CN113604365B - Ultrasonic wave control mycelium pellet size and method for producing citric acid by using same - Google Patents
Ultrasonic wave control mycelium pellet size and method for producing citric acid by using same Download PDFInfo
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Abstract
The invention discloses a method for controlling the particle size of mycelium pellets by ultrasonic waves and application thereof. The method of the invention comprises the following steps: (1) preparation of a seed culture medium; (2) Sterilizing and cooling the seed culture medium prepared in the step (1), inoculating aspergillus spores, and culturing to obtain a seed solution containing aspergillus mycelium pellets; (3) Carrying out ultrasonic treatment on the seed liquid containing the aspergillus mycelium pellets in the step (2) to obtain seed liquid containing mycelium pellets with different particle sizes; adding seed liquid containing mycelium pellets with different particle sizes into a fermentation medium, and stirring and fermenting to obtain citric acid fermentation liquid. The method is favorable for realizing unification of high concentration, high conversion rate and high efficiency of citric acid fermentation, and has important promotion effect on the technical improvement of the citric acid industry.
Description
Technical Field
The invention relates to the technical field of microbial culture, in particular to an ultrasonic wave control mycelium pellet size and a method for producing citric acid by using the same.
Background
Citric acid is an important six-carbon platform compound, is the organic acid with the largest production and use amount in the world, and has wide application. The citric acid is mainly used in food industry, such as sour agent, antioxidant, gelatinizer, etc., and has wide application in medicine, chemical industry, feed, electronic, textile, etc. industry fields, and the market demand is increasing year by year.
China is the largest country for producing and exporting citric acid, and occupies an important position in the citric acid market. Although the biotechnology is continuously developed in recent years, new fermentation technology is continuously emerging, the citric acid fermentation technology is still subject to various limitations and is slow to progress. A citric acid producing strain, aspergillus niger, is a multicellular filamentous fungus that forms mycelium pellets in submerged fermentation. Each mycelium pellet corresponds to a growth cell. In the culture, the quantity of mycelium pellets is not increased, but the volume of mycelium pellets is gradually increased, so that the transmission of oxygen and nutrient substances in the mycelium pellets is limited, and the improvement of acid production efficiency is seriously influenced.
Therefore, the development of a method for effectively controlling the grain size of Aspergillus niger mycelium pellet to improve the fermentation acid production efficiency is of great importance. The existing methods for controlling the particle size of mycelium mainly comprise controlling culture conditions, spore concentration, adding particles and the like, but the methods are all controlled in the initial stage of culture, and once mycelium pellets are formed, effects cannot be continuously produced, and repeated multi-batch control cannot be performed. Mycelium pellets can be crushed by grinding and extruding the glass beads, but the glass beads occupy a large amount of culture space, so that the effective volume is reduced, and the separation operation is increased. Cutting mycelium pellets by mechanical shear force has also been widely reported, such as controlling the size of mycelium pellet particle size by stirring rotation speed. Patent CN201410329652.X discloses a method for continuously culturing citric acid Aspergillus niger seeds based on mycelium pellet dispersion technology; patent CN201810986196.4 discloses a method for continuously culturing aspergillus niger seeds and producing citric acid by using the same, and both patents adopt a mechanical disperser to cut mycelium pellets and cut the mycelium pellets into fragments, thereby realizing the control of the mycelium pellet size. However, the mechanical shearing force has a large limitation on the initial size of the mycelium pellet, and when the particle size of the initial mycelium pellet is less than 150 μm, the cutting effect is not ideal; in addition, the mechanical rotating assembly easily forms a contamination risk point. Therefore, the search for a new method for controlling the particle size of Aspergillus niger mycelium pellets is a problem to be solved.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an ultrasonic wave control mycelium pellet size and a method for producing citric acid by using the same. The invention utilizes ultrasonic crushing to realize effective control of the grain size of the Aspergillus niger mycelium pellet, simultaneously ensures the activity of the mycelium, provides a new method for controlling the grain size of the filamentous fungi, can be beneficial to realizing unification of high concentration, high conversion rate and high efficiency by citric acid fermentation, and has important promotion effect on the technical promotion of the citric acid industry.
