CN114907984A - Production method of pleurotus djamor mycelium, produced pleurotus djamor mycelium and application thereof - Google Patents
Production method of pleurotus djamor mycelium, produced pleurotus djamor mycelium and application thereof Download PDFInfo
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Abstract
The invention provides a production method of pleurotus djamor mycelium, the produced pleurotus djamor mycelium and application thereof in preparing vegetarian meat. The production method of the pleurotus djamor mycelium comprises the steps of strain activation, seed liquid preparation, inoculation and fermentation, solid-liquid separation and mycelium collection; the preparation and inoculation fermentation of the seed liquid adopt the same liquid culture medium, the pH value of the liquid culture medium is 5.5-6.5, and the content of each component in the raw materials is as follows: with total sugar25-50 g/L of corn liquefied clear liquid, 0.02-3 g/L of magnesium sulfate, 0.1-2 g/L of ammonium sulfate, 0.02-3 g/L of monopotassium phosphate, 0.02-3 g/L of urea, 0.1-3 g/L of polynucleotide yeast powder and 0.05-4 g/L, V of bacterial cellulose B1 0.01-0.1 g/L of fresh carrot and 2-20 g/L of fresh carrot; wherein the mass percentage content of nucleotide in the high nucleotide yeast powder is more than or equal to 8 percent; the bacterial cellulose is a 110-220-mesh screen underflow.
Description
Technical Field
The invention belongs to the technical field of biological fermentation and food, and particularly relates to a production method of pleurotus eryngii mycelium, pleurotus eryngii mycelium produced by the method and application of the pleurotus eryngii mycelium.
Background
Protein is an essential nutrient for the human body. The global population in 2050 is close to 100 billion according to the prediction of united nations, and the demand of human beings for proteins is continuously rising. The traditional animal protein has the problems of occupying cultivated land, long growth period, easy epidemic disease occurrence, greenhouse gas emission, animal care and the like. Therefore, there is a need to develop alternative products to animal proteins. The substitute product not only provides the necessary nutrients, but also meets the consumer's needs for the texture and sensory attributes of meat products. The vegetarian meat product prepared by using the vegetable protein as the main base material has higher protein content and low fat content, and is suitable for the consumers with special requirements such as obesity, hyperlipidemia, vegetarian food and the like. Therefore, with the increasing awareness of environmental protection and the continuous pursuit of health and nutrition, the development and research of vegetarian meat are the trend-driven necessity.
The raw materials for producing vegetarian meat mainly comprise soybean protein, wheat protein, konjak, edible fungi and the like. Soy protein and wheat protein are susceptible to allergy and affect the range of its audibility. The edible fungi contain rich protein components, the mass fraction can reach 25 percent at most when the edible fungi are dried, and the content of the edible fungi is close to that of the protein in livestock and poultry products such as beef, pork and the like (Wang Yan Sheng, and the like; application prospect of the edible fungi protein and research hotspot analysis [ J ] food industry science and technology, 2019,40 (10): 339 one 344.). The edible fungi grow according to the cycle of 'spore → mycelium → sporocarp → spore'. The mycelium is a nutritive organ of edible fungi and is similar to the fruiting body in the young period in nutritional ingredients. The germination and growth of edible fungus sporocarp belongs to solid fermentation, and has high requirement on environment, long production period and low yield. The growth of the mycelium can be realized in a large scale by liquid fermentation; compared with solid fermentation, the method has the advantages of short production period, suitability for large-scale production, no seasonal restriction, simple production process, low cost, high strain purity, consistent seed age, low probability of being polluted by mixed bacteria and the like, and can purposefully obtain the required secondary metabolite. The large-scale production of the edible fungus mycelia can provide rich raw materials for the vegetable meat.
Pleurotus eryngii (Pleurotus djamor), also known as Pleurotus eryngii and Pleurotus eryngii, belongs to Pleurotus genus of Pleurotaceae of Agaricales of Hymenomycetes of Eumycota, and is a good variety introduced from India in recent years. It has good taste, crab flavor, and is rich in protein, amino acids, mineral elements and vitamins. The fruit body of the pleurotus djamor is bright in color and has ornamental value. Therefore, the research of early pleurotus djamor mainly focuses on the aspects of solid cultivation formula optimization and active ingredient research thereof, and the liquid fermentation production of mycelium and the mycelium application research report is less.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a method for producing pleurotus eryngii mycelium, which can improve the yield and the protein content of the pleurotus eryngii mycelium. The invention also provides application of the pleurotus djamor mycelium produced by the method in preparing vegetarian meat.
In order to realize the technical effects, the invention adopts the following technical scheme:
a method for producing Pleurotus djamor mycelium comprises activating strain, preparing seed solution, inoculating, fermenting, and performing solid-liquid separation to collect mycelium; wherein the same liquid culture medium is adopted for seed liquid preparation and inoculation fermentation; the pH value of the liquid culture medium is 5.5-6.5, and the content of each component in the raw materials is as follows:
25-50 g/L of corn liquefied clear liquid, 0.02-3 g/L of magnesium sulfate, 0.1-2 g/L of ammonium sulfate, 0.02-3 g/L of monopotassium phosphate, 0.02-3 g/L of urea, 0.1-3 g/L of high nucleotide yeast powder and 0.05-4 g/L, V of bacterial cellulose in terms of total sugar B1 0.01-0.1 g/L of fresh carrot and 2-20 g/L of fresh carrot;
wherein the mass percentage content of nucleotide in the high nucleotide yeast powder is more than or equal to 8 percent; the bacterial cellulose is 110-220 mesh screen underflow.
