WO2023165065A1 - 一种适用于大型发酵罐的挂壁式超声波强化发酵装置及其发酵方法 - Google Patents

一种适用于大型发酵罐的挂壁式超声波强化发酵装置及其发酵方法 Download PDF

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WO2023165065A1
WO2023165065A1 PCT/CN2022/104801 CN2022104801W WO2023165065A1 WO 2023165065 A1 WO2023165065 A1 WO 2023165065A1 CN 2022104801 W CN2022104801 W CN 2022104801W WO 2023165065 A1 WO2023165065 A1 WO 2023165065A1
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ultrasonic
fermentation
frequency
ultrasonic transducer
wall
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English (en)
French (fr)
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何荣海
马海乐
代春华
张�荣
吴本刚
许海宁
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江苏大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/02—Stirrer or mobile mixing elements
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M39/00—Means for cleaning the apparatus or avoiding unwanted deposits of microorganisms

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  • the invention belongs to the field of biological engineering equipment, and in particular relates to a wall-mounted ultrasonic intensified fermentation device suitable for large fermentation tanks.
  • Ultrasound as a physical means, is widely used in strengthening the value-added processing of agricultural products.
  • Ultrasonic has shown outstanding application value in the extraction of active ingredients of agricultural products, food sterilization, cell crushing, homogeneous emulsification, and promotion of heat and mass transfer. In particular, it also shows good application prospects in the promotion of fermentation by ultrasonic waves. Many reports at home and abroad show that low-intensity ultrasonic waves can promote the proliferation of fermenting microorganisms, improve fermentation efficiency and product yield.
  • the invention patent "A method for preparing oligopeptides from solid-state fermented cakes (CN201410390337.8)” discloses a method of using frequency-sweeping ultrasound to assist microbial fermentation, using frequency-sweeping ultrasound to assist in the cultivation of Bacillus subtilis, yeast and Aspergillus niger, under the same conditions, compared with no ultrasonic culture, the number of bacteria in the seed solution can be increased by 116-290%, 63%-152% and 160-264%, respectively, and the yield of oligopeptides produced by fermented cakes is higher than that of the control
  • the test increases by 13.5% to 46.4%, the protein content in the product increases by 8.97% to 15.2%, and the peptide content increases by 14.3% to 36.7%.
  • ultrasonic-assisted fermentation devices that have been reported so far are mainly divided into two types: external-attached and built-in.
  • an ultrasonic enhanced bioreactor belongs to the externally-attached ultrasonic action mode, and multiple sets of ultrasonic vibrators are attached to the outer wall of the fermentation tank, so that the ultrasonic wave can penetrate the tank wall and act on the material.
  • the disadvantages of this type of ultrasonic-assisted fermentation device are: (1) There is a hot water jacket on the outer wall of the fermenter for heat preservation during the fermentation process.
  • an ultrasonic vibrator is attached to the outer wall of the fermenter, it will destroy the continuity of the heat preservation jacket, affect the heat preservation effect, and increase (2)
  • the tank wall will cause ultrasonic attenuation, especially for large-scale fermentation tanks, which are relatively thick, and the sound energy attenuation is serious.
  • the diameter of industrial large-scale fermentation tanks can reach several meters, and the materials in the central area are difficult to be affected. Ultrasonic radiation treatment. Therefore, externally attached ultrasound is not suitable for large fermenters.
  • the built-in ultrasonic-assisted fermentation device such as the invention patent "fermentation tank with ultrasonic generating device (CN103952294B)” and the utility model patent “an ultrasonic-assisted air-lift fermentation device (CN208318170U)” have been reported.
  • the industrial fermentation tank is as high as 10m or even more than 20m, and the ultrasonic treatment is only performed from one end of the tank body, and the ultrasonic radiation area is only in a small range at that end, so the liquid in the tank is treated very unevenly, or The effect of promoting fermentation cannot be shown due to insufficient ultrasonic treatment. If the ultrasonic power is increased blindly, the problem of microbial death due to excessive ultrasonic radiation may occur.
  • the purpose of the present invention is to provide a wall-mounted ultrasonic intensified fermentation device suitable for large-scale fermentation tanks.
  • the problem of serious attenuation of sound energy and uneven ultrasonic effect due to the large diameter and height of the fermenter can avoid problems such as ineffective promotion of fermentation by traditional ultrasonic-assisted fermentation devices in large fermenters and unstable product quality.
  • a wall-mounted ultrasonic intensified fermentation device suitable for large fermentation tanks includes a fermentation tank, an ultrasonic transducer bracket, a bracket connector, an ultrasonic transducer, a high-frequency connecting line, a stirring shaft, and a stirring paddle , ultrasonic generator, ultrasonic vibrator and stainless steel welded short tube;
  • the stirring shaft is arranged inside the fermenter, and the stirring shaft is arranged coaxially with the fermenter; one end of the stirring shaft is provided with a motor for driving the stirring shaft; the other end is provided with a plurality of stirring paddles;
  • a plurality of ultrasonic transducer supports are arranged in an array inside the fermenter, and the ultrasonic transducer supports are arranged in parallel with the stirring shaft;
  • the ultrasonic transducer bracket is provided with multiple ultrasonic transducers; the inner wall of the ultrasonic transducer is provided with multiple ultrasonic vibrators; the ultrasonic vibrators are electrically connected to the ultrasonic generator through high-frequency connecting wires.
  • stirring blades there are specifically 4-6 stirring blades, which are arranged in parallel on the stirring shaft.
  • the ultrasonic transducer bracket is suspended from the top to the bottom along the inner wall of the fermenter; the ultrasonic transducer bracket is provided in multiple sections; it is specifically composed of multi-section stainless steel pipes, and there are flanges at both ends of each section of stainless steel pipe , sealed and connected by clamps; each section of the stainless steel tube contains 3 to 5 ultrasonic transducers, which are distributed equidistantly in the longitudinal direction.
  • the ultrasonic transducer bracket is made of 3-5 sections of 316L stainless steel tubes, and each section is sealed and connected by 316L stainless steel quick-fit clamps; the inner diameter of the stainless steel tubes is 2-5 cm.
  • the ultrasonic transducer is a cubic structure made of stainless steel sheet, which is suspended on the surface of the ultrasonic transducer support by welding short stainless steel pipes.
