Stable, efficient and environment-friendly silver nanoparticle lactic acid bacteria biosynthesis method and application

Abstract

The invention discloses a stable, efficient and environment-friendly silver nanoparticle lactic acid bacteria biosynthesis method and application, firstly, a nitrate reduction test is carried out to determine the ability of strains to reduce nitrate into nitrite, and optimized strains are screened from six lactic acid bacteria; then, a response surface method is used for optimizing culture medium components for nitrate reductase production, and synthesis conditions are optimized; and synthesizing the silver nanoparticles by using the screened optimized strains. Compared with physical and chemical methods, the lactobacillus synthesis of AgNPs has the advantages of cost effectiveness, environmental friendliness, low toxicity to human cells, easiness in large-scale production, no need of dangerous chemicals, high temperature and high pressure and the like. The invention provides an efficient, stable and environment-friendly method for biologically synthesizing silver nanoparticles by using lactic acid bacteria, the improvement of antibacterial packaging materials is a key strategy and method for controlling microorganisms of chilled meat putrefaction in China, and powerful technical support is provided for scientifically, systematically, efficiently and practically guaranteeing meat safety.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial application, and specifically relates to a stable, efficient and environmentally friendly silver nanoparticle lactic acid bacteria biosynthesis method and application. Background Art

[0002] Silver nanoparticles (AgNPs) have been identified as one of the most promising nanoparticles for biomedical and biological applications due to their unique physical and chemical properties, including antibacterial, anticancer, antiviral and drug delivery. In recent years, silver nanoparticles have attracted much attention due to their strong bactericidal ability. Silver ions can bind to bacterial proteins and DNA, preventing cell metabolism and division, thereby achieving a bactericidal effect. In addition, silver ions can also destroy the cell walls of viruses and fungi to prevent the spread of infection. However, traditional synthesis methods have high production costs and produce hazardous wastes that cause environmental pollution. Therefore, the process of using microorganisms to synthesize AgNPs as an alternative to conventional methods has received considerable attention. Lactic acid bacteria are Gram-positive non-pathogenic bacteria that are widely present in milk and fermented foods. They also have health-promoting probiotic properties and can be safely consumed by humans and animals. Studies have shown that lactic acid bacteria are a promising reducing agent for the production of AgNPs. The use of lactic acid bacteria supernatant as a reducing agent for the synthesis of AgNPs has been successful. By treating bacterial supernatant, downstream processing in the AgNPs synthesis process will become more direct and easy. In various studies on the production of AgNPs, lactic acid bacteria have been used as reducing agents and capping agents. However, in-depth studies on the role of lactic acid bacteria nitrate reductase have not been reported. At present, there are few studies on the biosynthesis of AgNPs using lactic acid bacteria. The process has disadvantages such as complex purification, low conversion efficiency, unstable yield, high energy consumption, and high cost. The potential of lactic acid bacteria cells as nanofactories needs further study and optimization. Therefore, more research is needed to optimize the reaction conditions and improve the yield and rate of nanoparticle biosynthesis. Summary of the invention

[0003] In view of the problems existing in the prior art, the present invention aims to establish a stable, efficient and environmentally friendly method for biosynthesis of silver nanoparticles by lactic acid bacteria, and to improve the composite antibacterial packaging for application in the preservation of fresh chilled meat to improve food safety.

[0004] The present invention conducts a nitrate reduction test to determine the ability of the strain to reduce nitrate to nitrite, screens out the optimized strain from 6 lactic acid bacteria, uses the response surface method to optimize the culture medium components for nitrate reductase production, and optimizes the synthesis conditions; lactic acid bacteria have a variety of metabolites that can reduce Ag ions to AgNPs, and can also cover and prevent agglomeration to reduce toxicity. Finally, a stable, efficient, and environmentally friendly lactic acid bacteria AgNPs biosynthesis method is developed.

[0005] Nanoparticles of different types and shapes used in the food industry must understand their interactions with food matrices and microorganisms. This invention characterizes AgNPs synthesized by lactic acid bacteria and evaluates their antibacterial activity against pathogenic and spoilage bacteria and their application value in food packaging. A research method is developed to clarify the antibacterial mechanism, and an in-depth study is conducted on whether AgNPs have an inhibitory or bactericidal effect on pathogenic and spoilage bacteria, the interaction mechanism that causes bacterial cell membrane rupture and cell lytic protein leakage is analyzed, and further research is conducted on methods for new antibacterial food packaging materials with composite AgNPs.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A stable, efficient and environmentally friendly method for biosynthesizing silver nanoparticles by lactic acid bacteria, comprising the following steps:

[0008] (1) conducting a nitrate reduction test to determine the ability of the strain to reduce nitrate to nitrite, and selecting the optimized strain from the six lactic acid bacteria strains;

[0009] (2) Optimize the medium composition for nitrate reductase production and optimize the synthesis conditions using response surface methodology;

[0010] (3) Synthesizing silver nanoparticles using the optimized strain screened in step (1).

[0011] Furthermore, in step (1), the screening method for optimizing the strain is as follows:

[0012] The nitrate reductase activity of 6 lactic acid bacteria strains was detected, and the blank group was used as a control. The nitrate reduction test was performed to determine the ability of microorganisms to reduce nitrate to nitrite. Lactic acid bacteria were cultured in MRS-nitrate medium at 37°C for 24h, and 100μL nitrate reagent A and 100μL nitrate reagent B were added to the culture in sequence, and the color changes that occurred were recorded;

[0013] The lactic acid bacteria strain with nitrate reductase activity was grown in MRS-nitrate medium at 37°C for 24 hours, and after shaking, 1.5 mL of the culture was extracted into a centrifuge tube with a pipette, and centrifuged at 10000 rpm for 20 minutes at 4°C. The supernatant was filtered using a 0.45 μm membrane filter, and the obtained cell-free supernatant was used as a crude enzyme. 1.8 mL of nitrate reductase substrate solution was added to 100 μL of crude enzyme, and then 100 μL of 2.0 mM β-nicotinamide adenine dinucleotide reducing solution (β-NADH) was added for reaction; the reaction mixture was incubated at 30°C without light for 1 hour, and then 1 mL of 58 mM sulfonamide solution was added, and then 1 mL of 0.77 mM N-(1-naphthyl)ethylenediamine dihydrochloride (NED) solution was added to terminate the reaction, and the solution was kept at 25°C for 20 minutes to obtain a lavender solution, and the absorbance of the solution was measured at a wavelength of 540 nm using a UV-visible spectrophotometer and recorded;

[0014] The six lactic acid bacteria strains are Lactobacillus plantarum, Lactococcus lactis subspecies, a mixture of Lactobacillus delbrueckii and Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus and Lactobacillus casei.

[0015] Further, the preparation method of nitrate reagent A is as follows: to prepare 250 mL of sulfanilic acid solution, 1.25 g of sulfanilamide should be weighed and added to a 250 mL volumetric flask containing an appropriate amount of 5N acetic acid, 5N acetic acid is added to make up the volume, the stopper is covered and shaken thoroughly.

[0016] Further, the preparation method of nitrate reagent B (N, N-dimethyl-1-naphthylamine solution) is as follows: to prepare 250 mL of N, N-dimethyl-1-naphthylamine solution, 1.5 mL of sulfonamide is needed, added to a 250 mL volumetric flask containing an appropriate amount of 5N acetic acid, 5N acetic acid is added to make up the volume, the stopper is covered and the solution is shaken thoroughly.

[0017] Furthermore, the preparation method of nitrate reductase substrate solution is as follows: prepare 500 mL of nitrate reductase substrate solution, weigh 2.65 g of potassium phosphate, 0.5 g of potassium nitrate, and 0.0043 g of ethylenediaminetetraacetic acid, add these reagents into a volumetric flask (500 mL) containing a small amount of distilled water, add distilled water to make up to 500 mL, cover the lid and shake well.

[0018] Furthermore, the preparation method of 2.0 mM β-nicotinamide adenine dinucleotide reducing solution (β-NADH) is as follows: to prepare 500 mL of 2.0 mM β-nicotinamide adenine dinucleotide reducing solution, 0.742 g of β-NADH needs to be weighed, and the β-NADH is added to a volumetric flask (500 mL) containing a small amount of distilled water, and the volume is made up to 500 mL with distilled water, and the lid is covered and shaken thoroughly.

