CN115804403A - Thermophilic bacteria liquid sterilization method by high-pressure microjet homogenization and radio frequency treatment - Google Patents
Thermophilic bacteria liquid sterilization method by high-pressure microjet homogenization and radio frequency treatment Download PDFInfo
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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Abstract
The invention discloses a thermophilic bacteria liquid sterilization method by high-pressure microjet homogenization and radio frequency treatment, which comprises the following steps: step 1, culturing and preparing bacterial liquid: using a specific culture medium to perform amplification culture on the Bacillus stearothermophilus for 18-24 h, wherein the initial volume number of the bacterial liquid is 10 7 ～10 8 CFU/mL; step 2, homogenizing the bacteria liquid by high-pressure micro-jet flow: carrying out high-pressure microjet homogenization treatment on the bacillus stearothermophilus liquid amplified and cultured in the step 1; step 3, low-temperature storage; and 4, combining radio frequency with hot water treatment. The invention combines the low-temperature storage technology on the basis of high-pressure micro-jet homogenization treatment, thereby enhancing the effect of high-pressure micro-jet homogenization sterilization; on the basis of radio frequency sterilizationGo up supplementary with the water bath environment of thermostatic control, overcome the short slab that singly uses traditional hot water sterilization or radio frequency sterilization, can make the internal temperature of fungus liquid reach the expectation target in the shorter time, show the programming rate and the sterilization efficiency that have promoted the fungus liquid.
Description
Technical Field
The invention belongs to the technical field of food radio frequency sterilization, and relates to a bacterial liquid sterilization method by high-pressure micro-jet homogenization and radio frequency treatment.
Background
The High Pressure Microfluidization (HPM) technology is a new High Pressure homogenization technology, and can be applied to the modification of protein and the extraction of active substances. The material is dispersed into valve groups capable of respectively adjusting the pressure by the action of a plunger, and after flowing through a working area, the material losing pressure is sprayed out at the speed of 1000-1500 m/s and then collides on an impact ring to generate the actions of shearing, cavitation and the like so as to destroy the cell walls or cell membranes of the material. Based on the above principle, high-pressure micro-jet is gradually used as a sterilization technique. HPM sterilization can avoid the quality change of food caused by the conventional heat sterilization technology treatment, and the natural characteristics of the food can be maintained to the maximum extent. Despite the above advantages of HPM technology, it is difficult to achieve the desired sterilization effect by using HPM technology alone, and especially, studies on thermophilic bacteria have been reported. The technology is more reasonable and efficient to be applied to a pilot plant so as to be finally popularized to the large-scale production of the food industry, and researchers need to combine other sterilization technologies to perform synergistic processing in a laboratory stage.
The radio frequency sterilization technology is a novel sterilization technology, and compared with the traditional heat sterilization technology, the radio frequency sterilization technology has a plurality of unique advantages. The traditional thermal sterilization technology utilizes heat transfer or radiation heating, has a better heating effect on the surface of an object, cannot realize rapid heating inside a material, can simultaneously heat the inside and the surface of the material by radio frequency, and has good heating uniformity. Moreover, because the frequency difference between the microwave and the radio frequency is large, the radio frequency is low, the wavelength of the generated electromagnetic wave is long, and the penetrability is better than that of the microwave. The radio frequency sterilization technology is adopted to kill common microorganisms such as salmonella, escherichia coli, mould and the like in food, the sterilization effect is good, and the food quality can be well maintained. Nevertheless, the research reports of selecting thermophilic microorganisms as research target strains in the field of food radio frequency sterilization (especially in the field of pasteurization) are rare, and experimental exploration of thermophilic microorganisms is very necessary in a laboratory stage.