  • ultrasonic vibrators are arranged on the inner wall of the ultrasonic transducer facing the center of the fermenter; one end of the high-frequency connection line is connected to the ultrasonic vibrator, and the other end passes through the stainless steel welded short tube and the ultrasonic transducer.
  • the transducer bracket protrudes through the upper top of the ultrasonic transducer bracket and is electrically connected with the ultrasonic generator.
  • the ultrasonic wave of the ultrasonic generator is a fixed frequency mode (fixed frequency mode) or a frequency sweep mode
  • the fixed frequency mode means that the frequency is constant during the ultrasonic work process
  • the frequency sweep mode means that the frequency is around the central frequency during the ultrasonic work process. fluctuate periodically;
  • the frequency range of the fixed frequency mode is 15 ⁇ 200kHz; the frequency range of the frequency sweep mode is 15 ⁇ 2 ⁇ 200 ⁇ 2kHz, which is center frequency+sweep frequency range; that is, the center frequency range is 15 ⁇ 200kHz, at each center
  • the range of frequency sweep range during frequency operation is ⁇ 0.5 ⁇ 2kHz.
  • the power range of a single ultrasonic vibrator is 5-20W.
  • the present invention also provides a fermentation method suitable for a wall-mounted ultrasonic intensified fermentation device of a large fermentation tank, specifically as follows:
  • Step 1 First, sterilize the fermenter tank, the sterilization conditions are 115°C, 8-15min; then inoculate the fermented seed liquid in the fermenter for fermentation, and set the corresponding fermentation conditions for fermentation according to the different fermented products :
  • Step 2 Turn on the ultrasonic generator 0 to 12 hours after the start of fermentation, and perform ultrasonic radiation treatment.
  • the ultrasonic action mode is fixed frequency or sweep mode, and the ultrasonic frequency range is 15 to 200kHz or 15 ⁇ 2kHz to 200 ⁇ 2kHz.
  • the power range of the ultrasonic transducer is 15-120W, the ultrasonic treatment time is 0.5-12h, and the ultrasonic generator is turned off after the treatment is completed;
  • Step 3 During and after the ultrasonic radiation treatment, the fermenter is fermented according to the conditions set in step 1 until the end of the fermentation;
  • Step 4 After the fermentation, empty the fermentation liquid in the tank and clean the tank body. During the cleaning process, turn on the ultrasonic generator, set the corresponding working frequency and working time for cleaning, and clean it for the next fermentation.
  • the fermented product in step 1 includes Bacillus subtilis, Streptomyces or Cordyceps sinensis.
  • the fermentation conditions described in step 1 are specifically: fermentation temperature 20-35° C., initial pH 6.5-7.0, stirring speed 100-200 r/min.
  • step 2 when the fermented product is a Bacillus subtilis microbial bacterial agent, the ultrasonic generator is turned on at the beginning of fermentation 6h, the ultrasonic mode is fixed frequency, the ultrasonic frequency is 28kHz, the power of a single ultrasonic transducer is 45W, and the ultrasonic treatment time is 12h;
  • step 2 when the fermented product is Streptomyces, the ultrasonic generator is turned on at the beginning of fermentation 6h, the ultrasonic mode is frequency sweep, the ultrasonic frequency is 15kHz ⁇ 0.5kHz, the power of a single ultrasonic transducer is 15W, and the ultrasonic treatment time is 3h;
  • step 2 when the fermented product is Cordyceps sinensis, the ultrasonic generator is turned on 12 hours after fermentation begins, the ultrasonic mode is frequency sweep, the ultrasonic frequency is 200kHz ⁇ 1kHz, the power of a single ultrasonic transducer is 120W, and the ultrasonic treatment time is 0.5 h.
  • the ultrasonic generator in step 4 selects a fixed frequency mode, the frequency is 20kHz, and the working time is 10-20min; the power of the single ultrasonic transducer is 60-120W.
  • the present invention Compared with the traditional ultrasonic enhanced fermentation device, the present invention has the following characteristics and beneficial effects:
  • a wall-mounted ultrasonic intensified fermentation device suitable for large-scale fermentation tanks By evenly mounting multiple ultrasonic transducers on the inner wall of the fermentation tank and directly acting on the fermentation liquid, it can overcome the traditional ultrasonic-assisted fermentation device in large-scale fermentation. When the tank is used, the ultrasonic wave is easy to attenuate or the effect is uneven, which leads to problems such as ineffective promotion of fermentation and unstable product quality.
  • a wall-mounted ultrasonic intensified fermentation device suitable for large-scale fermentation tanks When cleaning the fermentation tank after fermentation, it is not necessary to take out the ultrasonic transducer assembly. As long as the ultrasonic wave is turned on during cleaning, the ultrasonic vibration can make the The transducer assembly is cleaned and can speed up the cleaning process of the entire fermenter;
  • the ultrasonic transducer bracket is composed of 3 to 5 sections of stainless steel pipes, which are sealed and connected by quick joints. The bracket is too long and exceeds the height of the factory building and it is difficult to take it out;
  • a wall-mounted ultrasonic intensified fermentation device suitable for large-scale fermentation tanks which uses low-power ultrasonic waves to intensify the fermentation process, uses very low energy consumption, and belongs to a green, energy-saving and efficient physical intensified fermentation device;
  • a wall-mounted ultrasonic intensified fermentation device suitable for large-scale fermentation tanks which are respectively applied to the fermentation of Bacillus subtilis microbial inoculum, Streptomyces fermentation to produce transglutaminase (TG enzyme) and Cordyceps sinensis fermentation to produce mycelium
  • TG enzyme transglutaminase
  • Cordyceps sinensis fermentation to produce mycelium
  • concentration of Bacillus subtilis microbial agent, TG enzyme production and Cordyceps sinensis mycelium and polysaccharide production increased by 85.8%, 20.9%, 13.3% respectively and 16.1%.
  • Figure 1 is a front view of a wall-mounted ultrasonic intensified fermentation device suitable for large fermenters.
  • Fig. 2 is a top view of a wall-mounted ultrasonic enhanced fermentation device suitable for large fermenters.
  • Fig. 3 is an enlarged front view of the structure at A in Fig. 1 .
  • FIG. 4 is an enlarged top view of the structure at A in FIG. 1 .
