Method for reducing resistance of ultrahigh pressure deep dormant spores

Abstract

The invention provides a method for reducing resistance of ultrahigh-pressure deep dormant spores, which comprises the following steps: performing ultrahigh-pressure treatment on dormant spores to obtain ultrahigh-pressure treated substances; carrying out standing treatment on the ultrahigh pressure treatment substance to obtain a standing treatment substance; separating spores which are not germinated in the standing treatment substance to obtain ultrahigh-pressure deep dormancy spores; the temperature of the ultrahigh pressure treatment is 37-45 DEG C, and the pressure of the ultrahigh pressure treatment is 400-600 MPa; the standing treatment time is 1 to 3 hours. According to the method, ultrahigh pressure and standing treatment are combined, the ultrahigh pressure deep dormancy spore resistance can be effectively reduced, spore inactivation is better facilitated, and the method has the advantages of being easy and convenient to operate, short in consumed time, high in efficiency, low in cost and the like and is suitable for application and popularization.

Description

Technical Field

[0001] This application relates to the field of biology. Specifically, this application relates to a method for reducing the resistance of ultra-high pressure deep dormant spores. Background Technology

[0002] Spores are dormant forms of bacilli and clostridium bacteria that develop under nutrient-deficient conditions. They exhibit strong resistance to external adversities (such as heat, radiation, and compounds) and are difficult to kill through conventional food sterilization processes. Currently, the germination-inactivation strategy is an effective way to kill spores. This involves first inducing spore germination to eliminate their resistance, and then using a gentle method to inactivate the germinating spores.

[0003] Under the induction of germination factors, some spores germinate slowly or not at all; these spores that germinate slowly or not at all are called super dormant spores. Dormant (SD) spores. High hydrostatic pressure (HHP) technology can significantly induce spore germination, but deep dormant spores that germinate slowly or not at all under HHP still exist, namely high hydrostatic pressure super-dormant (HPSD) spores. The existence of HPSD spores hinders the implementation of HHP-based germination-inactivation strategies in food processing. Summary of the Invention

[0004] This application aims to at least partially address the technical problems existing in the prior art. To this end, this application proposes methods for reducing the resistance of ultra-high pressure deep-dormant spores and methods for improving the inactivation efficiency of ultra-high pressure deep-dormant spores. The methods of this application, by combining ultra-high pressure with static treatment, can effectively reduce the resistance of ultra-high pressure deep-dormant spores, thus making spore inactivation more effective. These methods have advantages such as simple operation, short processing time, high efficiency, and low cost, and are suitable for widespread application.

[0005] In one aspect of this application, a method for reducing the resistance of ultra-high pressure dormant spores is proposed, characterized by comprising: subjecting dormant spores to ultra-high pressure treatment to obtain an ultra-high pressure treated material; subjecting the ultra-high pressure treated material to static treatment to obtain a static treated material; separating ungerminated spores from the static treated material to obtain ultra-high pressure dormant spores; wherein the ultra-high pressure treatment temperature is 37℃~45℃ and the pressure is 400 MPa~600 MPa; and the static treatment time is 1 h~3 h.

[0006] The application carries out the standing treatment after the ultra-high pressure treatment, which can significantly reduce the resistance of the ultra-high pressure deep dormant spores in the spores treated by the ultra-high pressure treatment. Further, the ultra-high pressure treatment temperature and the standing treatment time can significantly affect the resistance of the ultra-high pressure deep dormant spores, and the resistance of the ultra-high pressure deep dormant spores is reduced by using the above-mentioned ultra-high pressure treatment temperature and standing treatment time, thereby being more conducive to the inactivation of the spores.

[0007] In some embodiments, the temperature of the ultra-high pressure treatment can be 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, 45℃. The time of the standing treatment can be 1 h, 1.5 h, 2 h, 2.5 h, 3 h.

[0008] According to the embodiments of the application, the temperature of the standing treatment is 30℃-40℃. In some embodiments, the temperature of the standing treatment is 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃. In this way, the resistance of the ultra-high pressure deep dormant spores can be further reduced.

[0009] According to the embodiments of the application, the pressure of the ultra-high pressure treatment is 400 MPa-600 MPa, and the time is 3 min-5 min. In some embodiments, the pressure of the ultra-high pressure treatment is 400 MPa, 420 MPa, 440 MPa, 450 MPa, 460 MPa, 480 MPa, 500 MPa, 520 MPa, 540 MPa, 550 MPa, 560 MPa, 580 MPa, 600 MPa; and the time is 3 min, 3.5 min, 4 min, 4.5 min, 5 min. In this way, the resistance of the ultra-high pressure deep dormant spores can be further reduced.

