CN114793753A - Edible fungus mycelium continuous fermentation method - Google Patents
Edible fungus mycelium continuous fermentation method Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01G18/00—Cultivation of mushrooms
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/20—Culture media, e.g. compost
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Abstract
The invention discloses a continuous fermentation method of edible fungus mycelia, which adopts a shake flask to prepare edible fungus mycelia seed liquid; transferring the edible fungus mycelium seed liquid obtained by culture into a fermentation tank for semi-continuous culture, discharging a part of fermentation liquid when the residual reducing sugar to be fermented in each batch is reduced to 1-5g/L, supplementing a new supplementary culture medium, and culturing for 3-5 batches in the same fermentation mode; adding fermentation medium into the fermentation liquid discharged from each batch of semi-continuous culture, and performing continuous fermentation. The invention adopts a semi-continuous fermentation mode to effectively enhance the starting efficiency and the fermentation intensity of continuous fermentation; the efficient and stable operation of continuous fermentation is realized by controlling the rotating speed and the sugar concentration, the strain is not easy to degenerate, and the fermentation intensity and the thallus yield are higher.
Description
Technical Field
The invention belongs to the field of edible fungus preparation, and particularly relates to a continuous fermentation method of edible fungus mycelia.
Background
Edible fungi are important economic crops in China. Since the reform is open, the domestic edible fungus industry rapidly develops, and the yield is rapidly increased from 5.8 ten thousand tons in 1978 to 3933.87 ten thousand tons in 2019, which accounts for more than 70 percent of the total amount of the whole world. The rapid development of the edible fungus industry makes important contributions to the economic and social development of China, and plays an important role in the aspects of improving the income of people, arranging redundant labor force, maintaining social stability and the like.
In the aspect of edible fungus production, the current production mode is mainly artificial cultivation, the production period is long (generally 40-60 days), and the production efficiency is low. The growth period of the edible fungi can be greatly reduced by adopting a deep liquid fermentation mode, the yield is 2-3 orders of magnitude of solid culture, and the fermentation cost is obviously reduced. With the breakthrough of the deep processing technology of edible mushrooms, the edible mushroom industry is rapidly developed, and the fermented edible mushroom protein can reform the traditional labor-intensive and resource-consuming production mode of the mushrooms, so that the mushroom production is replaced by less land resources and more efficient productivity. In addition, with the strengthening of the osmotic effect of 'plant replacement protein' of several vegetarian meat brand enterprises in the world, the improvement of the demand of people on healthy food and the scarcity of feed protein resources, the development and application potential of edible mushroom protein in the future is huge.
At present, domestic fungus mycelium is rarely applied in industrialization at foreign countries, and large-scale amplification fermentation is rare. Research reports mainly focus on formula optimization and fermentation condition optimization, and deep research on fermentation modes is rarely carried out. This patent adopts continuous fermentation process to realize the fermentation of the domestic fungus mycelium of high conversion rate and high production intensity to start in order to strengthen the mycelium activity with the semi-continuous fermentation mode, promote continuous fermentation stability and intensity, solved the production efficiency problem of traditional single batch fermentation and semi-continuous fermentation.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to solve the technical problem of production efficiency of traditional single batch fermentation and semi-continuous fermentation, and provides a production method of edible fungus mycelia with high conversion rate and high production strength.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a continuous fermentation method of edible fungus mycelia comprises the following steps:
(1) preparing edible fungus mycelium seed liquid by adopting a shake flask;
(2) transferring the edible fungus mycelium seed liquid obtained by culturing in the step (1) into a fermentation tank for semi-continuous culture, discharging a part of fermentation liquid when the fermentation residual reducing sugar in each batch is reduced to 1-5g/L, supplementing a new supplementary culture medium, and culturing for 3-6 batches in the same fermentation mode;
(3) and (3) supplementing a fermentation medium into the fermentation liquor discharged from each batch of semi-continuous culture in the step (2), and carrying out continuous fermentation to obtain the microbial fertilizer.
