CN104531561A - (Lactobacillus planetarium subsp. plantarum)Zhang-LL bacteriocin and Nisin composite bacteriostatic agent and its use in chilled meat - Google Patents

Abstract

The invention relates to a compound bacteriostatic agent of Lactobacillus plantarum subspecies bacteriocin and Nisin and its application in chilled fresh meat, which is suitable for controlling pathogenic bacteria in various foods and belongs to the field of food antisepsis and preservation. The present invention screens and obtains Lactobacillus plantarum subsp. plantarum Zhang-LL (CGMCC No. 6936) from bacon in Fujian farmers' market. The bacteriocin produced by the bacteria and Nisin were combined to prepare a compound bacteriostatic agent, which was applied to the preservation and preservation of cold fresh meat. The effect of inhibiting Listeria monocytogenes was 1000 times higher than that of using bacteriocin alone. The preservation period of meat is extended by 13 days. The composite bacteriostatic agent is applied to the antisepsis of chilled fresh meat, the treatment method is simple, the dosage of bacteriocin is small, and the cost is low, and it is suitable for antiseptic preservation of large-scale chilled fresh meat.

Description

A compound bacteriostatic agent of Lactobacillus plantarum subsp. planta bacteriocin and Nisin and its application in chilled fresh meat

technical field

The invention relates to a compound bacteriostatic agent of Lactobacillus plantarum subspecies bacteriocin and Nisin and its application in chilled fresh meat, which is suitable for controlling pathogenic bacteria in various foods and belongs to the field of food antisepsis and preservation.

Background technique

Meat products are different from other foods. Their own environmental conditions such as water activity, pH, and nutrient content are good substrates for microbial growth, so it is easy to breed a wide variety of microorganisms with a large content, including Salmonella, Staphylococcus aureus, and monocytogenes. Listeria, Escherichia coli, etc. According to data from the my country Foodborne Disease Surveillance Network, foodborne disease incidents and the largest number of people involved are caused by pathogenic microorganisms, and among animal foods, meat and meat products have the most disease incidents. At present, controlling the growth of Listeria monocytogenes in pork is a thorny issue. Eating pork infected with Listeria monocytogenes can cause infection in some people, especially pregnant women, infants and people with low immunity. The fatality rate in the crowd is high, between 27% and 34%. Listeria monocytogenes is a spoilage bacterium that widely exists in meat products. Because it can tolerate low temperature, refrigeration has little effect on its growth inhibition. The bacterium can grow under both aerobic and anaerobic conditions, so vacuum packaging or carbon dioxide controlled atmosphere storage can not inhibit the growth of the bacterium. In addition, the effects of pH, sodium chloride and nitrate on its growth were not obvious.

Adding preservatives is currently an important way of food preservation and preservation, and in the field of preservation and preservation, due to the potential side effects of chemically synthesized preservatives, the research on natural and safe biological preservatives is more extensive. Lactobacillus is a kind of protein or polypeptide with antibacterial activity produced by some lactic acid bacteria through the ribosome synthesis mechanism during the metabolic process. It can usually inhibit its close relative strains, and the producing bacteria are autoimmune. The reason why it can become the development object of natural biological preservatives is based on the following characteristics: ①The lactic acid bacteria produced by it grow and multiply quickly, the production cycle is short, and have industrial production prospects; ②It has human protease biodegradability, will not accumulate in the human body, and It will not affect the normal flora of human intestinal tract; ③Different from antibiotic resistance, it will not induce the resistance of pathogenic microorganisms to antibiotics; ④It is convenient for modern genetic engineering technology to modify and clone bacteriocin genes.

