CN108740684B - Pressure-heat sound composite inactivation spore method and equipment - Google Patents

Pressure-heat sound composite inactivation spore method and equipment Download PDF

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CN108740684B
CN108740684B CN201810421105.2A CN201810421105A CN108740684B CN 108740684 B CN108740684 B CN 108740684B CN 201810421105 A CN201810421105 A CN 201810421105A CN 108740684 B CN108740684 B CN 108740684B
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temperature
tank
electromagnetic valve
pressure
ultrasonic
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CN108740684A (en
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刘东红
吕瑞玲
丁甜
周建伟
王文骏
叶兴乾
曾天泽
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Zhejiang University ZJU
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/015—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with pressure variation, shock, acceleration or shear stress or cavitation
    • A23L3/0155—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with pressure variation, shock, acceleration or shear stress or cavitation using sub- or super-atmospheric pressures, or pressure variations transmitted by a liquid or gas
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/02—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by heating materials in packages which are progressively transported, continuously or stepwise, through the apparatus
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/10—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by heating materials in packages which are not progressively transported through the apparatus
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/16—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by heating loose unpacked materials
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/26—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by irradiation without heating
    • A23L3/30—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by irradiation without heating by treatment with ultrasonic waves
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
    • Y02P60/85—Food storage or conservation, e.g. cooling or drying

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  • Health & Medical Sciences (AREA)
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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Apparatus For Disinfection Or Sterilisation (AREA)
  • Food Preservation Except Freezing, Refrigeration, And Drying (AREA)

Abstract

The invention discloses a thermo-acoustic composite spore inactivation method, wherein a product to be sterilized is subjected to ultrasonic treatment for 25-35 min at the temperature of 75-85 ℃, the pressure of 450-550 kPa, the amplitude of 100% and the frequency of 20khz, and the spore inactivation effect is obvious. The invention adopts a pressure thermoacoustic composite method to treat to-be-sterilized treatment, has higher spore inactivation efficiency under the condition that the temperature is not more than 85 ℃, and compared with the prior art, not only can reduce energy consumption, but also can reduce the influence on the food quality and ensure the food safety. The invention also discloses a pressure-thermo-acoustic composite inactivated spore device which comprises a sterilization tank, a nitrogen tank, a cold water tank, a hot water tank, a temperature relay, an ultrasonic vibration device, a temperature probe, a pump assembly and a solenoid valve assembly, and meets the requirements of the device for ultrasonic, pressure and heat combined sterilization.

Description

Pressure-heat sound composite inactivation spore method and equipment
Technical Field
The invention relates to a sterilization method and equipment, in particular to a method and equipment for inactivating spores through thermo-compression and acoustic compounding.
Background
With the development of economy and the improvement of living standard, food becomes an increasingly active stage, consumers have higher requirements on food eaten by the consumers, and important attributes such as flavor, texture, appearance, nutrition and the like affecting the food have an inexplicable relationship with processing modes. Heat treatment is the most primitive and traditional way of sterilization and is currently the most widely used industrially, and much research has been devoted to the mechanism of heat sterilization and the effect on food quality. In recent years, various new food processing techniques and methods have been developed to meet the demand for personalized foods, but have not been industrially popularized for various reasons, and thus heat sterilization has been the most important and effective method for food sterilization so far.
Ultrasonic wave is an effective auxiliary sterilization method, has been successfully used in the fields of wastewater treatment, drinking water disinfection and the like, and has been widely researched in the application of liquid food sterilization, such as beer, orange juice, soy sauce and the like. The sterilization effect of the ultrasonic wave is mainly due to the cavitation generated by the ultrasonic wave, so that the food sterilization speed is accelerated. But the ultrasonic action alone is difficult to achieve the effect required for practical use.
Under the high pressure condition, the genetic material, the protein structure and other aspects of the microorganism are changed to a certain extent, the cell wall and the cell membrane are damaged to a certain extent, the physiological function of the microorganism is partially or completely lost, and even the microorganism is inactivated and even killed, but the single use of the high pressure technology has certain limitation, all bacteria cannot be completely killed, and the sterilization effect of combining the high pressure sterilization with other methods is more obvious.