The technical scheme of the invention is as follows:
a method for controlling particle size of mycelium pellets by ultrasonic waves, the method comprising the steps of:
(1) Preparing a seed culture medium;
(2) Sterilizing and cooling the seed culture medium prepared in the step (1), inoculating aspergillus spores, and culturing to obtain a seed solution containing aspergillus mycelium pellets;
(3) And (3) carrying out ultrasonic treatment on the seed liquid containing the aspergillus mycelium pellets in the step (2) to realize the control of the particle size of the mycelium pellets, thereby obtaining the seed liquid containing the mycelium pellets with different particle sizes.
Further, in the step (1), the total sugar of the seed culture medium is 100-140 g/L, and the C/N is 20-30.
Further, in the step (2), the sterilization temperature is 115-125 ℃ and the sterilization time is 15-30 min; the temperature after cooling is 35-39 ℃.
The aspergillus spores are aspergillus niger spores; the final concentration of the aspergillus access spores is 1 x 10 5 ～9*10 5 individual/mL; the final concentration is the concentration of aspergillus spores in the seed liquid before culture; the culture is carried out at the temperature of 35-39 ℃ and the speed of 150-300 r/min for 20-36h under stirring.
The grain diameter of the aspergillus mycelium pellet is larger than 70 mu m.
Further, in the step (3), the ultrasonic treatment conditions are as follows: the ultrasonic power is 150-300W, the temperature is 35-39 ℃, the total ultrasonic working time is 3-5 min, and the repeated working is carried out for 2s and 8s.
The repeated work 2s stop 8s is that after the ultrasonic 2s stop 8s, the ultrasonic 2s stop 8s is continuously repeated; the total ultrasonic working time only refers to the sum of the times of 2s of each repeated working, and the time of pause is not calculated.
Further, in the step (3), the proportion of mycelium pellets with the particle size of 30-50 μm in the mycelium pellets with different particle sizes is more than 80%.
The seed liquid containing mycelium pellets with different particle sizes prepared by the method.
Use of the seed liquid containing mycelium pellets of different particle sizes for the production of citric acid.
Further, the seed liquid containing mycelium pellets with different particle diameters is used for producing citric acid, and the steps are as follows:
(1) Preparing a fermentation medium; the fermentation medium is prepared from corn liquefied liquid and corn sugar liquid, the total sugar is 140-160 g/L, and the C/N is 55-90;
(2) Adding seed liquid containing mycelium pellets with different particle diameters into the fermentation medium prepared in the step (1), stirring and fermenting at the temperature of 35-39 ℃ and the speed of 100-300 r/min, and ending the fermentation when the concentration of reducing sugar is lower than 5g/L to obtain fermentation liquor containing citric acid; the seed liquid containing mycelium pellets with different particle diameters accounts for 6-15% of the volume fraction of the fermentation medium.
The principle of ultrasonic waves is that the instantaneous strong sound waves generate mechanical waves in liquid to achieve the effect of crushing, and ultrasonic crushing is a common means in the biological industry and is mainly used for sterilizing and crushing cells to promote the extraction of intracellular products. Typically, cells that have been disrupted by sonication lose growth activity, and even cell integrity, and thus ultrasound applications typically do not take into account the growth activity of the microbial cells being treated. Some documents report that the micro morphology of the thallus is changed by using ultrasonic waves with lower intensity, the integrity of the cell is still maintained, the permeability of the cell membrane is increased, and the secretion of intracellular products is improved. The method is applied to control the macroscopic morphology (namely the particle size) of the Aspergillus niger mycelium pellet in a breakthrough way, the mycelium pellet is split into mycelium pellets with small different particle sizes by utilizing moderate ultrasonic intensity, the macroscopic morphology of the mycelium pellet is obviously changed, the number of growth units is increased, and meanwhile, the growth vigor of the thalli is maintained, so that the conventional thinking is broken through.
The beneficial technical effects of the invention are as follows:
(1) The invention utilizes ultrasonic crushing to realize effective control of the particle size of the Aspergillus niger mycelium pellet, ensures the activity of the mycelium, and provides a novel method for controlling the particle size of the filamentous fungi. Under the same spore inoculum size condition, the number of the fermentation mycelium pellets is obviously increased, the average grain diameter of the mycelium pellets is obviously reduced, and the fermentation strength is obviously improved.