[0019] Further, the preparation method of 58mM sulfonamide solution is as follows: to prepare 58mM sulfonamide solution, 5g of sulfonamide should be weighed, and the sulfonamide should be added to a volumetric flask (500mL) containing a small amount of distilled water, and the volume should be made up to 500mL with distilled water, and the lid should be covered and shaken thoroughly.

[0020] Further, the preparation method of 0.77mM N-(1-naphthyl)ethylenediamine dihydrochloride (NED) solution is as follows: To prepare 0.77mM N-(1-naphthyl)ethylenediamine dihydrochloride (NED) solution, 0.1g N-(1-naphthyl)ethylenediamine dihydrochloride needs to be weighed, and N-(1-naphthyl)ethylenediamine dihydrochloride is added to a volumetric flask (500mL) containing a small amount of distilled water, and distilled water is added to make up to 500mL, and the lid is covered and shaken thoroughly.

[0021] Furthermore, in the step (1), the strain with the best nitrate reductase activity screened out from the 6 lactic acid bacteria is Lactobacillus plantarum subsp. plantarum CICC 20022, whose nitrate reductase activity reaches 27.53 U / mL.

[0022] Further, in the step (2), the response surface methodology is used to optimize the culture medium components for nitrate reductase production and the synthesis conditions. The composition of the optimized MRS-nitrate culture medium is: peptone 10.0 g / L, sodium acetate 5.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, magnesium sulfate 0.58 g / L, manganese sulfate 0.25 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, diammonium hydrogen citrate 2.0 g / L, glucose 8.5 g / L, Tween 80 1.0 mL / L, potassium nitrate 22.8 g / L, distilled water 1000 mL, adjusted to pH = 4.85.

[0023] Further, Lactobacillus plantarum was cultured in an optimized MRS-nitrate medium at 37°C for 24h; 1.5mL of the fermentation broth was centrifuged at 4°C and 10000rpm for 20min, and the supernatant was filtered using a 0.45μm membrane filter to obtain a cell-free supernatant as a crude enzyme, 1.8mL of a nitrate reductase substrate solution was added to 100μL of the crude enzyme, and then 100μL of a 2.0mM β-nicotinamide adenine dinucleotide reducing solution (β-NADH) was added to react; after the reaction mixture was incubated at 30°C without light for 1h, 1mL of a 58mM sulfonamide solution was added, and then 1mL of a 0.77mM N-(1-naphthyl)ethylenediamine dihydrochloride (NED) solution was added to terminate the reaction, and the solution was maintained at 25°C for 20min to obtain a lavender solution; the nitrate reductase activity of Lactobacillus plantarum was measured using a UV-Vis spectrophotometer at a wavelength of 540nm and successfully increased from 33.53U / mL to 39.13U / mL, an increase of 16.7%.

[0024] Further, the method for synthesizing silver nanoparticles in step (3) is as follows: taking out glycerol-preserved Lactobacillus plantarum subsp. plantarum CICC 20022 from a refrigerator, and after being completely thawed, taking 200 μL of the liquid with a pipette and inoculating it into 5 mL of sterilized MRS liquid culture medium on a sterile clean bench, culturing it in a 37° C. constant temperature incubator for 24 h, and then subculturing it with an inoculum amount of 5% to activate the strain twice; inoculating 5 mL of the twice-activated Lactobacillus plantarum bacterial liquid into an optimized MRS-nitrate culture medium and culturing it at 37° C. for 24 h, and then centrifuging it at 4° C. and 10000 rpm for 20 min to obtain a cell-free supernatant, adding an AgNO3 aqueous solution, and making the mixed solution silver ion (Ag + ) concentration is 1 mM; the mixed solution is incubated overnight at 25°C in the dark, centrifuged at 25°C 10000rpm for 10 min to remove excess supernatant and unreacted Ag, the precipitate is washed twice with sterile water and then centrifuged at 25°C 10000rpm for 5 min to collect biosynthesized AgNPs, the precipitate is dried to obtain powdered AgNPs.

[0025] The composition of the optimized MRS-nitrate medium is: peptone 10.0 g / L, sodium acetate 5.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, magnesium sulfate 0.58 g / L, manganese sulfate 0.25 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, diammonium hydrogen citrate 2.0 g / L, glucose 8.5 g / L, Tween 80 1.0 mL / L, potassium nitrate 22.8 g / L, distilled water 1000 mL, adjusted to pH = 4.85.

[0026] Furthermore, the absorption spectrum of AgNPs was measured at 430 nm using Fourier transform infrared spectrometer, and scanning electron microscopy showed that the biosynthesized silver nanoparticles AgNPs were spherical with an average diameter of 98.2±4.8 nm and uniform particles.

[0027] The present invention also provides the use of silver nanoparticles prepared by the lactic acid bacteria biosynthesis method in antibacterial packaging materials: a plurality of PE and PLA are sterilized by ultraviolet irradiation for 1 hour in a clean bench, 20 mL of a 40 mg / L silver nanoparticle (AgNPs) solution resuspended in sterile water is taken into a sterile spray pot, and evenly sprayed on the inside of a PE or PLA fresh-keeping bag, and the surface is waited for natural drying, and sprayed again to increase the attachment amount and uniformity of the silver nanoparticles. After the second spray drying is completed, the preparation of the nanosilver antibacterial packaging material is completed.

[0028] Furthermore, AgNPs were used as an antibacterial agent to improve PE and PLA food packaging materials for preserving chicken breast, and the antibacterial effects could be increased by 41.9% and 42.5%, respectively.

[0029] Beneficial effects of the present invention:

[0030] The present invention establishes a method for biosynthesis of silver nanoparticles by lactic acid bacteria, and improves the composite antibacterial packaging to successfully extend the shelf life of fresh meat. Different from the existing synthesis and preparation of silver nanoparticles by lactic acid bacteria, it is more efficient, stable and environmentally friendly. Using this synthesis method to improve the composite antibacterial packaging to extend the shelf life of fresh meat is conducive to the realization of food safety. In the present invention, the antibacterial properties and nitrate reduction abilities of 6 strains of lactic acid bacteria were qualitatively and quantitatively analyzed, and plant lactobacillus was determined to be the best production bacterium. The response surface method optimized the synthesis conditions of lactic acid bacteria nanosilver to 0.85% glucose, 2.28% KNO3, pH4.85 (corresponding to the composition of the optimized MRS-nitrate medium). After 24 hours of culture, due to Ag + Reduction and surface plasmon resonance excitation produced brown color, the nitrate reductase activity could reach 39.13U / mL, and the synthesis rate was increased by 16.7% compared with that before optimization.

[0031] The silver nanoparticles were characterized, and Fourier infrared spectroscopy analysis showed that the AgNPs synthesized by Lactobacillus plantarum showed a peak at 430nm. Scanning electron microscopy analysis showed that the AgNPs particles were spherical and uniform, with an average diameter of 98.2±4.8nm. Further analysis of the antibacterial spectrum revealed that AgNPs showed antibacterial activity against Gram-negative bacteria, but had no obvious effect on Gram-positive bacteria. AgNPs were used as an antibacterial agent to improve PE and PLA food packaging materials for preserving chicken breast, and the antibacterial effect could be increased by 41.9% and 42.5%, respectively.

[0032] Compared with physical and chemical methods, the lactic acid bacteria synthesis of AgNPs has the advantages of cost-effectiveness, environmental friendliness, low toxicity to human cells, easy large-scale production, and no need for the use of hazardous chemicals and high temperature and high pressure. AgNPs synthesized by lactic acid bacteria show stronger antibacterial activity than other bacteria. The present invention provides an efficient, stable and environmentally friendly method for synthesizing silver nanoparticles using lactic acid bacteria. Improving antibacterial packaging materials is a key strategy and method for controlling spoilage microorganisms of fresh meat, and provides strong technical support for scientifically, systematically, efficiently and practically ensuring the safety of meat products.

[0033] The invention develops a stable, efficient and environmentally friendly method for synthesizing silver nanoparticles by lactic acid bacteria, which has high technical content and popularization and application value, and is manifested in the following aspects: 1. AgNPs synthesized by Lactobacillus plantarum have the advantages of low toxicity to human cells, easy large-scale production, no need to use hazardous chemicals and high temperature and high pressure, and exhibit stronger antibacterial activity than other bacteria; 2. The synthesis cost is low, the environment is friendly, and no pollution is generated; 3. Silver nanoparticles synthesized by Lactobacillus plantarum have a killing effect on a variety of pathogenic bacteria and spoilage bacteria, and an environmentally friendly silver nano natural antibacterial agent suitable for fresh cold meat is developed to enhance food safety or extend the shelf life of food, which can effectively improve food safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The standard curve of nitrosamines.

