Fermentation composition for inhibiting food-borne harmful bacteria and preparation method thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicine fermentation, and particularly relates to a fermentation composition for inhibiting food-borne harmful bacteria and a preparation method thereof.The fermentation composition is prepared from traditional Chinese medicine and a leavening agent, and the traditional Chinese medicine comprises honeysuckle and golden camellia. The bacteriostatic fermentation composition provided by the invention has the characteristics of naturalness, safety and no side effect, and can effectively inhibit the growth of staphylococcus aureus, saccharomyces cerevisiae and aspergillus niger, so that the shelf life of food can be prolonged, and the safety and nutritional value of the food can be improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine fermentation, and particularly relates to a fermentation composition for inhibiting foodborne harmful bacteria and a preparation method thereof. Background Art

[0002] Honeysuckle, also known as Lonicera japonica Thunb., is sweet in taste and cold in nature, and has a strong antibacterial effect. Its main antibacterial components are chlorogenic acid, isochlorogenic acid, ginkgolic acid, luteolin, caffeic acid, etc. For example, Chinese Patent CN117257858A discloses a traditional Chinese medicine essential oil composition JY-1 with antibacterial and antiviral effects, its application and testing method, which includes components such as honeysuckle extract, camellia nitidissima extract, atractylodes extract, baicalein, rosemary essential oil, etc., and has the effect of antibacterial against bacteria such as Staphylococcus aureus and pathogenic fungi such as Candida.

[0003] Another example is that Chinese Patent CN106421855A discloses an in-vehicle air freshener with a bactericidal function and its production process, which is composed of raw materials such as honeysuckle, camellia nitidissima, atractylodes, mugwort, tea tree oil, etc., and has an antibacterial effect on Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans.

[0004] Staphylococcus aureus is a common foodborne pathogenic bacterium and is often contaminated during the food processing process. Yeast is also one of the most important microorganisms in beverages. Since yeast is acid-tolerant and mostly has high permeability, it can survive in general beverage products. The reproduction of yeast will produce carbon dioxide, causing the beverage product to expand the bottle and form unpleasant flavors such as ester smell and yeast smell; and mold is also highly acid-tolerant. When it appears in beverages, it can cause mold spots and musty smell in the product, and damage the color of the product. Some molds will also produce ethanol or other toxins, which are harmful to human health. There is currently no report on the antibacterial effect of honeysuckle on yeast and mold. Summary of the Invention

[0005] Therefore, on the one hand, the present invention provides a fermentation composition, which includes two raw materials, honeysuckle and camellia nitidissima, and can achieve the effect of inhibiting Staphylococcus aureus, yeast and mold after fermentation.

[0006] On the other hand, a method for preparing the above fermentation composition is provided, and this method is simple and can be used for industrial production.

[0007] To achieve the above object, the technical content of the present invention is as follows:

[0008] On the one hand, the present invention provides a fermentation composition for inhibiting foodborne harmful bacteria, which is mainly prepared from traditional Chinese medicine and a fermenting agent, and the traditional Chinese medicine includes honeysuckle and camellia nitidissima.

[0009] Preferably, the traditional Chinese medicine, by weight, comprises 30-50 parts of honeysuckle and 40-180 parts of golden camellia.

[0010] More preferably, the traditional Chinese medicine, by weight, comprises 35-45 parts of honeysuckle and 60-120 parts of golden camellia.

[0011] Even more preferably, the weight ratio of honeysuckle to golden camellia is 1:(1.3-4).

[0012] Preferably, the weight ratio of the traditional Chinese medicine to the fermenting agent is (625-800):1.

[0013] More preferably, the weight ratio of the traditional Chinese medicine to the fermenting agent is 680:1.

[0014] Preferably, the fermenting agent comprises at least one of Lactiplantibacillus plantarum, Lactobacillus acidophilus, and Lactobacillus mucosae fermentans.

[0015] Preferably, the fermenting agent comprises Lactiplantibacillus plantarum and Lactobacillus acidophilus.

[0016] More preferably, the fermenting agent comprises 0.08-0.2 parts of Lactiplantibacillus plantarum and 0.02-0.1 parts of Lactobacillus acidophilus.

[0017] More preferably, the weight ratio of Lactiplantibacillus plantarum to Lactobacillus acidophilus in the fermenting agent is (2-4):(1-2).

[0018] Even more preferably, the weight ratio of Lactiplantibacillus plantarum to Lactobacillus acidophilus is 3:2.

[0019] Even more preferably, the viable count of Lactiplantibacillus plantarum is 100-1000 billion CFU / g, and the viable count of Lactobacillus acidophilus is 100-1000 billion CFU / g.

