium of 100m L according to the inoculum size of 15%, culturing for 10h at 35 ℃ and 300r/min, and then screening and obtaining the domesticated Candida ethanolica Y1(Candida rugosa L Candida rugosa 7) and the domesticated Candida rugosa Y7;
d. expanding culture of acclimatized strain, namely taking 1m L acclimatized Candida ethanolica Y1(Candida acethamo L ica) and acclimatized Candida rugosa Y7(Candida rugosa) to respectively transfer into a 250m L triangular flask filled with 30m L second expanding culture solution, expanding culture for 11h in a constant-temperature shaking culture apparatus at the rotating speed of 185rpm at the temperature of 32 ℃ until the effective bacteria content of the Candida ethanolica per ml is 2.2 × 108-5.5×108CFU/m L, the effective bacteria content of the candida rugosa is 2.1 × 10 per milliliter8-4.8×108CFU/m L, wherein the second enlarged culture solution comprises herba Ephedrae extract 3.5 g/L, folium Bambusae extract 4 g/L, Tremella extract 4 g/L, ammonium sulfate 2 g/L, potassium dihydrogen phosphate 2 g/L, magnesium sulfate 0.8 g/L, pH 6.0, and distilled water.
In the step d, the ephedra extract is prepared by crushing ephedra, adding 17 times of water by mass, adding a potassium iodide solution with the volume 0.3 times of that of the water by mass, shearing in a high-speed shearing machine to obtain an ephedra soak solution, centrifuging, taking a filtrate, concentrating to 1.02 times of the original volume to obtain an extract, adding an ethanol solution with the mass concentration of 85% with the volume 3 times of the extract, stirring, standing for 48 hours, centrifuging at 1000r/min for 100S for 3 times, collecting precipitates, and freeze-drying to obtain an extract containing the ephedra polysaccharide with the content of 99%; adding fresh bamboo leaves into an ethanol solution with the mass concentration of 85%, quickly grinding the mixture into pulp, recovering ethanol, adding 10 times of mass water into the precipitate for ultrasonic extraction, filtering the obtained leaching liquor by an ultrafiltration membrane, concentrating the filtrate under reduced pressure to 1.1 times of the original volume, adding the ethanol solution with the mass fraction of 75% at 4 ℃, stirring, standing, filtering, taking the precipitate, adding ethyl acetate for extraction, taking the supernatant, concentrating to 1.1 times of the original volume, spray-drying to obtain an extract containing 99% of bamboo leaf polysaccharide, drying and crushing tremella, leaching 3 times by a hot bath alcohol extraction method, combining the leaching liquor for 3 times, adding a Sevage reagent to remove protein, concentrating the filtrate to 1/3 of the original volume, adding an ethanol solution with the mass concentration of 95% to ensure that the alcohol content of the concentrated solution reaches 75%, standing for 24 hr, vacuum filtering, and spray drying to obtain extract containing 99% of Tremella polysaccharide.
Example 5:
a method for reducing cyanide content in cassava residue comprises mixing yeast and actinomucor elegans at a volume ratio of 3:2 to obtain a mixed bacterial liquid, mixing the mixed bacterial liquid with 18m L and 225g of cassava residue, and fermenting at 30 ℃ for 75 h.
The yeasts comprise Candida ethanolica and Candida rugosa in a volume ratio of 2:1, the Candida ethanolica is Candida ethanolica Y1(Candida ethamo L ica) separated from buffalo milk and has a preservation number of CCTCCNO: M2016467, the Candida rugosa is Candida rugosa Y7(Candida rugosa) separated from the buffalo milk and has a preservation number of CCTCC NO: M2016468, and the Mucor elegans (Actinomucorens) is purchased from the China general microbiological culture Collection center and has a preservation number of CMCC NO. 3.2468.
The pre-yeast and actinomucor elegans are used for acclimatization culture in advance.