Specifically, the edible fungus strain is any one of oyster mushroom, agaricus bisporus, hypsizygus marmoreus, agaricus blazei, pleurotus geesteranus, panus giganteus, pleurotus citrinopileatus, agrocybe cylindracea, pleurotus citrinopileatus, russula vinosa, hyoplastron beech, shiitake mushroom, pleurotus eryngii, tremella, morel or flammulina velutipes. The edible fungus strains are all varieties which can be directly purchased in the market.
Preferably, in the step (1), the edible fungus mycelium seed culture medium consists of: 10-30g/L of soybean juice, 10-30g/L of corn steep liquor, 15-25g/L of glucose and MgSO 4 0.2-1g/L，FeSO 4 .7H 2 O 0.02-0.08g/L，KH 2 PO 4 0.2-1g/L，VB 1 0.01-0.05g/L，pH＝6.0。
Preferably, the soybean juice is obtained by cooking soybean powder to obtain juice, boiling the soybean powder for 20-40min, filtering with four layers of gauze, and preparing the filtrate into a culture medium.
Preferably, in step (2), the fermentation medium and the feed medium used in the semi-continuous culture process both consist of: molasses with sugar content of 15-30g/L, corn steep liquor 10-25g/L, high fiber raw material granule 0.5-5g/L, ammonium sulfate 2-5g/L, MgSO 4 0.2-1g/L，FeSO 4 .7H 2 O 0.02-0.08g/L，KH 2 PO 4 0.2-1g/L，VB 1 0.01-0.05g/L， pH＝4.2-6.5。
Preferably, the edible fungus mycelium seed liquid is transferred into a fermentation tank for semi-continuous fermentation culture according to the inoculation amount of 10-25% v/v, and the liquid filling amount of the fermentation tank is 60-75% v/v; stirring at 50-300r/min, preferably 200-250r/min, at 24-28 deg.C, with aeration rate of 2400L/h (aeration ratio of 0.4-1.5L/L/min), pressure of 0.06-0.1MPa, and pH adjusted to 4.2-6.5 with ammonia water; when the fermentation residual reducing sugar is reduced to 1-5g/L, discharging 20-80% volume (preferably 50-60%) of fermentation liquor, and supplementing new feeding culture medium with the same volume for subsequent continuous fermentation.
Preferably, in step (3), the fermentation medium used for the continuous fermentation culture consists of: molasses with sugar content of 15-30g/L, corn steep liquor 10-25g/L, high fiber raw material granule 0.2-2g/L, ammonium sulfate 2-5g/L, MgSO 4 0.2-1g/L，FeSO 4 .7H 2 O 0.02-0.08g/L，KH 2 PO 4 0.2-1g/L，VB 1 0.01-0.05g/L，pH＝4.2-6.5。
Preferably, in the step (3), the feeding speed of continuous fermentation is controlled to be 0.2-0.8L/h, 2-3g/L of reducing sugar in the fermentation tank is maintained, the stirring speed is 50-300r/min, the temperature is 24-28 ℃, the ventilation volume is 1000-2400L/h, the pressure is 0.06-0.1MPa, and the pH value is regulated to be 4.2-6.5 by adopting ammonia water.
Preferably, in the step (3), the concentration of the continuous fermentation thallus is maintained at 14-20g/L, preferably 16-17 g/L; when the cell concentration decreases by 20% or more, the operation is stopped.
Preferably, the high-fiber raw material particles are particles obtained by crushing one or more than two of corn husks, straws, microcrystalline cellulose and bacterial cellulose and sieving the crushed particles with a 40-120-mesh sieve.
Has the advantages that:
the invention adopts a semi-continuous fermentation mode to effectively enhance the starting efficiency and the fermentation intensity of continuous fermentation; the efficient and stable operation of continuous fermentation is realized by controlling the rotating speed and the sugar concentration, the strain is not easy to degenerate, and the fermentation intensity and the thallus yield are higher; the addition of high-fiber raw materials can obviously improve the activity of the thallus enzyme, maintain higher activity of the thallus and realize the biomass rich in high protein.
Drawings
The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.
FIG. 1 is a graph showing the variation of reducing sugar and cell concentration in two processes of example 5.