[0020] On the other hand, the present invention provides a method for preparing the above antibacterial fermented composition, comprising the following steps:

[0021] (1) Soak the traditional Chinese medicine in water and then decoct and extract to obtain a decoction;

[0022] (2) Sterilize the decoction to obtain a sterilized solution;

[0023] (3) Add the fermenting agent to the sterilized solution for fermentation to obtain a fermented liquid;

[0024] (4) Perform solid-liquid separation on the fermented liquid, and concentrate, spray-dry the fermented supernatant to obtain the product.

[0025] Preferably, it further includes a step of pulverizing traditional Chinese medicine, and the pulverizing conditions are: pulverizing temperature 20 - 40°C, pulverizing pressure 10 - 15 Mpa, air flow rate 300 - 400 m / s, and pulverizing time 1 - 2 h.

[0026] Preferably, in step (1), the weight ratio of water to traditional Chinese medicine is (8 - 12):1.

[0027] Preferably, in step (1), the soaking time of traditional Chinese medicine is 15 - 30 min; the decocting temperature is 80 - 110°C, and the decocting time is 30 - 80 min.

[0028] Preferably, in step (2), the sterilization temperature is 100 - 130°C, and the sterilization time is 10 - 30 min.

[0029] Preferably, in step (3), the fermentation temperature is 20 - 35°C, and the fermentation time is 12 - 24 h.

[0030] On the other hand, the present invention also provides an application of the above composition and the fermentation composition prepared by the above method in antibacterial products or as an antibacterial agent in the food field.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] 1. The fermentation composition for inhibiting foodborne harmful bacteria provided by the present invention selects honeysuckle and camellia nitidissima as raw materials, which are natural plant raw materials fermented by microorganisms, and have the characteristics of being natural, safe, and non-toxic. Moreover, the obtained fermentation composition can effectively inhibit the growth of Staphylococcus aureus, yeast, and mold, and can be used as a preservative in beverages or other foods.

[0033] 2. The fermentation composition for inhibiting foodborne harmful bacteria provided by the present invention can extend the shelf life of food, improve the safety and nutritional value of food.

[0034] 3. The preparation method of the traditional Chinese medicine antibacterial agent provided by the present invention is simple, with low cost, and is convenient for popularization. Detailed Embodiments

[0035] The following non-limiting embodiments can enable those of ordinary skill in the art to understand the present invention more comprehensively, but do not limit the present invention in any way. The following content is only an exemplary illustration of the scope claimed by the present invention. Those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and it should also fall within the scope claimed by the present invention.

[0036] When numerical ranges are given in the embodiments, it should be understood that unless otherwise specified in the present invention, either endpoint of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0037] The present invention will be further described below by way of specific embodiments. All kinds of chemical reagents used in the embodiments of the present invention are obtained through conventional commercial channels unless otherwise specified. Products from different manufacturers have no significant impact on the effects.

[0038] Lactobacillus plantarum was purchased from Xi'an Rongzhen Biotechnology Co., Ltd.; Lactobacillus acidophilus was purchased from Shandong Zhongke Jiayi Biotechnology Co., Ltd., Lactobacillus acidophilus JYLA - 191; Bifidobacterium adolescentis was purchased from Ningbo Mingzhou Biotechnology Co., Ltd., Bifidobacterium adolescentis B71914.

[0039] Example 1

[0040] This example provides an antibacterial fermentation composition, including 40 parts by weight of honeysuckle, 70 parts by weight of camellia flower, 0.097 parts by weight of Lactobacillus plantarum, 0.065 parts by weight of Lactobacillus acidophilus, and 1100 parts by weight of water. The viable count of Lactobacillus plantarum is 100 billion CFU / g, and the viable count of Lactobacillus acidophilus is 10 billion CFU / g.

[0041] Its preparation method is as follows:

[0042] (1) Crush 40 parts by weight of honeysuckle and 70 parts by weight of camellia flower at a temperature of 25°C, a pressure of 12 Mpa, an air flow rate of 350 m / s for 1.5 h to obtain traditional Chinese medicine powder; (2) Soak the traditional Chinese medicine powder in 1100 parts by weight of water for 20 min, and then decoct it at 100°C for 40 min to obtain a decoction; (3) Sterilize the decoction at 121°C for 15 min to obtain a sterilized solution; (4) After the sterilized solution is cooled to 30°C, add 0.097 parts by weight of Lactobacillus plantarum and 0.065 parts by weight of Lactobacillus acidophilus, and ferment at 30°C for 15 h to obtain a fermentation broth; (5) Separate the solid and liquid of the fermentation broth to obtain the upper fermentation supernatant, and concentrate, spray, powder, and dry the fermentation supernatant to obtain the fermentation composition.

[0043] Example 2

[0044] This example provides an antibacterial fermentation composition, including 30 parts by weight of honeysuckle, 40 parts by weight of camellia flower, 0.084 parts by weight of Lactobacillus plantarum, 0.028 parts by weight of Lactobacillus acidophilus, and 840 parts by weight of water. The viable count of Lactobacillus plantarum is 100 billion CFU / g, and the viable count of Lactobacillus acidophilus is 10 billion CFU / g.

