CN115820471A - Method for extracting chlorella pyrenoidosa protein by microbial fermentation method - Google Patents
Method for extracting chlorella pyrenoidosa protein by microbial fermentation method Download PDFInfo
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Abstract
The invention discloses a method for extracting chlorella pyrenoidosa protein by using a microbial fermentation method, which is characterized by comprising the following steps: (1) Inoculating Bacillus belgii in a culture medium for activation; (2) Mixing chlorella pyrenoidosa serving as a raw material with pure water, pretreating the raw material, and adjusting the pH value to 8.0-9.5 to obtain a fermentation substrate; (3) Inoculating Bacillus belgii into a fermentation substrate, and performing liquid fermentation to obtain a fermentation liquid; (4) Centrifuging the fermentation liquor, removing residues, concentrating, and drying to obtain the final product; its advantages are high extracting efficiency and purity.
Description
Technical Field
The invention relates to a preparation method of a microalgae extract, in particular to a method for extracting chlorella pyrenoidosa protein by using a microbial fermentation method.
Background
As a green nutrient source health food in the twenty-first century, the chlorella pyrenoidosa contains rich active substances such as protein, polysaccharide, polyunsaturated fatty acid, vitamins and the like, so that the chlorella pyrenoidosa has the functions of regulating lipid metabolism in vivo and preventing and improving related metabolic diseases. Wherein the protein content is 60%, which is higher than high protein food such as beef and soybean, and can be used as high quality raw material of protein extract. However, the development thereof has been in the first stage due to problems of strong algal smell, low protein utilization rate, and too hard cell wall to be handled. Research shows that the aromatic substances generated in the microbial fermentation process can endow the fermentation liquor with good flavor, so that the peculiar smell of algae is effectively reduced. Meanwhile, the microbial fermentation method has the advantages of milder reaction conditions, short production period, low cost, higher yield and the like. Avoids the influence of high pressure and high shearing force related to a repeated freezing and thawing method, an ultrasonic crushing method, an acid-base treatment method and the like which are commonly adopted in the research, and reduces the damage to active substances. And fermentation usually decomposes macromolecular polymers into small molecules through microbial metabolism and the action of enzymes, so that the nutrient utilization rate and the functional value of active ingredients can be improved. At present, no relevant research report of a method for extracting chlorella pyrenoidosa protein by using a microbial fermentation method is published at home and abroad.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for extracting chlorella pyrenoidosa protein by a microbial fermentation method, wherein the extraction efficiency and the purity are high.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for extracting Chlorella pyrenoidosa protein by using a microbial fermentation method comprises the following steps:
(1) Inoculating Bacillus belgii CICC 25171 in a culture medium for activation;
(2) Mixing the raw material chlorella pyrenoidosa with pure water, carrying out raw material pretreatment, and adjusting the pH value to 8.0-9.5 to obtain a fermentation substrate;
(3) Inoculating Bacillus beilesiensis CICC 25171 into a fermentation substrate, and performing liquid fermentation to obtain a fermentation liquid;
(4) Centrifuging the fermentation liquor, removing residues, concentrating, and drying to obtain the final product.
Specifically, the bacillus beilesensis CICC 25171 is inoculated on a YPD solid culture medium by adopting a plate-scribing method for strain purification, the culture medium is placed in a constant-temperature incubator at 37 ℃ for culture for 20h, a single colony is inoculated into a YPD liquid culture medium, the culture is carried out on a shaking table at 37 ℃ for 12h, the inoculation amount of 10% by volume is inoculated into the YPD liquid culture medium again, the amplification culture is carried out, the YPD liquid culture medium is placed on the shaking table at 37 ℃ and 180r/min for shake culture for 8-16h, and the OD value of a bacterial liquid is controlled to be 0.8-1.2, so that the bacillus seed liquid is obtained.