The non-thermal sterilization effectively kills microorganisms, simultaneously maintains the flavor and nutrient substances of the product to the maximum extent, and avoids the damage of the original structure and flavor of the food by the thermal sterilization. But non-thermal sterilization has certain limitation, and the effect is poor when the sterilization is carried out alone, so that the defects of the sterilization can be improved by combining heat and various non-thermal sterilization technologies, and the non-thermal technology has better application prospect.
Spores are formed as dormant bodies of spore-forming bacteria when the growing environment is starved of nutrients or harmful metabolic substances are accumulated excessively. The spore is the life body with the strongest stress resistance in the whole biological world, and is the first guideline in the aspects of heat resistance, chemical medicine resistance, radiation resistance, high pressure resistance and the like. The extremely strong resistance of the spores is a great challenge to the sterilization link in food processing, if the spores cannot be effectively killed or controlled, the spores can cause food spoilage after germination, and the generated toxins can also pose a potential threat to human health. The existing industrial common sterilization means is heat treatment, while the heat resistance of the spore is extremely high, for example, the D value of the bacillus subtilis spore at 88 ℃ is 30.2min, so the excessive heat treatment is necessary to ensure the food safety. Therefore, the search for an effective spore inactivation means is of great significance.
Thermoacoustic pressing is a novel processing technology and is mostly used for improving the food preservation effect, for example, non-patent documents, namely review on the influence of thermoacoustic pressing on enzyme activity, inform that thermoacoustic pressing can inactivate enzyme to achieve the preservation effect. The enzyme is used as a common protein, the activity of the protein can be reduced or even inactivated by simple treatment, such as separate heat treatment or ultrasonic treatment and the like, spores are used as special dormant structures of bacteria, corresponding treatment work cannot be carried out with reference enzymes, and documents related to the spore inactivation applying the thermo-compression acoustic technology are not inquired.
Disclosure of Invention
The invention aims to solve the technical problem of providing a thermo-compression and thermo-acoustic composite inactivated spore method and equipment.
In order to solve the problems, the invention provides a thermo-compression and thermo-acoustic combined inactivated spore device which comprises:
the thermoacoustic composite inactivated spore equipment comprises a sterilization tank, a jacket, a nitrogen tank, a cold water pool, a hot water pool and an ultrasonic vibration device;
the ultrasonic vibration device comprises an ultrasonic generator, an ultrasonic transducer and an ultrasonic probe which are connected in sequence;
the sterilization tank is communicated with the nitrogen tank;
the jacket is sleeved on the outer surface of the sterilization tank and is respectively communicated with the cold water pool and the hot water pool;
the ultrasonic transducer is arranged on the top of the sterilization tank, the ultrasonic probe is arranged on the ultrasonic transducer, and the end part of the ultrasonic probe penetrates through the sterilization tank in a sealing manner and then extends into the inner cavity I of the sterilization tank.
As an improvement of the thermo-compression sound composite inactivated spore device of the invention:
the thermoacoustic composite inactivated spore device further comprises a pump assembly, an electromagnetic valve assembly, a temperature relay and a temperature probe;
the temperature relay is respectively connected with the pump assembly, the electromagnetic valve assembly and the temperature probe;
the end part of the temperature probe extends into the inner cavity I.
As a further improvement of the thermo-compression composite inactivated spore device of the invention:
the pump assembly comprises a cold water pump and a hot water pump; the electromagnetic valve assembly comprises an electromagnetic valve I and an electromagnetic valve II;
and the cold water pump, the hot water pump, the electromagnetic valve I and the electromagnetic valve II are respectively connected with a temperature relay.
A water outlet and a water inlet are sequentially arranged on the jacket from top to bottom;
the water inlet is communicated with a cold water tank through a cold water pump and is also communicated with a hot water tank through a hot water pump;
the water outlet is communicated with the cold water tank through an electromagnetic valve I and is also communicated with the hot water tank through an electromagnetic valve II;
as a further improvement of the thermo-compression composite inactivated spore device of the invention:
the sterilization tank is provided with an air inlet and an air outlet;
the air inlet is provided with a pressure reducing valve and is communicated with the nitrogen tank through the pressure reducing valve;
a pressure relief valve is arranged at the air outlet;
the jacket is positioned below the air inlet and the air outlet.
In order to solve the problems, the invention also provides a thermo-acoustic composite inactivated spore method (which is realized by the device), which comprises the following steps:
putting a product to be sterilized into an inner cavity I of a sterilization tank, and carrying out ultrasonic treatment with the amplitude of 100% (114 microns) and the frequency of 20khz for 25-35 min at the heating temperature of 75-85 ℃ and the pressurizing pressure of 450-550 kPa.