Xiaoyang and Yupeng, et al (patent application No. CN 103704338A) disclose a method for producing ultrahigh pressure homogenized sterilized milk. According to the method, cow milk with different initial temperatures is subjected to high-pressure homogenization treatment for 1-2 times, the temperature is controlled below 80 ℃ in the homogenization process, and after the heat is preserved for 10-15 s, a product with a good sterilization effect, fresh cow milk flavor and an effectively prolonged shelf life can be obtained. However, the invention is only applicable to high pressure homogenization alone and is not mentioned in combination with other sterilization techniques. Besides, the indicator bacteria selected by the sterilization method are limited to the microorganisms commonly used for pasteurization, and have certain limitations.
Studies of Zhangihua et al (DOI No.: 10.11882/j. Issn.0254-5071.2018.12.021) compare autoclaving, pasteurization and NaHSO 3 And 4 sterilization modes of high-pressure micro-jet treatment on the quality of the fermented jujube wine. The results showed that only 7.67% of the ascorbic acid was lost in the HPM group at day 8 of fermentation, whereas NaHSO 3 The group, pasteurized group and autoclaved group then lost 9.35%, 13.43% and 18.71%, respectively. Pasteurization and NaHSO 3 The sensory score of the jujube wine in the treatment group is respectively reduced by 1.49 and 1.40 compared with that in the HPM group. The influence of HPM and other different sterilization modes on the quality of the fermented jujube wine is researched, but only different sterilization technologies are used independently, and the influence is not realizedThere are references to combinations with other sterilization techniques and no indication of the rule of inactivation of the sterilization.
In summary, the prior art has the following difficulties: 1. the sterilization mode is single and an effective combined sterilization mode is provided; 2. the sterilization efficiency is low; 3. thermophilic and refractory microorganisms have been less studied. Therefore, by effectively combining different sterilization modes, it is necessary to research an efficient sterilization method which can be widely applied to different types of microorganisms, especially high temperature resistant microorganisms.
Disclosure of Invention
The invention aims to provide a thermophilic bacteria liquid sterilization method by high-pressure micro-jet homogenization and radio frequency treatment, and aims to solve the technical problems that the traditional hot water or radio frequency sterilization method is low in sterilization efficiency and high in sterilization cost in the prior art.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a thermophilic bacteria liquid sterilization method with high-pressure micro-jet homogenization and radio frequency treatment cooperation comprises the following steps:
and 3, low-temperature storage: placing the bacterial liquid treated in the step 2 into an aseptic storage room, wherein the storage temperature is-30 ℃, the storage time is 12-36 h, and after the low-temperature storage is finished, placing the bacterial liquid into an aseptic storage room at the temperature of 0-4 ℃ for storage; placing the bacteria solution stored at low temperature in a 50mL centrifuge tube;
Further, in the step 1, the specific culture medium is nutrient broth NB; the number of cleaning and centrifuging times is 3; diluting the bacterial liquid to 10 initial volume number by using 0.8% normal saline as a medium 7 CFU/mL and cold storage at 4 ℃ for 24h.
Further, in the step 2, in the high-pressure homogenization process, a low-temperature cooling liquid circulating pump connected with the high-pressure micro-jet homogenization treatment device is adopted to control the temperature of the bacterial liquid to be below 30 ℃.
Further, in the step 2, in the single high-pressure homogenization treatment process, the control pressure value rapidly rises from 0Mpa to the target pressure value and then gradually falls back to 0Mpa, wherein the upper limit of the target pressure value of the high-pressure homogenization is 300Mpa.
Further, in the step 3, the bacterial liquid after the high-pressure micro-jet homogenization treatment in the step 2 is quickly poured into a 50mL centrifuge tube and is stored in an aseptic storage room at the temperature of-30 ℃.
Further, in the step 4, the sterilization kettle 2 is a cylindrical container; the centrifuge tube is made of polytetrafluoroethylene.
Further, in the step 4, a superheated water auxiliary radio frequency sterilization device is adopted to perform radio frequency combined hot water treatment; the distance between an upper polar plate and a lower polar plate connected with a radio frequency generator in the device is 180-200 mm.