The step (2) is specifically as follows: mixing chlorella pyrenoidosa and pure water according to the mass ratio of 1:8, soaking the mixture in an environment at 4 ℃ for 24h, and mixing the mixture according to the mass ratio of the chlorella powder to the glucose of 50:1, adding glucose, and adjusting the pH to 8.0-9.5 by using 0.1 mol/L NaoH to obtain a fermentation substrate.
The step (3) is specifically as follows: inoculating Bacillus beilesiensis CICC 25171 into a fermentation substrate according to an inoculation ratio of 8-10% in volume ratio, and performing liquid fermentation at 35-40 ℃ for 20-30h to obtain fermentation liquor.
The step (4) is specifically as follows: centrifuging the fermentation liquor at 4 deg.C and 5000 rpm for 10 min, and separating the fermentation liquor from the residue; and (4) carrying out rotary evaporation and concentration on the separated fermentation liquor to 15% of the original volume, and carrying out freeze-drying and powder preparation on the concentrated fermentation liquor to obtain a finished product.
Compared with the prior art, the invention has the advantages that
1. Compared with the traditional extraction mode, the traditional high-pressure high-shear physical wall breaking and acid-base chemical wall breaking can influence the structures and activities of proteins and other active ingredients, and the safety of the protein and the other active ingredients in food and medicine is not realized.
2. The invention adopts the self-sieving Bacillus belgii CICC 25171 (preserved in China center for culture Collection of industrial microorganisms) for fermentation, the method is simple, and large-scale mass production can be realized. The optimal fermentation extraction process parameters are determined by response surface optimization, and the protein extraction rate of the chlorella pyrenoidosa is improved to 61.20 percent while the extraction cost is controlled.
3. The invention adopts high-value and low-price chlorella pyrenoidosa as a raw material, and self-screened bacillus belgii CICC 25171 as a fermentation strain, so that the chlorella pyrenoidosa protein can be extracted with maximum efficiency. Compared with the extraction method which directly uses animal and plant protein powder as raw materials, the method can further reduce the production cost, reasonably develop and utilize ocean resources, and find a high-quality protein extraction raw material.
4. The method can realize the high-efficiency extraction of the protein in the chlorella pyrenoidosa, and simultaneously, the content of other active substances such as polysaccharide, polyphenol and flavonoid compounds in the chlorella pyrenoidosa is obviously increased after the fermentation of the method, so that the value of the chlorella pyrenoidosa can be fully exerted. Therefore, the chlorella pyrenoidosa extract not only can provide high-purity protein, but also has the functions of resisting oxidation, reducing blood fat, regulating body immunity and the like, and improves the nutritional and medical values of the product.
Drawings
FIG. 1 shows the effect of different amounts of Chlorella pyrenoidosa powder on the protein concentration of the fermented Chlorella pyrenoidosa powder;
FIG. 2 is a graph of the effect of initial pH on the protein concentration of fermented protein Chlorella powder;
FIG. 3 is the effect of the addition of the strain on the protein concentration of fermented protein chlorella powder;
FIG. 4 is a graph showing the effect of fermentation temperature on the protein concentration of fermented protein Chlorella powder;
FIG. 5 is a graph showing the effect of fermentation time on the protein concentration of fermented protein Chlorella powder.
Detailed Description
The invention is described in further detail below with reference to the following examples of the drawings.
1. Experimental methods
Method for measuring protein extraction rate
And (3) total protein-Kjeldahl nitrogen determination of algae powder: weighing a fully and uniformly mixed solid sample 1.36 g, adding 0.4 g copper sulfate, 6g potassium sulfate and 20 mL sulfuric acid, and digesting in a digestion furnace. After the temperature of the digestion furnace reaches 420 ℃, continuously digesting 1 h, taking out the liquid in the digestion tube to be green and transparent, cooling, adding 50 mL water, and realizing the processes of automatic liquid adding, distillation, titration and titration data recording on an automatic Kjeldahl apparatus (adding a sodium hydroxide solution, a hydrochloric acid or sulfuric acid standard solution and a boric acid solution containing a mixed indicator A or B before use). The specific process of measuring the protein content of the obtained centrifugal supernatant by using the BCA kit comprises the following steps: the supernatant was diluted 10-fold, and the protein content of the 10 uL dilution was measured by BCA kit at 562 nm wavelength.