[0035] Figure 2 Comparative analysis of nitrate reduction reaction of lactic acid bacteria (1-blank control; 2-Lactobacillus plantarum; 3-Lactococcus lactis subspecies; 4. Mixture of Lactobacillus delbrueckii and Lactobacillus bulgaricus; 5-Streptococcus thermophilus; 6-Lactobacillus acidophilus; 7-Lactobacillus casei).

[0036] Figure 3 The nitrite production of lactic acid bacteria was determined (1-control group; 2-Lactobacillus plantarum; 3-mixture of Lactobacillus delbrueckii and Lactobacillus bulgaricus; 4-Streptococcus thermophilus; 5-Lactobacillus casei).

[0037] Figure 4 The method is to determine the activity of nitrate reductase of lactic acid bacteria (1-control group; 2-Lactobacillus plantarum; 3-mixture of Lactobacillus delbrueckii and Lactobacillus bulgaricus; 4-Streptococcus thermophilus; 5-Lactobacillus casei).

[0038] Figure 5 Analysis of culture conditions for response surface optimization (a-response surface for the interaction between glucose and KNO3; b-response surface for the interaction between glucose and pH; c-response surface for the interaction between KNO3 and pH).

[0039] Figure 6 Comparison of the changes in AgNPs biosynthesis over 24 hours.

[0040] Figure 7 AgNPs particles (left: AgNPs precipitate, right: powdered AgNPs).

[0041] Figure 8 is the absorption spectrum of AgNPs.

[0042] Fig. 9Scanning electron microscopy analysis of AgNPs particles (a-morphology and average size of biosynthesized AgNPs under 30,000 times magnification; b-morphology and average size of biosynthesized AgNPs under 100,000 times magnification).

[0043] Fig.10 pH changes during the shelf life of antimicrobial packaging.

[0044] Fig.11 Changes in microbial indicators during the shelf life of antimicrobial packaging.

[0045] Fig.12 Comparison of sensory evaluation of PE+AgNPs antibacterial packaging during the shelf life (a-PE; b-PE+AgNPs).

[0046] Fig.13 Comparison of sensory evaluation of PLA+AgNPs antibacterial packaging during shelf life (a-PLA; b-PLA+AgNPs). DETAILED DESCRIPTION

[0047] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention, and those skilled in the art in this field can make some non-essential improvements and adjustments based on the content of the above invention.

[0048] 1. Materials and methods

[0049] 1.1 Materials

[0050] 1.1.1 Test strains

[0051] Lactobacillus casei, Lactobacillus plantarum, Lactococcus lactis subsp, Streptococcus thermophilus, Lactobacillus acidophilus, and Lactobacillus delbrueckii + Lactobacillus bulgaricus were provided by the laboratory of Zhengzhou University of Light Industry.

[0052] Bacteria for titer testing: Escherichia coli and Bacillus subtilis, both provided by the laboratory of Zhengzhou University of Light Industry. Chicken breast: Chicken breast purchased on the same day from Shuanghui Food Supermarket.

[0053] 1.1.2 Mainly required drugs and reagents

[0054] Table 1-1 Biochemical drugs and reagents used in the experiment

[0055]

[0056]

[0057] 1.1.3 Main instruments

[0058] Table 1-2 Manufacturers and models of instruments used in the experiment

[0059]

[0060]

[0061] 1.1.4 Other Materials

[0062] Beakers (100mL, 500mL, 1000mL), volumetric flasks (250mL, 500mL), test tubes (10ml, 20mL), measuring cylinders (50ml, 100mL), conical flasks (100mL, 250mL, 500ml, 1000ml), pipette tips (100μL, 200μL, 1000μL, 5mL), weighing paper, medicine spoon, inoculation loop, coating rod, glass rod, alcohol lamp, sterile gauze, cotton plugs, kraft paper, string, rubber band, newspaper and tin foil, etc.

[0063] 1.1.5 Culture medium

[0064] MRS liquid culture medium: peptone 10.0 g / L, sodium acetate 5.0 g / L, potassium dihydrogen phosphate 2.0 g / L, magnesium sulfate 0.58 g / L, manganese sulfate 0.25 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, diammonium hydrogen citrate 2.0 g / L, glucose 20.0 g / L, Tween 80 1.0 mL / L, distilled water 1000 mL, adjust to pH 6.2-6.6.

[0065] Detection medium: nutrient agar medium.

[0066] LB solid (agar) medium: 10 g / L tryptone, 20 g / L agar powder, 5 g / L yeast extract, 10 g / L sodium chloride, 1000 mL distilled water, adjusted to pH 7.2.

[0067] MRS-nitrate medium: peptone 10.0 g / L, sodium acetate 5.0 g / L, dipotassium phosphate 2.0 g / L, magnesium sulfate 0.58 g / L, manganese sulfate 0.25 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, diammonium hydrogen citrate 2.0 g / L, glucose 20.0 g / L, Tween 80 1.0 mL / L, potassium nitrate 1 g / L, distilled water 1000 mL, adjust to pH 6.2-6.6.

[0068] 1.1.6 Preparation of required solutions

[0069] 1) Nitrate reagent A (sulfanilic acid): To prepare 250 mL of sulfanilic acid solution, weigh 1.25 g of sulfanilamide and add it to a volumetric flask (250 mL) containing an appropriate amount of 5N acetic acid. Add 5N acetic acid to make up to volume, cover the stopper and shake thoroughly.

[0070] 2) Nitrate reagent B (N,N-dimethyl-1-naphthylamine solution): To prepare 250 mL of N,N-dimethyl-1-naphthylamine solution, measure 1.5 mL of sulfonamide and add it to a volumetric flask (250 mL) containing an appropriate amount of 5N acetic acid. Add 5N acetic acid to make up to volume, cover the stopper and shake thoroughly.

[0071] 3) Normal saline: To prepare 250 mL of 0.9% normal saline, weigh 2.25 g of sodium chloride. Add the sodium chloride into a volumetric flask (250 mL) containing a small amount of distilled water. Add distilled water to make up to 250 mL. Cover the flask with a lid and shake thoroughly.

[0072] 4) 5μg / mL sodium nitrite: To prepare 500mL of 5μg / mL sodium nitrite, measure 3.6mL of 0.01mol / L sodium nitrite solution, add the solution into a volumetric flask (500mL) containing a small amount of distilled water, add distilled water to make up to 500mL, cover the lid and shake thoroughly.

[0073] 5) Nitrate reductase substrate solution: Prepare 500mL of nitrate reductase substrate solution, weigh 2.65g potassium phosphate, 0.5g potassium nitrate, 0.0043g ethylenediaminetetraacetic acid, add these reagents to a volumetric flask (500mL) containing a small amount of distilled water, add distilled water to 500mL, cover with lid and shake well. 58mM sulfonamide solution: To prepare 58mM sulfonamide solution, weigh 5g sulfonamide, add sulfonamide to a volumetric flask (500mL) containing a small amount of distilled water, add distilled water to 500mL, cover with lid and shake well.

[0074] 6) 2.0 mM β-nicotinamide adenine dinucleotide reducing solution (β-NADH): To prepare 500 mL of 2.0 mM β-nicotinamide adenine dinucleotide reducing solution, weigh 0.742 g of β-NADH. Add β-NADH to a volumetric flask (500 mL) containing a small amount of distilled water. Add distilled water to make up to 500 mL. Cover the flask with a lid and shake thoroughly.

[0075] 7) 58mM sulfanilamide solution: To prepare 58mM sulfanilamide solution, weigh 5g sulfanilamide and add it to a volumetric flask (500mL) containing a small amount of distilled water. Add distilled water to make up to 500mL, cover with a lid and shake thoroughly.

[0076] 0.77mM N-(1-naphthyl)ethylenediamine dihydrochloride (NED) solution: To prepare 0.77mM N-(1-naphthyl)ethylenediamine dihydrochloride (NED) solution, weigh 0.1g N-(1-naphthyl)ethylenediamine dihydrochloride, add N-(1-naphthyl)ethylenediamine dihydrochloride to a volumetric flask (500mL) containing a small amount of distilled water, add distilled water to make up to 500mL, cover the lid and shake thoroughly.