Further, the distance between the upper polar plate and the lower polar plate is 180mm, and the height of the bacterial liquid is 80mm.
Further, the heating time in the step 4 is to keep the temperature for 0-10 min after the central temperature reaches the target temperature.
Further, the heating time is 10min.
Compared with the prior art, the invention has the following beneficial effects:
(1) On the basis of the traditional high-pressure homogenizing treatment of the bacterial liquid, a low-temperature storage technology and a radio frequency heating technology are combined, so that the high-pressure micro-jet homogenizing sterilization effect is enhanced, and the application range of the high-pressure micro-jet homogenizing sterilization is enriched; in the high-pressure homogenization treatment process, the bacterial liquid operating room is connected with the low-temperature cooling circulating device, and the sample temperature is controlled below 30 ℃ through rapid intermittent operation, so that the original properties of the bacterial liquid are greatly reserved. Meanwhile, due to the advantages, the bacteria liquid type is not limited too much.
(2) After HPM low-temperature storage treatment, the bacterium liquid is assisted by a constant-temperature water bath environment on the basis of radio-frequency sterilization, so that the defect that a traditional hot water sterilization or radio-frequency sterilization short plate is singly used is overcome, the internal temperature of the material can reach an expected target in a shorter time, and the heating rate and the sterilization efficiency of the bacterium liquid are obviously improved.
(3) Radio frequency combines the heat treatment integration mode of operation, can realize totally enclosed preheating-intensification-sterilization heat preservation-refrigerated one-key formula operation, has greatly shortened the time of handling, has reduced the required time of fungus liquid processing and apparatus consumptive material cost, has promoted the efficiency of whole sterilization process comparatively obviously.
Drawings
Fig. 1 is a schematic diagram of a sterilization kettle and a temperature measuring device in a conventional superheated water-assisted radio frequency sterilization device used in the present invention.
FIG. 2 shows the residual count of Bacillus stearothermophilus after different homogenization treatments in the examples.
FIG. 3 is a graph showing the effect of different treatments for homogenization times on the conductivity and protein content of B.stearothermophilus after different cryopreservation times in the examples.
FIG. 4 is a graph showing the temperature increase rate of the Bacillus stearothermophilus liquid after being treated with hot water at different target temperatures after being subjected to HPM treatment for 3 times and stored at low temperature for 24 hours in the examples.
FIG. 5 is a graph showing the temperature increase rate of Bacillus stearothermophilus liquid after 3 times of HPM treatment and 24 hours of cryopreservation in the examples after radio frequency combining with heat treatment of water at different target temperatures.
FIG. 6 is the inactivation curve of the Bacillus stearothermophilus after HPM treatment for 3 times cryopreservation for 24h and before reaching different target temperatures in the embodiment after radio frequency combined with heat treatment.
FIG. 7 is a scanning electron micrograph of Bacillus stearothermophilus treated with HPM in combination with RF in the examples.
The reference symbols in the drawings mean:
1. a temperature measuring optical fiber probe; 2. sterilizing the kettle; 3. a metal cover plate; 4. a temperature measuring flange plate.
Detailed Description
The invention is described in detail below with reference to the figures and the detailed description, without thereby limiting the invention to the described embodiments. The test methods of conditions not specified in the following examples were selected according to national standards or conventional methods and conditions.