FIG. 2 is a graph showing the change in reducing sugar and cell concentration in different batches of example 8.
FIG. 3 is the effect of different reducing sugar concentrations on yield in example 9.
Detailed Description
The invention will be better understood from the following examples.
In the following examples, the soybean juice was obtained by cooking soybean flour to obtain a juice, boiling the soybean flour for 20-40min, filtering with four layers of gauze, and collecting the filtrate. The high-fiber raw material particles are particles obtained by crushing one or more than two of corn husks, straws, microcrystalline cellulose or bacterial cellulose and sieving the crushed materials with a 40-120-mesh sieve.
Example 1 Effect of initial sugar concentration on cell yield (cell-to-sugar ratio)
Strain: pleurotus djamor is provided by vegetable research institute of agricultural academy of sciences of Jiangsu province.
Seed culture medium: 10-30g/L of soybean juice (cooking juice), 10-30g/L of corn steep liquor, 15-25g/L of glucose, MgSO 4 0.2-1g/L，FeSO 4 .7H 2 O 0.02-0.08g/L，KH 2 PO 4 0.2-1g/L，VB 1 0.01-0.05g/L，pH＝6.0；
fermentation medium: molasses (35 g/L of sugar content by conversion), corn steep liquor 25g/L, ammonium sulfate 3g/L, MgSO 4 0.6 g/L，FeSO 4 .7H 2 O 0.06g/L，KH 2 PO 4 0.5g/L，VB 1 0.02g/L，pH＝4.8。
The culture conditions are as follows: for each formulation, 6 parallel flasks of 150mL each were prepared, inoculated at 12%, and shake-cultured at 26 ℃ and 180 rpm. For each formula, 3 bottles of samples are destructively taken respectively for 2 days and 4 days of culture, glucose concentration is measured, and the size and total dry weight of the thalli are measured after each bottle of fermentation liquor is centrifugally cleaned once. The results are shown in Table 1.
TABLE 1
From experimental results, the initial sugar concentration is lower, which is beneficial to improving the thallus yield (percentage of the thallus dry weight concentration to the consumed sugar concentration), namely, the initial sugar concentration is controlled to be 5-15g/L, and the thallus yield can reach more than 90%.
Example 2 Effect of fiber-based Medium size and addition amount on fermentation
The seed medium and fermentation medium were the same as in example 1. The following components were added and cultured for 3 days under the conditions of example 1, and the results are shown in Table 2.
TABLE 2
From the above table, it can be found that, compared with the corn stalk, the corn husk powder can effectively increase the concentration of the thallus. The concentration is controlled between 0.5g/L and 1g/L, and the yield of the thalli can be improved by more than 20 percent.
Example 3 Effect of corn bran addition on microbial cell Activity
The strain is Pleurotus djamor. The control group and the experimental group were inoculated and cultured in the seed medium of example 1, and the fermentation medium was fermented as follows. Culturing at 26 deg.C and 220r/min for 9 days. After the completion of the culture, the fermentation media of the control and experimental groups were continuously supplemented with 10% of the remaining liquid, and the culture was continued for 2 days under the same conditions. The fermentation results are shown in Table 3.
Control group medium: molasses (35 g/L of sugar content by conversion), corn steep liquor 25g/L, ammonium sulfate 3g/L, MgSO 4 0.6g/L，FeSO 4 .7H 2 O 0.06g/L，KH 2 PO 4 0.5g/L，VB 1 0.02g/L，pH＝4.8。
Experimental group culture medium: molasses (35 g/L of sugar content by conversion), corn steep liquor 25g/L, ammonium sulfate 3g/L, MgSO 4 0.6g/L，FeSO 4 .7H 2 O 0.06g/L，KH 2 PO 4 0.5g/L，VB 1 0.02g/L, corn bran (60 mesh screen underflow) 1g/L, pH 4.8.
TABLE 3
| Item | Cell concentration after 9 days of culture | Cell concentration after liquid change |
| Control group | 13.11 | 8.12 |
| Experimental group | 13.27 | 11.27 |
From the results of the cell concentration after the liquid change, it can be found that the activity of the cells in the experimental group added with the corn bran is higher than that in the control group.