The improvement of the thermo-acoustic composite spore inactivation method of the invention is as follows:
the product to be sterilized is placed in the inner cavity I of a sterilization tank, and ultrasonic treatment with amplitude of 100% (114 μm) and frequency of 20khz is performed for 30min at a heating temperature of 80 ℃ and a pressurizing pressure of 500 kPa.
The invention is further improved by the thermo-compression composite inactivation spore method:
the heating method comprises the following steps:
firstly, setting heating temperature through a temperature relay;
the temperature probe detects the temperature of a product to be sterilized in real time and sends a temperature signal to the temperature relay, and the temperature relay controls the work of the electromagnetic valve assembly and the pump assembly according to the received temperature signal;
when the temperature of a product to be sterilized is lower than the set heating temperature, a temperature relay controls a hot water pump to work, and heated water (85-100 ℃) in a hot water pool enters an inner cavity II of the jacket; meanwhile, the temperature relay controls the opening of the electromagnetic valve II, and water in the inner cavity II of the jacket returns to the hot water pool through the electromagnetic valve II;
otherwise, the temperature relay controls the cold water pump to work, and cooling water (normal temperature water, 15-35 ℃) in the cold water pool enters the jacket inner cavity II; meanwhile, the temperature relay controls the solenoid valve I to be opened, and water in the jacket inner cavity II returns to the cold water tank through the solenoid valve I.
The invention is further improved by the thermo-compression composite inactivation spore method:
the ultrasonic treatment method comprises the following steps:
an ultrasonic generator is used for emitting ultrasonic signals, an ultrasonic transducer converts the ultrasonic signals into electric signals to be transmitted to an ultrasonic probe, and the ultrasonic probe generates vibration with specific amplitude in an inner cavity I of the sterilization tank according to the signals;
the distance between the end part of the ultrasonic probe and the bottom of the inner cavity I of the sterilization tank is 1 +/-0.05 cm.
The invention is further improved by the thermo-compression composite inactivation spore method:
the pressurizing method comprises the following steps:
and (3) injecting high-purity nitrogen into the inner cavity I of the sterilization tank by using a nitrogen tank, adjusting the pressure by using a pressure reducing valve, and opening a pressure release valve to release the nitrogen after sterilization is completed.
Compared with the prior art, the invention has the technical advantages that:
the invention adopts a method of combining ultrasonic wave, high pressure and heat to inactivate spores; compared with separate heat treatment and ultrasonic treatment, the thermo-compression sound can inactivate spores to a greater extent. On the other hand, the energy consumption can be reduced by reducing the sterilization time, and the important effects of energy conservation, consumption reduction and environmental protection are achieved.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a schematic structural view of a thermoacoustic composite inactivated spore device according to the present invention;
FIG. 2 is a schematic diagram of the spore ultrastructure in a blank control according to the invention;
FIG. 3 is a schematic diagram of the spore ultrastructure in example 1 of the present invention;
FIG. 4 is a schematic diagram showing the ultrastructure of spores in comparative example 1-1 of the present invention;
FIG. 5 is a schematic diagram of the spore ultrastructure in comparative examples 1-2 of the present invention;
FIG. 6 is a schematic diagram showing the ultrastructure of spores in comparative examples 1 to 3 of the present invention;
FIG. 7 is a schematic diagram showing the ultrastructure of spores in comparative examples 1 to 4 of the present invention.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.
Example 1, a thermo-acoustic pressure composite inactivated spore apparatus, as shown in fig. 1, includes a sterilization tank 1, a nitrogen tank 3, a cold water tank 4, a hot water tank 5, a temperature relay 6, an ultrasonic vibration device, a temperature probe 8, a pump assembly and a solenoid valve assembly.
The pump assembly includes a cold water pump 41 and a hot water pump 51; the solenoid valve assembly comprises a solenoid valve I42 and a solenoid valve II 52.
The ultrasonic vibration device comprises an ultrasonic generator 72, an ultrasonic transducer 70 and an ultrasonic probe 71 which are connected in sequence; the ultrasonic generator 72 is connected as a driving power source to the ultrasonic transducer 70 and the ultrasonic probe 71. When the ultrasonic generator 72 emits an ultrasonic signal, the ultrasonic transducer 70 converts the ultrasonic signal into an electrical signal, and transmits the electrical signal to the ultrasonic probe 71, and the ultrasonic probe 71 vibrates with a specific amplitude.