The method for domesticating and culturing the actinomucor elegans comprises the following specific steps:
(1) the strain activation comprises the steps of inoculating actinomucor elegans on a first activation slant culture medium, and culturing for 15h at 46 ℃, wherein the first activation slant culture medium comprises 17 g/L of peptone, 7 g/L of beef extract powder, 7 g/L0 of yeast extract powder, 7 g/L of aloe extract, 6 g/L of astragalus extract, 8 g/L of medlar extract, 0.7 g/L of dipotassium hydrogen phosphate, 0.45 g/L of diammonium hydrogen citrate, 0.45 g/L of magnesium sulfate, 0.09 g/L of manganese sulfate, 14 g/L of agar, 6.0 of pH value and distilled water;
(2) domesticating strains: put the inclined plane 0.3cm above2The lawn is inoculated into a 250m L triangular flask filled with 15m L domestication culture medium, the rotation speed of a shaking table is 290rpm, the shaking table is used for domestication culture at 29 ℃, a domestication period is formed every 3 days, 3 periods of domestication are carried out, after the domestication of each period is finished, a culture solution with the highest degradation rate of a cassava residue washing solution is taken as an inoculation source for the next domestication, and the domestication culture medium comprises 18 g/L of the cassava residue washing solution, 4.5 g/L of dipotassium phosphate, 3.5 g/L of sodium chloride, 0.4 g/L of magnesium sulfate heptahydrate, 1.0 g/L of diamine citrate, 0.25 g/L of ferric sulfate, the pH value is 6.0, and distilled water is prepared;
(3) expanding culture of bacterial liquid, transferring the actinomucor elegans obtained by screening after the last period of acclimation into the expanding culture liquid, expanding culture for 14h in a constant temperature shaking culture device at the rotating speed of 165rpm and the temperature of 30 ℃ until the effective bacterial content of the actinomucor elegans is 3.6 × 108-9.2×108CFU/m L, wherein the enlarged culture solution comprises sweet potato powder 9 g/L, Hericium erinaceus extract 4.5 g/L, Acanthopanax senticosus extract 2.5 g/L, Spirulina extract 2.5 g/L, yeast extract 2.5 g/L, sodium glutamate 2.5 g/L, dipotassium phosphate 4.5 g/L, vitamin B10.25g/L, magnesium sulfate 0.65 g/L, pH 6.5, and distilled water.
The aloe extract in the step (2) is prepared by crushing aloe leaves into pulp, filtering to obtain precipitate, adding 2 times (v/v) of water, extracting with water, taking supernatant, adding 75% by mass of ethanol solution, standing, taking precipitate, repeating ethanol extraction for 3 times, combining the precipitate, dissolving the combined precipitate in 4 times (v/v) of water, circulating and percolating in a percolation column, keeping the flow rate of the percolation liquid at 0.4ml/s, extracting for 1.5 hours, taking filtrate, and intercepting the molecular weight of the percolation liquid by an ultrafiltration membrane at 7600Da to obtain the aloe extract; wherein the content of polysaccharide in the aloe extract reaches 97%;
soaking astragalus into an ethanol solution with the mass fraction of 15%, heating for 5 minutes, standing overnight, filtering to obtain a precipitate, pulverizing the precipitate, drying and sieving with a 100-mesh sieve, adding 40 times of mass water and 0.1 time of mass chitosan flocculant, heating to 35 ℃ while stirring at the speed of 100r/min for 17 minutes, stopping heating, cooling, adding an ethanol solution with the mass fraction of 65% which is 0.5 times of the volume (v/v), stirring, standing for 27 hours, and performing ultrafiltration membrane to intercept the molecular weight of 4000Da to obtain the astragalus extract; wherein the content of polysaccharide in the radix astragali extract reaches 98%;
the wolfberry extract is prepared by adding 2 times (v/v) of water into wolfberry for pulping, then oscillating in an ultrasonic oscillator for 3.5 hours at 24KHZ, then filtering and collecting filtrate, then adding sodium carbonate and ethanol solution for extraction, collecting extract, and then concentrating the extract to 1.02 times of the original volume through a double-effect energy-saving concentrator. Wherein the content of polysaccharide in the fructus Lycii extract is 98%.