Although researchers at home and abroad are constantly exploring and researching the screening of natural antibacterial substances bacteriocins, some bacteriocins with broad-spectrum antibacterial activity against food spoilage microorganisms and pathogenic microorganisms have been found, but only Nisin is recognized as a natural natural substance in the world. It is the only lactic acid bacteria bacteriocin allowed to be used as a food preservative so far, and it has been widely used in about 50 countries and regions in the world so far. However, Nisin has a narrow antibacterial spectrum and is inactivated when the pH is above 8.0, so it can only play a role in acidic foods. Nisin mainly inhibits Gram-positive bacteria, but its antibacterial effect on Gram-negative bacteria is not ideal, thus limiting the application of Nisin. In the patent of the present invention, Lactobacillus plantarum subspecies bacteriocin and Nisin are compounded and used in proportion, which has good antibacterial effect, and the compound antibacterial agent can be applied to the control of pathogenic bacteria in various foods.

At present, there are reports on the application of lactic acid bacteria bacteriocins as preservatives in meat products in domestic and foreign literature. Liu Guorong and others studied that the surface lactic acid bacterium pentocin31-1 can effectively inhibit the growth of Pseudomonas and potential pathogenic bacteria Listeria in chilled meat. When the amount of pentocin31-1 added is 80AU/g, the premise of ensuring good sensory properties It can extend the shelf life of cold fresh meat to 15 days; Castellano et al. found that Lactobacillus curvatus CRL705 can be used as a biological preservative to keep vacuum-packed beef stored at 2°C for 60 days without deterioration; Rosalinda Urso et al. screened in naturally fermented sausage A strain of Lacticbacteria sakei, the bacteriocin Sakacin P produced by it can not only inhibit Enterococcus in sausage, but also has no adverse effect on the flavor of sausage. Reports on the application of lactic acid bacteria bacteriocins in meat products in domestic patents. "A bacteriocin-producing Lactobacillus oryzae strain and its application" applied by Nanjing Agricultural University, the patent application number is: CN03132323.5; "Bacteriocin and ultra-high pressure combined antiseptic preservation technology and its application" applied by China Agricultural University, The patent application number is: CN201110141938. Lactobacillus plantarum (Lactobacillus plantarum) is one of the strains expressly stipulated in the "List of strains that can be used for food" issued by the General Office of the Ministry of Health of the People's Republic of China on April 22, 2010, and the Lactobacillus plantarum plant in the present invention The subspecies is isolated from Fujian bacon, a traditional fermented food with probiotic properties, and its metabolites with antibacterial activity are considered basically safe. Therefore, the combination of Lactobacillus plantarum subsp. plantarum bacteriocin and Nisin is safe and reliable as a food preservative. Although there are reports on the antiseptic preservation of lactic acid bacteria bacteriocins in meat products at home and abroad, there are no literature and patent reports on the joint application of Lactobacillus plantarum subsp. planta bacteriocins and Nisin in meat product preservation.

Contents of the invention

The object of the present invention is to provide a kind of Lactobacillus plantarum subsp. planta bacteriocin and Nisin compound antibacterial agent and its application method in chilled fresh meat.

The source of bacteriocin in the composite bacteriostatic agent of the present invention is Lactobacillus plantarum subsp. plantarum bacteriocin and nisin (Nisin purchased from Sigma, USA). Lactobacillus plantarum subsp. plantarum bacteriocin is extracted from the fermentation broth of Lactobacillus plantarum subsp. plantarum Zhang-LL, which was released on December 4, 2012 It was deposited in the General Microorganism Center of China Committee for Culture Collection of Microorganisms (CGMCC for short), and the preservation number is: CGMCC No.6936.

The preparation method of compound bacteriostatic agent: take potency and be 1280AU/mL Lactobacillus plantarum subsp. plantarum bacteriocin and carry out two-fold gradient dilution to 320AU/mL with sterile distilled water; Nisin is formulated into 5% (W/V) working solution , and its titer was 320AU/mL by two-fold serial dilution method. The Lactobacillus plantarum subsp. plantarum bacteriocin and Nisin are mixed according to the volume ratio of 3:1 to obtain the compound antibacterial agent.