  • a wall-mounted ultrasonic intensified fermentation device suitable for large fermentation tanks comprising: a fermentation tank 1, an ultrasonic transducer bracket 2, a bracket connector 3, an ultrasonic transducer 4, a high-frequency connecting line 5, a stirring shaft 6, Stirring paddle 7, ultrasonic generator 8, ultrasonic vibrator 9 and stainless steel welded short tube 10;
  • the stirring shaft 6 is located inside the fermenter 1, and the stirring shaft 6 is coaxially arranged with the fermenter 1; one end of the stirring shaft 6 is provided with a motor for driving the stirring shaft 6 to run; the other end is provided with 4
  • the stirring paddle 7 is arranged in parallel on the surface of the stirring shaft 6;
  • ultrasonic transducer supports 2 are arranged in the internal array of the fermenter 1, and the ultrasonic transducer supports 2 and the stirring shaft 6 are arranged in parallel;
  • the ultrasonic transducer bracket 2 is made of 3 sections of 316L stainless steel thin tubes, each section has flanges at both ends, and the 316L stainless steel quick-fit clamps are sealed and connected, and the ultrasonic transducer bracket 2 is attached to the inner wall of the fermentation tank 1 and suspended from the top to the bottom;
  • Each section of the stainless steel tube includes 5 ultrasonic transducers 4, which are distributed equidistantly in the longitudinal direction;
  • the ultrasonic transducer 4 is made of a stainless steel sheet into a cube structure, and is suspended on the ultrasonic transducer support 2 by welding a short stainless steel welded tube 10 , the inner wall of the ultrasonic transducer facing the center of the tank is pasted with three ultrasonic vibrators 9, the ultrasonic vibrator is connected by a high-frequency connecting line 5, and passes through the stainless steel welded short tube 10 and the ultrasonic transducer bracket 2, and extends through the top of the upper part of the bracket Out, electrically connected with ultrasonic generator 8;
  • the ultrasonic wave of the ultrasonic generator 8 is a fixed frequency mode or a frequency sweep mode, the preferred frequency range of the fixed frequency mode is 15 ⁇ 200kHz, and the preferred frequency range of the frequency sweep mode is 15 ⁇ 2 ⁇ 200 ⁇ 2kHz;
  • the power range of a single ultrasonic vibrator 9 is 5-20W.
  • Step 1 First, carry out real-tank sterilization on the fermenter 1, the sterilization condition is 115°C, 8-15min; then inoculate the seed solution in the fermenter, and ferment according to the corresponding fermentation conditions;
  • Step 2 According to the different fermented products, turn on the ultrasonic generator 8 after 0-12 hours after the start of fermentation, and perform ultrasonic radiation treatment.
  • the power range of the transducer 4 is 15-120W
  • the ultrasonic treatment time is 0.5-12h
  • the ultrasonic generator is turned off after the treatment is completed.
  • Step 3 After the ultrasonic treatment process and the treatment are completed, the fermenter is fermented according to the set conditions until the end of the fermentation.
  • Step 4 Drain the fermentation liquid in the tank after fermentation, clean the tank according to the set procedure, turn on the ultrasonic generator for 10 minutes during the cleaning process, the ultrasonic frequency is 20kHz, and the power range of a single transducer is 60-120W; it can be carried out after cleaning Fermentation in the next tank.
  • the ultrasonic intensified fermentation of the Bacillus subtilis microbial agent is carried out in the 40m3 fermentation tank.
  • the fermentation tank 1 is equipped with four sets of wall-mounted ultrasonic transducer brackets 2, which are evenly arranged and suspended on the inner wall. Each bracket has three sections.
  • the stainless steel quick-install clamp seal bracket connecting piece 3 is connected, and each section is vertically arranged with 5 ultrasonic transducers 4, and the ultrasonic transducer bracket 2 is welded through the stainless steel welding short tube 10, and each transducer has 3 ultrasonic vibrators 9 , electrically connect the ultrasonic generator 8 through the high-frequency connecting wire 5;
  • Step 1 add fermented liquid to fermenter 1, except water in the fermented liquid composition composition, other substrates are 1.5% (w/v, all refer to mass-volume concentration below everything not indicated) soybean powder, 0.5% molasses , 0.8% ammonium chloride, 1.0% sodium chloride, 0.1% sodium citrate, 0.5% calcium carbonate, 0.1% magnesium sulfate heptahydrate, and the loading capacity of the fermenter is 80% volume.
  • the sterilization conditions are 115°C, 15min, inoculate 10% (v/v) Bacillus subtilis seed liquid into the fermenter 1 after cooling down, and ferment according to the set conditions: fermentation temperature 32°C , initial pH 7.0, start the fermenter to stir the rotating shaft 6, and the stirring speed is 200r/min.
  • Step 2 Turn on the ultrasonic generator 8 at 6 hours from the start of fermentation, the ultrasonic mode is fixed frequency, the ultrasonic frequency is 28kHz, the power of a single ultrasonic transducer 4 is 45W, the ultrasonic treatment time is 12h, and the ultrasonic generator 8 is turned off after the treatment.
  • Step 3 After the ultrasonic treatment process and the treatment, the fermenter 1 was fermented according to the conditions set in step 1 for 36 hours until the end of the fermentation; the cell biomass in the fermentation liquid was sampled and detected to be 2.75 ⁇ 10 10 cfu/mL.
  • Step 4 After the fermentation, empty the fermentation liquid in the fermentation tank 1, clean the tank body according to the set procedure, turn on the ultrasonic generator for 810 minutes during the cleaning process, the ultrasonic frequency is 20kHz, and the power range of a single transducer is 120W. After cleaning, it can be carried out Fermentation in the next tank.
  • Example 2 The fermentation conditions and fermentation process were the same as those in Example 2, except that ultrasonic treatment was not used. Same as Example 1, except that no ultrasonic treatment was used. At the end of the fermentation, samples were taken to detect that the cell biomass in the fermentation broth was 1.48 ⁇ 10 10 cfu/mL.
  • Example 1 Compared with the fermentation of Comparative Example 1, the ultrasonically enhanced fermentation of Example 1 increased the concentration of Bacillus subtilis microbial inoculum by 85.8%.
  • Streptomyces fermentation is carried out in a 40m3 fermenter to produce transglutaminase (TG enzyme) ultrasonically enhanced fermentation.