The hericium erinaceus extract in the step (3) is prepared by slicing hericium erinaceus, adding an ethanol solution with the mass fraction of 20%, soaking for 10 minutes under the assistance of ultrasonic waves, filtering to recover ethanol to obtain a precipitate, adding 28 times of water, heating to 40 ℃ for water bath boiling for 4 hours, filtering to obtain a filtrate, concentrating the filtrate to 2 times of the original volume, adding 75% ethanol, soaking for 48 hours, taking the precipitate, and drying to obtain the hericium erinaceus extract; wherein the content of polysaccharide in the Hericium erinaceus extract reaches 96%;
adding 2-fold water into acanthopanax senticosus, pulping, filtering to obtain a precipitate, adding 38 times of water, heating for water extraction, extracting for 20 minutes under the condition that the temperature of the water extraction is 75 ℃, performing water extraction with the frequency of 1000W of ultrasonic power during the water extraction, performing water extraction for 3 times, filtering to remove the precipitate, concentrating the filtrate obtained by 3 times to obtain an extract, performing spray drying on the extract to obtain dry powder, sieving with a 1000-mesh sieve, performing alcohol extraction for 4 hours by using 75% by mass of ethanol, and recovering the ethanol to obtain the precipitate, thereby obtaining the acanthopanax senticosus extract; wherein the content of polysaccharide in the radix Acanthopanacis Senticosi extract reaches 96%;
cleaning and crushing spirulina, performing water bath reflux extraction for 4.5 hours by using ethanol with the mass fraction of 85% in an amount which is 4 times that of the spirulina, filtering to obtain precipitate, adding water with the weight of 18 times that of the precipitate to perform water bath extraction for 4.5 hours, filtering to obtain filtrate, concentrating the filtrate to be 1.1 times that of the original volume, adding ethanol with the mass fraction of 65% to perform precipitation for 28 hours, centrifuging to remove the precipitate, dissolving the precipitate by using distilled water, intercepting liquid with the molecular weight of 5500Da through a filter membrane with the surface being D20 resin, concentrating, and performing spray drying to obtain the spirulina extract; wherein the content of polysaccharide in Spirulina extract is up to 96%.
Furthermore, the domestication and culture method of the candida ethanolica Y1 and the candida rugosa Y7 comprises the following specific steps:
(1) the strain activation, namely respectively culturing Candida ethanolica Y1(Candida ethamo L ica) and Candida rugosa Y7(Candida rugosa) which are separated from buffalo milk and are frozen and preserved in a glycerol tube form on YPD slant culture media at 35 ℃ for 23 hours, wherein the YPD slant culture media comprise potato extract powder 4.5 g/L, glucose 19 g/L, agar 14 g/L, pH value 5.8 and distilled water preparation;
(2) domesticating strains:
a. mixing cassava residue, agricultural wastewater and distilled water according to a mass ratio of 1: 1: 2 stirring and mixing to obtain a mixture for later use;
b. b, preparing potato extract powder by 4.5 g/L, glucose by 19 g/L, agar by 4.5 g/L, pH value by 5.5 and distilled water to obtain a domestication base liquid culture medium, adding the mixture obtained in the step a, and compounding to obtain a compound liquid culture medium, wherein the compound liquid culture medium is totally provided with 4 gradients, the volume of the added amount of the mixture in the first compound liquid culture medium is 10% of the volume of the domestication base liquid culture medium, the volume of the added amount of the mixture in the second compound liquid culture medium is 20% of the volume of the domestication base liquid culture medium, the volume of the added amount of the mixture in the third compound liquid culture medium is 40% of the volume of the domestication base liquid culture medium, and the volume of the added amount of the mixture in the fourth compound liquid culture medium is 80% of the volume of the domestication base liquid culture medium for later use;
c. respectively taking activated Candida ethanolica Y1(Candida ethanolica L ica) and Candida rugosa Y7(Candida rugosa) in the step (1), respectively inoculating the activated Candida ethanolica Y1 and Candida rugosa Y7(Candida rugosa) into a first compound liquid culture medium of 100m L according to the inoculation amount of 10%, culturing for 10h at 24 ℃ and 100r/min, respectively inoculating the cultured bacteria liquid into a second compound liquid culture medium of 100m L according to the inoculation amount of 10%, culturing for 10h at 28 ℃ and 200r/min, respectively inoculating the cultured bacteria liquid into a third compound liquid culture medium of 100m L according to the inoculation amount of 15%, culturing for 10h at 32 ℃ and 250r/min, respectively inoculating the cultured bacteria liquid into a fourth compound liquid culture medium of 100m 7 according to the inoculation amount of 15%, screening for 10h at 36 ℃ and 300r/min, and then performing acclimatization treatment to obtain acclimatized Candida ethanolica Y1(Candida ethanolica L) and Candida rugosa 7(Candida rugosa L);
d. expanding culture of acclimatized strain, namely taking 1m L acclimatized Candida ethanolica Y1(Candida acethamo L ica) and acclimatized Candida rugosa Y7(Candida rugosa) to respectively transfer into a 250m L triangular flask filled with 30m L second expanding culture solution, expanding culture for 12 hours in a constant-temperature shaking culture apparatus at the rotating speed of 190rpm at 34 ℃ until the effective bacteria content of the Candida ethanolica per ml is 2.2 × 108-5.5×108CFU/m L, the effective bacteria content of the candida rugosa is 2.1 × 10 per milliliter8-4.8×108CFU/m L, wherein the second enlarged culture solution comprises herba Ephedrae extract 4.5 g/L, folium Bambusae extract 4.5 g/L, Tremella extract 4.5 g/L, ammonium sulfate 2.5 g/L, potassium dihydrogen phosphate 2.5 g/L, magnesium sulfate 0.9 g/L, pH 6.0, and distilled water.