Preferably, in the raw material for liquid culture, the content of each component is as follows:
25-50 g/L of corn liquefied clear liquid, 0.1-1 g/L of magnesium sulfate, 0.1-0.5 g/L of ammonium sulfate, 0.1-1 g/L of monopotassium phosphate, 0.1-1 g/L of urea, 1-2 g/L of high nucleotide yeast powder and 1-2 g/L, V of bacterial cellulose in terms of total sugar B1 0.02-0.08 g/L and 15-20 g/L of fresh carrots.
Preferably, the corn liquefaction clear liquid is prepared by the following method:
mixing corn flour and water according to the mass ratio of 1: 1.5-1: 2.8 of the corn flour to the water, adjusting the pH value to be 5.5-6.0, adding 10-20U/g of alpha-amylase based on the mass of the corn flour, liquefying for 1.5-2 hours at the temperature of 80-100 ℃, then cooling to normal temperature, and carrying out solid-liquid separation to obtain clear liquid, namely the corn cleaning liquid.
In the preparation of the corn cleaning solution, after liquefaction is finished, the reaction solution can be cooled to room temperature at an accelerated speed or can be naturally cooled to the room temperature.
The total sugar content of the corn clear liquid prepared by the method is 150-200 g/L.
The high nucleotide yeast powder may be obtained from commercial sources, such as Nanjing Tongkaizhaoye Biotechnology, Inc.
Preferably, the bacterial cellulose is 200 mesh undersize.
Preferably, the bacterial cellulose is generated by fermenting Acetobacter xylinum, the generated bacterial cellulose is repeatedly boiled and washed by alkaline water until no obvious color exists, and is washed by pure water until the pH is neutral and the bacterial cellulose has no obvious color, and then is dried or freeze-dried in an oven, crushed and screened by a sieve of 110-220 meshes, and the screened material of 200 meshes is preferably selected, so that the antibacterial cellulose is obtained.
Preferably, the bacterial cellulose is produced by fermentation by:
1) scraping a ring acetobacter xylinum slant seed to be inoculated to a sterilized seed culture medium (20 g/L glucose, 2g/L yeast extract and 2g/L, CaCO peptone 3 0.5g/L), culturing at 28 ℃ and 100r/min for 16 hours, and carrying out amplification culture to obtain acetobacter xylinum seeds;
2) preparation of molasses: diluting cane molasses with water to 2 times of the mass of the cane molasses, adjusting the pH value to 2.0 with sulfuric acid, hydrolyzing for 30 minutes at 90 ℃, standing for one night, centrifuging or filtering, taking supernatant, diluting by 10 times, and adjusting the pH value to 5.5-6.5;
3) and (3) fermentation: inoculating 10% of the acetobacter xylinum seeds obtained in the step 1) into a 500mL triangular flask filled with 150mL of cane molasses culture medium (25 g/L of molasses, 0.5g/L of glycerol, 10g/L of corn steep liquor with the solid content of 35% -45%, 0.5g/L of ammonium sulfate, 1.0g/L of citric acid, 2.0g/L of disodium hydrogen phosphate and 0.2g/L of magnesium sulfate), standing and fermenting for 8 days at the fermentation temperature of 28 ℃ to obtain fermentation liquor, and performing solid-liquid separation by centrifugation or filtration to obtain bacterial cellulose.
Preferably, the liquid medium is prepared by the following method:
step I preparation of raw materials
Preparing the components in the raw materials according to the parts by weight;
step II carrot treatment
Cutting fresh carrot into blocks, crushing, mixing with partial water of the material, homogenizing, solid-liquid separating and collecting carrot juice;
step III, preparation and constant volume of liquid culture medium
And (3) mixing the carrot juice obtained in the step (II) with other raw materials, supplementing water to the total weight if necessary to ensure that the total sugar content of the corn liquefaction clear liquid is 25-50 g/L in the liquid culture medium, uniformly stirring, adjusting the pH value to 5.5-6.5, and sterilizing to obtain the carrot juice.
As a preferred embodiment, the present invention provides a method for producing pleurotus djamor mycelia, comprising the steps of:
step 1, strain activation
Cutting small pieces of pleurotus djamor slant strains preserved at low temperature, inoculating the cut small pieces of pleurotus djamor slant strains into the center of a PDA (personal digital assistant) plate, and performing strain activation culture under the culture condition of a constant-temperature incubator at 25 ℃ for 7-8 days until hyphae are fully distributed on the whole plate;
step 2. preparation of seed liquid
Using a puncher with the diameter of 0.5cm to punch 4-5 fungus blocks on a pleurotus eryngii hypha flat plate cultured for 7-8 days, inoculating the fungus blocks into a shake flask filled with the pleurotus eryngii liquid culture medium, wherein the shake flask specification is 500mL, the liquid filling amount is 150 +/-10 mL, and the shake culture is carried out in a constant-temperature incubator at 25 ℃ at 150rpm for 6 days to obtain pleurotus eryngii seed liquid;
step 3, inoculation and fermentation
Inoculating the pleurotus djamor seed solution obtained in the step 2 into a pleurotus djamor fermentation tank which is added with the liquid culture medium in advance by using a flame inoculation method according to the inoculation amount of 5-20%, wherein the parameters are as follows: the liquid filling amount is 50%, 20-100U/g of saccharifying enzyme is added based on the mass of corn flour used for preparing the corn liquefaction clear liquid in the liquid culture medium raw material, the stirring speed is 150-300 rpm, the temperature is 25 ℃, the dissolved oxygen is more than 50%, the pressure is 0.05-0.1 Mpa, and the culture and fermentation are carried out until the sugar concentration is reduced to be less than 1g/L and the shape of the mycelium is complete;
step 4, solid-liquid separation and collection of mycelium
And (3) processing the fermentation liquor obtained in the step (3) through centrifugation and/or filtration, and separating the obtained mycelium for later use.