The invention provides a sterilization method of high-pressure micro-jet homogenization and radio frequency treatment, which comprises the following steps:
And 3, storing the bacterial liquid treated by HPM at low temperature: placing the thermophilic bacteria liquid treated by HPM in the step 2 into an aseptic storage chamber, wherein the storage temperature is-30 ℃, the storage time is 12-36 h (as a control group without low-temperature storage treatment), and after the low-temperature storage is finished, placing the bacteria liquid into the aseptic storage chamber at 0-4 ℃ for storage; taking the bacterium liquid after low-temperature storage and HPM treatment (the bacterium liquid is selected and homogenized by 3 times of high-pressure micro-jet and stored for 24 hours at low temperature) in a 50mL centrifugal tube, and carrying out heat treatment for standby;
The technical scheme has the following advantages:
(1) The method has the advantages that the low-temperature storage technology is combined on the basis of the traditional high-pressure homogenizing treatment of the bacterial liquid, the high-pressure micro-jet homogenizing sterilization effect is enhanced, and the application range of the high-pressure micro-jet homogenizing sterilization is enriched; in addition, in the high-pressure micro-jet homogenization treatment process, the temperature of the sample is controlled to be below 30 ℃, and the original property of the bacterial liquid is greatly reserved. Meanwhile, due to the advantages, the bacteria liquid is not excessively limited.
(2) After the HPM low-temperature storage treatment, the bacterial liquid is assisted by a constant-temperature water bath environment on the basis of radio frequency sterilization, so that the defect that a traditional hot water sterilization or radio frequency sterilization short plate is singly used is overcome, the internal temperature of the material can reach an expected target in a shorter time, and the heating rate and the sterilization efficiency of the bacterial liquid are obviously improved.
(3) In the step 3, an integrated operation mode of radio frequency and hot water treatment is adopted, so that one-key operation of totally-enclosed preheating, temperature rise, sterilization and heat preservation and cooling can be realized, the treatment time is greatly shortened, the time required by bacterial liquid treatment and the cost of instrument consumables are reduced, and the efficiency of the whole sterilization process is obviously improved.
Specifically, the bacterial liquid after the high-pressure micro-jet homogenization treatment in the step 2 is quickly poured into a 50mL centrifuge tube and is stored in an aseptic storage room at the temperature of minus 30 ℃.
Preferably, in step 1, the Nutrient Broth (NB) is used as a culture medium for the amplification culture of bacillus stearothermophilus, the culture medium is washed and centrifuged 2 to 3 times, and then the bacterial solution is diluted to 10% with 0.8% physiological saline as a medium 7 ～10 8 CFU/mL and stored refrigerated at 4 ℃.
Specifically, in step 2, the high-pressure micro-jet homogenizing device is a high-pressure micro-jet homogenizer, such as NanoGenizer-30K high-pressure homogenizer, produced by Genizer company of America; the low-temperature cooling circulation device adopts a low-temperature constant-temperature tank, such as DW-2005, produced by Hangzhou David scientific and education instruments, inc. And 2, controlling the temperature of the bacterial liquid to be below 30 ℃ by adopting a low-temperature cooling liquid circulating pump connected with the high-pressure micro-jet homogenizing treatment device.
Preferably, in step 2, in the single high-pressure homogenization treatment process, the control pressure value rapidly rises from 0Mpa to the target pressure value and then gradually falls back to 0Mpa, wherein the upper limit of the target pressure value of the high-pressure homogenization is 300Mpa.
Specifically, in step 3, the centrifuge tube is sterilized at 121 ℃ for 15min before use.
Preferably, as shown in fig. 1, in step 4, the autoclave 12 is a cylindrical container, and the upper cover and the bottom plate are made of metal plates 19 (preferably aluminum alloy plates); the material of the middle container is preferably polytetrafluoroethylene, the material is high temperature and high pressure resistant, suitable for a radio frequency field and low in price, the sterilization kettle 19 is used, the constant temperature water bath and the temperature measuring optical fiber 25 are combined, the heating rate is faster and more uniform under the action of radio frequency, the operation is convenient, and the real-time temperature measurement can be realized.
Preferably, as shown in FIG. 1, in step 4, the RF-assisted RF sterilization device with superheated water is used to perform RF-combined hot water treatment, the specific composition and operation of the RF sterilization device with superheated water is disclosed in the patent application (patent No. ZL 202120356615.3) of the subject group, and the superheated water in the patent application can be replaced by hot water with temperature lower than 100 ℃.