The inner cavity I100 of the sterilization tank 1 is used for placing products to be sterilized; ultrasonic transducer 70 installs at retort 1 top, and ultrasonic probe 71 installs on ultrasonic transducer 70, and the sealed back that runs through retort 1 of the tip of ultrasonic probe 71 stretches into in its inner chamber I100, and in this embodiment, the tip of ultrasonic probe 71 is apart from about 1cm at the bottom of the inner chamber I100 of retort 1.
The sterilization tank 1 is provided with an air inlet 11 and an air outlet 12, the air inlet 11 is communicated with the nitrogen tank 3 through a pressure reducing valve 31, a pressure release valve 32 is arranged at the air outlet 12, and the air outlet 12 is communicated with the external environment through the pressure release valve 32.
In the actual use process, a worker injects high-purity nitrogen into the inner cavity I100 of the sterilization tank 1 by opening the nitrogen tank 3, adjusts the pressure to a required pressure through the pressure reducing valve 31, and opens the pressure release valve 32 to release the nitrogen after sterilization is completed (as shown by an arrow at the air outlet 12 in fig. 1, the nitrogen is discharged).
The sterilization tank 1 is also provided with a jacket 2, and the jacket 2 is sleeved on the outer surface of the sterilization tank 1 and is positioned below the air inlet 11 and the air outlet 12. The jacket 2 is sequentially provided with a water outlet 22 and a water inlet 21 from top to bottom; as shown by the thick solid line and the arrows on the thick solid line in fig. 1, the water inlet 21 is communicated with the cold water tank 4 through the cold water pump 41, and is also communicated with the hot water tank 5 through the hot water pump 51, that is, the cold water tank 4 is communicated with the inner cavity ii 101 of the jacket 2 through the cold water pump 41 and the water inlet 21 in sequence, and the hot water tank 5 is communicated with the inner cavity ii 101 of the jacket 2 through the hot water pump 51 and the water inlet 21 in sequence. The water outlet 22 is communicated with the cold water tank 4 through an electromagnetic valve I42 and is also communicated with the hot water tank 5 through an electromagnetic valve II 52.
The end part of the temperature probe 8 extends into the inner cavity I100 of the sterilization tank 1 and is immersed in a sample to be sterilized, and the temperature of the sample to be sterilized is detected in real time. The temperature probe 8 is connected with a temperature relay 6 through signals, and the temperature relay 6 is also electrically connected with the cold water pump 41, the hot water pump 51, the electromagnetic valve I42 and the electromagnetic valve II 52 (for the sake of the cleanness of the drawing, the connection of the temperature relay 6 with the hot water pump 51 and the electromagnetic valve II 52 is omitted in the figure 1).
In the actual working process, when the temperature probe detects that the temperature of the sample is higher than the set temperature, the temperature relay 6 sends a signal to turn on the cold water pump 41 (simultaneously turn on the electromagnetic valve I42), and the cooling water enters the inner cavity II 101 of the jacket 2 (the water in the inner cavity II 101 of the jacket 2 returns to the cold water tank 4 through the electromagnetic valve I42); when the temperature of the sample is lower than the set temperature, the temperature relay 6 turns on the hot water pump 51 (simultaneously turns on the solenoid valve II 52), and the heated water enters the cavity II 101 of the jacket 2 (the water in the cavity II 101 of the jacket 2 returns to the hot water pool 5 through the solenoid valve II 52).
Note: the heating water in this embodiment is 95 ℃ hot water, and in actual work, 85-100 ℃ hot water can be selected according to the heating temperature. The cooling water used in this example was water (25 ℃ C.).
The pressure thermoacoustic composite inactivated spore device is used for treating spores and comprises the following specific treatment steps:
the spore adopted by the invention is ATCC14579 Bacillus cereus of Qingdao Haibo biotechnology limited.
1. Heating treatment: a sample to be sterilized is put into the inner cavity I100 of the sterilization tank 1.
In this example, 25mL of sterile water was placed in the inner chamber I100 of the retort 1, and the preheating temperature was set to 80 ℃.