Preferably, in the step 3, repeated batch fermentation is adopted, and the specific operations are as follows:
and (3) discharging fermentation liquor accounting for 40-80% (v/v) of the total volume of the fermentation liquor at the end of each batch of fermentation, supplementing the liquid culture medium to the set liquid loading amount, and fermenting according to the parameters in the step (3).
The second object of the present invention is to provide a mycelium of pleurotus djamor prepared according to the above production method.
The third purpose of the invention is to provide the application of the pleurotus djamor mycelium in preparing vegetarian meat.
Specifically, the application refers to mixing the pleurotus djamor mycelium and the wiredrawing protein, extruding and forming, and drying to obtain the vegetarian meat.
The invention also provides a preparation method of the vegetarian meat, which comprises the following steps:
step A, pre-treatment of pleurotus eryngii mycelium
Washing the pleurotus djamor mycelium prepared by the method for 2-5 times by using water until no obvious culture medium component exists, centrifuging for 10min at 5000r/min, weighing, adding water with the wet weight of 5-50 times of the mycelium, stirring and homogenizing at the rotating speed of 1000-2500 r/min, preferably at the rotating speed of 2000r/minx until the mycelium has no obvious spherical shape, centrifuging and/or filtering, collecting the mycelium to obtain homogenized pleurotus djamor mycelium, and weighing for later use; wherein the water content of the homogenized pleurotus djamor mycelium is 89-92%;
step B, pretreatment of wiredrawing protein
Completely soaking the commercial finished wire drawing protein in water until the rehydration rate is not lower than 120-190%; removing the wire from the rehydrated wiredrawing protein to ensure that the removed wire is uniform, has no hard core and no large foreign matters and has a wire width of not more than 0.5cm, obtaining the wiredrawing protein after the wire removal, and weighing for later use;
step C, compounding and mixing materials
Adding water into the homogenized pleurotus djamor mycelium obtained in the step A for suspension, and mixing the homogenized pleurotus djamor mycelium with the wire drawing protein obtained in the step B after wire drawing to obtain a mixed material; the method comprises the following steps of (1) taking the total wet weight of the homogenized pleurotus djamor mycelium and the wire drawing protein after the wire drawing is removed as a reference, wherein the homogenized pleurotus djamor mycelium accounts for 10-60%, preferably 10-30%, optimally 30%, and the balance is the wire drawing protein after the wire drawing is removed;
step D, extrusion molding
Extruding and molding the mixed material obtained in the step C through a screw extruder to obtain a molded blank; wherein the water adding amount is adjusted in the extrusion process to ensure that the mass percentage content of water in the formed blank is 45-60%, and the temperature of an extruder die is 100-140 ℃;
step E. drying
And D, drying the formed blank obtained in the step D until the mass percentage of water is below 20%, and obtaining the vegetarian meat.
Still another object of the present invention is to provide vegetarian meat produced by the above-described method.
In the present specification, unless otherwise specified, the term "water" refers to water subjected to purification treatment, such as deionized water, distilled water, double distilled water, and the like.
The pleurotus djamor mycelium obtained by the production method of the pleurotus djamor mycelium provided by the invention has high yield, high protein content and rich nutrition. The vegetarian meat prepared by taking the pleurotus djamor mycelium as the raw material has good appearance, good flavor and low cost, and can meet the requirements of people on the taste, flavor and nutrition of the vegetarian meat. The invention provides a valuable path for the commercial development of the pleurotus djamor mycelium.
Drawings
The invention is further described below with reference to the accompanying drawings.
FIG. 1 is a photograph showing the mycelia of Pleurotus djamor obtained at three homogenization speeds in example 3, observed under a 40-fold microscope, wherein:
a: the components of the formula are 14-500,
b: the formulations are in the range of 14 to 2000,
c: the formula is 14-10000.
FIG. 2 is a photograph showing a photograph of a finished vegetarian meat product prepared from formulations 14-2000 of mycelium and spun protein of example 3, wherein the formulation 14-2000 of mycelium comprises 30% of the total wet weight of the vegetarian meat material.
Detailed Description
The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials, reagent materials and the like used in the following examples are all commercially available products unless otherwise specified. Wherein, the source conditions of part strains and reagents are as follows:
the pleurotus djamor strain: the bacterial culture collection center of agricultural institute of Jiangsu province presents, and the collection unit is self-built with number NKY _ H2020_ 15;
acetobacter xylinum, ATCC, strain deposit No. ATCC 700178;
drawing protein: anyang Beijia food Co., Ltd;
angel yeast powder: FM888, wherein the nucleotide content is less than 3%; angel Yeast Inc.;
high nucleotide yeast powder (nucleotide content is more than or equal to 8%): nanjing Tongkaizhao Biotechnology Co.
The corn liquification serum described in the following examples was prepared by the following method:
mixing corn flour and water according to the mass ratio of 1: 2.5 of the corn flour to the water, adjusting the pH value to 5.5, adding 10-20U/g of alpha-amylase based on the mass of the corn flour, liquefying at 80-100 ℃ for 1.5-2 hours, then cooling to normal temperature, and carrying out solid-liquid separation to obtain clear liquid, namely the corn cleaning liquid; wherein the total sugar content is 150-200 g/L.