Finally, in the above method, preferably, the thermophilic bacteria solution is amplified and cultured for 24 hours by using Nutrient Broth (NB), and the initial volume number of the solution is 10 7 CFU/mL; the upper limit of the high-pressure homogenization pressure is 300MPa, and the high-pressure homogenization frequency is set to be 3 times for subsequent heat treatment tests; the temperature of the bacterial liquid in the whole high-pressure homogenization process is controlled below 30 ℃, and 200mL of bacterial liquid can be produced at most after each homogenization; the bacteria liquid treated by HPM is stored at low temperature (-30 ℃) for 24 hours; and (3) combining radio frequency with hot water treatment, wherein the distance between the radio frequency cavity electrode plates is 180mm, and the heating time is 10min.
The heating time in the step 4 means that the central temperature is kept for 0-10 min after reaching the target temperature. The distance between the polar plates is 180mm, and the height of the bacterial liquid is 80mm.
Example 1 measurement of the number of Bacillus stearothermophilus remaining bacteria after treatment with different homogenization procedures
Bacillus stearothermophilus ATCC 7953 (G. Stearothermophilus) (available from Shanghai science and technology, inc.)Limited company) for 24h of amplification culture by using NB medium, reaching a stationary phase, cleaning and centrifuging for 3 times to make the initial volume number of the bacterial liquid be 10 7 CFU/mL. Carrying out high-pressure microjet homogenization treatment on 200mL of Bacillus stearothermophilus liquid, wherein the upper limit of the pressure of high-pressure homogenization is 300Mpa, the temperature of the bacterial liquid is controlled below 30 ℃ in the homogenization process, sampling is carried out after 1-5 times of homogenization, 160-200 mL of bacterial liquid can be produced after each high-pressure homogenization treatment, and for the sampling dilution gradient, carrying out microbial determination on a flat plate poured with a Nutrient Agar (NA) culture medium, wherein each dilution degree is 3 in parallel, and each sample is 3 in parallel. The plates were incubated in a 56 ℃ incubator for 24h and then counted.
As shown in FIG. 2, the number of times of homogenization treatment (1, 2, 3, 4, and 5 times) with high-pressure microjet decreased from the initial 7 orders of magnitude to 6.83, 6.20, 5.52, 5.49, and 5.40 orders of magnitude; accordingly, the lethality was 2.47%, 11.48%, 21.19%, 21.61%, and 22.89% after the different treatment times, respectively. This indicates that the high-pressure microjet homogenization treatment has a bactericidal effect on the bacillus stearothermophilus, but the bactericidal effect is not obvious; this necessitates the use of high pressure micro-jet homogenisation sterilisation in combination with other techniques.
Example 2 high pressure microjet homogeneous combination Germination of Bacillus stearothermophilus liquid conductivity and protein assay after cryopreservation
The bacillus stearothermophilus liquid treated by the high-pressure micro-jet homogenization treatment for 3 times in the embodiment 1 is poured into a sterilized 50mL centrifuge tube by 45mL, is stored in an aseptic storage room at-30 ℃ for 12h, 24h and 36h respectively, is rapidly placed in an aseptic storage room at 4 ℃ and is measured for conductivity and protein when the liquid is completely liquid, and the heterogeneous liquid is used as a control. Wherein, the bacteria liquid operating room is connected with a low-temperature cooling circulating device, and the temperature of the sterile storage room is kept at minus 30 ℃ by rapid intermittent operation on a touch screen adjusting plate.