Example 4 Effect of different rotation speeds on semi-continuous fermentation (cell concentration, morphology and fermentation Rate to the following batch)
The strain adopts pleurotus eryngii and is provided by vegetable research institute of agricultural academy of sciences of Jiangsu province. The seed medium and the fermentation medium were identical to the experimental group medium of example 3, and 28L of the medium was metered out of the tank (each component was weighed in a 30L system), adjusted to pH6, and added with 20mL of a defoaming agent. Sterilized at 121 ℃ for 20 minutes, cooled and then flame-inoculated into a seeding tank at 10% inoculum size (2 bottles of large shake flasks). 200-500r/min (specifically controlled according to the following table), 2000L/h of ventilation, 0.08MPA of tank pressure, pH4.2-6.5, culturing at 26 ℃, supplementing sterile water to the scale mark every day, and finishing fermentation after 4 days. After the fermentation, samples were taken to determine the dry weight concentrations of glucose and biomass, and the results are shown in Table 4.
TABLE 4
During the fermentation process, starting at a lower flow rate, larger size mycelium pellets and yields can be obtained. As shown in the above table, the cell yield was significantly higher than 400r/min at 200r/min, which may be detrimental to cell growth due to the easy rupture of mycelium under high shear. However, when the biomass is started at a high rotating speed and operated at a low rotating speed, the yield of thalli can be properly improved, probably because the initially inoculated seed state presents mycelium balls with uniform size, the growth of the mycelium balls needs to form mycelium and reproduce to form balls, and a certain shearing force is provided at a high rotating speed to promote the dispersion and regrowth of the mycelium balls. In addition, the rotating speed is properly increased in the later stage of fermentation, so that the dissolved oxygen environment can be improved, and the mass transfer effect is improved.
Example 550L tank conventional fermentation vs. feed fermentation
Conventional fermentation basal medium: molasses (35 g/L of sugar content by conversion), corn steep liquor 25g/L, high-fiber raw material particles 0.5g/L, ammonium sulfate 3g/L, MgSO 4 0.6g/L，FeSO 4 .7H 2 O 0.06g/L，KH 2 PO 4 0.5g/L，VB 1 0.01-0.05g/L，PH＝4.8。
Feeding fermentation culture medium: molasses (20 g/L of sugar content by conversion), corn steep liquor 25g/L, high-fiber raw material particles 0.5g/L, ammonium sulfate 3g/L, MgSO 4 0.6g/L，FeSO 4 .7H 2 O 0.06g/L，KH 2 PO 4 0.5g/L，VB 1 0.02g/L，PH＝4.8。
And (3) a supplemented medium in supplemented fermentation: raw molasses (5L after 1 time dilution, 600mL of corn syrup and 30g of ammonium sulfate).
The strain is Pleurotus djamor. And (3) quantifying 28L outside the tank, (weighing each component according to a 30L system, adding a supplemented medium for 2 days of fermentation, adding 20mL of a defoaming agent, and adjusting the pH value to 6, wherein the process 1 is conventional fermentation, the process 2 is supplemented fermentation, and the supplemented medium is supplemented after 2 days of fermentation, and the converted total sugar amount is consistent with that of the process 1). Sterilized at 121 ℃ for 20 minutes, cooled and then flame-inoculated into a seeding tank at 10% inoculum size (2 bottles of large shake flasks). 200r/min, the ventilation rate is 1800L/h, the tank pressure is 0.08MPA, the pH value is 4.2-6.5, the culture is carried out for 6 days at 26 ℃, and the fermentation is finished. Performing solid-liquid separation by gauze, pouring the collected thallus into a 50L barrel, adding 30L of water, stirring and cleaning for 10 minutes, and then separating by gauze; the washing process was repeated 4 times. The collected biomass was finally weighed, and the results are shown in FIG. 1 and Table 5.
TABLE 5
From the above table, it can be seen that the fed-batch fermentation rate is relatively fast, i.e. the fermentation yield is large; in addition, the yield of the saccharomycetes is obviously improved.