  • Fermenter 1 is equipped with four sets of wall-mounted ultrasonic transducer brackets 2, which are evenly arranged and suspended on the inner wall. Each bracket There are three sections, which are connected by stainless steel quick-install clamp seal bracket connectors 3, and five ultrasonic transducers 4 are arranged longitudinally in each section, and ultrasonic transducer brackets 2 are welded through stainless steel welding short tubes 10, and each transducer has 3
  • An ultrasonic vibrator 9 is electrically connected to the ultrasonic generator 8 through a high-frequency connecting wire 5 .
  • Step 1 Add fermented liquid to fermenter 1. Except for water, other substrates of fermented liquid are 2.4% soybean meal hydrolyzate, 2.4% glycerin, 0.5% yeast extract, 0.3% ammonium sulfate, packed in fermented tank The loading is 75% volume.
  • fermenter 1 is sterilized, the sterilization condition is 121°C, 15min, inoculate 8% (v/v) streptomyces seed liquid in fermenter 1 after cooling, ferment according to set conditions: fermentation temperature 30°C, The initial pH is 7.0, and the fermentation tank is started to stir the rotating shaft 6, and the stirring speed is 180r/min.
  • Step 2 Turn on the ultrasonic generator 8 at 6 hours from the start of fermentation, the ultrasonic mode is frequency sweep, the ultrasonic frequency is 15kHz ⁇ 0.5kHz, the power of a single ultrasonic transducer 4 is 15W, the ultrasonic treatment time is 3h, and the ultrasonic generator 8 is turned off after the treatment is completed;
  • Step 3 After the ultrasonic treatment process and the treatment, the fermenter was fermented for 54 hours according to the conditions set in step 1 until the end of the fermentation, and the TG enzyme activity in the fermentation broth was sampled and detected to be 6.87U/mL.
  • Step 4 After the fermentation, empty the fermentation liquid in the fermentation tank 1, clean the tank body according to the set procedure, turn on the ultrasonic generator for 810 minutes during the cleaning process, the ultrasonic frequency is 20kHz, and the power range of a single transducer is 120W; it can be carried out after cleaning Fermentation in the next tank.
  • the fermentation conditions and fermentation process were the same as those in Example 2, except that ultrasonic treatment was not used.
  • the TG enzyme activity in the fermentation broth was sampled and detected to be 5.68U/mL.
  • the fermentation tank 1 is equipped with four sets of wall-mounted ultrasonic transducer brackets 2, which are evenly arranged and suspended on the inner wall. Each bracket has three sections.
  • the stainless steel quick-install clamp seal bracket connecting piece 3 is connected, and each section is vertically arranged with 5 ultrasonic transducers 4, and the ultrasonic transducer bracket 2 is welded through the stainless steel welding short tube 10, and each transducer has 3 ultrasonic vibrators 9 , electrically connected to the ultrasonic generator 8 through the high-frequency connecting wire 5 .
  • Step 1 Add fermented liquid to fermenter 1. Except for water, other substrates of fermented liquid are rice bran powder 3.6%, bran powder 4.8%, KH 2 PO 4 0.27%, MgSO 4 ⁇ 7H 2 O 0.27 %, cysteine hydrochloride 0.05%, sodium copper chlorophyllin 0.01%, and the loading capacity of the fermenter is 75% volume.
  • the fermenter 1 is sterilized, the sterilization condition is 121°C, 15min, after cooling, 8% (v/v) Cordyceps sinensis seed liquid is inoculated in the fermenter 1, and fermented according to the set conditions: fermentation temperature 23°C, The initial pH is 7.0, start the fermenter to stir the shaft 6, and the stirring speed is 100r/min;
  • Step 2 Turn on the ultrasonic generator 8 12 hours after the start of fermentation, the ultrasonic mode is frequency sweep, the ultrasonic frequency is 200kHz ⁇ 1kHz, the power of a single ultrasonic transducer 4 is 120W, the ultrasonic treatment time is 0.5h, and the ultrasonic generator 8 is turned off after the treatment is completed;
  • Step 3 After the ultrasonic treatment process and the treatment, the fermenter was fermented for 5 days according to the conditions set in step 1 until the end of the fermentation.
  • the fermentation broth was taken for detection, the mycelia dry weight was 48.5g/L fermentation broth, and the exopolysaccharide was 2.67g/L fermentation broth;
  • Step 4 After the fermentation, empty the fermentation liquid in the fermentation tank 1, clean the tank body according to the set procedure, turn on the ultrasonic generator for 810 minutes during the cleaning process, the ultrasonic frequency is 20kHz, and the power range of a single transducer is 120W; it can be carried out after cleaning Fermentation in the next tank.
  • the fermentation conditions and fermentation process were the same as in Example 3, except that ultrasonic treatment was not used. Fermentation finishes sampling detection mycelia dry weight 42.8g/L fermented liquid in fermented liquid, exopolysaccharide is 2.30g/L fermented liquid;
  • Example 3 Compared with the fermentation of Comparative Example 3, the ultrasonically enhanced fermentation of Example 3 increased the dry weight of mycelium by 13.3%, and the exopolysaccharide increased by 16.1%.