As shown in fig. 3, the conductivity and protein content of the bacteria solution after different homogenization treatments were higher than those of the control group, and increased with the increase of the homogenization times. Wherein, the conductivity and the protein content of the bacterial liquid after being homogenized for 3 times by the high-pressure micro-jet are respectively 18.2 percent and 122.2 percent higher than those of a control group. This is because the cell membrane and other protective barriers of bacillus stearothermophilus are destroyed with the increase of the number of homogenization times, so that the electrolytes, proteins and the like inside the cells leak into the bacterial liquid, and the conductivity and protein content of the bacterial liquid tend to increase; after the same homogenization treatment, the conductivity and the protein content of the bacterial liquid are increased along with the increase of the low-temperature storage time, and the effect is most obvious when the storage time is longer than 24 hours. The result shows that after the high-pressure microjet homogenization and the low-temperature storage, protective barriers such as cell membranes of the bacillus stearothermophilus can be further destroyed, and the overall sterilization efficiency can be improved. Therefore, the bacillus stearothermophilus liquid which is sterilized effectively, homogenized by high-pressure micro-jet for 3 times and stored for 24 hours at low temperature is selected as a test object to perform subsequent operation.
Example 3 measurement of the rate of temperature rise of Bacillus stearothermophilus liquid after high-pressure microjet homogenization in cooperation with radio frequency and water bath heat treatment at different temperatures
45mL of the Bacillus stearothermophilus liquid obtained in the embodiment 2 after being homogenized for 3 times by high-pressure microfluidics and stored at low temperature for 24h is poured into a sterilized 50mL centrifuge tube (the liquid level height is 80 mm), the liquid is cultured for 1h at the constant temperature of 25 ℃ in a constant temperature incubator, the liquid is placed at the geometric center of a sterilization kettle 12 as shown in FIG. 1, the target temperature set by constant temperature water bath is 70, 80 and 90 respectively, the distance between an upper electrode plate and a lower electrode plate of a radio frequency heating system is set to be 180mm according to previous experiments, the heating time is 10min, the temperature rise data of the Bacillus stearothermophilus liquid in the centrifuge tube is monitored in real time by connecting a temperature measuring optical fiber 7 with a computer, and the liquid without radio frequency treatment is used as a control.
As shown in FIG. 4, the temperature rising trends of the Bacillus stearothermophilus bacterial liquid after 3 times of high-pressure microjet homogenization and low-temperature storage for 24h are similar in different water bath (W) temperatures, forming an S-shaped curve, and 500S, 520S and 610S are respectively used when the central temperature of the bacterial liquid reaches 70, 80 and 90 ℃; the results of the same cases combined with the radio frequency (RF + W) treatment are shown in fig. 5, and the results of 270s, 270s and 290s for the bacteria liquid center temperature of 70, 80 and 90 ℃ respectively are nearly half of the time required for the group without the radio frequency treatment, which indicates that the present invention can significantly increase the temperature increase rate of the bacillus stearothermophilus liquid.
Example 4 inactivation curve of Bacillus stearothermophilus liquid after high-pressure microjet homogenization in cooperation with radio frequency and water bath heat treatment at different temperatures
According to the temperature rise rate data of the bacillus stearothermophilus bacterial liquid obtained in the embodiment 3, the geometric center position of the centrifugal tube sterilization kettle 12 is shown in fig. 1, the temperature measuring optical fiber 7 is used for controlling the central temperature of the bacterial liquid to be 70 ℃, 80 ℃ and 90 ℃, the centrifugal tube is placed into the sterilization kettle 12 for instant timing, when different target temperatures are reached, the radio frequency is closed, the sterilization kettle 12 is opened to take out the centrifugal tube after timing is finished, the radio frequency is combined with constant-temperature water bath treatment, and then the centrifugal tube is rapidly placed into ice water for cooling for 5min. At each time point, 3 replicates were taken and the microbial population of the treated Bacillus stearothermophilus liquid was measured as described in example 1, and homogenized Bacillus stearothermophilus liquid without RF treatment was used as a control.