[0010] According to the embodiments of the application, the standing treatment is directly carried out after the ultra-high pressure treatment. After the ultra-high pressure treatment, no other steps such as adding other chemical substances are needed, and the standing treatment is directly carried out, which can better reduce the resistance of the ultra-high pressure deep dormant spores.

[0011] According to the embodiments of the application, no additional chemical substances are added in the method. Exemplarily, the chemical substance can be a chemical substance for promoting germination.

[0012] According to the embodiments of the application, the spores are Bacillus subtilis or Bacillus cereus.

[0013] According to the embodiments of the application, the spores are derived from food.

[0014] In another aspect of the present application, the present application provides a method for improving the inactivation efficiency of ultra-high pressure deep-dormant spores. According to an embodiment of the present application, the method comprises: treating spores by the method for reducing the resistance of ultra-high pressure deep-dormant spores described above to obtain ultra-high pressure deep-dormant spores; and performing inactivation treatment on the pretreated spores. Thus, the ultra-high pressure deep-dormant spores obtained by the method described above have low resistance, and can be killed by inactivation treatment, thereby achieving higher inactivation efficiency of spores, and the inactivation method is simple, low in cost, and suitable for large-scale promotion.

[0015] According to an embodiment of the present application, the inactivation treatment comprises moist heat treatment, hydrochloric acid treatment, sodium hypochlorite treatment, or formaldehyde treatment. Thus, the inactivation effect of spores is better.

[0016] According to an embodiment of the present application, no additional chemical substance is added in the method. Exemplarily, the chemical substance can be a chemical substance for promoting germination or a chemical substance for promoting inactivation.

[0017] According to an embodiment of the present application, the temperature of the moist heat treatment is 90-95°C, and the time is 60-100 min. In some embodiments, the temperature of the moist heat treatment is 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C, and the time is 60 min, 65 min, 70 min, 75 min, 80 min, 85 min, 90 min, 95 min, or 100 min. Thus, the ultra-high pressure deep-dormant spores can be efficiently killed.

[0018] According to an embodiment of the present application, the concentration of hydrochloric acid in the hydrochloric acid treatment is 200-240 mM, the temperature is 20-25°C, and the time is 10-20 min. In some embodiments, the concentration of hydrochloric acid in the hydrochloric acid treatment is 200 mM, 210 mM, 220 mM, 230 mM, or 240 mM, the temperature is 20°C, 21°C, 22°C, 23°C, 24°C, or 25°C, and the time is 10 min, 12 min, 14 min, 15 min, 16 min, 18 min, or 20 min. Thus, the ultra-high pressure deep-dormant spores can be efficiently killed.

[0019] According to the embodiments of the present application, the sodium hypochlorite concentration of the sodium hypochlorite treatment is 0.1 mM-0.5 mM, the temperature is 20°C-25°C, and the time is 20 min-40 min. In some embodiments, the sodium hypochlorite concentration of the sodium hypochlorite treatment is 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, the temperature is 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, and the time is 20 min, 25 min, 30 min, 35 min, 40 min. In this way, the ultra-high pressure deep-dormant spores can be efficiently killed.

[0020] According to the embodiments of the present application, the formaldehyde concentration of the formaldehyde treatment is 0.5 mM-1 mM, the temperature is 25°C-35°C, and the time is 40 min-80 min. In some embodiments, the formaldehyde concentration of the formaldehyde treatment is 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, the temperature is 25°C, 26°C, 28°C, 30°C, 32°C, 34°C, 35°C, and the time is 40 min, 45 min, 50 min, 55 min, 60 min, 65 min, 70 min, 75 min, 80 min. In this way, the ultra-high pressure deep-dormant spores can be efficiently killed.

[0021] Advantages (1) The method of ultra-high pressure and standing is first proposed, which significantly reduces the resistance of ultra-high pressure deep-dormant spores. The content of Bacillus subtilis HPSD spores after standing is reduced to one-tenth of that of the traditional method.

[0022] (2) The ultra-high pressure and standing conditions are first optimized. The treatment of 500 MPa / 37°C for 4 min and standing at 37°C for 2 h can significantly reduce the HPSD spores. The HPSD spores of Bacillus subtilis after standing can achieve a killing effect of about 6-7 logs after heat treatment, and the HPSD spores of Bacillus cereus can be completely inactivated.