2. Detailed description of the preferred embodiments
Method for extracting chlorella pyrenoidosa protein by microbial fermentation method
1. Preparation of seed liquid
The Bacillus belgii CICC 25171 is preserved in an ultralow temperature environment of-80 ℃ and glycerol is used as a protective agent. Taking Bacillus belief CICC 25171 preserved in glycerinum tubing, quickly unfreezing in a water bath kettle at 37 ℃, inoculating on a solid culture medium by adopting a plate scribing method, purifying strains, placing the culture medium in a constant-temperature incubator at 37 ℃ for culturing for 20 hours, taking a single colony to be inoculated into a liquid culture medium, culturing 12h on a shaker at 37 ℃, inoculating into the liquid culture medium again according to 10 percent of inoculation amount, performing amplification culture, placing on a shaker at 37 ℃ and 180r/min for shake culture of 8-16h, and controlling the OD value of a bacterial liquid to be 0.8-1.2.
The solid culture medium used in the preparation process of the seed liquid is YPD solid culture medium, each liter of the culture medium is prepared, and peptone 10.0 g, glucose 20.0 g, yeast extract powder 5g and agar 14.0 g are added into deionized water of 900 mL. Adding deionized water to a constant volume of 1L, heating for dissolving, and autoclaving at 121 deg.C for 15 min.
The liquid culture medium used in the preparation process of the seed liquid is YPD liquid culture medium, each liter of the liquid culture medium is prepared, and peptone 20.0 zxft 3252, glucose 20.0 g and yeast extract powder 10 g are added into deionized water of 900 ml. The pH was adjusted to 6.5 with 5 mol/L NaOH. Adding deionized water to a constant volume of 1L, heating for dissolving, and autoclaving at 121 deg.C for 15 min.
2. Preparing raw materials: mixing chlorella pyrenoidosa algae powder and pure water according to the mass ratio of 1:8, soaking the mixture in an environment at 4 ℃ for 24h, and mixing the mixture according to the mass ratio of the chlorella powder to the glucose of 50:1, adding glucose, and adjusting the pH to 8.0-9.5 by using 0.1 mol/L NaoH. The total amount of protein in the chlorella pyrenoidosa was found to be 64.8% by nitrogen fixation with kjeldahl method.
3. Fermentation: inoculating bacillus beleisi CICC 25171 into a fermentation substrate according to an inoculation proportion (volume fraction, the same below) of 8-10%, wherein the fermentation temperature is 35-40 ℃, placing the fermentation substrate into a shaking table, and the fermentation time is 20-30 h. The protein concentration of the fermentation broth was determined to be 61.2% by BCA kit.
4. Centrifuging the fermentation liquor at 4 deg.C and 5000 rpm for 10 min to separate the fermentation liquor from the residue; and (3) carrying out rotary evaporation and concentration on the separated fermentation liquor to 15% of the original volume, and carrying out freeze-drying and powder preparation on the concentrated fermentation liquor to obtain a finished product.
3. Analysis of test results
1. Single factor experiment determination of protein extraction rate of chlorella pyrenoidosa
(1) Comparative example 1: influence of Chlorella pyrenoidosa powder addition amount on protein concentration in fermentation broth
The Bacillus belgii CICC 25171 is activated according to the steps of the specific embodiment, and the OD value of the bacterial liquid is controlled to be 0.8. Fixing the initial pH of the fermentation liquor to 7, the fermentation temperature to 37 ℃, and the fermentation time to 24h. Respectively setting the feed-liquid ratio (water: chlorella pyrenoidosa) to be 5, 10, 20, 30 and 40 (mL/g), respectively inoculating the Bacillus belgii CICC 25171 into fermentation substrates with different feed-liquid ratios according to the inoculation proportion of 10 percent in volume ratio. The rest of the operations were carried out in accordance with the procedure of the specific examples. The protein extraction was determined using the method of example 3. As shown in FIG. 1, it can be seen from FIG. 1 that the protein concentration in the fermentation broth increases with the feed-to-liquid ratio at the feed-to-liquid ratio, and the protein extraction rate reaches 44.20% at the maximum when the feed-to-liquid ratio reaches 10 ml/g. When the feed-liquid ratio is increased continuously, the protein concentration in the fermentation liquor is reduced, so that the optimum extraction level is obtained when the feed-liquid ratio is 10 mL/g.