[0077] 1.2 Experimental methods

[0078] 1.2.1 Screening of nitrate reductase-producing bacteria

[0079] The nitrate reductase activity of six lactic acid bacteria (Lactobacillus plantarum, Lactococcus lactis subsp., Lactobacillus delbrueckii and Lactobacillus bulgaricus mixed, Streptococcus thermophilus, Lactobacillus acidophilus and Lactobacillus casei) was tested, and the blank group was used as a control. The nitrate reduction test was performed to determine the ability of microorganisms to reduce nitrate to nitrite. The lactic acid bacteria were cultured in MRS-nitrate medium at 37°C for 24h, and 100μL of nitrate reagent A and 100μL of nitrate reagent B were added to the culture in sequence, and the color changes that occurred were recorded. In order to avoid false negatives in nitrite reduction reactions, nitrite negative reactions were verified by adding zinc powder to the culture medium, and uninoculated MRS-nitrate medium was used as a negative control. The nitrate reduction test used is based on the principle of the Griess diazotization reaction described by Peter Griess. Due to the reaction between nitrite and sulfuric acid, the appearance of pink azo dye indicates the presence of nitrite, followed by α-naphthylamine. The intensity of the color formed by the reaction is proportional to the amount of nitrite formed. In the present invention, the original formula of nitrate reagent B, α-naphthylamine, is replaced by N,N-dimethyl-α-naphthylamine due to its potential carcinogenicity.

[0080] 1.2.2 Study on the antibacterial activity of lactic acid bacteria

[0081] 1.2.2.1 Preparation of indicator bacteria suspension

[0082] The antibacterial properties of the above 6 lactic acid bacteria were compared by agar diffusion method. Escherichia coli (G-) and Bacillus subtilis (G+) were inoculated on the slope of nutrient agar solid culture medium and cultured for 24 hours under constant temperature. Use a sterile inoculation loop to scrape an appropriate amount of colonies from the slope and transfer them to a conical flask containing 20mL sterilized saline. Shake the conical flask to fully mix the colonies with the saline, and use glass beads to further break up the cells to prepare a single cell suspension. The biomass of the bacterial suspension was determined by turbidimetry to ensure that the concentration of each batch of bacterial suspensions was consistent for subsequent experimental operations.

[0083] 1.2.2.2 Determination of indicator bacteria suspension concentration

[0084] Under the condition of absorbance of 540nm, Bacillus subtilis suspensions (0.153, 0.135, 0.109 and 0.076, respectively) and Escherichia coli suspensions (0.131, 0.119, 0.094 and 0.077, respectively) with different absorbance values ​​were selected for agar diffusion method antibacterial test. During the test, 200 μL of lactic acid bacteria 24-hour fermentation supernatant was added to each Oxford cup, and the antibacterial situation was carefully observed.

[0085] The experimental results showed that when the absorbance of the Bacillus subtilis suspension was 0.076, it could form a layer of stable colonies with moderate density and uniform size on the surface of the culture medium, and present a more obvious inhibition zone, and its transparency was also more significant. Therefore, in the subsequent antibacterial test, we decided to use OD 540nm A suspension of Bacillus subtilis with a value of 0.076 was used as a standard.

[0086] When the absorbance of the E. coli suspension is 0.077, it can also form a layer of stable colonies with moderate density and uniform size on the surface of the culture medium, and show a more obvious inhibition zone and transparency. Therefore, OD should be used in subsequent antibacterial tests. 540nm The value of Escherichia coli suspension is 0.077.

[0087] 1.2.2.3 Comparison of antibacterial properties

[0088] Under aseptic operation, pour about 20mL of sterilized test culture medium into a sterilized plate, place it on a clean bench, and wait for the culture medium to solidify naturally. Use a pipette to accurately draw 100μL of indicator bacteria suspension and evenly drop it onto the prepared plate. Use a sterile coating stick to evenly spread the bacterial suspension to ensure that the bacterial liquid is evenly distributed on the entire surface of the plate.

[0089] After coating, gently place the sterile Oxford cups on the plate, making sure the distance between the Oxford cups is at least 20 mm to avoid mutual influence. Then, use a pipette to accurately add 200 μL of the sample to be tested into each Oxford cup.

[0090] Place the inoculated plate in a 37°C constant temperature incubator for 24 hours to observe and analyze the antibacterial effect of the sample to be tested. Then take out the plate and measure the diameter of the inhibition zone with a ruler. The antibacterial performance of the strains can be compared by the size of the inhibition zone.

[0091] 1.2.3 Determination of nitrate reductase activity

[0092] 1.2.3.1 Determination of nitrite standard curve

[0093] Take 9 50mL stoppered colorimetric tubes, keep the concentration of sodium nitrite standard solution (5μg / mL) unchanged, only change the amount of sodium nitrite standard solution, add 0.00mL, 1.00mL, 1.50mL, 2.00mL, 2.50mL, 3.00mL, 4.00mL, 6.00mL, 8.00mL (equivalent to 0.0μg, 5.0μg, 7.5μg, 10.0μg, 12.5μg, 15.0μg, 20.0μg, 30.0μg, 40.0μg), respectively, and then add 2mL of 4g / L p-aminobenzenesulfonic acid solution, mix well, let stand for 3-5min, then add 1 mL of 2g / L naphthylethylamine hydrochloride solution, add water to the scale, mix well, and prepare a standard series of solutions. After color development at room temperature for 15 minutes, use tube 0 as the blank reference, measure the absorbance at a wavelength of 540nm, and draw a standard curve.

[0094] 1.2.3.2 Determination of nitrite absorbance in bacterial culture fluid

[0095] The above bacteria were grown in MRS-nitrate medium at 37°C for 24h. After shaking, 1.5mL of the culture was extracted with a pipette into a centrifuge tube and centrifuged at 10000rpm for 20min at 4°C. The supernatant was filtered using a 0.45μm membrane filter, and the cell-free supernatant obtained was used as a crude enzyme. 1.8mL of nitrate reductase substrate solution was added to 100μL of crude enzyme, and 100μL of 2.0mM β-nicotinamide adenine dinucleotide reducing solution (β-NADH) was added for reaction. The reaction mixture was incubated at 30°C without light for 1h. The reaction was terminated by adding 1 mL of 58mM sulfonamide solution and then 1 mL of 0.77mM N-(1-naphthyl)ethylenediamine dihydrochloride (NED) solution. The solution was mixed and incubated at 25°C for 20min to obtain a lavender solution. The absorbance of the solution was measured at a wavelength of 540nm using a UV-visible spectrophotometer and recorded.

[0096] The micromoles of unknown nitrite formed are estimated by comparing the unknown nitrite to standards of known micromoles of nitrite. Enzyme activity is calculated based on the micromoles of nitrite formed using the standard curve and expressed as units / mL enzyme using the following formula:

[0097] Units / mLenzyme=(μmole Nitrite formed)(df) / (r)(V) (1-1)

[0098] where df represents the dilution factor, T represents the assay time (in minutes), and V represents the volume of enzyme used (in mL).

[0099] 1.2.4 Optimization of culture medium components for nitrate reductase-producing bacteria by response surface methodology

[0100] The lactic acid bacteria with the highest nitrate reductase activity were selected for enzyme activity optimization. Different conditions of glucose (0.5%, 1.0%, 2.0%, 3.0%), potassium nitrate (1.0%, 2.0%, 3.0%) and pH (4.0, 5.0, 6.0, 7.0) were selected to culture lactic acid bacteria to determine the nitrate reductase activity. The selected ranges of the three factors obtained from the preliminary study were applied to the central composite rotation design (Design Expert 13, Stat-Ease Inc, USA) to evaluate the interaction between the independent variables to optimize the enzyme activity. As shown in Table 2-3, each variable was adjusted at five coding levels (-α, -1, 0, +1, +α). The central composite design generated a total of 20 experimental runs, including 8 factorial design runs, 6 axial point runs (2 for each variable) and 6 center point replicate runs. Nitrate reductase activity (dependent variable) is represented as the response to be analyzed. The response value (Y) of each run is the average of triplicates. The experimental data were fitted according to the following polynomial equation:

[0101] Y=β0+β1A+β2B+β3C+β1β1A 2 +β2β2B 2 +β3β3C 2 +β1β2AB+β1β3AC+β2β3BC (1-2)

[0102] Where Y is the reaction (nitrate reductase activity), A = glucose, B = KNO3, C = pH, the values ​​of A, B, and C represent the coded values ​​of the independent variables; β0 represents the constant regression coefficient; β1, β2, and β3 are linear regression coefficients; β1β1, β2β2, and β3β3 are quadratic regression coefficients; β1β2, β1β3, and β2β3 are cross-product regression coefficients (interactions).