At the moment, the temperature relay 6 opens the hot water pump 51 (simultaneously opens the electromagnetic valve II 52), and the hot water in the hot water pool 5 enters the inner cavity II 101 of the jacket 2 through the hot water pump 51 for heating treatment (the water in the inner cavity II 101 of the jacket 2 returns to the hot water pool 5 through the electromagnetic valve II 52).
The temperature probe 8 in the inner cavity I100 of the sterilization tank 1 can detect the temperature of a sample to be sterilized in real time, and the temperature relay 6 can open corresponding electromagnetic valve assemblies and pump assemblies in real time when the temperature is higher than or lower than a set temperature. Therefore, the temperature of the inner cavity I100 of the sterilization tank 1 is kept within 80 +/-2 ℃ in the whole sterilization process (the signal transmission and the water bath still need a certain time, so the temperature still has certain fluctuation).
Note: in the actual working process, the product to be sterilized can be directly placed into the I100 inner cavity of the sterilization tank 1 for direct heating sterilization. In the embodiment, the spore suspension is directly heated to 80 ℃, and the spore is inactivated during heating, so that quantification is difficult. Therefore, the effect of the method can be more accurately quantified by adopting a preheating method, so that the influence of different parameters on the inactivation effect of the spores can be conveniently analyzed.
2. Pressurizing and ultrasonic processing: adding 2.0 × 10 of sterile water when the preheating temperature reaches 80 deg.C85mL of CFU/mL spore suspension. The retort 1 was closed, the nitrogen gas tank 3 was opened to increase the pressure in the inner chamber I100 of the retort 1 to a pressure of 500kPa (atmospheric pressure 100kPa, pressure of 400kPa was additionally increased), and sonication was performed with an amplitude of 100% (in the present example, amplitude of 114 μm) and a frequency of 20khz for 30 min.
Note: the heating, ultrasound and pressurization operations described above occur simultaneously.
The effect is better when the pressure is higher, but in the practical process, the higher the pressure is, the higher the requirement on the container (sterilization tank 1) is, and when the pressure is 450-550 kPa, the requirement of a common container is met, and the sterilization effect is better.
The higher the temperature is, the better the bactericidal effect is, but the sample and the instrument damage degree of treating the sterilization are great to too high temperature, and when the sterilization temperature was 75 ~ 85 ℃, can not treat promptly that the sterilization sample quality causes great influence, had better bactericidal effect again, compares with prior art, still reduces energy loss.
The longer the ultrasonic treatment is, the better the sterilization effect is, the comprehensive consideration of three treatment modes of temperature, pressure and ultrasonic is that the sterilization effect is obvious when ultrasonic treatment with the amplitude of 100% (114 microns) and the frequency of 20khz is carried out for 25-35 min at the temperature of 75-85 ℃ and the pressure of 450-550 kPa. Compared with the prior art that spore inactivation is realized through excessive heat treatment, the method can realize the inactivation of the spores without increasing the sterilization temperature or prolonging the sterilization time, can ensure the food safety and the food quality, and can also ensure that the sterilization temperature is low, the sterilization time is short, and the energy consumption is reduced.
3. And (3) after the step (2) is finished, stopping ultrasonic treatment, and opening the pressure release valve 32 to restore the normal pressure (100kPa) of the I100 in the inner cavity of the sterilization tank 1. At this time, the temperature is set to room temperature (25 ℃), the cold water pump 41 is turned on by a temperature relay transmitting a signal (the electromagnetic valve I42 is simultaneously turned on), and cooling water enters the inner cavity II 101 of the jacket 2 (the water in the inner cavity II 101 of the jacket 2 returns to the cold water tank 4 through the electromagnetic valve I42) to be cooled. Taking out after the temperature is cooled to room temperature (25 ℃).
Comparative example 1-1, the pressure of step 2 in example 1 was changed to 100kPa (normal pressure), i.e., only heat ultrasonic composite sterilization was performed; the rest is equivalent to embodiment 1.
Comparative examples 1-2, example 1 preheating temperature in step 1 was changed to 25 ℃ (room temperature), i.e., only pressure ultrasonic composite sterilization was performed; the rest is equivalent to embodiment 1.
Comparative examples 1-3, 25mL sterile Water Pre-heated to 80 deg.C and 2.0X 1085mL of CFU/mL spore suspension, uniformly mixing, and treating at 80 ℃ for 30min, namely, only carrying out heat sterilization.
Note: the enzyme can be inactivated by the heat treatment.