[0023] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0024] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description of embodiments, taken in conjunction with the following drawings in which: Figure 1 The content analysis diagram of deep-dormant spores after ultra-high pressure and standing treatment is shown; Figure 2Graphs showing the effect of ultra-high pressure treatment temperature and treatment time on the number of B. subtilis HPSD spores (A-C) and B. cereus HPSD spores (D-F); Figure 3 Graphs showing the resistance properties of HPSD spores to (A) heat and moisture (93°C), (B) 220 mM hydrochloric acid (23°C), (C) 0.34 M sodium hypochlorite (23°C) and (D) 0.83 M formaldehyde (30°C); Figure 4 Graphs showing the effect of standing time on the number of B. subtilis HPSD spores (A) and B. cereus HPSD spores (B); Figure 5 Graphs showing the effect of standing treatment on the number of B. subtilis and B. cereus HPSD spores in skimmed milk (A) and vegetable juice (B) systems. DETAILED DESCRIPTION

[0025] The solutions of the present application will be explained hereinafter in connection with embodiments. Those skilled in the art will appreciate that the following embodiments are merely illustrative of the present application and should not be viewed as limiting the scope of the present application. Unless specific techniques or conditions are mentioned, techniques or conditions described in the literature or product specifications are used. When the manufacturer of a product is not mentioned, a conventional product available from the market is used.

[0026] Example 1 In this example, B. subtilis spores were treated according to the following method: Reference Figure 1 A sterile water system containing 10 11~12 CFU of B. subtilis spores was divided into two groups for the following experiments: Experimental group: The dormant spore sample was treated with ultra-high pressure at 500 MPa / 20°C for 5 min, then subjected to standing treatment at 37°C for 12 h, and the non-germinated ultra-high pressure deep-dormant spores were isolated and named HP 5min +IC 12h -SD spores.

[0027] Control group: The dormant spore sample was treated with ultra-high pressure at 500 MPa / 20°C for 5 min, and then the non-germinated ultra-high pressure deep-dormant spores were directly isolated and named HP 5min -SD spores.

[0028] The separation method is as follows: after the ultra-high pressure treatment, the spores are centrifuged and washed with 4°C sterile water for 2-3 times, and then suspended in 20% Nycodenz. Part of the suspension is placed on 50% Nycodenz, centrifuged at 20627xg for 20 min, so that the HPSD spores are precipitated and the germinated spores are floated. Then the HPSD spores are washed with 4°C sterile water to remove Nycodenz, and subjected to two Nycodenz gradient centrifugations to obtain high-purity HPSD spores. Finally, the HPSD spores are suspended in sterile water and stored at -80°C for subsequent analysis. Figure 1 The light and dark spores after the ultra-high pressure treatment are slow-germinating deep-dormant spores and germinated spores, respectively.

[0029] The results show that after 500 MPa treatment at 20°C for 5 minutes, about 40.23% of the spores germinate. The proportion of HPSD spores is calculated by [OD 600 HPSD芽孢 ] / [ OD 600初始休眠芽孢 ]x100%. Correspondingly, the separation rate of HP 5 min -SD spores is about 29.67%, while the separation rate of HP 5 min +IC 12 h -SD spores is only about one-tenth of that of HP 5 min -SD spores.

[0030] Example 2 In this example, the B. subtilis spores and B. cereus spores are treated according to the following method: Reference Figure 2 , 10 8~9 CFU / ml of B. subtilis spores / B. cereus spores in sterile water is treated at a pressure of 500 MPa, a temperature of 20°C-45°C for 1 min-7 min, and then incubated at 37°C for 12 h, followed by further heat treatment at 80°C / 30 min.

[0031] The results are shown in Figure 2 , which shows that under the condition of 500 MPa / 37°C treatment for 4 min, there is no significant difference between the results of increasing the treatment temperature and time and 500 MPa / 37°C treatment for 4 min. After heat treatment under this condition, the survival rate of B. subtilis spores is reduced to 0.002%, and B. cereus spores can be completely inactivated.

[0032] Example 3 The ultra-high pressure deep-dormant spores obtained from the experimental group and the control group of Example 1 are respectively treated with moist heat (93°C), 220 mM hydrochloric acid (23°C), 0.34 M sodium hypochlorite (23°C), and 0.83 M formaldehyde (30°C)Figure 3 The results showed that HP 5min Compared to SD spores, HP spores separated by ultra-high pressure followed by settling... 5min +IC 12h - SD spores showed significantly reduced resistance to the four treatments (P < 0.05). This method reduced the resistance of HPSD spores to moist heat, hydrochloric acid, sodium hypochlorite, and formaldehyde by 1.58, 1.22, 1.22, and 1.34 times, respectively (Table 1), with moist heat showing the best effect.