(2) According to the invention, the active damage of the mycelium pellets can be reduced while the effective division of the mycelium pellets is ensured, so that the obtained mycelium pellets with different particle diameters have better seed culture time with good growth activity, particle diameters of mycelium pellets before crushing and ultrasonic power, and the research shows that the mycelium pellets just formed have high growth speed, relatively thin cell walls and cell membranes, are more easily damaged to lose cell integrity, and the mycelium pellets obtained by ultrasonic treatment are difficult to keep activity; the mature mycelium pellet has relatively thick cell walls and cell membranes, strong damage resistance, better cell integrity and intact activity of the mycelium pellet obtained by ultrasonic treatment. Therefore, in order to maintain the number and growth vigor of mycelium pellets, seed culture is limited to 20 to 36 hours and mycelium pellet size is larger than 70 μm before ultrasonic treatment.
(3) The invention defines the ultrasonic conditions as follows: the power is 150-300W, the total ultrasonic treatment time is 3-5 min, the working period is 10s, each period works for 2s, and 8s is stopped. Through limiting the working conditions of ultrasonic waves, the phenomenon that the mycelium pellets obtained by ultrasonic treatment lose vitality due to overlarge ultrasonic power or overlong treatment time is avoided.
(4) In order to ensure that the particle size of the mycelium pellets obtained by ultrasonic treatment is relatively stable, the ultrasonic power and the treatment time are dynamically adjusted aiming at mycelium pellets with different initial particle sizes, and the larger the particle size of the mycelium pellets is, the larger the corresponding power is, and the longer the total ultrasonic treatment time is.
(5) According to the invention, under the condition that the initial total sugar concentration of the fermentation liquid is 140g/L and the same spore inoculation amount is adopted, after ultrasonic treatment, the number of mycelium pellets is increased by 12.6 times, the particle size is also reduced to 33.8%, and as the mycelium pellets are smaller, nutrient substances and oxygen are more easily absorbed, the final fermentation strength is improved by 22.9%, the fermentation conversion rate is improved by 5.86%, and the fermentation efficiency and conversion rate of citric acid are remarkably improved.
(6) As the particle size of the mycelium pellets after ultrasonic treatment is obviously reduced, the specific surface area is increased, and the number of the growth units is obviously increased, the invention has larger advantage under the condition of high-concentration fermentation. When the initial total sugar concentration of fermentation is increased to 160g/L, the fermentation strength of the invention is improved by 28.0%, and the fermentation conversion rate is improved by 6.88%. Therefore, the method can be beneficial to realizing unification of high concentration, high conversion rate and high efficiency of citric acid fermentation, and has important promotion effect on the technical improvement of the citric acid industry.
(7) The invention firstly controls the grain diameter of mycelium to be more than 70 mu m, then controls the grain diameter to be 30-50 mu m after ultrasonic treatment, and the mycelium grows to be more than 50 mu m in the process of fermenting citric acid, compared with the process of directly fermenting citric acid without ultrasonic treatment, the seed liquid achieves the purpose of controlling the grain diameter of the final mycelium pellet by ultrasonic treatment, and the mycelium pellet obtained by ultrasonic treatment can also keep better growth activity; the overall number of mycelium pellets was also increased over that without ultrasound.
Drawings
FIG. 1 is a comparison of seed mycelium pellets before and after sonication in example 1 of the present invention.
In the figure: (1) mycelium pellet in seed liquid before ultrasonic treatment; (2) mycelium pellet in seed liquid after ultrasonic treatment.
FIG. 2 is a comparison of the fermented mycelium pellets prepared in example 3 of the present invention and comparative example 3.
In the figure: (1) fermented mycelium pellets of example 3; (2) A fermented mycelium pellet of comparative example 3.
Detailed Description
The invention will now be described in detail with reference to the drawings and specific examples.
The embodiments and specific operation procedures are given on the premise of implementing the technical scheme of the invention, but the protection scope of the invention is not limited to the following embodiments.
The raw materials and reagents involved in the following examples are all commercially available; aspergillus niger is derived from China center for type culture Collection (CICC), accession number CICC 40021. The determination of total sugar and reducing sugar adopts film titration method; the determination of the nitrogen source adopts a Kjeldahl nitrogen determination method; the citric acid is determined by titration with NaOH of 0.1429 mol/L; spore counting adopts a blood cell counting plate; the mycelium particle size distribution was determined using a laser particle size analyzer. Mycelium viability was characterized by the proportion of mycelium pellets greater than 50 μm after cultivation. After ultrasonic treatment, the particle size of the mycelium pellet which loses activity is not increased and is kept at 30-50 mu m; the mycelium pellet with retained activity can form new mycelium pellet, and the final particle size is larger than 50 μm. Thus, a higher proportion of mycelium pellets greater than 50 μm after cultivation indicates a higher proportion of mycelium pellets that remain viable after sonication. Unless otherwise indicated, all equipment and processes commonly used in the art are employed.