The bacterial cellulose described in the following examples was prepared by the following method:
1) scraping a ring acetobacter xylinum slant seed to be inoculated to a sterilized seed culture medium (20 g/L glucose, 2g/L yeast extract and 2g/L, CaCO peptone) 3 0.5g/L), culturing at 28 ℃ and 100r/min for 16 hours, and carrying out amplification culture to obtain acetobacter xylinum seeds;
2) preparation of molasses: diluting cane molasses with water to 2 times of the mass of the cane molasses, adjusting the pH value to 2.0 with sulfuric acid, hydrolyzing for 30 minutes at 90 ℃, standing for one night, centrifuging or filtering, taking supernatant, diluting by 10 times, and adjusting the pH value to 5.5-6.5;
3) fermentation: inoculating 10% of the acetobacter xylinum seeds obtained in the step 1) into a 500mL triangular flask filled with 150mL of molasses culture medium (step 2) to prepare 25g/L molasses, 0.5g/L glycerol, 10g/L corn steep liquor, 0.5g/L ammonium sulfate, 1.0g/L citric acid, 2.0g/L disodium hydrogen phosphate and 0.2g/L magnesium sulfate, standing and fermenting for 8 days at the fermentation temperature of 28 ℃ to obtain fermentation liquor, and performing solid-liquid separation by centrifugation or filtration to obtain bacterial cellulose;
4) and (3) fermentation post-treatment: repeatedly boiling and washing the bacterial cellulose obtained in the step 3) by using alkaline water until no obvious color exists, washing the bacterial cellulose by using pure water until the pH value is neutral, and then drying or freeze-drying the bacterial cellulose in an oven and crushing the bacterial cellulose for later use.
EXAMPLE 1 examination and optimization of Pleurotus djamor mycelium liquid Medium
The liquid culture medium is prepared by the following method:
step I. preparation of raw materials
The components (unit: g/L) in the raw materials are respectively prepared according to the following formula:
formula 1: glucose 10, corn starch 15, magnesium sulfate 0.4, ammonium sulfate 0.2, potassium dihydrogen phosphate 0.6, urea 0.5, Angel Yeast powder (FM888)2, V B1 0.05；
And (2) formula: 25 parts of corn liquefied clear liquid, 0.4 part of magnesium sulfate, 0.2 part of ammonium sulfate, 0.6 part of potassium dihydrogen phosphate, 0.5 part of urea, 2 parts of Angel yeast powder (FM888) and V parts of corn liquefied clear liquid calculated by total sugar B1 0.05；
And (3) formula: 25 parts of glucose, 0.4 part of magnesium sulfate, 0.2 part of ammonium sulfate, 0.6 part of potassium dihydrogen phosphate, 0.5 part of urea, 2 parts of Angel yeast powder (FM888) and V parts of yeast B1 0.05；
And (4) formula: 25 parts of corn liquefied clear liquid, 0.4 part of magnesium sulfate, 0.2 part of ammonium sulfate, 0.6 part of potassium dihydrogen phosphate, 0.5 part of urea, 2 parts of Angel yeast powder (FM888) and V parts of total sugar B1 0.05, bacterial cellulose (18 mesh screen underflow) 1;
and (5) formula: 25 parts of corn liquefied clear liquid, 0.4 part of magnesium sulfate, 0.2 part of ammonium sulfate, 0.6 part of potassium dihydrogen phosphate, 0.5 part of urea, 2 parts of Angel yeast powder (FM888) and V parts of corn liquefied clear liquid calculated by total sugar B1 0.05, bacterial cellulose (110 mesh screen underflow) 1;
and (6) formula: 25 parts of corn liquefied clear liquid, 0.4 part of magnesium sulfate and sulfuric acid in terms of total sugarAmmonium 0.2, potassium dihydrogen phosphate 0.6, urea 0.5, Angel Yeast powder (FM888)2, and V B1 0.05, bacterial cellulose (200 mesh screen underflow) 1;
and (3) formula 7: 25 parts of corn liquefied clear liquid, 0.4 part of magnesium sulfate, 0.2 part of ammonium sulfate, 0.6 part of potassium dihydrogen phosphate, 0.5 part of urea, 2 parts of Angel yeast powder (FM888) and V parts of corn liquefied clear liquid calculated by total sugar B1 0.05, bacterial cellulose (200 mesh screen underflow) 2;
and (4) formula 8: 25 parts of corn liquefied clear liquid, 0.4 part of magnesium sulfate, 0.2 part of ammonium sulfate, 0.6 part of potassium dihydrogen phosphate, 0.5 part of urea, 2 parts of Angel yeast powder (FM888) and V parts of corn liquefied clear liquid calculated by total sugar B1 0.05, bacterial cellulose (200 mesh screen underflow) 4;
formula 9: 25 parts of corn liquefied clear liquid, 0.4 part of magnesium sulfate, 0.2 part of ammonium sulfate, 0.6 part of potassium dihydrogen phosphate, 0.5 part of urea, 2 parts of Angel yeast powder (FM888) and V parts of corn liquefied clear liquid calculated by total sugar B1 0.05, corn stover (110 mesh screen minus) 1;
formula 10: 25 parts of corn liquefied clear liquid, 0.4 part of magnesium sulfate, 0.2 part of ammonium sulfate, 0.6 part of potassium dihydrogen phosphate, 0.5 part of urea, 2 parts of Angel yeast powder (FM888) and V parts of corn liquefied clear liquid calculated on total sugar B1 0.05, bagasse (110 mesh screen underflow) 1;
formula 11: 25 parts of corn liquefied clear liquid, 0.4 part of magnesium sulfate, 0.2 part of ammonium sulfate, 0.6 part of potassium dihydrogen phosphate, 0.5 part of urea, 2 parts of Angel yeast powder (FM888) and V parts of corn liquefied clear liquid calculated by total sugar B1 0.05, bacterial cellulose (200 mesh screen underflow) 1, carrot 18;
formula 12: 25 parts of corn liquefied clear liquid, 0.4 part of magnesium sulfate, 0.2 part of ammonium sulfate, 0.6 part of potassium dihydrogen phosphate, 0.5 part of urea, 2 parts of Angel yeast powder (FM888), 1 part of high nucleotide yeast powder and V parts of high nucleotide yeast powder B1 0.05；
Formula 13: 25 parts of corn liquefied clear liquid, 0.4 part of magnesium sulfate, 0.2 part of ammonium sulfate, 0.6 part of monopotassium phosphate, 0.5 part of urea, 2 parts of high nucleotide yeast powder and V parts of high nucleotide yeast powder B1 0.05。
Step II carrot treatment
Cutting and crushing the fresh carrots in parts by weight, mixing the fresh carrots with part of water in the raw materials, homogenizing, performing solid-liquid separation, and taking carrot juice for later use;
step III, preparation and constant volume of liquid culture medium
Mixing the carrot juice obtained in the step (II) with other raw materials, adding water if necessary to enable the components to reach the concentration, uniformly stirring, adjusting the pH value to be 6.0, and sterilizing to obtain the carrot juice.