[0045] Its preparation method is as follows:

[0046] (1) Mix 30 parts by weight of honeysuckle and 40 parts by weight of golden camellia, pulverize them at a temperature of 20°C, a pressure of 15 Mpa, and an air flow rate of 400 m / s for 1 h to obtain traditional Chinese medicine powder; (2) Soak the traditional Chinese medicine powder in 840 parts by weight of water for 15 min, then decoct it at 80°C for 60 min to obtain a decoction; (3) Sterilize the decoction at 100°C for 30 min to obtain a sterilized liquid; (4) After the sterilized liquid cools to 25°C, add 0.084 parts by weight of Lactiplantibacillus plantarum and 0.028 parts by weight of Lactobacillus acidophilus, and ferment at 25°C for 24 h to obtain a fermentation liquid; (5) Separate the solid and liquid of the fermentation liquid to obtain the upper-layer fermentation supernatant, and concentrate, spray-dry the fermentation supernatant to obtain the fermentation composition.

[0047] Example 3

[0048] This example provides an antibacterial fermentation composition, including 35 parts by weight of honeysuckle, 60 parts by weight of golden camellia, 0.109 parts by weight of Lactiplantibacillus plantarum, 0.027 parts by weight of Lactobacillus acidophilus, and 1045 parts by weight of water. The viable count of Lactiplantibacillus plantarum is 100 billion CFU / g, and the viable count of Lactobacillus acidophilus is 10 billion CFU / g.

[0049] Its preparation method is as follows:

[0050] (1) Mix 35 parts by weight of honeysuckle and 60 parts by weight of golden camellia, pulverize them at a temperature of 25°C, a pressure of 12 Mpa, and an air flow rate of 350 m / s for 1.5 h to obtain traditional Chinese medicine powder; (2) Soak the traditional Chinese medicine powder in 1045 parts by weight of water for 15 min, then decoct it at 90°C for 50 min to obtain a decoction; (3) Sterilize the decoction at 110°C for 25 min to obtain a sterilized liquid; (4) After the sterilized liquid cools to 25°C, add 0.109 parts by weight of Lactiplantibacillus plantarum and 0.027 parts by weight of Lactobacillus acidophilus, and ferment at 25°C for 20 h to obtain a fermentation liquid; (5) Separate the solid and liquid of the fermentation liquid to obtain the upper-layer fermentation supernatant, and concentrate, spray-dry the fermentation supernatant to obtain the fermentation composition.

[0051] Example 4

[0052] This example provides an antibacterial fermentation composition, including 45 parts by weight of honeysuckle, 180 parts by weight of golden camellia, 0.2 parts by weight of Lactiplantibacillus plantarum, 0.1 parts by weight of Lactobacillus acidophilus, and 2025 parts by weight of water. The viable count of Lactiplantibacillus plantarum is 100 billion CFU / g, and the viable count of Lactobacillus acidophilus is 10 billion CFU / g.

[0053] Its preparation method is as follows:

[0054] (1) Take 45 parts by weight of honeysuckle and 180 parts by weight of golden camellia, pulverize them at a temperature of 40 °C, a pressure of 10 Mpa, an air flow rate of 350 m / s for 2 h to obtain traditional Chinese medicine powder; (2) Soak the traditional Chinese medicine powder in 2025 parts by weight of water for 25 min, and then decoct it at 105 °C for 30 min to obtain a decoction; (3) Sterilize the decoction at 115 °C for 20 min to obtain a sterilized liquid; (4) After the sterilized liquid is cooled to 35 °C, add 0.2 parts by weight of Lactiplantibacillus plantarum and 0.1 parts by weight of Lactobacillus acidophilus, and ferment at 35 °C for 16 h to obtain a fermentation broth; (5) Separate the solid and liquid of the fermentation broth to obtain the upper-layer fermentation supernatant, and concentrate and spray-dry the fermentation supernatant to obtain the fermentation composition.

[0055] Example 5

[0056] This example provides an antibacterial fermentation composition, including 50 parts by weight of honeysuckle, 120 parts by weight of golden camellia, 0.142 parts by weight of Lactiplantibacillus plantarum, 0.071 parts by weight of Lactobacillus acidophilus and 1360 parts by weight of water. The viable count of Lactiplantibacillus plantarum is 100 billion CFU / g, and the viable count of Lactobacillus acidophilus is 10 billion CFU / g.