Example 1
A method of controlling mycelium pellet size by ultrasonic waves, the method comprising the steps of:
mixing corn flour and tap water at a mass ratio of 1:3, and adding Ca (OH) 2 Adjusting the pH of the slurry to 5.8-6.0, and adding alpha-high temperature amylase according to the adding amount of 20U/g corn flour; liquefying in a water bath kettle at 97 ℃ to obtain qualified corn liquefied liquid after iodine test is light brown; filtering 70% of the corn liquefied liquid through a buchner funnel to remove filter residues, and obtaining corn sugar liquid. Preparing seed culture medium (total sugar 100g/L, C/N of 30) from semen Maydis liquefied solution and ammonium sulfate, sterilizing at 115deg.C for 30min, cooling to 35deg.C, inoculating Aspergillus niger spores to sterilized and cooled seed culture medium, and final concentration of spores is 1×10 5 The culture was carried out at 35℃and 300rpm for 20 hours per mL to obtain a seed solution having an average mycelium pellet diameter of 78. Mu.m.
And (3) treating the prepared seed liquid by utilizing ultrasonic waves to split mycelium pellets into mycelium pellets with different particle sizes. The ultrasonic conditions are as follows: the power is 150W, the total ultrasonic treatment time is 3min, the work is 2s, and the stop is 8s. The temperature of the seed liquid is controlled at 35 ℃ in the treatment process, and the particle size distribution ratio of 30-50 mu m in mycelium pellets with different particle sizes obtained by ultrasonic treatment is more than 80%.
The seed liquid containing mycelium pellets with different particle sizes obtained by the ultrasonic treatment is used for fermenting citric acid, and specifically comprises the following steps:
inoculating seed liquid containing mycelium pellets with different particle sizes obtained by ultrasonic treatment into a fermentation medium according to the volume ratio of 15%, fermenting and culturing at 35 ℃ and the rotating speed of 300rpm, and ending fermentation when the concentration of reducing sugar is lower than 5g/L to obtain the citric acid fermentation liquid.
The fermentation medium was prepared from corn liquefaction and corn sugar solution (total sugar 140g/L, C/N90).
Example 2
A method of controlling mycelium pellet size by ultrasonic waves, the method comprising the steps of:
the preparation of the corn flour liquefaction solution and the corn sugar solution is the same as in example 1. Preparing seed culture medium (total sugar 120g/L, C/N25) from semen Maydis liquefied solution and ammonium sulfate, sterilizing at 121deg.C for 20min, cooling to 37deg.C, inoculating Aspergillus niger spores to sterilized and cooled seed culture medium, and final concentration of spores is 5×10 5 The culture was carried out at 37℃and 200rpm for 26 hours per mL to obtain a seed solution having an average mycelium pellet diameter of 91. Mu.m.
And (3) treating the prepared seed liquid by utilizing ultrasonic waves to split mycelium pellets into mycelium pellets with different particle sizes. The ultrasonic conditions are as follows: the power is 225W, the total ultrasonic treatment time is 4min, the operation is 2s, and the operation is stopped for 8s. The temperature of the seed liquid is controlled at 37 ℃ in the treatment process, and the particle size distribution ratio of 30-50 mu m in mycelium pellets with different particle sizes obtained by ultrasonic treatment is more than 80%.
The mycelium pellets with different particle diameters obtained by ultrasonic treatment are used for fermenting citric acid, and specifically comprise the following steps:
inoculating mycelium pellets with different particle diameters obtained by ultrasonic treatment into a fermentation culture medium according to the volume proportion of 10%, fermenting and culturing at 37 ℃ and a rotating speed of 200rpm, and ending fermentation when the concentration of reducing sugar is lower than 5 g/L.
The fermentation medium was prepared from corn liquefaction and corn sugar solution (total sugar 150g/L, C/N70).