[0103] Table 1-3 Experimental ranges and levels of independent variables used in RSM in terms of real and coded factors

[0104]

[0105] Note: In the optimization of the culture medium components of nitrate reductase-producing bacteria by response surface methodology, % represents the content of each component in 100 mL of culture medium. That is, different conditions of glucose (0.5%, 1.0%, 2.0%, 3.0%) and potassium nitrate (1.0%, 2.0%, 3.0%) were selected. The glucose concentrations corresponding to glucose (0.5%, 1.0%, 2.0%, 3.0%) were 5g / L, 10g / L, 20g / L, 30g / L, and the potassium nitrate concentrations corresponding to potassium nitrate (1.0%, 2.0%, 3.0%) were 10g / L, 20g / L, 30g / L, and the same below.

[0106] 1.2.5 Biosynthesis of AgNPs

[0107] The selected lactic acid bacteria were cultured in optimized nitrate medium and MRS-nitrate medium at 37°C for 24 hours. After incubation, the cell supernatant was obtained by centrifugation (10000rpm, 4°C, 20min) and used for the synthesis of AgNPs. Then, the cell supernatant was added to the silver nitrate aqueous solution (AgNO3) to make the mixed solution silver ion (Ag + ) concentration was 1 mM. The reaction mixture was then incubated overnight at 25°C in the dark. The optimized and non-optimized culture media for the synthesis of AgNPs were compared. The specific method is as follows:

[0108] 5 mL of the activated twice Lactobacillus plantarum culture was inoculated into 95 mL of optimized nitrate medium and cultured at 37°C for 24 h. The supernatant was centrifuged at 10000 rpm for 20 min at 4°C to obtain a cell-free supernatant. AgNO3 aqueous solution was added to make the mixed solution silver ion (Ag + ) concentration was 1 mM. The reaction mixture was incubated overnight at 25°C in the dark. Excess supernatant and unreacted Ag were removed by centrifugation at 25°C and 10,000 rpm for 10 min. The precipitate was washed twice with sterile water and centrifuged at 25°C and 10,000 rpm for 5 min to collect the biosynthesized AgNPs. The precipitate was dried to obtain powdered AgNPs.

[0109] The composition of the optimized nitrate medium is: peptone 10.0 g / L, sodium acetate 5.0 g / L, dipotassium phosphate 2.0 g / L, magnesium sulfate 0.58 g / L, manganese sulfate 0.25 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, diammonium hydrogen citrate 2.0 g / L, glucose 8.5 g / L, Tween 80 1.0 mL / L, potassium nitrate 22.8 g / L, distilled water 1000 mL, adjusted to pH 4.85.

[0110] The composition of MRS-nitrate medium is: peptone 10.0 g / L, sodium acetate 5.0 g / L, dipotassium phosphate 2.0 g / L, magnesium sulfate 0.58 g / L, manganese sulfate 0.25 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, diammonium hydrogen citrate 2.0 g / L, glucose 20.0 g / L, Tween 80 1.0 mL / L, potassium nitrate 1 g / L, distilled water 1000 mL, adjusted to pH 6.2-6.6.

[0111] 1.2.6 Characterization of biosynthesized AgNPs

[0112] The synthesis of AgNPs can be initially observed by visually observing the color change in the reaction mixture. For further characterization, the biosynthesized AgNPs were collected by centrifugation (10000rpm, 10 min, 25°C), then washed twice with sterile water and centrifuged (10000rpm, 5min, 25°C) to remove excess supernatant and unreacted Ag. The precipitate was dried to obtain powdered AgNPs. The absorption spectrum of AgNPs was measured using a Fourier transform infrared spectrometer. The surface morphology of biosynthesized AgNPs was studied using a scanning electron microscope. A small amount of AgNPs sample was placed on a conductive carbon tape and air-dried for 20min. Before viewing the sample, the sample surface was cleaned with a sample cleaner to eliminate hydrocarbon contamination for imaging.

[0113] 1.2.7 Antibacterial activity of biosynthesized AgNPs

[0114] The Oxford cup inhibition test was used to evaluate the antibacterial activity of biosynthesized AgNPs against Gram-positive (Bacillus subtilis) and Gram-negative bacteria (Escherichia coli). Using a colony suspension of a determined concentration, use a pipette to extract 100 μL onto LB agar medium, apply it evenly with a sterile coating rod and place it in a sterile Oxford cup. The spacing between the Oxford cups should not be too small, at least 20 mm. A known volume (100 μL) of biosynthesized AgNPs with a fixed concentration of 100 μg / mL was placed in the Oxford cup. The plate was then incubated at 37 ° C for 24 hours, and the diameter of the inhibition zone was recorded. The activity of AgNPs was calculated using the following formula:

[0115] AgNPs activity=(Lz-Ls) / V (1-3)

[0116] Where Lz represents the clear zone area (mm 2 ), Ls represents the well area (mm 2 ), V represents the sample volume (mL).

[0117] 1.2.8 Antibacterial effect of AgNPs in the preservation of fresh meat

[0118] 1.2.8.1 Preparation of different antibacterial packaging films

[0119] (1) Blank control group:

[0120] Prepare packaging films made of polyethylene (PE) and polylactic acid (PLA), place them on a clean bench, and irradiate them with ultraviolet light for 1 hour to ensure their sterility, providing standard control group conditions for subsequent experiments.

[0121] (2) Experimental group:

[0122] Use alcohol to disinfect a 50mL spray bottle, and also place it in a clean bench and irradiate it with ultraviolet light for 1 hour to ensure sterility. Add 20mL of AgNPs (silver nanoparticles) solution resuspended in sterile water to the spray bottle. Spray the AgNPs solution evenly on the inside of the PE and PLA packaging films. After spraying, wait for the surface to dry naturally, and spray again to increase the adhesion and uniformity of the silver nanoparticles. After waiting for the second spray to dry, these packaging films can be used as samples for the experimental group.

[0123] 1.2.8.2 Fresh Chicken Breast Processing

[0124] Prepare fresh chicken breast and divide it into 32 portions, each weighing 5g. Choose four different types of fresh-keeping bags: PE, PLA, PE+AgNPs (polyethylene combined with silver nanoparticles), and PLA+AgNPs (polylactic acid combined with silver nanoparticles), and put the chicken breast into these four types of fresh-keeping bags respectively.

[0125] The chicken breasts in each fresh-keeping bag were further divided into four storage time groups, namely T0 (immediate testing), T2 (testing after 2 days of storage), T5 (testing after 5 days of storage) and T7 (testing after 7 days of storage). To ensure the accuracy and reliability of the experimental results, a parallel control group was set up for each storage time group.

[0126] After grouping and bagging, all the bags were sealed and stored in a refrigerator at 4°C. Through this experimental setting, we can comprehensively evaluate the effects of fresh-keeping bags of different materials on the preservation of chicken breast at different storage times.

[0127] A series of quality assessments were conducted at four time points: day 0, day 2, day 5, and day 7, including sensory evaluation, total bacterial count, and pH determination, in order to fully understand the quality changes of chicken breast during the preservation process.

[0128] 1.2.8.3 Effects of different antibacterial packaging films on total bacterial count and pH during storage of chicken breast

[0129] Mix 45mL of pre-sterilized saline with 5g of chicken breast to be tested on the same day, ensure that the two are evenly mixed, and then put them into a special sealed bag. Use a beating homogenizer to beat the mixture for 2min at a frequency of 8 times / s to make it more uniform. Transfer the treated mixture to a centrifuge tube. In order to determine the total number of colonies, 0.5mL of the mixed solution was drawn from the centrifuge tube, and 4.5mL of saline was added to perform multiple 10-fold gradient dilutions. During the dilution process, three appropriate dilutions were selected to ensure that the number of colonies was within the countable range. 100μL of solution was drawn from each dilution and evenly spread on the nutrient agar plate, and two parallel experiments were performed for each dilution. The coated plate was placed in an incubator at 37°C for 24h, and the total number of colonies on the plate was determined. At the same time, in order to determine the pH value of the mixed solution, the mixed solution in the centrifuge tube was directly measured using a pH meter and the results were recorded.