The application method of compound antibacterial agent in cold fresh meat preservation: live pigs, stunned, bloodletting, head removal, skinning, removal of internal organs, disinfection, cooling, segmentation and deboning, spraying compound antibacterial agent, packaging. Spray 320AU/mL compound bacteriostatic agent after segmentation, spraying amount is 0.1mL/cm ² , spray about 10mL bacteriostatic agent per kilogram of chilled fresh meat, and store after air-drying at 4°C.

The preparation method of the above-mentioned Lactobacillus plantarum subsp. plantarum bacteriocin and Nisin composite antibacterial agent and its application in chilled fresh meat belong to the protection scope of the present invention.

Description of drawings

The process flow chart of the application of Lactobacillus plantarum subspecies bacteriocin and Nisin composite bacteriostatic agent in cold fresh meat preservation is as follows:

(1) Live pigs: pass inspection and quarantine; (2) Stunning: voltage 85V, time 3-4s. (3) Bloodletting: Hanging method, time 4min; (4) Head removal, peeling, internal organs removal; (5) Disinfection: dehydroacetic acid (0.001%), 5s; (6) Cooling: 0～4℃, 10h , wind speed 1m/s; (7) split and deboned; (8) spray compound antibacterial agent, the potency is 320AU/mL, and the spraying amount is 0.1mL/cm ² ; (9) packaging: the product is stored at 4°C.

Lactobacillus plantarum subsp.plantarum Zhang-LL was isolated and screened from bacon products in Fujian farmers' market, and the bacteriocin produced by it can be degraded by proteases in human body. In the present invention, the lactic acid bacteriocin composite bacteriostatic agent is applied to fresh-keeping of chilled fresh meat, and the processing method is simple, the dosage of bacteriocin is small, and the cost is low, and it is suitable for large-scale preservation of chilled fresh meat.

Detailed ways

The experimental methods in the following examples are conventional methods unless otherwise specified.

The percentages in the following examples are volume percentages unless otherwise specified.

Embodiment 1, the inhibitory effect of Lactobacillus plantarum subspecies bacteriocin and Nisin in the culture medium system on Listeria monocytogenes

1. Determination of antibacterial activity of Lactobacillus plantarum subsp. plantarum bacteriocin and Nisin

The crude extract of Lactobacillus plantarum subsp. plantarum bacteriocin was prepared by pH adsorption-desorption method, and Nisin was formulated into a 5% (m/V) working solution. Using Listeria monocytogenes ATCC54003 as an indicator bacterium, the antibacterial activity of Lactobacillus plantarum subsp. The antibacterial activity of Nisin is 1280AU/mL, and the antibacterial activity of Nisin working solution is 320AU/mL.

2. Inhibitory effect of Lactobacillus plantarum subsp. plantarum bacteriocin and Nisin in the culture medium system on Listeria monocytogenes

Within the limit of 0.5g/kg of Nisin required by the national standard GB/T2760-2007, dilute Lactobacillus plantarum subsp. 10mL of Listeria monocytogenes culture solution was added with 6 groups of bacteriocin bacteriostatic agents in different proportions, and the growth status of Listeria monocytogenes was determined by using a nephelometric analyzer WBS-100.

From the growth trend of Listeria monocytogenes in Table 2, it can be obtained that the growth of Listeria monocytogenes was inhibited to varying degrees after the addition of antibacterial agents, and when the total antibacterial activity of antibacterial agents was the same, both being 32AU, Z :N (3:1) group and Z:N (1:1) group had better inhibitory effects on Listeria monocytogenes than bacteriocin alone (Zhang-LL group and Nisin group), Z:N (3:1) group bacteriocin compound bacteriostatic agent can completely inhibit the growth of Listeria monocytogenes within 22 hours of its growth, and Z:N (3:1) group is determined to be the best ratio of bacteriocin antibacterial.