Example 3
A method of controlling mycelium pellet size by ultrasonic waves, the method comprising the steps of:
the preparation of the corn flour liquefaction solution and the corn sugar solution is the same as in example 1. Preparing seed culture medium (total sugar 140g/L, C/N20) from liquefied semen Maydis and ammonium sulfate, sterilizing at 125deg.C for 15min, cooling to 39deg.C, inoculating Aspergillus niger spores to seed culture medium, and final concentration of spores is 9×10 5 The culture was carried out at 39℃and 150rpm for 36 hours per mL to obtain a seed solution having an average mycelium pellet diameter of 142. Mu.m.
The obtained seed liquid is treated by ultrasonic wave, and mycelium pellets are split into mycelium pellets with different particle sizes. The ultrasonic conditions are as follows: the power is 300W, the total ultrasonic treatment time is 5min, the work is 2s, and the stop is 8s. The temperature of the seed liquid is controlled at 39 ℃ in the treatment process, and the particle size distribution ratio of 30-50 mu m in mycelium pellets with different particle sizes obtained by ultrasonic treatment is more than 80%.
The mycelium pellets with different particle diameters obtained by ultrasonic treatment are used for fermenting citric acid, and specifically comprise the following steps:
inoculating mycelium pellets with different particle diameters obtained by ultrasound into a fermentation medium according to the volume proportion of 6%, fermenting and culturing at 39 ℃ and a rotating speed of 100rpm, and ending fermentation when the concentration of reducing sugar is lower than 5 g/L.
The fermentation medium was prepared from corn liquefaction and corn sugar solution (total sugar 160g/L, C/N55).
Comparative example 1
The preparation of the corn flour liquefaction liquid and the corn sugar liquid is the same as in example 1. Preparing seed culture medium (total sugar 100g/L, C/N of 30) from semen Maydis liquefied solution and ammonium sulfate, sterilizing at 115deg.C for 30min, cooling to 35deg.C, inoculating Aspergillus niger spores to seed culture medium, and final concentration of spores is 1×10 5 And culturing at 35 ℃ and 300rpm for 20h per mL to obtain seed liquid.
Inoculating the seed liquid into a fermentation culture medium according to the volume proportion of 15% for fermentation culture.
The fermentation medium is prepared from corn liquefied solution and corn sugar solution (total sugar 140g/L, C/N is 90); the fermentation conditions were the same as in example 1.
Comparative example 2
The preparation of the corn flour liquefaction liquid and the corn sugar liquid is the same as in example 1. Preparing seed culture medium (total sugar 120g/L, C/N25) from liquefied semen Maydis and ammonium sulfate, sterilizing at 121deg.C for 20min, cooling to 37deg.C, inoculating Aspergillus niger spores to seed culture medium, and final concentration of spores is 5×10 5 The seed solution is obtained by culturing the seed solution for 26 hours at 37 ℃ and 200 rpm.
And (3) inoculating the seed liquid into a fermentation medium according to the volume ratio of 10% for fermentation culture.
The fermentation medium is prepared from corn liquefaction liquid and corn sugar liquid (total sugar 150g/L, C/N is 70); the fermentation conditions were the same as in example 2.
Comparative example 3
The preparation of the corn flour liquefaction liquid and the corn sugar liquid is the same as in example 1. Preparing seed culture medium (total sugar 140g/L, C/N20) from liquefied semen Maydis and ammonium sulfate, sterilizing at 125deg.C for 15min, cooling to 39deg.C, inoculating Aspergillus niger spores to seed culture medium, and final concentration of spores is 9×10 5 The seed solution is obtained by culturing the seed solution for 36 hours at the temperature of 39 ℃ and the rotating speed of 150 rpm.
And (3) inoculating the seed liquid into a fermentation medium according to the volume ratio of 6% for fermentation culture.
The fermentation medium is prepared from corn liquefaction liquid and corn sugar liquid (total sugar 160g/L, C/N is 55); the fermentation conditions were the same as in example 3.
Comparative example 4
The preparation of the corn flour liquefaction liquid and the corn sugar liquid is the same as in example 1. Preparing seed culture medium (total sugar 120g/L, C/N25) from liquefied semen Maydis and ammonium sulfate, sterilizing at 121deg.C for 20min, cooling to 37deg.C, inoculating Aspergillus niger spores to seed culture medium, and final concentration of spores is 5×10 5 The culture was carried out at 37℃and 200rpm for 16 hours per mL to obtain a seed solution having an average mycelium pellet diameter of 58. Mu.m.