Comparative examples 1-4, 25mL sterile water preheating to 25 deg.C and 2.0 addition1085mL of CFU/mL spore suspension was sonicated for 30min at an amplitude of 100% and a frequency of 20khz, i.e., sonication alone was performed.
Note: the above-mentioned ultrasonic treatment can inactivate the enzyme part.
Comparative example 2-1, the treatment temperature in steps 1 and 2 of example 1 was changed to 60 ℃, and the rest was identical to example 1.
Comparative example 2-2, the ultrasonic duration in step 2 of example 1 was changed to 10min, and the rest was the same as example 1.
Experiment 1, spore inactivation determination:
0.1mL of the solution obtained after sterilization in example 1 was uniformly applied to a 20mLNA + 01% soluble starch medium, incubated at 37 ℃ for 24 hours, counted, and subjected to the same experiment with a blank (25mL of sterile water mixed with 5mL of spore suspension as a blank), and the specific data are shown in Table 1.
The solutions obtained in all the above comparative examples were subjected to the procedure of the experiment described in example 1, and the specific data are shown in table 1.
Table 1 plate technique colony counts units after different treatments: log (CFU/mL)
Colony count/log (CFU/mL)
Blank control 7.30±0.02
Example 1 4.18±0.03
Comparative examples 1 to 1 6.78±0.08
Comparative examples 1 to 2 7.00±0.02
Comparative examples 1 to 3 6.92±0.12
Comparative examples 1 to 4 7.18±0.03
Comparative example 2-1 6.25±0.08
Comparative examples 2 to 2 5.71±0.01
As can be seen from the data in Table 1, the inactivation effect of the independent ultrasonic treatment and the heat treatment which can cause enzyme inactivation on spores is not good, the inactivation effect of the ultrasonic combined pressure treatment on spores is not obvious, although the synergistic effect of the ultrasonic combined heat treatment on the spore inactivation is still not obvious, the ultrasonic combined heat treatment has a better synergistic effect on the spore inactivation after the combined action of the pressure and the heat, and the number of the inactivated spores after the treatment is greatly increased.
Experiment 2: and (3) spore ultrastructure determination:
the treated and control samples (25mL sterile water mixed with 5mL spore suspension as blank) were centrifuged at 8000r/min for 10min, and the pellet was washed 3 times with 0.85% physiological saline (centrifugation same as above). Secondly, fixing the sample by using 2.5 percent glutaraldehyde solution at 4 ℃ overnight, pouring out the fixing solution, and rinsing the sample by using 0.1mol/L phosphate buffer solution with pH of 7.0 for three times, 15min each time; after fixing the sample with 1% osmate solution for 1-2h, rinsing the sample with 0.1mol/L phosphate buffer solution pH7.0 three times for 15min each time; then, dehydrating the sample by using ethanol solutions with gradient concentrations (comprising six concentrations of 30%, 50%, 70%, 80%, 90% and 95%), treating for 15min at each concentration, treating for 20min by using 100% ethanol, and then transitioning to pure acetone treatment for 20 min; embedding the sample by an embedding medium, and heating the sample at 70 ℃ overnight to obtain the embedded sample. A sample is sliced in an LEICA EMUC7 type ultrathin slicer to obtain 70-90nm slices, and the slices are respectively dyed for 5-10min by a lead citrate solution and a uranyl acetate 50% ethanol saturated solution, so that the change of the spore ultrastructure after different treatments can be observed in a JEM-1230 transmission electron microscope.
As shown in figures 2-7, the ultrasonic treatment port can destroy spore ultrastructures, but the damage to the content and the shape of the spore ultrastructures after the pressure-heat-sound treatment is more obvious, and the spore content leaks as shown in figure 3.