As shown in FIG. 6a, the volume number of the bacillus stearothermophilus liquid after 3 times of high-pressure micro-jet homogenization and low-temperature storage for 24h is reduced from the moment the bacillus stearothermophilus liquid is placed in the sterilization kettle 3 at different water bath (W) temperatures, the change range of the first 60s is not large, the inactivation rate is gradually increased after the bacillus stearothermophilus liquid is treated for 60s, and the bacillus stearothermophilus liquid is not changed after reaching a certain stable value, which is basically the same as the temperature rise rule of the bacillus stearothermophilus liquid in the embodiment 2. Finally, when the Bacillus stearothermophilus liquid is heated for 600s by water bath (70, 80 and 90 ℃) (W), the initial 7.04, 7.13 and 7.08 orders of magnitude are respectively reduced to 3.45, 3.47 and 3.53 orders of magnitude; accordingly, the lethality was 50.64%, 50.74% and 49.52%, respectively.
The inactivation rule of the bacillus stearothermophilus bacterial liquid which is homogenized by 3 times of high-pressure microjet and stored for 24 hours at low temperature is similar to that of a non-radio frequency cooperative treatment group under the condition of combining different water baths with radio frequency (RF + W), but the time of the bacterial liquid reaching a stable value is 300s, the sterilization efficiency is greatly improved and is about one time of that of the non-radio frequency cooperative treatment group; finally, the thermophilic fat bacillus liquid is reduced to 3.45, 3.45 and 3.55 orders of magnitude from the initial 7.06, 7.08 and 7.12 orders of magnitude; the lethality was 51.07%, 50.72% and 50.66%, respectively.
Example 5 Effect of HPM Mass-synergistic RF treatment on the Permitral Structure of Bacillus stearothermophilus
In order to further verify that the method has a good sterilization effect, the cell surface is observed in a scanning electron microscope mode. FIG. 7 shows the morphology of B.stearothermophilus after and after homogenization with high pressure microjet combined with RF treatment. In FIG. 7a, the untreated Bacillus stearothermophilus has a full and plump shape of the cells, uniform distribution and smooth surface, and is in the shape of a long rod. FIG. 7b shows the morphology of Bacillus stearothermophilus after 3 times homogenization with high pressure microjet, from which it is apparent that the cells are largely cut off by the transverse plane, and that part of the cells are deformed and separated from the original cells. FIG. 7c shows the morphology of Bacillus stearothermophilus after 3 times cryopreservation for 24h by high pressure microjet homogenization, wherein the cells are broken into larger cross sections and more cells are deformed and fragmented compared to FIG. 7 b. This shows that the high-pressure microjet homogenization can effectively destroy the cell membrane and cell wall of the bacillus stearothermophilus, thereby causing the inactivation of the thalli; after 3 times of homogenization by high-pressure microfluidization and 24 hours of cryopreservation, the cell membrane and cell wall of B.stearothermophilus were further destroyed, which corresponds to the results in example 2.
FIG. 7d shows the shape of the Bacillus stearothermophilus after 3 times of high pressure microjet homogenization and 24 hours of low temperature storage and treatment in a water bath (W) at 90 ℃ for 5min, from which it is apparent that the cells are almost completely cut off by the transverse plane and deformed and completely fragmented. FIG. 7e shows the high pressure micro-jet homogenization for 3 times of cryopreservation for 24h, and the Bacillus stearothermophilus liquid after being treated in a water bath (W) at 90 ℃ for 10min in combination with RF treatment, it is also evident that the cells are almost completely cut off laterally, deformed and completely fragmented, and have a greater degree of destruction than FIG. 7 d. This shows that the cell membrane and cell wall of the Bacillus stearothermophilus can be further destroyed by the high-pressure microjet homogenization in cooperation with the radio frequency treatment, so that the thallus is inactivated. This corresponds to the result in embodiment 4.