The obtained seed liquid is treated by ultrasonic wave, and mycelium pellets are split into mycelium pellets with different particle sizes. The ultrasonic conditions are as follows: the power is 150W, the total ultrasonic treatment time is 3min, the work is 2s, and the stop is 8s. The temperature of the seed liquid is controlled at 37 ℃ in the treatment process, and the particle size distribution ratio of 30-50 mu m in mycelium pellets with different particle sizes obtained by ultrasonic treatment is more than 80%.
And inoculating mycelium pellets with different particle diameters obtained by ultrasonic treatment into a fermentation medium according to the volume proportion of 10% for fermentation culture.
The fermentation medium is prepared from corn liquefaction liquid and corn sugar liquid (total sugar 150g/L, C/N is 70); the fermentation conditions were the same as in example 2.
Comparative example 5
The corn meal liquefaction and corn sugar solution preparation were the same as in example 1, and the seed culture medium formulation and seed culture were the same as in example 2.
The obtained seed liquid is treated by ultrasonic wave, and mycelium pellets are split into mycelium pellets with different particle sizes. The ultrasonic conditions are as follows: the power is 400W, the total ultrasonic treatment time is 4min, the work is 2s, and the stop is 8s. The temperature of the seed liquid is controlled at 37 ℃ in the treatment process, and the particle size distribution ratio of 30-50 mu m in mycelium pellets with different particle sizes obtained by ultrasonic treatment is more than 80%.
And inoculating mycelium pellets with different particle diameters obtained by ultrasonic treatment into a fermentation medium according to the volume proportion of 10% for fermentation culture.
The fermentation medium is prepared from corn liquefaction liquid and corn sugar liquid (total sugar 150g/L, C/N is 70); the fermentation conditions were the same as in example 2.
Comparative example 6
The corn meal liquefaction and corn sugar solution preparation were the same as in example 1, and the seed culture medium formulation and seed culture were the same as in example 2.
The obtained seed liquid is treated by ultrasonic wave, and mycelium pellets are split into mycelium pellets with different particle sizes. The ultrasonic conditions are as follows: the power is 225W, the total ultrasonic treatment time is 6min, the operation is 2s, and the operation is stopped for 8s. The temperature of the seed liquid is controlled at 37 ℃ in the treatment process, and the particle size distribution ratio of 30-50 mu m in mycelium pellets with different particle sizes obtained by ultrasonic treatment is more than 80%.
And inoculating mycelium pellets with different particle diameters obtained by ultrasonic treatment into a fermentation medium according to the volume proportion of 10% for fermentation culture.
The fermentation medium is prepared from corn liquefaction liquid and corn sugar liquid (total sugar 150g/L, C/N is 70); the fermentation conditions were the same as in example 2.
Test example 1
The comparison of the citric acid fermentation effects of mycelium pellets prepared in examples 1 to 3 and comparative examples 1 to 3 is shown in Table 1. Wherein the calculation formula of the fermentation conversion rate is shown in the formula (1), and the calculation formula of the fermentation strength is shown in the formula (2).
TABLE 1
| Project | Example 1 | Example 2 | Example 3 | Comparative example 1 | Comparative example 2 | Comparative example 3 |
| Fermentation initial total sugar (g/L) | 140 | 150 | 160 | 140 | 150 | 160 |
| Fermentation acid yield (g/L) | 145.5 | 153.1 | 162.2 | 137.3 | 143.6 | 151.2 |
| Fermentation period (h) | 50 | 56 | 62 | 58 | 64 | 74 |
| Fermentation conversion (%) | 103.93 | 102.07 | 101.38 | 98.07 | 95.73 | 94.50 |
| Fermentation intensity (g/h/L) | 2.91 | 2.73 | 2.62 | 2.37 | 2.24 | 2.04 |
| Concentration of mycelium pellet (10) 4 personal/mL) | 10.9 | 11.8 | 12.3 | 0.8 | 3.4 | 5.8 |
| Mycelium pellet size (mum) | 107 | 103 | 98 | 317 | 226 | 184 |
As can be seen from Table 1, the method of the present invention uses ultrasonic waves to control the particle size of mycelium pellets, and has the most remarkable effect that the ultrasonic treatment significantly increases the number of mycelium pellets for fermentation, the average diameter of mycelium pellets is significantly reduced, and the fermentation strength is significantly improved. The comparison shows that under the same spore inoculation amount condition, the ultrasonic wave can obviously improve the fermentation efficiency and the conversion rate by controlling the particle size of mycelium pellets.