[0130] 1.2.8.4 Effects of different antibacterial packaging films on sensory evaluation of chicken breast during storage

[0131] The chicken breasts that were previously stored in a 4°C refrigerator were taken out and placed at room temperature for 5 minutes. The sensory evaluation was performed with appropriate modifications according to the method of Stojanovic-Radic (Stojanovic-Radic et al., 2018). The sensory evaluation reference table is as follows.

[0132] Table 1-4 Sensory reference table for chicken breast samples

[0133]

[0134]

[0135] 2. Research Results

[0136] 2.1 Determination of nitrosamine standard curve

[0137] Figure 1 The standard curve was fitted to obtain the absorbance values ​​of 9 groups of solutions with different contents of nitrosamine.

[0138] Depend on Figure 1 It can be seen that the standard curve of nitrosamine is y=0.0113x+0.0102, R 2 =0.9998, indicating that the standard curve has a high degree of confidence and can be used for subsequent measurement of the nitrite mass of solutions with unknown nitrite content.

[0139] 2.2 Screening of nitrate reductase activity of lactic acid bacteria

[0140] When screening 6 lactic acid bacteria cultures, 4 lactic acid bacteria (Lactobacillus plantarum, a mixture of Lactobacillus delbrueckii and Lactobacillus bulgaricus, Streptococcus thermophilus, and Lactobacillus casei) were identified as having nitrate reducing properties. This result was verified by a nitrate reduction test after adding nitrate reagents A and B in sequence ( Figure 2 ).

[0141] Nitrate reduction test was performed on MRS-nitrate medium. A red color appeared after the addition of sulfanilic acid and N,N-dimethyl-1-naphthylamine, indicating the presence of nitrites in the presence of nitrate reduction. The intensity of the pink color was proportional to the amount of nitrite formed, with Lactobacillus plantarum, Lactobacillus delbrueckii and Lactobacillus bulgaricus mixed and Lactobacillus casei showing intense red color.

[0142] According to the results, the color observed in the nitrate reduction test was not intense, indicating partial reduction of nitrate. This may be due to the partial reduction of nitrite to other nitrogen-containing compounds such as ammonia and nitric oxide.

[0143] 2.3 Study on the antibacterial activity of lactic acid bacteria

[0144] After culturing for 24 hours using the agar diffusion method, it can be observed that there is a clear antibacterial zone. The diameter of the antibacterial zone is shown in Table (2-1), and the results are expressed as mean and standard deviation, where the test was performed in triplicate. Plant lactobacillus has a significant antibacterial effect on both Gram-positive and Gram-negative bacteria, with the largest antibacterial diameter, followed by Lactococcus lactis subsp. lactis, which also has a good antibacterial effect, and other species also have a strong antibacterial effect.

[0145] Table 2-1 Comparison of antibacterial effects of 6 lactic acid bacteria strains

[0146]

[0147]

[0148] 2.4 Screening of high-yield nitrate reductase-producing bacteria

[0149] The nitrite content and nitrate reductase activity of the four lactic acid bacteria were compared. Among the four lactic acid bacteria, Lactobacillus plantarum had the highest nitrite formation, with a nitrate reductase activity of 27.53 U / mL. In addition, Lactobacillus casei also showed a considerable amount of enzyme activity (26.47 U / mL). A smaller but still considerable amount of nitrite production was observed in the mixture of Lactobacillus delbrueckii and Lactobacillus bulgaricus and Streptococcus thermophilus ( Figure 4 ).

[0150] Figure 3 Indicates the production of nitrite in different lactic acid bacteria cultures, Figure 4Represents the nitrate reductase activity of different lactic acid bacteria cultures. The results are expressed as mean and standard deviation, and the test is performed in triplicate. The culture without inoculated strains is used as a blank control. The nitrite formation amount (8.26 μM) and nitrate reductase activity (27.53 U / mL) of plant lactobacillus are the highest. Therefore, in the present invention, the lactic acid bacteria selects the plant lactobacillus with the highest reductase activity among the 4 lactic acid bacteria to optimize the nitrate reductase production.

[0151] 2.5 Optimization of culture medium components for nitrate reductase production by response surface methodology

[0152] The central composite design (CCD) statistical model in the response surface analysis method was used to study the main interaction effects of three significant factors, glucose, potassium nitrate and pH, on nitrate reductase activity. The results obtained by CCD were analyzed using standard analysis of variance (ANOVA). Table 2-2 shows the experimental and predicted data of nitrate reductase activity obtained under various culture medium conditions designed from CCD. The actual experimental data values ​​ranged from 30.78U / mL to 39.18U / mL. On the other hand, the predicted data values ​​ranged from 31.01U / mL to 38.95U / mL. The highest nitrate reductase activity (39.18U / mL) was achieved under the independent variables of 0.75% glucose and 2% KNO3, with a pH of 5.0. In contrast, the lowest expected nitrate reductase activity (30.18U / mL) was obtained under the independent variables of 0.75% glucose and 3.7% KNO3, with a pH of 5.0.

[0153] Table 2-2 CCD experimental design for optimizing nitrate reductase culture conditions

[0154]

[0155]

[0156] The experimental results of variance analysis are shown in Table 2-3. The variance analysis of nitrate reductase activity showed that the F value of the model was 182.39 and the "Probability (F)" value was <0.0001, indicating that the model was highly significant. During the experiment, the probability of the model F value occurring due to human or environmental errors was only 0.01%. The "lack of fit F value" of 2.02 indicated that the model fit due to lack of fit was not significant compared with pure error. P value <0.05 indicated that the model terms A, B, C, AB, A 2 , B 2 and C 2 The second-order polynomial model equation generated by nitrate reductase as a function of glucose (A), KNO3 (B), and pH (C) is as follows:

[0157] Activity=

[0158] 38.95+1.76A-0.4218B-0.4836C-0.3473AB-0.1722AC-0.2228BC-1.19AA-

[0159] 2.73BB-0.6930CC

[0160] (2-1)

[0161] Table 2-3 Analysis of variance of response surface quadratic model for nitrate reductase culture conditions

[0162]

[0163]

[0164] According to the fitting statistics, the coefficient of determination (R 2 ) was calculated to be 0.9939, indicating that the model well explains 99.39% of the total deviation of the experimental results. The predicted R 2 0.9660 and the adjusted R 2 The 0.9885 is reasonably consistent, as the difference is <0.2, sufficient accuracy to measure the signal (response) to noise ratio (bias). The ratio is 36.9526, which is greater than 4, indicating that the signal is sufficient, so the model is significant for this process. Therefore, the fit statistics indicate that equation (2-1) is adequately used as a predictive model for this experiment.

[0165] By plotting the three-dimensional response surface curves of any two independent variables while keeping the other independent variables at the central level, the effect of the interaction of three important factors (glucose, potassium nitrate and pH) on nitrate reductase production (z axis) was studied. Therefore, in formula (1-2), the prediction model generated three response surfaces expressing the interaction between the independent variables. The interaction term between A (glucose) and B (KNO3) is significant (p < 0.05). In contrast, the interaction term between AC and BC is not significant (p greater than 0.05). From the reaction surface ( Figure 5 ) It can be seen that component A (glucose) has an effect on the production of nitrate reductase. As the glucose concentration increases, the nitrate reductase activity increases steadily. On the other hand, the reaction surface shows a slight increase in the production of nitrate reductase, an initial increase in component B (potassium nitrate) (1-2%), and then a steady decrease in the production of nitrate reductase with further increases in concentration (2-3%). At the same time, component C (pH) has little effect on nitrate reductase activity and therefore plays an insignificant role in the production of the enzyme.

[0166] The response surface plot is a graph that intuitively represents the relationship between two factors in the response. The 3D surface plot can be used to study the required response values ​​and conditions. Relative to the nitrate reductase activity, other components were kept at the central level. There was a significant interaction between glucose and KNO3. Through the response surface methodology, the predicted optimal conditions for nitrate reductase production were 0.85% glucose, 2.28% KNO3, and pH 4.85, that is, the composition of the optimized nitrate medium was: peptone 10.0g / L, sodium acetate 5.0g / L, dipotassium hydrogen phosphate 2.0g / L, magnesium sulfate 0.58g / L, manganese sulfate 0.25g / L, beef extract 10.0g / L, yeast extract 5.0g / L, diammonium hydrogen citrate 2.0g / L, glucose 8.5g / L, Tween 80 1.0mL / L, potassium nitrate 22.8g / L, distilled water 1000mL, adjusted to pH 4.85.