(2) Comparative example 2: effect of initial pH on protein concentration of fermented protein Chlorella powder
The Bacillus belgii CICC 25171 is activated according to the steps of the specific embodiment, and the OD value of the bacterial liquid is controlled to be 0.8. The fixed fermentation temperature is 37 ℃, the fermentation time is 24h, the ratio of chlorella pyrenoidosa feed to liquid is 8mL/g, the initial pH is respectively set to be 7, 8, 9, 10 and 11, bacillus belgii CICC 25171 is respectively inoculated into fermentation substrates with different pH values according to the inoculation proportion of 10 percent in volume ratio, and the rest operations are fermented according to the steps of the specific embodiment to determine the protein extraction rate. As shown in FIG. 2, it is understood from FIG. 2 that the protein concentration initially increased with the increase in pH, and the protein extraction rate reached 50.55% at a maximum at pH 8. When the pH is further increased, the protein extraction rate decreases, so that pH 8 is the optimum extraction level at which the protein utilization rate of Chlorella pyrenoidosa is highest.
(3) Comparative example 3: influence of addition amount of strain on protein concentration in fermentation liquid of protein nucleus chlorella powder
Bacillus belgii CICC 25171 was activated according to the procedure of example 1, and the OD value of the bacterial liquid was controlled to 0.8. The initial pH value is fixed to be 7, the fermentation temperature is 37 ℃, the fermentation time is 24h, and the feed-liquid ratio of the chlorella pyrenoidosa is 8mL/g. The Bacillus belgii CICC 25171 is inoculated into the fermentation substrate according to the inoculation ratio of 7%, 8%, 9%, 10% and 11%. The rest of the operations were performed according to the procedure in the specific examples to determine the protein extraction yield. To investigate the effect of the addition of the strain on the yield of protein produced from fermented chlorella pyrenoidosa powder, the results are shown in fig. 3, and it can be seen from fig. 3 that the protein concentration initially increases with the increase in the addition of the strain, the protein extraction rate reaches a maximum of 41.19% when the addition reaches 9%, and the protein concentration decreases when the addition continues to increase, so that the addition of the strain of 9% is the optimum extraction level, at which time the utilization rate of the protein in chlorella pyrenoidosa is highest.
(4) Comparative example 4: influence of fermentation temperature on protein concentration in protein nucleus chlorella powder fermentation liquor
The Bacillus belgii CICC 25171 is activated according to the steps of the specific embodiment, and the OD value of the bacterial liquid is controlled to be 0.8. Fixing the initial pH value to 7, fermenting for 24h, and the feed-liquid ratio of chlorella pyrenoidosa to 8mL/g, and inoculating bacillus belief 25171 into the fermentation substrate according to the inoculation ratio of 10%. The fermentation temperatures were set to 28, 32, 35, 37 and 40 ℃ respectively, and the influence of the fermentation temperatures on the protein yield of the fermented protein chlorella powder was investigated. The rest of the operations were performed according to the procedure in the specific examples to determine the protein extraction yield. As a result, as shown in FIG. 4, it is understood from FIG. 4 that the protein concentration increases with the increase in temperature at the beginning, and the protein extraction rate reached 49.62% at the maximum when the temperature reached 37 ℃. When the fermentation temperature is increased, the protein concentration is reduced, so that the temperature of 37 ℃ is the optimal extraction level, and the utilization rate of the protein in the chlorella pyrenoidosa is highest.