In the step d, the ephedra extract is prepared by crushing ephedra, adding 19 times of water by mass, adding potassium iodide solution with the volume 0.25 times of that of the water by mass, shearing in a high-speed shearing machine to obtain ephedra soak solution, centrifuging, taking filtrate, concentrating to 1.02 times of the original volume to obtain extract, adding 3 times of ethanol solution with the mass concentration of 85% into the extract, stirring, standing for 48 hours, centrifuging at 1000r/min for 100S, collecting precipitate for 3 times, and freeze-drying to obtain the extract containing the ephedra polysaccharide of 99%. Adding fresh bamboo leaves into an ethanol solution with the mass concentration of 85%, quickly grinding the mixture into pulp, recovering ethanol, adding 10 times of mass water into the precipitate for ultrasonic extraction, filtering the obtained leaching liquor by an ultrafiltration membrane, concentrating the filtrate under reduced pressure to 1.1 times of the original volume, adding the ethanol solution with the mass fraction of 75% at 4 ℃, stirring, standing, filtering, taking the precipitate, adding ethyl acetate for extraction, taking the supernatant, concentrating to 1.1 times of the original volume, spray-drying to obtain an extract containing 99% of bamboo leaf polysaccharide, drying and crushing tremella, leaching 3 times by a hot bath alcohol extraction method, combining the leaching liquor for 3 times, adding a Sevage reagent to remove protein, concentrating the filtrate to 1/3 of the original volume, adding an ethanol solution with the mass concentration of 95% to ensure that the alcohol content of the concentrated solution reaches 75%, standing for 24 hr, vacuum filtering, and spray drying to obtain extract containing 99% of Tremella polysaccharide.
Example 6;
the test for fermentation degradation of cyanide content in cassava residue was carried out on Candida ethanolica Y1(Candida ethano L ica) with preservation number of CCTCC NO: M2016467, Candida rugosa Y7(Candida rugosa) with preservation number of CCTCC NO: M2016468, and Rhizomucor elegans (Actinomucorellas) with preservation number of CMCC NO.3.2468, which were purchased from China general microbiological culture Collection, and examples 2-5, which were respectively acclimatized Candida ethanolica Y1(Candida ethano L ica), Candida rugosa Y7(Candida rugosa) and Rhizomucor elegans (Actinomucorellas) as study subjects.
Shaking culturing the microorganisms of the control group and the examples in a liquid enrichment medium, inoculating 2m L bacterial liquid into 250m L the same culture solution containing KCN 100 mg/L when the culture OD value is 1.0, culturing for 48 hours under the same conditions, centrifuging for 10min at 3000r/min, collecting the supernatant 100m L, and measuring CN-And (4) calculating the cyanogen reduction rate according to the cyanogen concentration before and after the culture solution treatment, wherein the cyanogen reduction rate is (the cyanogen concentration in the culture solution before the reaction-the cyanogen concentration in the culture solution after the reaction)/the cyanogen concentration in the culture solution before the reaction, and performing three parallel detections for each detection, and taking an average value.