[0167] 2.6 Verification of the optimal culture medium components for nitrate reductase production

[0168] A validation test was performed using the predicted values ​​of the optimal conditions for nitrate reductase production (0.85% glucose, 2.28% KNO3 and pH 4.85) to check the adequacy of the prediction model of equation (1-2). The experiment used was a two-tailed test with a confidence level of 95% to test whether the enzyme activity was greater than or less than a certain range of values. The actual nitrate reductase activity obtained in this experiment was 39.13±0.05U / mL, which was similar to the predicted value of 39.19U / mL. In the 95% low prediction interval (PI) and high prediction interval, the actual data value (39.13±0.05U / mL) was in the range of (39.14~39.46U / mL), with a standard error of 0.1864 and a standard deviation of 0.4591, respectively. The two-tailed p value was 0.808, which is greater than 0.05. Therefore, the mean value of the actual nitrate reductase activity was not significantly different from the predicted value. Therefore, this model is an ideal choice for predicting nitrate reductase production values. In the optimization study, nitrate reductase activity was successfully increased from 33.53 U / mL (unoptimized) to 39.13 U / mL (optimized), an increase of 16.7%.

[0169] 2.7 Biosynthesis of AgNPs

[0170] The reduction of Ag to AgNPs was recorded by visually observing the color change in the reaction mixture. The reaction mixture was observed to turn brown after 24 h of incubation. Figure 6As shown, indicating the formation of AgNPs. The appearance of brown color is due to the effects of Ag reduction and surface plasmon resonance (SPR) excitation. In metal nanoparticles such as Ag, electrons can move freely due to the proximity of the conduction band and the valence band. The collective oscillation of the electrons of AgNPs in resonance with the light waves produces a unique SPR absorption band, which is also the origin of the observed brown color. It has been previously noted that the biosynthesis of AgNPs using the cell supernatant of lactic acid bacteria also showed similar findings, and a brown color was observed in the reaction mixture after incubation. Figure 6 In the figure, the formation of AgNPs is visually observed from 0h to 24h from the left to the right. Brown color appears due to the reduction of Ag and the stimulation of SPR effect. The synthesized AgNPs solution is centrifuged to remove the supernatant, and the precipitated water is dried to obtain powdered AgNPs particles such as Figure 7 shown.

[0171] By comparing the biosynthesized AgNPs yields before and after optimizing the culture medium, the yield ratio improved by optimizing the culture medium was determined as shown in Table 2-4.

[0172] Table 2-4 Optimization of silver nanoparticle yield

[0173]

[0174]

[0175] The comparison showed that the increase in the biosynthesized AgNPs yield was 16.69%, which was basically equal to the increase in nitrate reductase activity of 16.7%, ensuring the rigor of the culture medium optimization and verifying the optimal conditions for nitrate reductase production.

[0176] 2.8 Fourier transform infrared spectroscopy analysis

[0177] Fourier transform infrared spectroscopy analysis showed that the biosynthesized AgNPs exhibited a peak at 430 nm ( Figure 8 ), the peaks obtained from this study are similar to the typical peak range of AgNPs. Due to their unique optical properties, AgNPs interact strongly with specific wavelengths of light. AgNPs have been shown to absorb electromagnetic radiation in the visible light region of 380-450nm.

[0178] 2.9 SEM analysis

[0179] Cold-field scanning electron microscopy (SEM) was used to study the surface morphology of biosynthesized AgNPs at the micro-nanoscale. Fig. 9). SEM analysis showed that the AgNPs surface was approximately spherical, the particles were uniform, the particle size was small, and they were easy to agglomerate, with an average diameter of 98.2±4.8nm. It should be noted that the AgNPs observed in the SEM analysis were in an aggregated state, and the aggregation of biosynthesized AgNPs may be due to the loss of water after complete drying on the conductive carbon tape during the sample preparation for FE-SEM analysis.

[0180] 2.10 Antibacterial activity of biosynthesized AgNPs

[0181] The experimental results show that 100 μg / mL of biosynthesized AgNPs can inhibit Gram-negative bacteria (Escherichia coli). However, Gram-positive bacteria (Bacillus subtilis) are less susceptible to AgNPs than Gram-negative bacteria (Table 2-5).

[0182] Table 2-5 Antibacterial activity of biosynthesized AgNPs

[0183]

[0184] 2.11 Study on the preservation of chicken breast by AgNPs antibacterial packaging

[0185] 2.11.1 pH change curve during storage of chicken breast

[0186] The pH curves of chicken breast meat stored at 4°C under four types of plastic wrap: T0, T2, T5, and T7. Fig.10 As shown:

[0187] like Fig.10 As shown in the figure, during the storage of chicken breast, its pH value gradually increased with the extension of storage time. Especially in the time period from T2 to T5, the pH value of chicken breast increased significantly with the increase of storage time. This phenomenon is mainly due to the fact that aerobic bacteria on the surface of the meat began to grow and multiply during the storage process. These bacteria produced protein metabolites through metabolic activities, mainly releasing alkaline amines (such as ammonia, primary amines, secondary amines and tertiary amines), as well as decomposition products of other organic matter. The release of these alkaline substances led to a significant increase in the pH value of chicken breast.

[0188] In addition, studies have shown that when sealed packaging is used for storage, as the oxygen in the environment is gradually consumed, a relatively anaerobic environment will be formed. In this environment, the growth of aerobic microorganisms will be inhibited to a certain extent. This helps to slow down the increase in the pH value of chicken breast, thereby extending the shelf life of chicken breast to a certain extent.

[0189] 2.11.2 Changes in total bacterial count in chicken breast during storage

[0190] The change curve of total bacteria count in chicken breast meat stored under four kinds of plastic wrap at 4℃ for four days: T0, T2, T5 and T7 Fig.11 shown.

[0191] like Fig.11 As shown in the figure, during the storage period from T0 to T7, the total number of bacteria showed a continuous upward trend. Regardless of whether the combination of PE+AgNPs or PLA+AgNPs was used, their total bacterial counts were reduced compared with ordinary packaging films, showing a significant antibacterial effect. Especially before T2, the two combinations were most effective in delaying the growth of spoilage microorganisms. Throughout the experiment, the total number of bacteria in the packaging using the PE+AgNPs and PLA+AgNPs combinations was reduced by 41.9% and 42.5% respectively compared with ordinary commercial packaging. This result shows that the spray-coated silver nanocomposite antibacterial packaging can effectively inhibit the growth of bacteria, thereby extending the shelf life of chicken breast. Further comparison of the two combinations shows that the combination of PLA+AgNPs is slightly better than the combination of PE+AgNPs in antibacterial efficacy.

[0192] 2.11.3 Sensory changes during storage

[0193] Sensory changes of chicken breasts stored in four kinds of plastic wrap at 4°C at T0, T2, T5 and T7 Fig.12 and 13 shown.

[0194] according to Fig.12 and 13 Data analysis shows that compared with commercial packaging, the packaging film sprayed with AgNPs can significantly improve the sensory score of chicken breast during the first 5 days of storage. The chicken breast under this packaging film has less sensory changes during storage, the odor is relatively weak, and the tissue morphology remains relatively compact, which effectively delays the degree of rancidity of the chicken breast.

[0195] In contrast, the preservation effect of ordinary packaging film is poor. At T2, the chicken breasts using ordinary packaging film can smell a distinct rancid smell. However, no rancid smell was detected at T2 time point, whether it was PE+AgNPs cling film or PLA+AgNPs cling film, indicating that these two packaging films have a good effect in inhibiting bacterial growth and delaying spoilage in the first two days.

[0196] 3 Conclusion

[0197] (1) Four lactic acid bacteria strains (Lactobacillus plantarum, a mixture of Lactobacillus delbrueckii and Lactobacillus bulgaricus, Streptococcus thermophilus and Lactobacillus casei) showed their ability to produce nitrate reductase activity, among which the best producing strain was Lactobacillus plantarum, with a nitrate reductase activity of up to 27.53 U / mL;

[0198] (2) Through response surface methodology analysis, the nitrate reductase activity of Lactobacillus plantarum was successfully increased from 33.53U / mL to 39.13U / mL under the optimal culture conditions (0.85% glucose, 2.28% KNO3 and pH 4.85), an increase of 16.7%;

[0199] (3) Characterization of silver nanoparticles revealed that the biosynthesized AgNPs exhibited a peak at 430 nm. The biosynthesized AgNPs were spherical and nanosized, with an average diameter of 98.2 ± 4.8 nm and uniform particles.