Compared with the prior art, the method for treating the product to be sterilized by using the pressure thermoacoustic compounding method can greatly improve the inactivation efficiency of spores, reduce the influence on the food quality compared with the prior art and ensure the food safety. The invention can effectively sterilize without increasing the sterilization temperature or prolonging the sterilization time, thereby reducing the cost and reducing the energy consumption.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

Claims (2)

1. The thermoacoustic pressure composite spore inactivation method is characterized by comprising the following steps:
the pressure thermo-acoustic composite inactivated spore equipment comprises a sterilization tank (1), a jacket (2), a nitrogen tank (3), a cold water pool (4), a hot water pool (5) and an ultrasonic vibration device;
the ultrasonic vibration device comprises an ultrasonic generator (72), an ultrasonic transducer (70) and an ultrasonic probe (71) which are connected in sequence;
the sterilization tank (1) is communicated with the nitrogen tank (3);
the jacket (2) is sleeved on the outer surface of the sterilization tank (1), and the jacket (2) is respectively communicated with the cold water pool (4) and the hot water pool (5);
the ultrasonic transducer (70) is arranged at the top of the sterilization tank (1), the ultrasonic probe (71) is arranged on the ultrasonic transducer (70), and the end part arranged on the ultrasonic transducer penetrates through the sterilization tank (1) in a sealing manner and then extends into an inner cavity I (100) of the sterilization tank;
the thermoacoustic pressure composite inactivated spore equipment further comprises a pump assembly, an electromagnetic valve assembly, a temperature relay (6) and a temperature probe (8);
the temperature relay (6) is respectively connected with the pump assembly, the electromagnetic valve assembly and the temperature probe (8);
the end part of the temperature probe (8) extends into the inner cavity I (100);
the pump assembly comprises a cold water pump (41) and a hot water pump (51); the electromagnetic valve assembly comprises an electromagnetic valve I (42) and an electromagnetic valve II (52);
the cold water pump (41), the hot water pump (51), the electromagnetic valve I (42) and the electromagnetic valve II (52) are respectively connected with the temperature relay (6);
a water outlet (22) and a water inlet (21) are sequentially arranged on the jacket (2) from top to bottom;
the water inlet (21) is communicated with the cold water tank (4) through a cold water pump (41) and is also communicated with the hot water tank (5) through a hot water pump (51);
the water outlet (22) is communicated with the cold water tank (4) through an electromagnetic valve I (42) and is also communicated with the hot water tank (5) through an electromagnetic valve II (52);
the spore is ATCC14579 Bacillus cereus;
the thermoacoustic pressure composite inactivated spore method comprises the following steps:
1) and (3) heating treatment: putting a sample to be sterilized into an inner cavity I (100) of the sterilization tank (1);
placing 25mL of sterile water into an inner cavity I (100) of a sterilization tank (1), and setting the preheating temperature to 80 ℃;
at the moment, the temperature relay (6) opens the hot water pump (51) and simultaneously opens the electromagnetic valve II (52), hot water in the hot water tank (5) enters the inner cavity II (101) of the jacket (2) through the hot water pump (51) for heating treatment, and water in the inner cavity II (101) of the jacket (2) returns to the hot water tank (5) through the electromagnetic valve II (52);
the temperature probe (8) in the inner cavity I (100) of the sterilization tank (1) can detect the temperature of a sample to be sterilized in real time, and the temperature relay (6) can open the corresponding electromagnetic valve component and pump component in real time when the temperature is higher or lower than the set temperature; therefore, the temperature of the inner cavity I (100) of the sterilization tank (1) is kept within 80 +/-2 ℃ in the whole sterilization process;
2) and pressurizing and ultrasonic processing: adding 2.0 × 10 of sterile water when the preheating temperature reaches 80 deg.C85mL of CFU/mL spore suspension; closing the sterilization tank (1), opening the nitrogen tank (3) to increase the pressure in the inner cavity I (100) of the sterilization tank (1) to 500kPa, and carrying out ultrasonic treatment with the amplitude of 100% and the frequency of 20khz for 30 min;
3) after the step 2), stopping ultrasonic treatment, and opening a pressure release valve (32) to restore the normal pressure of an inner cavity I (100) of the sterilization tank (1); setting the temperature to room temperature, transmitting a signal by a temperature relay to turn on a cold water pump (41), simultaneously turning on an electromagnetic valve I (42), enabling cooling water to enter an inner cavity II (101) of the jacket (2), and returning the water in the inner cavity II (101) of the jacket (2) to a cold water pool (4) through the electromagnetic valve I (42) for cooling treatment; cooling to room temperature and taking out.
2. Thermo-acoustic composite inactivated spores method as defined in claim 1, characterized in that:
the sterilization tank (1) is provided with an air inlet (11) and an air outlet (12);
a pressure reducing valve (31) is arranged at the air inlet (11), and the air inlet (11) is communicated with the nitrogen tank (3) through the pressure reducing valve (31);
a pressure release valve (32) is arranged at the air outlet (12);
the jacket (2) is positioned below the air inlet (11) and the air outlet (12).
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