TABLE 6 cyanogen reduction
As can be seen from the above table, the cyanogen reduction rate of the domesticated Candida ethanolica Y1 is improved by more than 20% compared with the cyanogen reduction rate of the Candida ethanolica Y1 which is not domesticated by the method, the cyanogen reduction rate of the domesticated Candida rugosa Y7 is improved by more than 22% compared with the cyanogen reduction rate of the Candida rugosa Y7 which is not domesticated by the method, the cyanogen reduction rate of the domesticated Mucor elegans is improved by more than 22% compared with the cyanogen reduction rate of the domesticated Mucor elegans which is not domesticated by the method, firstly, the Aloe extract 5-8 g/L, the radix astragali extract 3-7 g/L and the medlar extract 5-9 g/L are used for replacing the conventional power element of glucose when the strains of the actinomyces elegans are activated, the Aloe extract, the radix astragali extract and the medlar extract all contain high-purity Aloe polysaccharide, the lycium barbarum extract, the polysaccharide extracted by contrast, not only contains one energy power element of glucose, but also contains galactose, xylose, the extract can be used as a medium for enhancing the activated culture of the activated bacterium, the bacterium can be used for enhancing the growth resistance of the bacterium of the actinomyces after the bacterium, the bacterium can be used for enhancing the bacterium, the bacterium can be used for enhancing the bacterium, the bacterium can be used for enhancing the bacterium8-9.2×108If the CFU/m L is cultured for 12-15h by using glucose instead of other culture media with unchanged proportion, the effective bacteria content can only reach 1.02 × 108-1.59×108CFU/m L Candida ethanolicaA mode of gradient domestication and screening is adopted in the domestication process of the yeast Y1 and the Candida rugosa Y7, cassava residue washing liquid and agricultural wastewater are added into a domestication liquid culture medium, the cassava residue washing liquid adopted in the application contains a large amount of harmful substances such as sulfide and cyanide, the agricultural wastewater also contains a large amount of harmful substances such as sulfide and cyanide, the pH value is far higher than a safety value, after the domestication of the concentration of the harmful substances is increased in a gradient manner, the domesticated Candida rugosa Y1 and Candida rugosa Y7 have better capacity of resisting severe environment and degrading sulfide and cyanide, a new way for treating the harmful substances in the cassava residue washing liquid and the agricultural wastewater is provided, the respective reduction rate of the hydrogen sulfide concentration in the cassava residue washing liquid and the agricultural wastewater after the microbial domestication is detected to reach 10.5 percent, 22.5%, the reduction rate of cyanide concentration reached 23.1%, 32.2%, wherein the reduction rate = (cyanogen/sulfate-containing concentration in the culture solution before reaction-cyanogen/sulfate-containing concentration in the culture solution after reaction)/cyanogen/sulfate-containing concentration in the culture solution before reaction.
Example 7:
control 1. 18m L Yazhi Mucor was mixed with 225g cassava residue and fermented at 30 ℃ for 75 hours.
Control group 2, 18m L Candida ethanolica Y1 was mixed with 225g cassava dregs and fermented at 30 ℃ for 75 h.
Control 3. 18m L Candida rugosa Y7 was mixed with 225g cassava dregs and fermented at 30 ℃ for 75 h.
Control group 4. 9m L Candida ethanolica Y1, 9m L Candida rugosa Y7 and 225g cassava dregs were mixed and fermented at 30 ℃ for 75 hours.
Control group 5, 9m L Yazhi Mucor, 9m L Candida ethanolica Y1 and 225g cassava residue were mixed and fermented at 30 ℃ for 75 hours.
Control 6, 9m L Yazhi Mucor, 9m L Candida rugosa Y7 and 225g cassava dregs were mixed and fermented at 30 ℃ for 75 hours.
Control group 7, 6m L Yazhi Mucor, 6m L Candida ethanolica Y1, 6m L Candida rugosa Y7 and 225g cassava residue were mixed and fermented at 30 ℃ for 75 hours.
None of the control groups 1-7 microorganisms were acclimatized.
CN is carried out on the cassava dregs treated by the modes of the control groups 1 to 7 and the examples 2 to 5-And (4) residual concentration detection, namely calculating the cyanogen reduction rate according to the cyanogen content of the cassava residue before and after fermentation treatment, wherein the cyanogen reduction rate is (the cyanogen concentration in the cassava residue before reaction-the cyanogen concentration in the cassava residue after reaction)/the cyanogen concentration in the cassava residue before reaction, and performing three parallel detections for each detection, and taking an average value.