[0200] (4) AgNPs showed a significant antibacterial effect against Escherichia coli (Gram-negative bacteria), but had no significant antibacterial effect against Bacillus subtilis (Gram-positive bacteria), which is consistent with previous research results that Gram-negative bacteria are more susceptible to AgNPs due to the thickness of their cell wall and cell composition.

[0201] (5) The combination of AgNPs and plastic wrap can improve the antibacterial and preservation capabilities of plastic wrap. The total bacterial count of chicken breast preserved with AgNPs combined with PE and PLA plastic wrap in the first seven days was reduced by 41.9% and 42.5%, respectively, compared with that of ordinary commercial packaging. The effect of PLA+AgNPs was slightly better than that of PE+AgNPs.

[0202] The present invention establishes an efficient and stable method for biosynthesizing nano silver particles by lactic acid bacteria, and improves the composite antibacterial packaging to extend the shelf life of fresh cold meat, achieving a relatively ideal result.

[0203] Note: The optimal culture conditions (0.85% glucose, 2.28% KNO3 and pH 4.85) in the article refer to the composition of the optimized MRS-nitrate medium: peptone 10.0g / L, sodium acetate 5.0g / L, potassium hydrogen phosphate 2.0g / L, magnesium sulfate 0.58g / L, manganese sulfate 0.25g / L, beef extract 10.0g / L, yeast extract 5.0g / L, diammonium hydrogen citrate 2.0g / L, glucose 8.5g / L, Tween 80 1.0mL / L, potassium nitrate 22.8g / L, distilled water 1000mL, adjusted to pH = 4.85. The glucose concentration corresponding to 0.85% glucose is 8.5g / L, and the concentration corresponding to 2.28% KNO3 is 22.8g / L.

[0204] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A stable, efficient and environmentally friendly method for the biosynthesis of silver nanoparticles by lactic acid bacteria, characterized in that The following steps are involved: (1) A nitrate reduction test was conducted to determine the ability of the strain to reduce nitrate to nitrite, and the optimized strain was selected from the six lactic acid bacteria strains; (2) Optimize the culture medium composition and synthesis conditions for nitrate reductase production using response surface methodology; (3) Synthesizing silver nanoparticles using the optimized strain screened in step (1).

2. The stable, efficient and environmentally friendly method for lactic acid bacteria biosynthesis of silver nanoparticles according to claim 1, characterized in that: In step (1), the screening method for optimizing the strain is as follows: The nitrate reductase activity of the six lactic acid bacteria strains was detected, and the blank group was used as a control. The nitrate reduction test was performed to determine the ability of microorganisms to reduce nitrate to nitrite. The lactic acid bacteria were cultured in MRS-nitrate medium at 37°C for 24h, and 100 μL of nitrate reagent A and 100 μL of nitrate reagent B were added to the culture in sequence, and the color changes that occurred were recorded; The lactic acid bacteria strain with nitrate reductase activity was grown in MRS-nitrate medium at 37°C for 24 h. After shaking, 1.5 mL of the culture was extracted into a centrifuge tube with a pipette, and centrifuged at 10,000 rpm for 20 min at 4°C. The supernatant was filtered using a 0.45 μm membrane filter. The obtained cell-free supernatant was used as the crude enzyme. 1.8 mL of nitrate reductase substrate solution was added to 100 μL of the crude enzyme, and then 100 μL of 2.0 mM β-nicotinamide adenine dinucleotide reducing solution (β-NADH) was added for reaction. The reaction mixture was incubated at 30°C in the dark for 1 h, and then 1 mL of 58 mM sulfonamide solution was added, followed by 1 mL of 0.77 mM The reaction was terminated by adding N-(1-naphthyl)ethylenediamine dihydrochloride (NED) solution and maintained at 25°C for 20 min to obtain a lavender solution. The absorbance of the solution was measured at a wavelength of 540 nm using a UV-visible spectrophotometer and recorded.

3. The stable, efficient and environmentally friendly method for biosynthesis of silver nanoparticles by lactic acid bacteria according to claim 1, characterized in that: In the step (1), the six lactic acid bacteria strains are Lactobacillus plantarum, Lactococcus lactis subsp., a mixture of Lactobacillus delbrueckii and Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus and Lactobacillus casei.

4. The stable, efficient and environmentally friendly method for lactic acid bacteria biosynthesis of silver nanoparticles according to claim 2, characterized in that: In the step (1), the strain with the best nitrate reductase activity was selected from the six lactic acid bacteria, and the strain was Lactobacillus plantarum. Lactiplantibacillus plantarum subsp. plantarum CICC 20022, nitrate reductase activity reached 27.53 U / mL.

5. The stable, efficient and environmentally friendly method for lactic acid bacteria biosynthesis of silver nanoparticles according to claim 1, characterized in that: In the step (2), the response surface methodology is used to optimize the culture medium components for nitrate reductase production and the synthesis conditions. The optimized MRS-nitrate culture medium has the following composition: peptone 10.0 g / L, sodium acetate 5.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, magnesium sulfate 0.58 g / L, manganese sulfate 0.25 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, diammonium hydrogen citrate 2.0 g / L, glucose 8.5 g / L, Tween 80 1.0 mL / L, potassium nitrate 22.8 g / L, and distilled water 1000 mL, adjusted to pH = 4.

85.

6. The stable, efficient and environmentally friendly method for lactic acid bacteria biosynthesis of silver nanoparticles according to claim 4, characterized in that: The method for synthesizing silver nanoparticles in step (3) is as follows: Lactobacillus plantarum ( Lactiplantibacillus plantarum subsp. plantarum CICC 20022) was taken out of the refrigerator, and after it was completely thawed, 200 μL was taken out with a pipette and inoculated into 5 mL of sterilized MRS liquid culture medium on a sterile clean bench, and cultured in a constant temperature incubator at 37°C for 24 h, and then subcultured at a 5% inoculation volume to activate the strain twice; 5 mL of the twice activated Lactobacillus plantarum culture liquid was inoculated into the optimized MRS-nitrate culture medium at 37°C for 24 h, and then centrifuged at 4°C and 10000 rpm for 20 min to obtain a cell-free supernatant, and an AgNO3 aqueous solution was added to make the mixed solution silver ion (Ag + ) concentration was 1 mM; the mixed solution was incubated at 25 °C in the dark overnight, and the excess supernatant and unreacted Ag were removed by centrifugation at 25 °C and 10,000 rpm for 10 min. The precipitate was washed twice with sterile water and centrifuged at 25 °C and 10,000 rpm for 5 min to collect the biosynthesized AgNPs. The precipitate was dried to obtain powdered AgNPs. The composition of the optimized MRS-nitrate medium is: peptone 10.0 g / L, sodium acetate 5.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, magnesium sulfate 0.58 g / L, manganese sulfate 0.25 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, diammonium hydrogen citrate 2.0 g / L, glucose 8.5 g / L, Tween 80 1.0 mL / L, potassium nitrate 22.8 g / L, distilled water 1000 mL, adjusted to pH = 4.

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7. The stable, efficient and environmentally friendly method for lactic acid bacteria biosynthesis of silver nanoparticles according to claim 6, characterized in that: The absorption spectrum of AgNPs was measured by Fourier infrared spectrometer at 430 nm, and scanning electron microscopy showed that the biosynthesized silver nanoparticles AgNPs were spherical with an average diameter of 98.2±4.8nm and uniform particles.

8. Use of silver nanoparticles obtained by the lactic acid bacteria biosynthesis method according to any one of claims 1 to 7 in antibacterial packaging materials.

9. The use according to claim 8, characterized in that: In the clean bench, PE and PLA were sterilized by ultraviolet irradiation for 1 h. 20 mL of 40 mg / L silver nanoparticle (AgNPs) solution resuspended in sterile water was taken into a sterile spray bottle and sprayed evenly on the inside of the PE or PLA fresh-keeping bag. The surface was allowed to dry naturally and sprayed again to increase the adhesion and uniformity of the silver nanoparticles. After the second spray drying was completed, the preparation of the nanosilver antibacterial packaging material was completed.

10. The use according to claim 9, characterized in that: AgNPs were used as an antibacterial agent to improve PE and PLA food packaging materials for preserving chicken breast, and the antibacterial effects could be increased by 41.9% and 42.5%, respectively.

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