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Abstract

本申请提供一种适用于大型发酵罐的挂壁式超声波强化发酵装置及其发酵方法，属于生物工程装备领域，该发酵装置包括：发酵罐、超声波换能器支架、支架连接件、超声波换能器、高频连接线、搅拌轴、搅拌桨、超声波发生器、超声波振子和不锈钢焊接短管；所述搅拌轴设于罐体内部，且与发酵罐同轴设置；搅拌轴的一端设有电机，另一端设有搅拌桨；在发酵罐的内部阵列设置多个超声波换能器支架，超声波换能器支架上设有多个超声波换能器，超声波换能器内壁设有多个超声波振子；超声波振子通过高频连接线与超声波发生器电性连接。本申请还公开了利用该发酵装置进行发酵的方法。本申请的装置解决了传统超声发酵装置超声波易衰减、作用不均匀而导致的促进发酵效果不明显和产品品质不稳定的问题。

Description

一种适用于大型发酵罐的挂壁式超声波强化发酵装置及其发酵方法 技术领域
本发明属于生物工程装备领域，具体涉及一种适用于大型发酵罐的挂壁式超声波强化发酵装置。
背景技术
由于微生物生长繁殖周期长、发酵底物与微生物的物质交换速率低等原因，导致发酵周期很长，产物的得率也较低，这是生物发酵转化的重要阻碍因素。
超声波作为一种物理学手段在强化农产品加工增值加工中有广泛应用，超声波在农产品有效成分提取、食品杀菌、细胞破碎、均质乳化、促进传热传质等方面已经显示了突出的应用价值，特别是在超声波促进发酵方面也表现出良好的应用前景，国内外许多报道表明，低强度超声波可促进发酵微生物增殖、提高发酵效率和产物得率。例如发明专利“一种固态发酵饼粕制备低聚肽的方法(CN201410390337.8)”中公开了一种采用扫频超声辅助微生物发酵的方法，采用扫频超声辅助培养枯草芽孢杆菌、酵母菌及黑曲霉，在同等条件下与无超声培养相比，种子液中菌数分别可提高116～290％、63％～152％以及160～264％，发酵饼粕生产的低聚肽得率比对照试验提高13.5％～46.4％，产品中蛋白质含量提高8.97％～15.2％，肽含量提高14.3％～36.7％。在超声辅助发酵设备方面也有不少报道，如发明专利“一种超声强化生物反应器(CN201110033778.9)”、“设有超声波发生装置的发酵罐(CN103952294B)”以及实用新型专利“一种超声辅助气升式发酵装置(CN208318170U)”等，均显示利用这些超声辅助发酵装置可有效提高发酵效率。
目前已报道的各类超声辅助发酵装置，其超声波的作用方式主要分为外贴式和内置式两类。如发明专利CN201110033778.9所述的“一种超声强化生物反应器”即属于外贴式超声作用方式，在发酵罐外壁贴上多组超声振子，使超声波透过罐壁作用于物料。这类超声辅助发酵装置的弊端在于：(1)发酵罐外壁有热水夹套用于发酵过程保温，如在发酵罐外壁贴超声振子，会破坏保温夹套的连续性，影响保温效果，并增加了发酵罐设备制造的工艺复杂性；(2)罐壁会导致超声波衰减，尤其是大型发酵罐比较厚，声能衰减严重，工业化大型发酵罐直径可达数米，中心区域的物料很难受到超声辐射处理。因此外贴式超声不适用于大 型发酵罐。
关于内置式超声辅助发酵装置，目前已报道的如发明专利“设有超声波发生装置的发酵罐(CN103952294B)”以及实用新型专利“一种超声辅助气升式发酵装置(CN208318170U)”等，一般采用圆盘的形式，置于罐底或罐顶。这类装置存在的主要问题在于，工业化发酵罐高达10m甚至20m以上，只从罐体一端进行超声处理，超声辐射的区域只在那一端较小范围内，对罐内液体处理非常不均匀，或因超声处理不足而不能显现促进发酵的效果，如果一味提高超声功率，则可能产生因超声过度辐射导致微生物死亡的问题。
因此，要将超声强化发酵技术应用于大型工业发酵罐，面临着要克服超声传导效率低或作用不均匀等技术难题，是目前国内生物工程领域迫切需要突破的卡脖子问题。
发明内容
本发明的目的是提供一种适用于大型发酵罐的挂壁式超声波强化发酵装置，通过在发酵罐内壁均衡挂置多组超声换能器，直接作用于发酵液，克服因大型发酵罐壁厚使得声能衰减严重、以及因发酵罐直径和高度大，使得超声作用不均匀的难题，从而避免传统超声辅助发酵装置在大型发酵罐的促进发酵效果不明显、以及产品品质不稳定等问题发生。
为了实现以上目的，本发明采用的技术方案如下：
首先提供一种适用于大型发酵罐的挂壁式超声波强化发酵装置，所述装置包括发酵罐、超声波换能器支架、支架连接件、超声波换能器、高频连接线、搅拌轴、搅拌桨、超声波发生器、超声波振子和不锈钢焊接短管；
所述搅拌轴设于发酵罐的内部，且搅拌轴与发酵罐同轴设置；所述搅拌轴的一端设有电机，用于驱动搅拌轴运转；另一端设有多个搅拌桨；
在发酵罐的内部阵列设置多个超声波换能器支架，所述超声波换能器支架和搅拌轴平行设置；
所述超声波换能器支架上设有多个超声波换能器；所述超声波换能器内壁设有多个超声波振子；所述超声波振子通过高频连接线与超声波发生器电性连接。
优选的，所述搅拌桨具体为4-6个，平行设置在搅拌轴上。
优选的，所述超声波换能器支架沿着发酵罐内壁从顶部悬挂至底部；所述超 声波换能器支架为多段式设置；具体由多节不锈钢管组成，每节不锈钢管的两头有法兰，通过卡箍密封连接；所述每节不锈钢管上包含3～5个超声波换能器，纵向等距分布。
优选的，所述超声波换能器支架由3～5节316L不锈钢管制成，各节之间通过316L不锈钢快装卡箍密封连接；所述不锈钢管的内径为2～5cm。
优选的，所述超声波换能器为不锈钢薄板制成的立方体结构，通过不锈钢焊接短管焊接悬挂于超声波换能器支架表面。
优选的，所述超声波换能器朝向发酵罐中心的一侧内壁上设有3～6个超声波振子；所述高频连接线的一端连接超声波振子，另一端穿过不锈钢焊接短管和超声波换能器支架，经超声波换能器支架的上部顶端伸出，与超声波发生器电性连接。