Table 1 The ratio of different concentrations of bacteriocin

Table 2 The growth trend of Listeria monocytogenes after adding different antibacterial agents

Note: Z: N (3: 1): the concentration ratio of Lactobacillus plantarum subsp. plantarum to Nisin is 3:1; Z: N (1:1): the concentration ratio of Lactobacillus plantarum subsp. plantarum to Nisin is 1:1; Z:N (1:3): The concentration ratio of Lactobacillus plantarum subsp plantarum bacteriocin to Nisin is 1:3.

3. Preparation of Lactobacillus plantarum subsp. plantarum bacteriocin and Nisin composite bacteriostatic agent

The optimal proportioning ratio of Lactobacillus plantarum subspecies bacteriocin and Nisin compounded by above-mentioned test optimization is obtained, and the preparation method of its compound bacteriostatic agent is as follows:

The titer is 1280AU/mL Lactobacillus plantarum subsp. plantarum bacteriocin was diluted twice to 320AU/mL with sterile distilled water; Its potency is 320AU/mL. The Lactobacillus plantarum subsp. plantarum bacteriocin and Nisin are mixed according to the volume ratio of 3:1 to obtain the compound antibacterial agent.

Embodiment 2, the application of Lactobacillus plantarum subspecies bacteriocin and Nisin compound antibacterial agent to Listeria monocytogenes in chilled fresh meat

1. Referring to the sampling method and quantity requirements of GB/T 4789.1-2003, divide the freshly purchased chilled pork that has been cooled and deacidified for 24 hours into small pieces of about 25g, and inoculate 4.5×10 ² CFU/g monocytes on the surface of the meat pieces Listeria monocytogenes. The meat pieces were divided into 4 groups, and the following treatments were carried out respectively: uniform spraying of sterile water, addition of 10 mL/kg of Lactobacillus plantarum subsp. The compound antibacterial agent (Z:N=3:1) was placed in the sterile room for 30 minutes, turned over to dry, and put into a sterile fresh-keeping bag. All operations were performed in the sterile room. The packaged meat pieces were placed in a 4°C refrigerator and stored under light irradiation to simulate actual sales conditions. Samples were taken regularly on the 1d, 4d, 7d, 10d, 13d, and 20d of chilled meat storage. Take 25g samples each time, place them in 225mL 0.9% normal saline, homogenize them, and dilute them tenfold to an appropriate concentration. Use PALCAM Listeria Selective Medium to count Listeria monocytogenes, with 3 parallels in each group .

2. Evaluation of the antibacterial effect of compound antibacterial agents in the application of cold fresh meat preservation

Add two kinds of bacteriocins (Lactobacillus plantarum subsp. plantarum bacteriocin and Nisin) and their compound antibacterial agent to chilled meat separately, and take no treatment as a control, and calculate the test group regularly during the 20d storage period. and the total number of Listeria monocytogenes colonies in the blank control group, and calculate the bacteriostatic rate.

The formula for calculating the antibacterial rate is:

X=(A-B)/A×100%

In the formula, X is the bacteriostatic rate (%);

A is the total number of colonies (CFU/mL) of the blank control group Listeria monocytogenes;

B is the total number of colonies (CFU/mL) of Listeria monocytogenes in the test group.

Evaluation criteria: antibacterial rate ≥ 50% to 90%, bacteriocin has antibacterial effect, antibacterial rate ≥ 90%, bacteriocin has strong antibacterial effect.

Table 3 Inhibitory effects of different bacteriostatic agents on Listeria monocytogenes in chilled fresh meat

Note: Z:N (3:1): The concentration ratio of Lactobacillus plantarum subsp. plantarum bacteriocin to Nisin is 3:1.

Table 4 The antibacterial rate of different antibacterial agents against Listeria monocytogenes in chilled fresh meat

Note: Z:N (3:1): The concentration ratio of Lactobacillus plantarum subsp. plantarum bacteriocin to Nisin is 3:1.