(5) Comparative example 5: influence of fermentation time on protein concentration of fermented protein chlorella powder
Bacillus belgii CICC 25171 was activated according to the procedure of example 1, and the OD value of the bacterial liquid was controlled to 0.8. Fixing the initial pH value to be 7, setting the feed-liquid ratio of the chlorella pyrenoidosa to be 8ml/g, inoculating bacillus belgii CICC 25171 into a fermentation substrate according to the inoculation proportion of 10%, controlling the fermentation temperature to be 37 ℃, and respectively setting the fermentation time to be 12h, 18 h, 24h, 30h and 36 h, and researching the influence of the fermentation time on the protein yield of the fermented chlorella pyrenoidosa powder. The rest of the operations were performed according to the procedure in the specific examples to determine the protein extraction yield. As a result, as shown in FIG. 5, it is understood from FIG. 5 that the protein concentration increases with the increase of the fermentation time at the beginning, the protein extraction rate reaches 39.27% at the maximum when the time reaches 30h, and the protein concentration decreases when the time continues to increase, so that the fermentation time of 30h is the optimum extraction level, and the utilization rate of the protein in the Chlorella pyrenoidosa is the highest at this time.
2. Determination of optimum parameters for extraction process
On the basis of a single-factor test result, selecting pH, inoculum size and liquid-material ratio as variable factors, and optimizing the process for preparing the protein by fermenting the protein chlorella powder by adopting a Box-menken central combined test design in a response surface analysis method. The influence of respective variables and interaction thereof on the protein extraction rate is studied, and three fermentation conditions of pH value, liquid-material ratio and inoculation amount are optimized on three heights of tables 1 and 2.
The design of the response surface experiment and the analysis of the results are shown in Table 1
And (3) performing multiple regression fitting on the experimental data by applying Design-Expert to obtain a quadratic multiple regression model of the extraction rate and the selected 3 factors, wherein the quadratic multiple regression model comprises the following steps: y =37.17-0.53938 × A-2.18 × B-0.2263 × C-7.87 × A2-4.17 × B2-5.03 × C2+0.1423 × AB-1.92 × AC-3.73 × BC;
table 1 response surface experimental design and influence of pH value, liquid-solid ratio and inoculum size on protein extraction of chlorella fermentation broth
Analysis of variance was performed on the regression model, and the results are shown in table 2:
table 2 chlorella protein extraction yield response surface quadratic model analysis of variance optimization.
The variance test of the regression equation model on the experimental results thereof showed that the model has significance (P = 0.1326). The F value (3.4300) indicates a high agreement between the measured and predicted values for the model. The results of the anova showed that the factors had an influence on the protein concentration in the order of initial pH > liquid-to-feed ratio > inoculum size. Determining the coefficients (R2 = 0.9896) indicates a higher degree of fit. The corrected coefficient of certainty (0.9763) is close to the coefficient of certainty, indicating that the model has good statistical significance under comprehensive evaluation.
Through the selection of the protein extraction strain, the single factor experiment and the determination experiment of the optimal parameters of the extraction process, the following steps can be determined: bacillus belgii CICC 25171 is a fermentation strain, and the optimal process conditions are as follows: pH =9.30, liquid-to-feed ratio =8.7, inoculum size =10.00%, validation experiment was performed under optimal conditions, maximum protein concentration was 40.8 mg/mL, maximum protein extraction =61.20%. The regression model of the protein rate of the chlorella pyrenoidosa extracted by microbial fermentation is as follows: y =37.17-0.53938 × A-2.18 × B-0.2263 × C-7.87 × A2-4.17 × B2-5.03 × C2+0.1423 × AB-1.92 × AC-3.73 × BC.
