Plasma-activated lactic acid preparation and method for inhibiting bacillus

Abstract

The present application relates to a kind of plasma activated lactic acid preparation and its inhibiting bacillus method, belong to food microbiological control technical field, to solve the microbial contamination and prevention and control problem in the process of food processing and storage.Specifically, the present application discloses a kind of preparation method of plasma activated lactic acid, by plasma activation technology reduces the addition amount of lactic acid while inhibiting the growth of a variety of thermophilic bacillus, and plays the effect of killing spore.The present application provides a kind of green safe, simple operation technical means for the prevention and control of food microbial contamination, has important practical application value and broad market prospect.

Description

Technical Field

[0001] This invention belongs to the field of food microbial control technology, specifically relating to a method for preparing lactic acid using plasma activation and inhibiting Bacillus. Background Technology

[0002] Bacillus species include various foodborne spoilage and pathogenic bacteria, such as Bacillus subtilis and Bacillus cereus, which are widely found in fruits, vegetables, fish, meat, grains, and dairy products, causing food spoilage and even food poisoning. Bacillus is difficult to eradicate primarily because it forms spores under adverse conditions. Spores exhibit strong resistance and survival ability to high temperatures, ultraviolet light, chemical preservatives, and pH levels, making them difficult to completely eliminate using conventional sterilization methods. Given suitable conditions, they germinate and multiply, causing food spoilage and posing a significant challenge to the food industry. Traditional methods for inhibiting spores include high-temperature sterilization and chemical disinfection, but these methods suffer from problems such as affecting food quality, high energy consumption, and chemical residues. With increasing awareness of green and healthy consumption, these traditional methods can no longer meet current market demands. Developing green and natural antibacterial methods aligns with national policies, market demands, and environmental requirements.

[0003] Lactic acid, as a natural antibacterial agent, is highly safe and readily available, and has been widely used in the food industry. Lactic acid increases cell membrane permeability, leading to leakage of intracellular substances and affecting bacterial activity and survival. It plays a role in pH regulation, antibacterial action, preservation, flavoring, and maintaining food color. However, excessive amounts of lactic acid can negatively impact food flavor. Therefore, employing green physical field technology to enhance the inhibitory effect of lactic acid on spores, allowing for sufficient inhibition of spore growth with small amounts of lactic acid, is of great significance to the food processing industry. In recent years, plasma technology has received widespread attention due to its highly efficient antibacterial capabilities and environmental friendliness. Its antibacterial activity is the result of the combined effects of active substances, acidic pH, and charged particles, making it a green, safe, and effective sterilization and preservation technology. Currently, plasma treatment mainly involves deionized water, phosphate buffer, and hydrogen peroxide solution. This study aims to establish a green and efficient antibacterial method by treating lactic acid solutions with plasma.

[0004] A search revealed three patents closely related to this patent: The domestic invention patent "A plasma-activated lactic acid solution with high-efficiency antibacterial properties and its preparation method and application" (CN201910196952.8) discloses a plasma-activated lactic acid solution prepared by treating 300 mL of lactic acid solution (0.05%, 0.10%, 0.15%, and 0.20%) with a plasma spray gun for 80 seconds. The preparation parameters for the plasma-activated lactic acid are different, and it does not involve the control of foodborne spoilage bacteria or pathogenic bacteria of the Bacillus genus. The domestic invention patent "A method for temperature-assisted plasma combined with sterilization of Bacillus cereus spores" (CN202311697443.6) discloses a method for temperature-assisted plasma combined with sterilization, but it does not involve the synergistic application of lactic acid and plasma technology. The domestic invention patent "A method for plasma-activated water and inhibition and removal of Bacillus biofilm" discloses a method for plasma-activated water to inhibit Bacillus biofilm, but it does not involve the synergistic application of lactic acid and plasma technology. The method for preparing lactic acid and inhibiting Bacillus by plasma activation involved in this invention has not been reported before.

[0005] Based on this, the present invention provides a new technology for inhibiting Bacillus by activating lactic acid with plasma, which not only broadens the application scope of organic acids and plasma technology, but also provides a new solution for the prevention and control of Bacillus contamination. Summary of the Invention

[0006] Technical issues

[0007] Conventional food processing methods are insufficient to completely eliminate Bacillus contamination, and spore reactivation can still lead to food spoilage. This invention addresses the problem of Bacillus contamination in food processing by providing a green, safe, efficient, convenient, and economical method for activating lactic acid and inhibiting Bacillus spores using plasma. This method inhibits the growth of Bacillus, reduces its survival rate, and broadens the application scope of lactic acid and plasma technologies. It represents a new technology for microbial control.

[0008] Technical solution

[0009] The objective of this invention can be achieved through the following technical solutions:

[0010] A method for preparing plasma-activated lactic acid solution involves using an atmospheric pressure cold plasma jet generator with air as the discharge gas, an output voltage of 220V, a gas flow rate of 20-30L / min, and a probe extending 1-10cm below the surface of the lactic acid solution. The plasma-activated lactic acid is obtained by discharging for 10-20min.

[0011] Furthermore, the volume of the lactic acid solution is 300-1000 mL, and the concentration is 0.2-2.0% (w / v); preferably, the concentration is 0.6-1.0% (w / v).

[0012] Furthermore, this application provides plasma-activated lactic acid prepared using the above method.

[0013] Furthermore, this application provides a method for inhibiting Bacillus spores with plasma-activated lactic acid, wherein the plasma-activated lactic acid prepared above is contacted with Bacillus spores for 4-24 hours.