The liquid culture medium prepared by the method is used for shake flask culture of pleurotus djamor, and the specific operation is as follows:
step 1, strain activation
Cutting small pieces of pleurotus djamor slant strains preserved at a low temperature, inoculating the cut small pieces of pleurotus djamor slant strains into the center of a PDA agar plate, and performing strain activation culture under the culture condition of a constant-temperature incubator at 25 ℃ for 7-8 days until hyphae are fully distributed on the whole plate;
step 2. Shake flask culture
Using a hole puncher with the diameter of 0.5cm to punch 4-5 fungus blocks on a pleurotus djamor hypha flat plate cultured for 7-8 days, respectively inoculating the fungus blocks into a shake flask containing the pleurotus djamor liquid culture medium prepared in the embodiment, wherein the shake flask specification is 500mL, the liquid loading amount is 150mL, adding 70U/g of saccharifying enzyme according to the mass of corn starch in raw materials or corn flour used for preparing the corn liquefied clear solution, carrying out shake culture for 6 days in a constant-temperature incubator at the temperature of 25 ℃, centrifuging to carry out solid-liquid separation, washing the obtained mycelium for 2 times by using pure water, drying the centrifuged solid mycelium at the temperature of 100 ℃ to constant weight, and calculating the dry weight of the mycelium. And detecting the nitrogen content by adopting a Kjeldahl apparatus, and calculating the content of the crude protein. The results are shown in Table 1.
TABLE 1 EXAMPLE 1 Effect of various liquid media on the growth of Pleurotus djamor mycelia
| Formulation of | Dry weight of cell (g/L) | Crude protein content (%, g/g) |
| 1 | 13.20 | 29.52 |
| 2 | 15.23 | 31.89 |
| 3 | 11.23 | 31.03 |
| 4 | 16.92 | 31.22 |
| 5 | 18.75 | 31.43 |
| 6 | 20.66 | 31.98 |
| 7 | 21.76 | 31.72 |
| 8 | 17.52 | 30.23 |
| 9 | 16.81 | 30.67 |
| 10 | 17.23 | 31.13 |
| 11 | 22.8 | 34.27 |
| 12 | 16.12 | 35.44 |
| 13 | 17.85 | 36.89 |
Comparing the data in the table shows that:
1) comparing formulas 1, 2 and 3, it can be seen that the corn liquefaction clear liquid as a carbon source has a significant effect of improving the yield of mycelia, and therefore the corn liquefaction clear liquid is preferably used as the carbon source in the invention.
2) Comparing the formulas 2 and 4-10, the proper amount of bacterial cellulose has obvious promotion effect on the yield of the mycelium, and the smaller the particle size in the range of 110-200 meshes is, the more beneficial the growth of the mycelium is; however, the bacterial cellulose is used in an excessive amount to adversely affect the growth of mycelia. Therefore, the bacterial cellulose is preferably undersize with the particle size of 110-200 meshes, and more preferably undersize with 200 meshes; the content of the bacterial cellulose in the liquid culture medium is 1-2 g/L, and more preferably 2 g/L.
3) Comparing the formulas 6 and 11, it can be seen that the growth of the mycelia can be promoted by adding carrot juice.
4) Comparing formulas 2, 12 and 13, it can be seen that the addition of a proper amount of high nucleotide yeast powder has a significant improvement effect on the synthesis of mycelium protein, compared with the common yeast powder. Therefore, the preferred yeast powder is a high nucleotide yeast powder.
Through the experiments, the preferable liquid culture medium of the pleurotus djamor mycelium comprises the following raw materials:
25g/L of corn liquefied clear liquid, 0.4g/L of magnesium sulfate, 0.4g/L of ammonium sulfate, 0.6g/L of monopotassium phosphate, 0.5g/L of urea, 2g/L of high nucleotide yeast powder,Bacterial cellulose 2g/L, V screened by a sieve of 110-200 meshes B1 0.05g/L and 18g/L of fresh carrots.
Example 2 liquid fermentation of Pleurotus eryngii mycelia
The liquid culture medium used in this example has the following formulation:
formulation 2 (g/L): 25 parts of corn liquefied clear liquid, 0.4 part of magnesium sulfate, 0.2 part of ammonium sulfate, 0.6 part of potassium dihydrogen phosphate, 0.5 part of urea, 2 parts of Angel yeast powder (FM888) and V parts of corn liquefied clear liquid calculated on total sugar B1 0.05；
Formulation 14 (g/L): 25 percent of corn liquefied clear solution, 0.4 percent of magnesium sulfate, 0.4 percent of ammonium sulfate, 0.6 percent of monopotassium phosphate, 0.5 percent of urea, 2 percent of high nucleotide yeast powder and 2 percent of bacterial cellulose and V under the sieve of 200 meshes in terms of total sugar B1 0.05, fresh carrots 18.