EXAMPLE 6 semi-continuous fermentation retentate ratio impact on fermentation
The culture method of the process 2 of the example 5 is the same, and when the sugar is reduced to below 2g/L, the liquid is changed according to different proportions, and the results are shown in the table 6.
TABLE 6
It can be seen that the yield and productivity of the thallus can be obviously improved by the proper ratio of the liquid retention, and the liquid retention ratio is preferably controlled to be about 55%.
Example 7 semi-continuous fermentation batch fermentation index
The first batch was prepared by referring to the culture method of example 6 (retentate ratio of 55%). The reducing sugar was reduced to below 2g/L and the solution was changed, the results are shown in Table 7.
TABLE 7
As can be seen, 6 liquid changes are carried out, the yield and the yield of the thalli tend to be stable from the 4 th batch, and the average values in the stationary phase are 74.8 percent and 0.197g/L/h respectively, so the 4 th to 6 th semi-continuous fermentation liquids are preferably used for subsequent continuous fermentation.
Example 8 semi-continuous fermentation batch versus continuous fermentation index
A continuous fermentation test was carried out in a 50L fermenter, and the initial liquid loading was 30L. Using the procedure of example 7, continuous fermentation was started when the first and fourth batches of fermenters reached a concentration of 17g/L, respectively. The continuous fed-batch fermentation medium comprises: molasses (20 g/L of sugar content by conversion), corn steep liquor 22g/L, corn bran 0.5g/L (80 mesh), ammonium sulfate 3g/L, MgSO 4 0.4g/L，FeSO 4 .7H 2 O 0.05g/L，KH 2 PO 4 0.4g/L, VB10.01g/L, PH 5.0. The feed flow rate was 0.3L/h. The fermentation results are shown in FIG. 2.
From the operation effect, the first batch finishes the starting continuous fermentation, the reducing sugar is not completely consumed, and although the reducing sugar concentration in the later period shows a descending trend, the period is longer. In the continuous fermentation started in batch 4, the reducing sugar concentration was kept at 2.5g/L or less, and the average cell concentration was about 17.5 g/L. Meanwhile, the integral thallus size of the fermentation process of the latter is smaller and is in the range of 2.5-2.9mm, while the external dimension of the thallus of the fermentation process of the former is larger and is generally 2.9-3.3 mm. In addition, the average yield of the former cells was about 70%, and the sugar loss was large. The average yield of the fermentation thalli of the latter is more than 90 percent.
Example 9 Effect of different reducing sugar concentrations on the intensity (yield) of continuous fermentation
On the basis of example 8, after 408 hours of fermentation, the sugar concentration of the continuous feed was increased to 30g/L with a constant flow rate. The operation was continued for a period of 5 days, and the results are shown in FIG. 3.
From the fermentation curve, when the initial sugar is increased to 30g/L, the reducing sugar concentration of the fermentation liquor is obviously improved, the thallus concentration is also obviously improved, but after 2 days of fermentation, the thallus concentration tends to be reduced, and the thallus concentration is maintained to be about 19 g/L. The average yield is 81.8 percent after 5 days of fermentation. This is probably due to the fact that at higher biomass concentrations, more sugar is diverted to secondary metabolites and the higher biomass concentration affects mass transfer effects.
The present invention provides a method and a concept for continuous fermentation of edible fungus mycelia, and a plurality of methods and ways for implementing the technical solution, and the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, a plurality of modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention. All the components not specified in this embodiment can be implemented by the prior art.
Claims (10)
1. A continuous fermentation method of edible fungus mycelia is characterized by comprising the following steps:
(1) preparing edible fungus mycelium seed liquid by adopting a shake flask;
(2) transferring the edible fungus mycelium seed liquid obtained by culturing in the step (1) into a fermentation tank for semi-continuous culture, discharging a part of fermentation liquid when the fermentation residual reducing sugar in each batch is reduced to 1-5g/L, supplementing a new supplementary culture medium, and culturing for 3-6 batches in the same fermentation mode;
(3) and (3) supplementing a fermentation medium into the fermentation liquor discharged from each batch of semi-continuous culture in the step (2), and carrying out continuous fermentation to obtain the microbial fertilizer.