[0014] Furthermore, the Bacillus is one or more of the common spore-forming putrefactive bacteria such as Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, Bacillus lysine, Bacillus spp., or Bacillus thiamine.

[0015] Beneficial effects

[0016] (1) This invention successfully developed an innovative plasma-activated lactic acid (PALA). Experimental results show that PALA has more stable physicochemical properties than plasma-activated water (PAW) and is more capable of generating reactive oxygen and nitrogen substances with antibacterial effects. Figure 1 ).

[0017] (2) The present invention successfully prepared a plasma-activated lactic acid (PALA). The method is simple to operate, low in cost, has little environmental pollution, and is easy to industrialize.

[0018] (3) This invention improves the inhibitory effect of lactic acid on Bacillus spores through plasma technology, thereby reducing the amount of lactic acid used. Experimental results show that the lactic acid concentration required to inhibit spore growth is 0.80-1.50%, and the required lactic acid concentration after plasma activation is 0.65-1.00%. Figure 2 ).

[0019] (4) The PALA of the present invention can not only effectively inhibit the growth of various Bacillus species, but also show significant inhibition and elimination effects on typical Bacillus spores in two kinds of food. It provides a brand-new solution for the prevention and control of microbial contamination in the food processing field, and has important practical application value and broad market prospects.

[0020] In summary, the PALA of this invention has significant scientific value and broad application prospects, and is of great importance for disinfection and cleaning in food production lines. Attached Figure Description

[0021] Figure 1 Physicochemical properties of plasma-activated lactic acid

[0022] Figure 2Lactic acid and plasma-activated lactic acid: minimum concentration to inhibit spore growth

[0023] Figure 3 The decrease in the total number of Bacillus subtilis spores in different treatment groups

[0024] Figure 4 The decrease in the total number of Bacillus cereus spores in different treatment groups Detailed Implementation

[0025] The present invention will be further described below with reference to embodiments, but these are not intended to limit the scope of the invention.

[0026] Unless otherwise specified, all materials, reagents, and instruments used in the embodiments of this invention are commercially available. Experimental methods not specifically specified in the embodiments are generally performed according to conventional methods skilled in the art.

[0027] Example 1

[0028] Prepare 1000 mL of lactic acid solutions of different concentrations (0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.5%, and 2.0% w / v). Treat the solutions using an atmospheric pressure cold plasma jet generator (PG-1000Z / D), with air as the discharge gas, an operating voltage of 220 V, a gas flow rate of 30 L / min, and the probe extended 10 cm below the liquid surface for 10 min. Next, prepare nutrient broth using the above-mentioned different concentrations of PALA. Transfer 180 μL of each broth to a 96-well plate, add 20 μL (5-6 log CFU / mL) of spore suspension to each well, and incubate at 30 °C for 30 days. Each experimental condition has at least six replicates. Spore growth is assessed by monitoring changes in OD600 values. The spore suspension was prepared from Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, Bacillus spp., Bacillus lysineus, or Bacillus thiamineus cultured to the logarithmic phase.

[0029] Example 2

[0030] Prepare 300 mL of lactic acid solutions of different concentrations (0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.5%, and 2.0% w / v). Treat the solutions using an atmospheric pressure cold plasma jet generator (PG-1000Z / D), with air as the discharge gas, an operating voltage of 220 V, a gas flow rate of 30 L / min, and the probe extended 10 cm below the liquid surface for 20 min. Next, prepare nutrient broth using the above-mentioned different concentrations of PALA. Transfer 180 μL of each broth to a 96-well plate, and add 20 μL (5-6 log CFU / mL) of spore suspension to each well. Incubate at 30°C for 30 days, with at least 6 replicates for each experimental condition. Spore growth is assessed by monitoring changes in OD600 values. The spore suspension is prepared from Bacillus subtilis or Bacillus cereus cultured to the logarithmic growth phase. The spores were suspended in 0.5% PALA and 1% PALA and exposed at 30°C for 4, 8, 12 and 24 h. The killing effect of plasma-activated lactic acid on Bacillus subtilis and Bacillus cereus spores was analyzed by plate counting. Plasma-activated water, 0.5% lactic acid solution and 1% lactic acid solution were used as control groups.

[0031] The above description only details the embodiments of the present invention, but the present invention is not limited to the specific embodiments shown herein. Those skilled in the art can make various modifications and improvements to the embodiments without departing from the scope defined by the claims, and all such modifications and improvements should fall within the scope of protection of the present invention.

Claims

1. A method for preparing plasma-activated lactic acid solution, wherein the solution is prepared by an atmospheric pressure cold plasma jet generator, using air as the discharge gas, with an output voltage of 220V and a gas flow rate of 20-30L / min, and the probe is extended to 1-10cm below the surface of the lactic acid solution, and the plasma-activated lactic acid is obtained by discharging for 10-20min.

2. The lactic acid solution of claim 1 has a volume of 300-1000 mL and a concentration of 0.2-2.0% (w / v); preferably, the concentration is 0.6-1.0% (w / v).

3. A plasma-activated lactic acid, characterized in that, It is prepared by the method described in claim 1 or 2.

4. A method for inhibiting and eliminating Bacillus spores, characterized in that, The plasma-activated lactic acid described in claim 3 is contacted with Bacillus spores for 4-24 hours. The Bacillus spores are one or more common spore-forming putrefactive bacteria, such as Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, Bacillus lysine, Bacillus spp., or Bacillus thiamine.

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