TABLE 7
As can be seen from the above table, the cyanogen reduction rate of the control groups 1-3 almost coincides with the detection value of the control groups 1-3 in example 6, and the coincidence rate is more than 95%; the cyanide reducing capability of the bacteria liquid of the comparison groups 4-6 is reduced by 10 percent compared with that of the comparison group 7 by pairwise combination; the cyanide reducing capability of the bacteria liquid of the comparison groups 4-6 is slightly better than that of the comparison groups 1-3 in pairwise combination, and the cyanide reducing rate is at least 15% lower than that of the bacteria liquid of the examples 2-5; the difference of cyanogen reduction of comparative group 7 compared with examples 2-5 is at least 5%; therefore, the combined bacterial liquid domesticated by the method has more excellent CN in the cassava residues-The content of (a).
The above examples are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but not to be construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.
Claims (1)
1. A method for reducing cyanide content in cassava residue is characterized in that yeast and actinomucor elegans are mixed according to a volume ratio of 1-5:1-3 to obtain mixed bacterial liquid, then the mixed bacterial liquid of 10-20m L is mixed with 250g of cassava residue, and fermentation is carried out for 48-96h at 25-31 ℃;
the yeast and the actinomucor elegans are both subjected to acclimation culture before use;
the method for domesticating and culturing the actinomucor elegans comprises the following specific steps:
(1) inoculating actinomucor elegans to a first activated slant culture medium, and culturing for 8-16h at 40-50 ℃, wherein the first activated slant culture medium comprises peptone 12-18 g/L, beef extract powder 5-8 g/L, yeast extract powder 5-8 g/L0, aloe extract 5-8 g/L, astragalus extract 3-7 g/L, medlar extract 5-9 g/L, dipotassium hydrogen phosphate 0.5-0.8 g/L, diammonium hydrogen citrate 0.3-0.5 g/L, magnesium sulfate 0.3-0.5 g/L, manganese sulfate 0.05-0.1 g/L, agar 10-15 g/L, pH 6.0, and distilled water;
(2) domesticating strains: put the inclined plane 0.3cm above2The lawn is inoculated into a 250m L triangular flask filled with 15m L domestication culture medium, the rotating speed of a shaking table is 280 plus 300rpm, the shaking table is used for domestication culture at 29-31 ℃, each 3 days is a domestication period, domestication is carried out for 3 periods, and after the domestication of each period is finished, a culture solution with the highest degradation rate of a cassava residue washing solution is taken as an inoculation source for the next domestication, wherein the domestication culture medium comprises 10-20 g/L of the cassava residue washing solution, 3-5 g/L of dipotassium phosphate, 2-5 g/L of sodium chloride, 0.2-0.45 g/L of magnesium sulfate heptahydrate, 0.5-1.2 g/L of diamine citrate, 0.1-0.3 g/L of ferric sulfate, the pH value is 6.0, and distilled water is prepared;
(3) expanding culture of bacterial liquid, transferring the actinomucor elegans obtained by screening treatment after the last period acclimation into the expanding culture liquid, expanding culture for 12-15h in a constant temperature shaking culture device at the rotating speed of 160-170rpm and the temperature of 29-31 ℃ until the effective bacterial content of the actinomucor elegans is 3.6 × 108-9.2×108CFU/m L, wherein the enlarged culture solution comprises sweet potato powder 8-10 g/L, Hericium Erinaceus extract 3-5 g/L, radix Acanthopanacis Senticosi extract 1-3 g/L, Spirulina extract 1-3 g/L, yeast extract 1-3 g/L, sodium glutamate 1-3 g/L, dipotassium phosphate 2-5 g/L, and vitamin B10.1-0.3 g/L, magnesium sulfate 0.3-0.8 g/L, pH 6.5, and distilled water;
the yeasts comprise candida ethanolica and candida rugosa, and the volume ratio of the candida ethanolica to the candida rugosa is 1-3: 1-2;
the Candida ethanolica is Candida ethanolica Y1 separated from buffalo milk (C. ethanolica:)Candida ethamoLica) M2016467 with the preservation number of CCTCC NO; the Candida rugosa is Candida rugosa Y7 separated from buffalo milk (a)Candida rugosa) M2016468 with the preservation number of CCTCC NO; the actinomucor elegans (A), (B), (CActinomucorelegans) Purchased from China general microbiological culture Collection center with the preservation number of CMCCNO.3.2468;
the domestication and culture method of the candida ethanolica Y1 and the candida rugosa Y7 comprises the following specific steps of:
(1) activating strains: candida ethanolica Y1 (isolated from buffalo milk and cryopreserved in the form of glycerol tubes)Candida ethamoLica) And Candida rugosa Y7 (C.rugosa Y7)Candida rugosa) Respectively culturing on YPD slant culture medium at 34-36 deg.C for 22-24 hr, wherein the YPD slant culture medium comprises potato extract powder 3-5 g/L, glucose 18-20 g/L, agar 12-15 g/L, pH 5.8, and distilled water;
(2) domesticating strains:
a. mixing cassava residue, agricultural wastewater and distilled water according to a mass ratio of 1: 1: 2 stirring and mixing to obtain a mixture for later use;
b. 3-5 g/L of potato extract powder, 18-20 g/L of glucose, 3-5 g/L of agar, 5.5 of pH value and distilled water are prepared to obtain a domestication base liquid culture medium, then the mixture obtained in the step a is added to be compounded to obtain a compound liquid culture medium, 4 gradients are prepared in the compound liquid culture medium, the volume of the adding amount of the mixture in the first compound liquid culture medium is 10% of the volume of the domestication base liquid culture medium, the volume of the adding amount of the mixture in the second compound liquid culture medium is 20% of the volume of the domestication base liquid culture medium, the volume of the adding amount of the mixture in the third compound liquid culture medium is 40% of the volume of the domestication base liquid culture medium, and the volume of the adding amount of the mixture in the fourth compound liquid culture medium is 80% of the volume of the domestication base liquid culture medium for standby;
c. respectively taking the Candida ethanolica Y1 (activated in the step (1))Candida ethamoLica) And Candida rugosa Y7 (C.rugosa Y7)Candida rugosa) Inoculating into 100m L first compound liquid culture medium according to 10% inoculum size, culturing at 20-25 deg.C and 100r/min for 10h, inoculating into 100m L second compound liquid culture medium according to 10% inoculum size, culturing at 25-30 deg.C and 200r/min for 10h, inoculating 15% inoculum size into 100m L third compound liquid culture medium, culturing at 31-33 deg.C and 250r/min for 10h, inoculating 15% inoculum size into 100m L fourth compound liquid culture medium, culturing at 33-37 deg.C and 300r/min for 10h, and screening to obtain acclimatized Candida ethanolica Y1(Candida ethanolica Y1)Candida ethamoLica) And acclimatized Candida rugosa Y7(Candida rugosa）；
d. Expanding culture of acclimatized strain, namely acclimatizing 1m L to obtain Candida ethanolica Y1 (C)Candida ethamoLica) And acclimatized Candida rugosa Y7(Candida rugosa) Transferring into 250m L triangular flask containing 30m L second amplification culture solution, and performing amplification culture at 30-35 deg.C for 10-13h in constant temperature shaking culture apparatus at 180-200rpm until the effective bacteria content of Candida ethanolica per ml is 2.2 × 108-5.5×108CFU/m L, the effective bacteria content of the candida rugosa is 2.1 × 10 per milliliter8-4.8×108CFU/m L, wherein the second enlarged culture solution comprises herba Ephedrae extract 3-5 g/L, folium Bambusae extract 3-5 g/L, Tremella extract 3-5 g/L, ammonium sulfate 1-3 g/L, potassium dihydrogen phosphate 1-3 g/L, magnesium sulfate 0.5-1.0 g/L, pH 6.0, and distilled water.
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| CN108651162A (en) * | 2018-04-25 | 2018-10-16 | 山东老来寿生物工程有限公司 | A kind of preparation method and products thereof of Antialcoholic liver-protecting Agricus blazei zymotic fluid |
| CN109735460B (en) * | 2019-03-01 | 2022-02-22 | 深圳市诚致生物开发有限公司 | Detoxification method for flax cake by solid state fermentation of composite strain |
| CN111500471B (en) * | 2020-05-10 | 2022-09-06 | 浙江澄宇环保新材料股份有限公司 | Repairing agent for treating cyanide-containing tailing slag |
| CN111855853A (en) * | 2020-07-27 | 2020-10-30 | 海南省农业科学院热带园艺研究所 | Method for quickly and efficiently extracting cyanogenic glycosides from cassava leaves and root tubers |
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Application publication date: 20171226 Assignee: Guangxi Yulin Yiliang Technology Co.,Ltd. Assignor: GUANGXI ZHUANG AUTONOMOUS REGION BUFFALO INSTITUTE Contract record no.: X2023980045755 Denomination of invention: A Method for Reducing the Cyanide Content of Cassava Residue Granted publication date: 20200728 License type: Common License Record date: 20231107 |
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