2. The method of claim 1, wherein the edible fungus is any one of oyster mushroom, agaricus bisporus, hypsizygus marmoreus, pleurotus geesteranus, panus giganteus, lepista bulbophylla, agrocybe aegerita, pleurotus citrinopileatus, russula, beefsteak mushroom, shiitake mushroom, pleurotus eryngii, tremella, morchella esculenta or flammulina velutipes.
3. The continuous fermentation method of edible fungus mycelia according to claim 1, wherein in the step (1), the edible fungus mycelia seed culture medium comprises: 10-30g/L of soybean juice, 10-30g/L of corn steep liquor, 15-25g/L of glucose and MgSO 4 0.2-1g/L，FeSO 4 .7H 2 O 0.02-0.08g/L，KH 2 PO 4 0.2-1g/L，VB 1 0.01-0.05g/L，pH＝6.0。
4. The method for continuously fermenting edible fungus mycelia according to claim 3, wherein the soybean juice is obtained by cooking soybean flour to obtain juice, boiling the soybean flour for 20-40min, filtering with four layers of gauze, and preparing the filtrate into a culture medium.
5. The continuous fermentation method of edible fungus mycelia according to claim 1, wherein in step (2), the fermentation medium and the feeding medium adopted in the semi-continuous culture process both comprise: molasses with sugar content of 15-30g/L, corn steep liquor 10-25g/L, high fiber raw material granule 0.5-5g/L, ammonium sulfate 2-5g/L, MgSO 4 0.2-1g/L，FeSO 4 .7H 2 O 0.02-0.08g/L，KH 2 PO 4 0.2-1g/L，VB 1 0.01-0.05g/L，pH＝4.2-6.5。
6. The continuous fermentation method of edible fungus mycelia according to claim 5, wherein in the step (2), the seed solution of edible fungus mycelia is transferred to a fermentation tank according to an inoculation amount of 10-25% v/v for semi-continuous fermentation culture, and the liquid loading amount of the fermentation tank is 60-75% v/v; stirring at a rotation speed of 50-300r/min, at a temperature of 24-28 ℃, with a ventilation volume of 1000-2400L/h and a pressure of 0.06-0.1Mpa, and regulating the pH value to 4.2-6.5 by adopting ammonia water; when the fermentation residual reducing sugar is reduced to 1-5g/L, discharging 20-80% of fermentation liquid, and supplementing new supplemented medium with the same volume for subsequent continuous fermentation.
7. The method for continuously fermenting edible fungus mycelia according to claim 1, wherein the fermentation medium used in the continuous fermentation culture in step (3) comprises: molasses with sugar content of 15-30g/L, corn steep liquor 10-25g/L, high fiber raw material granule 0.2-2g/L, ammonium sulfate 2-5g/L, MgSO 4 0.2-1g/L，FeSO 4 .7H 2 O0.02-0.08g/L，KH 2 PO 4 0.2-1g/L，VB 1 0.01-0.05g/L，pH＝4.2-6.5。
8. The method for continuously fermenting edible fungus mycelia according to claim 7, wherein in the step (3), the feeding speed of the continuous fermentation is controlled to be 0.2-0.8L/h, the reducing sugar in the fermentation tank is maintained to be 2-3g/L, the stirring speed is 50-300r/min, the temperature is 24-28 ℃, the ventilation rate is 1000-.
9. The method according to claim 8, wherein the continuous fermentation is stopped when the concentration of the mycelia decreases by 20% or more while the concentration of the mycelia in the continuous fermentation is maintained at 14 to 20g/L in the step (3).
10. The method for continuously fermenting edible fungus mycelia according to claim 5 or 7, wherein the high-fiber raw material particles are particles obtained by crushing any one or a combination of more than two of corn husks, straws, microcrystalline cellulose and bacterial cellulose and sieving the crushed particles with a 40-120-mesh sieve.
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