The technical effects of example 1 and comparative example 1 are compared to see: when the initial total sugar concentration of fermentation is 140g/L, the concentration of mycelium pellets of the comparative example is lower, the average particle size is larger, and the corresponding fermentation strength is lower; after ultrasonic treatment in example 1, the number of mycelium pellets is increased by 12.6 times, the particle size is also reduced to 33.8%, the fermentation strength is improved by 22.9%, and the fermentation conversion rate is improved by 5.86%. Comparison of the technical effects of example 2 and comparative example 2 shows that: at an initial total sugar concentration of 150g/L, the fermentation strength of example 2 was increased by 21.8% and the fermentation conversion rate was increased by 6.33% as compared to comparative example 2. Comparison of the technical effects of example 3 and comparative example 3 shows: at an initial total sugar concentration of 160g/L, the fermentation strength of example 3 was increased by 28.0% and the fermentation conversion rate was increased by 6.88% as compared to comparative example 3. The result shows that the method has larger advantages under the condition of high-concentration fermentation, is favorable for realizing high-concentration, high-conversion rate and high-efficiency fermentation production of the citric acid, and has important promotion effect on the technical improvement of the citric acid industry.
In example 1, the comparison of mycelium pellets in seed liquid before and after ultrasonic treatment is shown in FIG. 1. As can be seen from FIG. 1, the diameter of the seed mycelium pellet is in the range of 77-80 μm before ultrasonic treatment; after ultrasonic treatment, the particle size range of mycelium pellets in the seed liquid is reduced to 31-47 mu m, and the particle size of mycelium pellets is obviously changed.
Comparing the particle size of mycelium pellets of fermented citric acid in example 3 with that of comparative example 3, as shown in fig. 2, the particle size of mycelium pellets after fermentation of citric acid is significantly smaller than that of mycelium pellets without ultrasonic treatment in comparative example. As can be seen from fig. 1 and 2, the seed liquid is subjected to ultrasonic treatment, the particle size of the seed mycelium pellet is obviously reduced, and after the seed mycelium pellet is transferred to a fermentation tank and cultured, the diameter of the corresponding fermentation mycelium pellet is increased to 75-115 μm; the seed liquid is not treated by ultrasonic wave, the particle size of initial seed mycelium pellet is larger, and after transferring to a fermentation tank and culturing, the diameter of the corresponding fermentation mycelium pellet is further increased to 173-212 mu m. The result shows that the seed liquid achieves the purpose of controlling the particle size of the final mycelium pellet through ultrasonic treatment, and the small mycelium pellet obtained through ultrasonic treatment can also keep better growth activity. The number of mycelium pellets was also significantly increased compared to that without sonication.
Test example 2
The fermentation effects of examples 1 to 3 and comparative examples 4 to 6 were compared as shown in Table 2.
TABLE 2
As is clear from Table 2, in comparative example 4, the mycelium pellet was just formed at 16 hours, was more easily damaged and lost in activity, and the mycelium pellet ratio after cultivation was only 26.3% at a size of more than 50 μm; meanwhile, too high ultrasonic power or too long ultrasonic treatment time can also cause the obtained mycelium pellets to lose activity, the power in comparative example 5 is 400W, and the proportion of mycelium pellets with the power of more than 50 μm after culture is only 42.6%; in comparative example 6, the ultrasonic treatment time was 6min, and the proportion of mycelium pellets larger than 50 μm after cultivation was 47.1%. In examples 1 to 3, the seed age was extended to 20 to 36 hours to form mature mycelium pellets, the anti-damage ability was strong, the ultrasonic power and the treatment time were both in the appropriate ranges, the ratio of mycelium pellets larger than 50 μm after cultivation was more than 90%, and the final mycelium pellet concentration was more than 10.0 x 10 4 And the indexes such as fermentation intensity, conversion rate and the like are obviously higher than those of the comparative example.
Therefore, in order to maintain the number and growth vigor of mycelium pellets, it was confirmed that seed culture was performed for 20 to 36 hours and mycelium pellet diameter was greater than 70 μm before ultrasonic treatment. Meanwhile, the optimal ultrasonic conditions are as follows: the power is 150-300W, the total ultrasonic treatment time is 3-5 min, the single ultrasonic operation is carried out for 2s, and then 8s are stopped.