优选的，所述超声波发生器的超声波为固定频率模式(定频模式)或扫频模式，固定频率模式是指超声波工作过程中频率不变，扫频模式是指超声波工作过程中频率围绕中心频率作周期性上下波动；
所述定频模式的频率范围为15～200kHz；所述扫频模式频率范围为15±2～200±2kHz，为中心频率+扫频幅度；即中心频率范围为15～200kHz，在每一个中心频率工作时的扫频幅度范围为±0.5～±2kHz。
优选的，所述超声波振子单个的功率范围为5～20W。
本发明还提供一种适用于大型发酵罐的挂壁式超声波强化发酵装置的发酵方法，具体如下：
步骤1：首先对发酵罐实罐灭菌，灭菌条件为115℃、8～15min；然后在发酵罐内接种发酵物种子液进行发酵，根据发酵物的不同，设定相应的发酵条件进行发酵：
步骤2：在发酵开始后的0～12h后开启超声波发生器，进行超声波辐射处理，超声波作用模式为定频或扫频模式，超声波频率范围为15～200kHz或15±2kHz～200±2kHz，单个超声波换能器功率范围15～120W，超声波处理时间为0.5～12h，处理结束关闭超声波发生器；
步骤3：在超声波辐射处理过程中及处理结束后，发酵罐均按照步骤1设定条件进行发酵，直至发酵结束；
步骤4：发酵结束后排空罐内发酵液，进行罐体的清洗，清洗过程中开启超声波发生器，设定相应的工作频率、工作时间进行清洗，清洗完毕以备下一次发酵使用。
优选的，步骤1中所述发酵物包括枯草芽孢杆菌、链霉菌或冬虫夏草菌。
优选的，步骤1中所述发酵条件具体为：发酵温度20～35℃、初始pH6.5～7.0、搅拌转速100～200r/min。
优选的，步骤2中当发酵物为枯草芽孢杆菌微生物菌剂时，在发酵开始6h开启超声波发生器，超声波模式为定频，超声波频率为28kHz，单个超声波换能器功率45W，超声波处理时间为12h；
优选的，步骤2中当发酵物为链霉菌时，在发酵开始6h开启超声波发生器，超声波模式为扫频，超声波频率为15kHz±0.5kHz，单个超声波换能器的功率15W，超声波处理时间为3h；
优选的，步骤2中当发酵物为冬虫夏草菌时，在发酵开始12h开启超声波发生器，超声波模式为扫频，超声波频率为200kHz±1kHz，单个超声波换能器的功率120W，超声波处理时间为0.5h。
优选的，步骤4中所述超声波发生器选择定频模式，频率为20kHz，工作时间为10～20min；所述单个超声波换能器的功率为60～120W。
与传统超声波强化发酵装置相比，本发明具有如下特点和有益效果：
(1)一种适用于大型发酵罐的挂壁式超声波强化发酵装置，通过在发酵罐内壁均衡挂置多架超声换能器，直接作用于发酵液，可克服传统超声辅助发酵装置在大型发酵罐应用时超声波易衰减或作用不均匀，从而导致的促进发酵效果不明显以及产品品质不稳定等问题。
(2)一种适用于大型发酵罐的挂壁式超声波强化发酵装置，在发酵结束后对发酵罐清洗时，不需要取出超声波换能器组件，只要清洗时开启超声波，由于超声波振动就能使换能器组件清洗干净，并且可加快整个发酵罐的清洗进程；
(3)一种适用于大型发酵罐的挂壁式超声波强化发酵装置，超声波换能器支架由3～5段不锈钢管组成，利用快接头密封连接，在检修超声装置时方便拆卸，不会因支架太长超出厂房高度而难以取出；
(4)一种适用于大型发酵罐的挂壁式超声波强化发酵装置，采用低功率超 声波对发酵过程进行强化，使用的能耗很低，属于绿色节能高效的物理强化发酵装置；
(5)一种适用于大型发酵罐的挂壁式超声波强化发酵装置，分别应用于枯草芽孢杆菌微生物菌剂发酵、链霉菌发酵生产转谷氨酰胺酶(TG酶)以及冬虫夏草菌发酵生产菌丝体及多糖，与对照试验(不使用超声，其他条件相同)的发酵相比，枯草芽孢杆菌微生物菌剂浓度、TG酶产量及冬虫夏草菌丝体及多糖产量分别提高85.8％、20.9％、13.3％和16.1％。
附图说明
图1为一种适用于大型发酵罐的挂壁式超声波强化发酵装置的正视图。
图2为一种适用于大型发酵罐的挂壁式超声波强化发酵装置的俯视图。
图3为图1中A处结构放大后的正视图。
图4为图1中A处结构放大后的俯视图。
图1～4中：1-发酵罐，2-超声波换能器支架，3-支架连接件，4-超声波换能器，5-高频连接线，6-搅拌轴，7-搅拌桨，8-超声波发生器，9-超声波振子，10-不锈钢焊接短管。
具体实施方式
下面结合附图和具体实施方式对本发明的应用方法作进一步详细说明，但本发明的保护范围并不限于此。
一种适用于大型发酵罐的挂壁式超声波强化发酵装置，包括：发酵罐1、超声波换能器支架2、支架连接件3、超声波换能器4、高频连接线5、搅拌轴6、搅拌桨7、超声波发生器8、超声波振子9和不锈钢焊接短管10；
所述搅拌轴6设于发酵罐1的内部，且搅拌轴6与发酵罐1同轴设置；所述搅拌轴6的一端设有电机，用于驱动搅拌轴6运转；另一端设有4个搅拌桨7，平行设置在搅拌轴6的表面；
在发酵罐1的内部阵列设置6个超声波换能器支架2，所述超声波换能器支架2和搅拌轴6平行设置；
超声波换能器支架2由3节316L不锈钢细管制成，每节两头有法兰，316L不锈钢快装卡箍密封连接，超声波换能器支架2贴着发酵罐1内壁从顶部悬挂至底部；
所述每节不锈钢管上包含5个超声波换能器4，纵向等距分布；超声波换能器4为不锈钢薄板制成立方体结构，通过不锈钢焊接短管10焊接悬挂于超声波换能器支架2上，超声换能器朝向罐中心一侧内壁贴有3个超声波振子9，采用高频连接线5连接超声波振子，并穿过不锈钢焊接短管10和超声波换能器支架2，经支架上部顶端延伸出，与超声波发生器8电性连接；
超声波发生器8的超声波为定频模式或扫频模式，定频模式优选的频率范围为15～200kHz，扫频模式优选的频率范围为15±2～200±2kHz；
超声波振子9单个的功率范围为5～20W。
利用上述一种适用于大型发酵罐的挂壁式超声波强化发酵装置进行发酵的方法，按照下述步骤进行：
步骤1：首先对发酵罐1进行实罐灭菌，灭菌条件为115℃、8～15min；然后在发酵罐内接种种子液，按照相应的发酵条件进行发酵；