Claims (10)
1. A thermophilic bacteria liquid sterilization method by high-pressure microjet homogenization and radio frequency treatment is characterized by comprising the following steps:
step 1, culturing and preparing bacterial liquid: using a specific culture medium to amplify and culture the thermophilic bacteria liquid to be treated for 18-24 h to reach a stationary phase, and cleaning and centrifuging to ensure that the initial volume number of the thermophilic bacteria liquid is 10 7 ～10 8 CFU/mL；
Step 2, homogenizing the bacteria liquid by high-pressure micro-jet flow: carrying out high-pressure microjet homogenization treatment on the bacterial liquid subjected to amplification culture in the step 1, wherein the pressure of the high-pressure microjet homogenization treatment is 0-300 Mpa, the times are 1-5 times, and the temperature of the bacterial liquid is controlled below 30 ℃;
and 3, low-temperature storage: placing the bacterial liquid treated in the step 2 into an aseptic storage room, wherein the storage temperature is-30 ℃, the storage time is 12-36 h, and after the low-temperature storage is finished, placing the bacterial liquid into an aseptic storage room at 0-4 ℃ for storage; placing the bacteria solution stored at low temperature in a 50mL centrifuge tube;
step 4, radio frequency combined hot water treatment: placing the centrifugal tube into a sterilization kettle, and sterilizing the bacterial liquid in the sterilization kettle in a hot water and radio frequency combined mode; wherein the temperature of the hot water bath is 70-90 ℃; the heating time is 0-10 min.
2. The method for sterilizing thermophilic bacteria liquid by high-pressure microjet homogeneous synergistic radio frequency treatment as claimed in claim 1, wherein in the step 1, the specific culture medium is nutrient broth NB; the number of cleaning and centrifuging times is 3; diluting the bacterial liquid to 10 initial volume number by using 0.8% normal saline as a medium 7 CFU/mL and cold storage at 4 ℃ for 24h.
3. The method as claimed in claim 1, wherein in step 2, a low-temperature cooling liquid circulating pump connected to the high-pressure micro-jet homogenization processing apparatus is used to control the temperature of the bacteria liquid below 30 ℃ during the high-pressure homogenization process.
4. The method for sterilizing a thermophilic bacteria liquid by high pressure micro jet homogenization in cooperation with radio frequency treatment as claimed in claim 1, wherein in the step 2, the control pressure value is rapidly increased from 0Mpa to a target pressure value and then gradually decreased back to 0Mpa in a single high pressure homogenization process, wherein the upper limit of the target pressure value of the high pressure homogenization is 300Mpa.
5. The method for sterilizing thermophilic bacteria liquid through high-pressure micro-jet homogenization and radio frequency treatment as claimed in claim 1, wherein in the step 3, the bacteria liquid after the high-pressure micro-jet homogenization treatment in the step 2 is quickly poured into a 50mL centrifuge tube and is stored in a sterile storage room at-30 ℃.
6. The method for sterilizing a thermophilic bacteria liquid by high-pressure micro-jet homogenization in cooperation with radio frequency treatment as claimed in claim 1, wherein in the step 4, the sterilizing kettle 2 is a cylindrical container; the centrifuge tube is made of polytetrafluoroethylene.
7. The method for sterilizing the thermophilic bacteria liquid by the high-pressure micro-jet homogenization and radio frequency concurrent treatment as claimed in claim 1, wherein in the step 4, a superheated water assisted radio frequency sterilization device is adopted for carrying out radio frequency combined hot water treatment; the distance between an upper polar plate and a lower polar plate connected with a radio frequency generator in the device is 180-200 mm.
8. The method according to claim 7, wherein the distance between the upper and lower plates is 180mm, and the height of the bacteria liquid is 80mm.
9. The method for sterilizing thermophilic bacteria liquid by high-pressure micro-jet homogenization in cooperation with radio frequency treatment as claimed in claim 1, wherein the heating time in the step 4 is that the temperature is kept for 0-10 min after the central temperature reaches the target temperature.
10. The method for sterilizing a thermophilic bacteria liquid by high-pressure microjet homogenization in cooperation with radio frequency treatment according to claim 1 or 9, characterized in that the heating time is 10min.
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