Liquid media were prepared according to the preparation methods described in example 1, respectively; then preparing the pleurotus djamor mycelium through liquid fermentation according to the following steps:
step 1, strain activation
Cutting small pieces of pleurotus djamor slant strains preserved at a low temperature, inoculating the cut small pieces of pleurotus djamor slant strains into the center of a PDA agar plate, and performing strain activation culture under the culture condition of a constant-temperature incubator at 25 ℃ for 7-8 days until hyphae are fully distributed on the whole plate;
step 2. Shake flask culture
Using a puncher with the diameter of 0.5cm to punch 4-5 fungus blocks on a pleurotus eryngii hypha flat plate cultured for 7-8 days, respectively inoculating the fungus blocks into shake flasks containing the pleurotus eryngii liquid culture medium prepared in the embodiment, wherein the shake flask specification is 500mL, the liquid filling amount is 150mL, and the pleurotus eryngii seed liquid is obtained by shake culture in a constant-temperature incubator at 25 ℃ at 150rpm for 6 days;
step 3, inoculation and fermentation
And (3) respectively inoculating the pleurotus djamor seed liquid obtained in the step (2) into pleurotus djamor fermentation tanks (3L) which are pre-added with the liquid culture medium by using a flame inoculation method according to the inoculation amount of 15%, and setting parameters as follows: the liquid filling amount is 50%, 20-100U/g of saccharifying enzyme is added by taking the mass of corn flour in the liquid culture medium raw material as a reference, the stirring speed is 150-300 rpm, the temperature is 25 ℃, the dissolved oxygen is more than 50%, the pressure is 0.05-0.1 Mpa, and the culture and fermentation are carried out until the sugar content is less than 1g/L and the shape of the mycelium is complete; discharging 70% of the total volume of the fermentation liquid after the fermentation is finished, then supplementing a new liquid fermentation culture medium, and continuing to culture and ferment under the same conditions.
Step 4, solid-liquid separation and collection of mycelium
The fermentation broth obtained in step 3 was treated by centrifugation and/or filtration, and the mycelium obtained by separation was assayed for crude protein content as described in example 1, with the results shown in Table 2.
TABLE 2 Effect of different liquid media and fermentation modes on the protein synthesis of mycelia
The data in table 2 show: the liquid medium of formula 14 has better effects on promoting mycelium growth and protein synthesis than formula 2. The fed-batch fermentation can greatly shorten the time for reaching the fermentation end point and improve the production efficiency.
Example 3 preparation of vegetarian meat
1. Post-treatment of pleurotus eryngii mycelium
The two formulas of fermented mycelia obtained in the example 2 are subjected to solid-liquid separation through centrifugation, water 40 times the wet weight of the mycelia is added to prepare a suspension, three stirring speeds of 500r/min, 2000r/min and 10000r/min are adopted in a homogenization process, solid-liquid separation is performed through centrifugation to obtain homogenized pleurotus djamor mycelia, and the weight is respectively calculated as follows:
formula 2-500, formula 2-2000, formula 2-10000;
14-500 of formula, 14-2000 of formula and 14-10000 of formula.
The state of the mycelium was observed under 40 times microscope, wherein the photographs of the mycelium of formulas 14-500, 14-2000 and 14-10000 were taken under 40 times microscope, see FIG. 1. Both formulations fermented mycelium presented similar status:
formula 2-500 and formula 14-500 (homogenizing rotation speed 500 r/min): part of the mycelium is agglomerated;
formulas 2-2000 and formulas 14-2000 (homogenizing rotation speed 2000 r/min): the dispersion was uniform, no mycelium break was observed;
formula 2-10000 and formula 14-10000 (homogenizing rotation speed 10000 r/min): the dispersion was uniform, but the mycelium broke significantly.
Therefore, the rotation speed of the homogenate is preferably more than 500r/min but less than 10000 r/min; more preferably 1000 to 2500r/min, most preferably 2000 r/min.
2. Preparation of vegetarian meat
Completely soaking the commercial finished product wire-drawing protein in water until the rehydration rate is not lower than 120%; removing the wire from the rehydrated wiredrawing protein to ensure that the removed wire is uniform, has no hard core and no large foreign matters and has a wire width of not more than 0.5cm, obtaining the wiredrawing protein after the wire removal, and weighing for later use; adding water into the homogenized pleurotus djamor mycelium for suspension, and mixing the pleurotus djamor mycelium with the wire drawing protein after wire drawing to obtain a mixed material; wherein the homogenized pleurotus djamor mycelium accounts for 30 or 60 percent respectively based on the total mass of the homogenized pleurotus djamor mycelium and the fiber-drawing protein after fiber removal, and the rest is the fiber-drawing protein after fiber removal;
extruding and molding the mixed material by a screw extruder to obtain a molded blank; wherein the water adding amount is adjusted in the extrusion process to ensure that the mass percentage of water in the formed blank is 50-60%, and the temperature of a die head of an extruder is 100-140 ℃; and drying the obtained molded blank until the mass percentage of water is below 20 percent, thus obtaining the vegetarian meat.
Each of the prepared samples was subjected to a texture analysis (TPA) test using a texture analyzer, and an AB/E-d35 cylindrical probe was selected to analyze the cohesion and elasticity. And simultaneously, the relevant indexes of a commercial vegetarian meat are measured. The finished product with 60% of mycelium added has poor meat compactness, and the prepared vegetarian meat has loose texture and is not suitable for subsequent industrial processing. The finished product with 30% of added mycelium has good meat texture, compact texture, mushroom fragrance and fresh and delicious taste. Therefore, in the present invention, the mass ratio of the mycelium (wet weight) in the vegetarian meat material is preferably 10% to 30%, more preferably 30%.