[0033] Table 1. Time required (D value) to kill 90% of spores using different treatment methods

[0034] Example 4 In this embodiment, Bacillus subtilis spores and Bacillus cereus spores are treated respectively according to the following methods: refer to Figure 3 , will contain 10 8~9 The sterile water system of CFU Bacillus subtilis spores / Bacillus cereus spores was treated at 500 MPa / 37℃ for 4 min, then allowed to stand at 37℃ for 0 h, 1 h, 2 h, 4 h, 7 h, and 12 h, and subsequently subjected to heat treatment at 80℃ / 30 min.

[0035] The results are as follows Figure 4 As shown, a static treatment of 2 hours, and further extending the time, did not significantly improve the inactivation efficiency of spores. Under these conditions, the survival rate of Bacillus subtilis spores decreased to 0.00049% after heat treatment, while the survival rate of Bacillus cereus spores decreased to 0.003%.

[0036] Example 5 Skim milk containing Bacillus subtilis spores and Bacillus cereus spores, respectively, and celery and cucumber compound vegetable juice were treated at 500 MPa / 37℃ for 4 min, then allowed to stand at 37℃ for 2 h, and subsequently subjected to heat treatment at 80℃ / 30 min.

[0037] The results are as follows Figure 5 As shown, this method can reduce the survival rate of Bacillus subtilis spores and Bacillus cereus spores in skim milk to 0.0037% and 0.14%, respectively. Simultaneously, this method can also reduce the survival rate of Bacillus subtilis spores in vegetable juice to 2.5 × 10⁻⁶. -5 %, and inactivate all Bacillus cereus spores in the system.

[0038] The endpoints of the ranges and any values disclosed herein are not limited to the precise values recited as exactly that endpoint. Any values that are between two herein- disclosed values are considered as being included in this range, even if not explicitly listed. These are only examples of what is provided by this application, and in light thereof changes in form and detail can be made without departing from the true scope of the application.

[0039] In this document, the terms "comprise" or "comprising" are open- ended, that is, they mean "including, but not limited to".

[0040] Although the embodiments of the present application have been shown and described above, it should be understood by those having ordinary skill in the art that such embodiments are presented by way of example only, and not limitation, and that a variety of changes in the shape, size, materials, and components etc. can be made to the above-described embodiments without departing from the scope of the present application.

Claims

1. A method of reducing the resistance of an ultra-high pressure deep-dormant spore, characterized in that, The application relates to a method for reducing the resistance of deep-dormant spores to ultra-high pressure, and a method for inactivating deep-dormant spores. The method comprises the following steps: The method comprises the following steps: The method comprises the following steps: The temperature of the ultra-high pressure treatment is 37 DEG C-45 DEG C, and the pressure is 400 MPa-600 MPa. The time of the static treatment is 1 h-3 h.

2. The method of claim 1, wherein, The temperature of the static treatment is 30 DEG C-40 DEG C.

3. The method of claim 1, wherein, The time of the ultra-high pressure treatment is 3 min-5 min.

4. The method of claim 1, wherein, The static treatment is directly performed after the ultra-high pressure treatment.

5. The method of claim 1, wherein, The spores are Bacillus subtilis or Bacillus cereus. The spores are derived from food.

6. A method for improving the inactivation efficiency of ultra-high pressure deep-dormant spores, characterized by, The application relates to a method for reducing the resistance of deep-dormant spores to ultra-high pressure, and a method for inactivating deep-dormant spores. The method comprises the following steps: The inactivation treatment comprises moist heat treatment, hydrochloric acid treatment, sodium hypochlorite treatment or formaldehyde treatment.

7. The method of claim 6, wherein, The temperature of the moist heat treatment is 90 DEG C-95 DEG C, and the time is 60 min-100 min.

8. The method of claim 7, wherein, The concentration of the hydrochloric acid in the hydrochloric acid treatment is 200 mM-240 mM, the temperature is 20 DEG C-25 DEG C, and the time is 10 min-20 min. The concentration of the sodium hypochlorite in the sodium hypochlorite treatment is 0.1 mM-0.5 mM, the temperature is 20 DEG C-25 DEG C, and the time is 20 min-40 min. The concentration of the formaldehyde in the formaldehyde treatment is 0.5 mM-1 mM, the temperature is 25 DEG C-35 DEG C, and the time is 40 min-80 min. ​

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