The above is only a preferred embodiment of the present invention, and the present invention is not limited to the above examples. It is to be understood that other modifications and variations which may be directly derived or contemplated by those skilled in the art without departing from the spirit and concepts of the present invention are deemed to be included within the scope of the present invention.
Claims (6)
1. A method for controlling the particle size of mycelium pellets by ultrasonic waves, which is characterized by comprising the following steps:
(1) Preparing a seed culture medium;
(2) Sterilizing and cooling the seed culture medium prepared in the step (1), inoculating aspergillus spores, and culturing to obtain a seed solution containing aspergillus mycelium pellets;
(3) Carrying out ultrasonic treatment on the seed liquid containing the aspergillus mycelium pellets in the step (2) to realize the control of the particle size of the mycelium pellets, thereby obtaining seed liquid containing mycelium pellets with different particle sizes;
in the step (2), the aspergillus spores are aspergillus niger spores; the final concentration of the aspergillus access spores is 1 x 10 5 ~9*10 5 individual/mL; the culture is carried out at the temperature of 35-39 ℃ and the speed of 150-300 r/min, and the culture is carried out for 20-36h under stirring;
in the step (2), the grain size of the aspergillus mycelium pellet is larger than 70 mu m;
in the step (3), the ultrasonic treatment conditions are as follows: the ultrasonic power is 150-300W, the temperature is 35-39 ℃, the total ultrasonic time is 3-5 min, and the ultrasonic operation is stopped for 8s each time;
in the step (3), the proportion of mycelium pellets with the particle size of 30-50 μm in the mycelium pellets with different particle sizes is more than 80%.
2. The method of claim 1, wherein in step (1), the total sugar of the seed medium is 100-140 g/L and the C/N is 20-30.
3. The method according to claim 1, wherein in the step (2), the sterilization temperature is 115-125 ℃ for 15-30 min; the temperature after cooling is 35-39 ℃.
4. A seed solution containing mycelium pellets of different particle sizes prepared by the method of any one of claims 1 to 3.
5. Use of the seed liquid containing mycelium pellets of different particle sizes according to claim 4 for producing citric acid.
6. The use according to claim 5, wherein the seed liquid containing mycelium pellets of different particle sizes is used for the production of citric acid by the steps of:
(1) Preparing a fermentation medium; the fermentation medium is prepared from corn liquefied liquid and corn sugar liquid, the total sugar is 140-160 g/L, and the C/N is 55-90;
(2) Adding seed liquid containing mycelium pellets with different particle diameters into the fermentation medium prepared in the step (1), stirring and fermenting at the temperature of 35-39 ℃ at the speed of 100-300 r/min, and ending the fermentation when the concentration of reducing sugar is lower than 5g/L to obtain fermentation liquor containing citric acid; the seed liquid containing mycelium pellets with different particle sizes accounts for 6-15% of the volume fraction of the fermentation medium.
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| CN104087629A (en) * | 2014-07-04 | 2014-10-08 | 江苏大学 | Method for increasing yield of intercellular polysaccharide in phellinus igniarius mycelium fermentation process by utilizing ultrasonic technique |
| CN104099253A (en) * | 2014-07-11 | 2014-10-15 | 江南大学 | Citric acid aspergillus niger seed continuous culture method based on mycelium pellet dispersion technology |
| CN108277240A (en) * | 2018-04-26 | 2018-07-13 | 齐齐哈尔龙江阜丰生物科技有限公司 | A kind of technique of preparation of citric acid by fermentation |
| CN112608956A (en) * | 2020-12-10 | 2021-04-06 | 浙江大学 | Method for improving yield of extracellular polysaccharide of bacteria by using ultrasonic waves |
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|---|---|---|---|---|
| CN104087629A (en) * | 2014-07-04 | 2014-10-08 | 江苏大学 | Method for increasing yield of intercellular polysaccharide in phellinus igniarius mycelium fermentation process by utilizing ultrasonic technique |
| CN104099253A (en) * | 2014-07-11 | 2014-10-15 | 江南大学 | Citric acid aspergillus niger seed continuous culture method based on mycelium pellet dispersion technology |
| CN108277240A (en) * | 2018-04-26 | 2018-07-13 | 齐齐哈尔龙江阜丰生物科技有限公司 | A kind of technique of preparation of citric acid by fermentation |
| CN112608956A (en) * | 2020-12-10 | 2021-04-06 | 浙江大学 | Method for improving yield of extracellular polysaccharide of bacteria by using ultrasonic waves |
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