The results of the measurement of 30% mycelium of the vegetarian meat are shown in Table 3, wherein photographs of the finished vegetarian meat prepared according to formulas 14-2000 are shown in FIG. 2.
TABLE 3 quality of different vegetarian meats
| Sample (I) | Cohesion property | Elasticity | Feeling of surface graininess |
| Commercial vegetarian meat | 0.97 | 0.99 | Not found out |
| Formulation 2-500 | 0.91 | 0.92 | Is remarkable in that |
| Formulations 2 to 2000 | 0.95 | 1.16 | Not found out |
| Formulation 2-10000 | 0.96 | 1.02 | Not found out |
| Formulations 14 to 500 | 0.93 | 0.97 | Is remarkable in |
| Formulations 14 to 2000 | 0.96 | 1.35 | Not found out |
| Formula 14-10000 | 0.98 | 1.18 | Not found out |
The data in Table 3 show that insufficient homogenization of the mycelium (500 r/min homogenization speed) resulted in a distinct graininess of the finished product; in contrast, homogenizing mycelium well results in a significant improvement in finished product appearance. The vegetarian meat prepared by mycelium obtained at a homogenate rotating speed of 2000r/min is better than other rotating speeds by measuring the elasticity index of the vegetarian meat. In addition, the protein content of the mycelium of the formula 14 is higher than that of the mycelium of the formula 2, and the vegetarian meat prepared by the mycelium of the formula 14 has higher elasticity under the condition that the protein content of the mycelium of the formula 14 is the same in the vegetarian meat. The invention particularly preferably adopts the formula 14-2000 as the edible fungus raw material of vegetarian meat.
Claims (10)
1. A method for producing Pleurotus djamor mycelium comprises activating strain, preparing seed solution, inoculating, fermenting, and performing solid-liquid separation to collect mycelium; wherein the same liquid culture medium is adopted for seed liquid preparation and inoculation fermentation; the pH value of the liquid culture medium is 5.5-6.5, and the content of each component in the raw materials is as follows:
25-50 g/L of corn liquefied clear liquid, 0.02-3 g/L of magnesium sulfate, 0.1-2 g/L of ammonium sulfate, 0.02-3 g/L of monopotassium phosphate, 0.02-3 g/L of urea, 0.1-3 g/L of high nucleotide yeast powder and 0.05-4 g/L, V of bacterial cellulose in terms of total sugar B1 0.01-0.1 g/L of fresh carrot and 2-20 g/L of fresh carrot;
wherein the content of nucleotide in the high nucleotide yeast powder is more than or equal to 8 percent by mass; the bacterial cellulose is 110-220-mesh screen underflow.
2. The production method according to claim 1, wherein the liquid culture raw material contains the following components:
25-50 g/L of corn liquefied clear liquid, 0.1-1 g/L of magnesium sulfate, 0.1-0.5 g/L of ammonium sulfate, 0.1-1 g/L of monopotassium phosphate, 0.1-1 g/L of urea, 1-2 g/L of high nucleotide yeast powder and 1-2 g/L, V of bacterial cellulose in terms of total sugar B1 0.02-0.08 g/L and 15-20 g/L of fresh carrots.
3. The production method according to claim 1 or 2, characterized in that the corn liquification serum is prepared by the following method:
mixing corn flour and water according to the mass ratio of 1: 1.5-1: 2.8 of the corn flour to the water, adjusting the pH value to 5.5-6.0, adding 10-20U/g of alpha-amylase based on the mass of the corn flour, liquefying at the temperature of 80-100 ℃ for 1.5-2 hours, then cooling to normal temperature, and carrying out solid-liquid separation to obtain clear liquid, namely the corn cleaning liquid.
4. The production method according to claim 1 or 2, wherein the bacterial cellulose is 200-mesh-screen-underflow;
preferably, the bacterial cellulose is generated by fermenting Acetobacter xylinum, the generated bacterial cellulose is repeatedly boiled and washed by alkaline water until no obvious color exists, and is washed by pure water until the pH is neutral, and then is dried by an oven or freeze-dried and crushed, and a material with a sieve of 110-220 meshes, preferably 200 meshes, is taken out to obtain the bacterial cellulose;
preferably, the bacterial cellulose is produced by fermentation by:
1) preparing acetobacter xylinum seeds: scraping a ring of acetobacter xylinum slant seed, inoculating to a sterilized seed culture medium, culturing at 28 ℃ and 100r/min for 16 hours, and performing amplification culture to obtain acetobacter xylinum seed; wherein the raw materials of the seed culture medium comprise:
glucose 20g/L, yeast extract 2g/L, peptone2g/L、CaCO 3 0.5g/L；
2) Preparation of molasses: diluting cane molasses with water to 2 times of the mass of the cane molasses, adjusting the pH value to 2.0 with sulfuric acid, hydrolyzing for 30 minutes at 90 ℃, standing for one night, centrifuging or filtering, taking supernatant, diluting by 10 times, and adjusting the pH value to 5.5-6.5;
3) fermentation: inoculating 10% of the acetobacter xylinum seeds obtained in the step 1) into a 500mL triangular flask filled with 150mL of cane molasses culture medium, standing and fermenting for 8 days at the fermentation temperature of 28 ℃ to obtain fermentation liquor, and performing solid-liquid separation by centrifugation or filtration to obtain bacterial cellulose; wherein, the raw materials of the sugar cane molasses culture medium comprise:
25g/L of molasses prepared in the step 2), 0.5g/L of glycerol, 10g/L of corn steep liquor with the solid content of 35-45%, 0.5g/L of ammonium sulfate, 1.0g/L of citric acid, 2.0g/L of disodium hydrogen phosphate and 0.2g/L of magnesium sulfate.