When different antibacterial agents were applied to chilled fresh meat, the inhibitory effects on Listeria monocytogenes were shown in Table 3 and Table 4. Adding Lactobacillus plantarum subsp. Reduce the concentration of Listeria monocytogenes by 10 times; and add the compound antibacterial agent Z:N (3:1) of the two, and completely inhibit the growth of Listeria monocytogenes on the 4th to 13th day of storage, which is reduced by 10,000 times. It can be seen from Table 4 that the inhibitory effect of Lactobacillus plantarum subsp. plantarum bacteriocin and Nisin on the growth of L. monocytogenes in pork was gradually enhanced, and the bacteriostatic rate was less than 50% in the first 7 days of storage; Bacterial rate ≥ 50%, with obvious inhibitory effect. The compound antibacterial agent Z:N (3:1) has a strong inhibitory effect on the growth of Listeria monocytogenes in pork, and the antibacterial rate is less than 50% within 1 day of storage. ≥90%. The test results show that Lactobacillus plantarum subspecies bacteriocin and Nisin compound antibacterial agent added to chilled meat can inhibit the growth of spoilage bacteria Listeria monocytogenes within 20 days of storage. It can be seen that Lactobacillus plantarum Plant subspecies bacteriocin and Nisin compound antibacterial agent have obvious antiseptic effect in chilled meat.

In summary, the application of Lactobacillus plantarum subsp. planta bacteriocin and Nisin compound bacteriostatic agent to chilled fresh meat can inhibit Listeria monocytogenes by 1000 times compared with the effect of using bacteriocin alone. The preservation period was extended by 13 days.

Project 1 of this patent: the sub-project of the "12th Five-Year" National High-tech Research and Development Plan (863) project of the Ministry of Science and Technology "Research on Accurate Detection and Control Technology of Food Biological Hazards" sub-project "Safety Control Technology of Pathogens in Livestock Products"

Item number: 2012AA101606-05

Project start and end time: 2012.01-2015.12

Project leader: Zhang Hongxing

Project 2 of this patent: "Research on the antibacterial mechanism and effect of natural lactic acid bacteria on important pathogenic bacteria in livestock products" for the introduction and training of high-level talents in Beijing Municipal Universities.

Project No.: CIT&TCD20140315

Project start and end time: 2014.01-2016.12

Project leader: Zhang Hongxing

Project 3 of this patent: The Ministry of Science and Technology's major national science and technology project "Cultivation of new breeds of disease-resistant transgenic sheep" sub-project "Establishment of multiplication system for disease-resistant transgenic sheep / disease resistance, production performance and safety evaluation of disease-resistant transgenic sheep"

Item number: 2013ZX08008-005

Project start and end time: 2013.01-2013.12

Project leader: Liu Hui.

Claims (3)

1. plant lactobacillus plant subspecies (Lactobacillus plantarum subsp.plantarum) Zhang-LLCGMCC No.6936.

2. a preparation method for plant lactobacillus plant subspecies bacteriocin Zhang-LL and Nisin compound preservative, is characterized in that: get and tire as 1280AU/mL plant lactobacillus plant subspecies bacteriocin sterile distilled water carries out two times of gradient dilutions to 320AU/mL; Nisin is mixed with the working fluid of 5% (W/V), and it is tired as 320AU/mL.Plant lactobacillus plant subspecies bacteriocin and Nisin mix according to 3: 1 volume ratios, obtain compound preservative.

3. a plant lactobacillus plant subspecies bacteriocin Zhang-LL and Nisin compound preservative application method in cold fresh meat, is characterized in that: live hog, cause dusk, bloodletting, decaptitate, peeling, excision internal organ, sterilization, cooling, segmentation pick a bone, spray compound preservative, packaging.Spraying after singulation tires as 320AU/mL compound preservative, and fountain height is 0.1mL/cm ², the cold fresh meat of per kilogram need spray about 10mL fungistat, 4 DEG C of air-dry rear preservations.