步骤2：根据发酵物的不同，在发酵开始后的0～12h后开启超声波发生器8，进行超声波辐射处理，超声波作用模式为扫频或定频模式，超声波频率范围为15～200kHz，单个超声波换能器4的功率范围15～120W，超声波处理时间为0.5～12h，处理结束关闭超声波发生器。
步骤3：超声处理过程及处理结束后，发酵罐均按照设定条件进行发酵，直至发酵结束。
步骤4：发酵结束后排空罐内发酵液，按设定程序清洗罐体，清洗过程中开启超声波发生器10min，超声波频率为20kHz，单个换能器功率范围60～120W；清洗完即可进行下一罐发酵。
实施例1：
按照上述步骤在40m 3发酵罐进行枯草芽孢杆菌微生物菌剂的超声波强化发酵，发酵罐1配置四组挂壁式超声波换能器支架2，均匀排布悬挂于内壁，每个支架有三节，通过不锈钢快装卡箍密封支架连接件3连接，每节纵向排列5个超声波换能器4，通过不锈钢焊接短管10焊接超声波换能器支架2，每个换能器内有3个超声波振子9，通过高频连接线5电性连接超声波发生器8；
具体发酵按如下步骤进行：
步骤1：向发酵罐1加注发酵液，发酵液组成成分中除水外，其他底物为1.5％ (w/v，以下凡未注明皆是指质量体积浓度)黄豆粉、0.5％糖蜜、0.8％氯化铵、1.0％氯化钠、0.1％柠檬酸钠、0.5％碳酸钙、0.1％七水合硫酸镁，发酵罐装载量为80％容积。
首先对发酵罐1实罐灭菌，灭菌条件为115℃、15min，降温后向发酵罐1内接种10％(v/v)枯草芽孢杆菌种子液，按照设定条件发酵：发酵温度32℃，初始pH7.0，开动发酵罐搅转轴6，搅拌转速200r/min。
步骤2：在发酵开始6h开启超声波发生器8，超声波模式为定频，超声波频率为28kHz，单个超声波换能器4功率为45W，超声波处理时间为12h，处理结束关闭超声波发生器8。
步骤3：超声处理过程及处理结束后，发酵罐1均按照步骤1设定条件进行发酵36h，直至发酵结束；取样检测发酵液中细胞生物量为2.75×10 10cfu/mL。
步骤4：发酵结束后排空发酵罐1内发酵液，按设定程序清洗罐体，清洗过程中开启超声波发生器810min，超声波频率为20kHz，单个换能器功率范围120W，清洗完即可进行下一罐发酵。
对比例1:
发酵条件及发酵过程与实施例2相同，区别是不使用超声处理。与实施例1相同，区别是不使用超声处理。发酵结束取样检测发酵液中细胞生物量为1.48×10 10cfu/mL。
结果对比：与对比例1的发酵相比，实施例1的超声强化发酵使枯草芽孢杆菌微生物菌剂浓度提高了85.8％。
实施例2：
在40m 3发酵罐进行链霉菌发酵生产转谷氨酰胺酶(TG酶)的超声波强化发酵，发酵罐1配置四组挂壁式超声波换能器支架2，均匀排布悬挂于内壁，每个支架有三节，通过不锈钢快装卡箍密封支架连接件3连接，每节纵向排列5个超声波换能器4，通过不锈钢焊接短管10焊接超声波换能器支架2，每个换能器内有3个超声波振子9，通过高频连接线5电性连接超声波发生器8。
具体发酵按如下步骤进行：
步骤1：向发酵罐1加注发酵液，发酵液组成成分中除水外，其他底物为2.4％豆粕粉水解物、2.4％甘油、0.5％酵母浸膏、0.3％硫酸铵，发酵罐装载量为75％ 容积。首先对发酵罐1实罐灭菌，灭菌条件为121℃、15min，降温后向发酵罐1内接种8％(v/v)链霉菌种子液，按照设定条件发酵：发酵温度30℃，初始pH7.0，开动发酵罐搅转轴6，搅拌转速180r/min。
步骤2：在发酵开始6h开启超声波发生器8，超声波模式为扫频，超声波频率为15kHz±0.5kHz，单个超声波换能器4功率15W，超声波处理时间为3h，处理结束关闭超声波发生器8；
步骤3：超声处理过程及处理结束后，发酵罐均按照步骤1设定条件进行发酵54h，直至发酵结束，取样检测发酵液中TG酶活力为6.87U/mL。
步骤4：发酵结束后排空发酵罐1内发酵液，按设定程序清洗罐体，清洗过程中开启超声波发生器810min，超声波频率为20kHz，单个换能器功率范围120W；清洗完即可进行下一罐发酵。
对比例2:
发酵条件及发酵过程与实施例2相同，区别是不使用超声处理。发酵结束取样检测发酵液中TG酶活力为5.68U/mL。
结果对比：与对比例2的发酵相比，实施例2的超声强化发酵使TG酶产量提高了20.9％。
实施例3：
在40m 3发酵罐进行冬虫夏草菌发酵生产菌丝体及多糖的超声波强化发酵，发酵罐1配置四组挂壁式超声波换能器支架2，均匀排布悬挂于内壁，每个支架有三节，通过不锈钢快装卡箍密封支架连接件3连接，每节纵向排列5个超声波换能器4，通过不锈钢焊接短管10焊接超声波换能器支架2，每个换能器内有3个超声波振子9，通过高频连接线5电性连接超声波发生器8。
具体发酵按如下步骤进行：
步骤1：向发酵罐1加注发酵液，发酵液组成成分中除水外，其他底物为米糠粉3.6％、麸皮粉4.8％、KH 2PO 4 0.27％、MgSO 4·7H 2O 0.27％、半胱氨酸盐酸盐0.05％、叶绿素铜钠盐0.01％，发酵罐装载量为75％容积。首先对发酵罐1实罐灭菌，灭菌条件为121℃、15min，降温后向发酵罐1内接种8％(v/v)冬虫夏草菌种子液，按照设定条件发酵：发酵温度23℃，初始pH7.0，开动发酵罐搅转轴6，搅拌转速100r/min；
步骤2：在发酵开始12h开启超声波发生器8，超声波模式为扫频，超声波频率为200kHz±1kHz，单个超声波换能器4功率120W，超声波处理时间为0.5h，处理结束关闭超声波发生器8；
步骤3：超声处理过程及处理结束后，发酵罐均按照步骤1设定条件进行发酵5d，直至发酵结束。取发酵液检测，菌丝干重48.5g/L发酵液，胞外多糖为2.67g/L发酵液；
步骤4：发酵结束后排空发酵罐1内发酵液，按设定程序清洗罐体，清洗过程中开启超声波发生器810min，超声波频率为20kHz，单个换能器功率范围120W；清洗完即可进行下一罐发酵。
对比例3:
发酵条件及发酵过程与实施例3相同，区别是不使用超声处理。发酵结束取样检测发酵液中菌丝干重42.8g/L发酵液，胞外多糖为2.30g/L发酵液；
结果对比：与对比例3的发酵相比，实施例3的超声强化发酵使菌丝干重提高了13.3％，胞外多糖增加了16.1％。
说明：以上实施例仅用以说明本发明而并非限制本发明所描述的技术方案；因此，尽管本说明书参照上述的各个实施例对本发明已进行了详细的说明，但是本领域的普通技术人员应当理解，仍然可以对本发明进行修改或等同替换；而一切不脱离本发明的精神和范围的技术方案及其改进，其均应涵盖在本发明的权利要求范围内。

Claims (10)

  1. 一种适用于大型发酵罐的挂壁式超声波强化发酵装置，其特征在于，所述装置包括发酵罐(1)、超声波换能器支架(2)、支架连接件(3)、超声波换能器(4)、高频连接线(5)、搅拌轴(6)、搅拌桨(7)、超声波发生器(8)、超声波振子(9)和不锈钢焊接短管(10)；
    所述搅拌轴(6)设于发酵罐(1)的内部，且搅拌轴(6)与发酵罐(1)同轴设置；所述搅拌轴(6)的一端设有电机，用于驱动搅拌轴(6)运转；另一端设有多个搅拌桨(7)；
    在发酵罐(1)的内部阵列设置多个超声波换能器支架(2)，所述超声波换能器支架(2)和搅拌轴(6)平行设置；
    所述超声波换能器支架(2)上设有多个超声波换能器(4)；所述超声波换能器(4)内壁设有多个超声波振子(9)；所述超声波振子(9)通过高频连接线(5)与超声波发生器(8)电性连接。
  2. 根据权利要求1所述的一种适用于大型发酵罐的挂壁式超声波强化发酵装置，其特征在于，所述搅拌桨(7)具体为4-6个，平行设置在搅拌轴(6)上。
  3. 根据权利要求1所述的一种适用于大型发酵罐的挂壁式超声波强化发酵装置，其特征在于，所述超声波换能器支架(2)沿着发酵罐(1)内壁从顶部悬挂至底部；所述超声波换能器支架(2)为多段式设置；具体由多节不锈钢管组成，每节不锈钢管的两头有法兰，通过卡箍密封连接；所述每节不锈钢管上包含3～5个超声波换能器(4)，纵向等距分布。
  4. 根据权利要求3所述的一种适用于大型发酵罐的挂壁式超声波强化发酵装置，其特征在于，所述超声波换能器支架(2)由3～5节316L不锈钢管制成，各节之间通过316L不锈钢快装卡箍密封连接；所述不锈钢管的内径为2～5cm。
  5. 根据权利要求1所述的一种适用于大型发酵罐的挂壁式超声波强化发酵装置，其特征在于，所述超声波换能器(4)为不锈钢薄板制成的立方体结构，通过不锈钢焊接短管(10)焊接悬挂于换能器支架(2)表面；所述超声波换能器(4)朝向发酵罐(1)中心的一侧内壁上设有3～6个超声波振子(9)；所述高频连接线(5)的一端连接超声波振子(9)，另一端穿过不锈钢焊接短管(10)和超声波换能器支架(2)，经超声波换能器支架(2)的上部顶端伸出，与超声波发生器(8)电性连接。
  6. 根据权利要求1所述的一种适用于大型发酵罐的挂壁式超声波强化发酵装置，其特征在于，所述超声波发生器(8)的超声波为固定频率模式或扫频模式；固定频率模式即定频模式，是指超声波工作过程中频率不变；扫频模式是指超声波工作过程中频率围绕中心频率作周期性上下波动；
    所述定频模式的频率范围为15～200kHz；所述扫频模式频率范围为15±2～200±2kHz，为中心频率+扫频幅度，即中心频率范围为15～200kHz，在每一个中心频率工作时的扫频幅 度范围为±0.5～±2kHz；
    所述超声波振子(9)单个的功率范围为5～20W。
  7. 根据权利要求1～6任一项所述的一种适用于大型发酵罐的挂壁式超声波强化发酵装置进行发酵的方法，其特征在于，包括以下步骤:
    步骤1：首先对发酵罐(1)实罐灭菌，灭菌条件为115℃、8～15min；然后在发酵罐(1)内接种发酵物种子液进行发酵，根据发酵物的不同，设定相应的发酵条件进行发酵：
    步骤2：在发酵开始后的0～12h后开启超声波发生器(8)，进行超声波辐射处理，超声波作用模式为定频或扫频模式，超声波频率范围为15～200kHz或15±2kHz～200±2kHz，单个超声波换能器(4)功率范围15～120W，超声波处理时间为0.5～12h，处理结束关闭超声波发生器；
    步骤3：在超声波辐射处理过程中及处理结束后，发酵罐均按照步骤1设定条件进行发酵，直至发酵结束；
    步骤4：发酵结束后排空罐内发酵液，进行罐体的清洗，清洗过程中开启超声波发生器(8)，设定相应的工作频率、工作时间进行清洗，清洗完毕以备下一次发酵使用。
  8. 根据权利要求7所述的发酵方法，其特征在于，步骤1中所述发酵物包括枯草芽孢杆菌、链霉菌或冬虫夏草菌；所述发酵条件具体为：发酵温度20～35℃、初始pH6.5～7.0、搅拌转速100～200r/min。
  9. 根据权利要求7所述的发酵方法，其特征在于，步骤2中当发酵物为枯草芽孢杆菌微生物菌剂时，在发酵开始6h开启超声波发生器(8)，超声波模式为定频，超声波频率为28kHz，单个超声波换能器(4)功率45W，超声波处理时间为12h；
    当发酵物为链霉菌时，在发酵开始6h开启超声波发生器(8)，超声波模式为扫频，超声波频率为15kHz±0.5kHz，单个超声波换能器(4)的功率15W，超声波处理时间为3h；
    当发酵物为冬虫夏草菌时，在发酵开始12h开启超声波发生器(8)，超声波模式为扫频，超声波频率为200kHz±1kHz，单个超声波换能器(4)的功率120W，超声波处理时间为0.5h。
  10. 根据权利要求7所述的发酵方法，其特征在于，步骤4中超声波发生器(8)选择定频模式，频率为20kHz，工作时间为10～20min；所述单个超声波换能器(4)的功率为60～120W。
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