5. The production method according to any one of claims 1 to 4, wherein the liquid medium is prepared by:
step I preparation of raw materials
Preparing the components in the raw materials according to the parts by weight;
step II carrot treatment
Cutting and crushing the fresh carrots in parts by weight, mixing the fresh carrots with part of water in the raw materials, homogenizing, carrying out solid-liquid separation, and taking carrot juice for later use;
step III, preparation and constant volume of liquid culture medium
And (3) mixing the carrot juice obtained in the step (II) with other raw materials, supplementing water to the total weight if necessary to ensure that the total sugar content of the corn liquefaction clear liquid is 25-50 g/L in the liquid culture medium, uniformly stirring, adjusting the pH value to 5.5-6.5, and sterilizing to obtain the carrot juice.
6. The production method according to any one of claims 1 to 5, wherein the production method of the Pleurotus djamor mycelium comprises the steps of:
step 1, strain activation
Cutting small pieces of pleurotus djamor slant strains preserved at a low temperature, inoculating the cut small pieces of pleurotus djamor slant strains into the center of a PDA agar plate, and performing strain activation culture under the culture condition of a constant-temperature incubator at 25 ℃ for 7-8 days until hyphae are fully distributed on the whole plate;
step 2. preparation of seed liquid
Using a hole puncher with the diameter of 0.5cm to punch 4-5 fungus blocks on a pleurotus djamor hypha flat plate cultured for 7-8 days, inoculating the fungus blocks into a shake flask filled with a pleurotus djamor liquid culture medium defined in claim 1 or 2, wherein the size of the shake flask is 500mL, the liquid filling amount is 150 +/-10 mL, and the pleurotus djamor seed liquid is obtained by shake culture in a constant-temperature incubator at 25 ℃ at 150rpm for 6 days;
step 3, inoculation and fermentation
Inoculating the pleurotus djamor seed solution obtained in the step 2 into a fermentation tank which is pre-added with a pleurotus djamor liquid culture medium defined in claim 1 or 2 by using a flame inoculation method according to the inoculation amount of 5-20%, wherein the parameters are as follows: the liquid loading amount is 50% +/-10%, 20-100U/g saccharifying enzyme is added based on the mass of corn flour used for preparing the corn liquefaction clear liquid in the liquid culture medium raw material, the stirring speed is 150-300 rpm, the temperature is 25 ℃, the dissolved oxygen is more than 50%, the pressure is 0.05-0.1 Mpa, and the culture and fermentation are carried out until the sugar concentration is reduced to be less than 1g/L and the mycelium is complete in shape;
step 4, solid-liquid separation and collection of mycelium
Centrifuging and/or filtering the fermentation liquor obtained in the step 3, and separating the obtained mycelium for later use;
preferably, the step 3 adopts repeated batch fermentation, and the specific operation is as follows:
and (3) discharging fermentation liquor accounting for 40-80% of the total volume of the fermentation liquor at the end of each batch of fermentation, supplementing the liquid culture medium to a set liquid loading amount, and fermenting according to the parameters in the step (3).
7. A mycelium of Pleurotus djamor prepared by the production method of any one of claims 1 to 6.
8. Use of the mycelium of Pleurotus djamor prepared by the production method according to any one of claims 1 to 6 or the mycelium of Pleurotus djamor according to claim 7 for preparing vegetarian meat;
specifically, the application refers to mixing the pleurotus djamor mycelium with the wiredrawing protein, extruding and forming, and drying to obtain the vegetarian meat.
9. A preparation method of vegetable vegetarian meat comprises the following steps:
step A, pre-treatment of pleurotus eryngii mycelium
Washing the pleurotus eryngii mycelium prepared by the production method according to any one of claims 1 to 6 or the pleurotus eryngii mycelium according to claim 7 with water for 2-5 times until no obvious culture medium components exist, centrifuging for 10min at 5000r/min, weighing, adding water which is 5-50 times of the wet weight of the mycelium, stirring and homogenizing at the rotating speed of 1000-2500 r/min, preferably at the rotating speed of 2000r/minx until the mycelium has no obvious spherical shape, centrifuging and/or filtering, collecting the mycelium, obtaining the homogenized pleurotus eryngii mycelium, and weighing for later use; wherein the water content of the homogenized pleurotus djamor mycelium is 89-92%;
step B, pretreatment of wiredrawing protein
Completely soaking the commercial finished wire drawing protein in water until the rehydration rate is not lower than 120-190%; removing the wire from the rehydrated wiredrawing protein to ensure that the removed wire is uniform, has no hard core and no large foreign matters and has a wire width of not more than 0.5cm, obtaining the wiredrawing protein after the wire removal, and weighing for later use;
step C, compounding and mixing materials
Adding water into the homogenized pleurotus djamor mycelium obtained in the step A for suspension, and mixing the homogenized pleurotus djamor mycelium with the wire drawing protein obtained in the step B after wire drawing to obtain a mixed material; the method comprises the following steps of (1) taking the total wet weight of homogenized pleurotus djamor mycelium and wire drawing protein after wire removal as a reference, wherein the homogenized pleurotus djamor mycelium accounts for 10-60%, preferably 10-30%, optimally 30%, and the balance is the wire drawing protein after wire removal;
step D, extrusion molding
Extruding and molding the mixed material obtained in the step C through a screw extruder to obtain a molded blank; wherein the water adding amount is adjusted in the extrusion process to ensure that the mass percentage content of water in the formed blank is 45-60%, and the temperature of an extruder die is 100-140 ℃;
step E. drying
And D, drying the formed blank obtained in the step D until the mass percentage of water is below 20%, and obtaining the vegetarian meat.
10. A vegetarian meat produced by the production process of claim 9.
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