In addition, the invention also measures the change of other active substances in the chlorella pyrenoidosa in the fermentation process. In the fermentation process, the chlorella pyrenoidosa is used as a fermentation substrate to provide energy and nutrition for the bacillus belgii CICC 25171, so that the composition and the physicochemical property of the fermentation liquid are changed. For example: the polysaccharide and one of the main active ingredients of the chlorella have the functions and values of regulating the immunity of the organism, reducing blood sugar, resisting aging, resisting viruses and the like. The analysis of the active ingredients after fermentation is shown in table 3 below.
TABLE 3 fermented and unfermented active ingredient content
The results show that the polysaccharide content after fermentation is increased from 21.65 mg/mL to 40.12 mg/mL. And polyphenol and flavonoid compounds have the effects of resisting oxidation, delaying aging, enhancing immunity, inhibiting tumor, reducing cholesterol and the like, and are important components of vegetable food. The results show that the total polyphenol content and the total flavone content are respectively increased from 10.19 mu g/mL and 12.18 mg/mL to 24.41 mu g/mL and 27.50 mg/mL. Besides the protein which is the main extract of the chlorella pyrenoidosa, the active ingredients fully exert the utilization value of the chlorella pyrenoidosa and improve the nutrition and medical value of the product.
The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art should also realize that changes, modifications, additions and substitutions can be made without departing from the true spirit and scope of the invention.
Claims (5)
1. A method for extracting chlorella pyrenoidosa protein by using a microbial fermentation method is characterized by comprising the following steps:
(1) Inoculating Bacillus belgii CICC 25171 in a culture medium for activation;
(2) Mixing chlorella pyrenoidosa serving as a raw material with pure water, pretreating the raw material, and adjusting the pH value to 8.0-9.5 to obtain a fermentation substrate;
(3) Inoculating Bacillus beilesiensis CICC 25171 into a fermentation substrate, and performing liquid fermentation to obtain a fermentation liquid;
(4) Centrifuging the fermentation liquor, removing residues, concentrating, and drying to obtain the final product.
2. The method for extracting chlorella pyrenoidosa protein by using a microbial fermentation method as claimed in claim 1, wherein the step (1) is to inoculate bacillus belgii CICC 25171 on a YPD solid culture medium by adopting a plate-scribing method for strain purification, the culture medium is placed in a constant-temperature incubator at 37 ℃ for culture for 20h, then a single colony is inoculated into the YPD liquid culture medium, the culture is carried out on a shaker at 37 ℃ for 12h, the inoculation amount according to the volume ratio of 10% is inoculated into the YPD liquid culture medium again for amplification culture, the culture medium is placed on a shaker at 37 ℃ and 180r/min for shake culture for 8-16h, and the OD value of the bacterial liquid is controlled to be 0.8 to 1.2, so as to obtain the bacillus seed liquid.
3. The method for extracting the chlorella pyrenoidosa protein according to claim 1, wherein the step (2) is specifically as follows: mixing chlorella pyrenoidosa and pure water according to the mass ratio of 1:8, soaking the mixture in an environment at 4 ℃ for 24h, and mixing the powder of the chlorella and the glucose according to the mass ratio of 50:1, adding glucose, and adjusting the pH to 8.0-9.5 by using 0.1 mol/L NaoH to obtain the fermentation substrate.
4. The method for extracting the chlorella pyrenoidosa protein according to claim 1, wherein the step (3) is specifically as follows: inoculating Bacillus beilesiensis CICC 25171 into a fermentation substrate according to an inoculation ratio of 8-10% in volume ratio, and performing liquid fermentation at 35-40 ℃ for 20-30h to obtain fermentation liquor.
5. The method for extracting chlorella pyrenoidosa protein by microbial fermentation according to claim 1, wherein the step (4) is specifically as follows: centrifuging the fermentation liquor at 4 deg.C and 5000 rpm for 10 min, and separating the fermentation liquor from the residue; and (4) carrying out rotary evaporation and concentration on the separated fermentation liquor to 15% of the original volume, and carrying out freeze-drying and powder preparation on the concentrated fermentation liquor to obtain a finished product.
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