[0057] Its preparation method is as follows:

[0058] (1) Take 40 parts by weight of honeysuckle and 70 parts by weight of golden camellia, pulverize them at a temperature of 25 °C, a pressure of 12 Mpa, an air flow rate of 300 m / s for 1.5 h to obtain traditional Chinese medicine powder; (2) Soak the traditional Chinese medicine powder in 1360 parts by weight of water for 30 min, and then decoct it at 110 °C for 30 min to obtain a decoction; (3) Sterilize the decoction at 130 °C for 10 min to obtain a sterilized liquid; (4) After the sterilized liquid is cooled to 35 °C, add 0.142 parts by weight of Lactiplantibacillus plantarum and 0.071 parts by weight of Lactobacillus acidophilus, and ferment at 35 °C for 12 h to obtain a fermentation broth; (5) Separate the solid and liquid of the fermentation broth to obtain the upper-layer fermentation supernatant, and concentrate and spray-dry the fermentation supernatant to obtain the fermentation composition.

[0059] Comparative Example 1

[0060] This comparative example provides an antibacterial fermentation composition, which is different from Example 1 only in that it does not include honeysuckle, that is, it includes 110 parts by weight of golden camellia, 0.097 parts by weight of Lactiplantibacillus plantarum, 0.065 parts by weight of Lactobacillus acidophilus and 1100 parts by weight of water. The viable count of Lactiplantibacillus plantarum is 100 billion CFU / g, and the viable count of Lactobacillus acidophilus is 10 billion CFU / g.

[0061] Its preparation method is the same as that of Example 1.

[0062] Comparative Example 2

[0063] This comparative example provides an antibacterial fermentation composition, which is different from Example 1 only in that it does not include Camellia nitidissima, that is, it includes 110 parts by weight of honeysuckle, 0.097 parts by weight of Lactiplantibacillus plantarum, 0.065 parts by weight of Lactobacillus acidophilus, and 1100 parts by weight of water. Among them, the viable count of Lactiplantibacillus plantarum is 100 billion CFU / g, and the viable count of Lactobacillus acidophilus is 10 billion CFU / g.

[0064] Its preparation method is the same as that of Example 1.

[0065] Comparative Example 3

[0066] This comparative example provides an antibacterial fermentation composition, which is different from Example 1 in that it does not undergo the decocting process.

[0067] Its preparation method is as follows:

[0068] (1) 40 parts by weight of honeysuckle and 70 parts by weight of Camellia nitidissima are pulverized at a temperature of 25°C, a pressure of 12 Mpa, an air flow rate of 350 m / s for 1.5 h to obtain traditional Chinese medicine powder; (2) the traditional Chinese medicine powder is soaked in 1100 parts by weight of water for 20 min and then sterilized at 121°C for 15 min to obtain a sterilized solution; (3) after the sterilized solution is cooled to 30°C, 0.097 parts by weight of Lactiplantibacillus plantarum and 0.065 parts by weight of Lactobacillus acidophilus are added, and fermentation is carried out at 30°C for 15 h to obtain a fermentation broth; (4) the fermentation broth is subjected to solid-liquid separation to obtain the upper-layer fermentation supernatant, and the fermentation supernatant is concentrated, spray-dried to obtain the fermentation composition.

[0069] Comparative Example 4

[0070] This comparative example provides an antibacterial composition, which is different from Example 1 in that it does not undergo the fermentation process.

[0071] Its preparation method is as follows:

[0072] (1) 40 parts by weight of honeysuckle and 70 parts by weight of Camellia nitidissima are pulverized at a temperature of 25°C, a pressure of 12 Mpa, an air flow rate of 350 m / s for 1.5 h to obtain traditional Chinese medicine powder; (2) the traditional Chinese medicine powder is soaked in 1100 parts by weight of water for 30 min and then decocted at 110°C for 30 min to obtain a decocted solution; (3) the decocted solution is sterilized at 130°C for 10 min to obtain a sterilized solution; (4) the sterilized solution is subjected to solid-liquid separation to obtain the supernatant, and the supernatant is concentrated, spray-dried to obtain the antibacterial composition.

[0073] Comparative Example 5

[0074] This comparative example provides an antibacterial fermentation composition, which is different from Example 1 in that the fermentation agent only contains 0.162 parts by weight of Lactiplantibacillus plantarum. The remaining components and the preparation method are the same as those of Example 1. Among them, the viable count of Lactiplantibacillus plantarum is 100 billion CFU / g.

[0075] Comparative Example 6

[0076] This comparative example provides an antibacterial fermentation composition, which is only different from Example 1 in that the starter contains 0.097 parts by weight of Lactiplantibacillus plantarum and 0.065 parts by weight of Bifidobacterium adolescentis. The remaining components and preparation method are the same as those in Example 1. The viable count of Lactiplantibacillus plantarum is 100 billion CFU / g, and the viable count of Bifidobacterium adolescentis is 100 billion CFU / g.

[0077] Experimental Example 1 Antibacterial Experiment

[0078] Experimental strains and sources: Staphylococcus aureus was purchased from Qingdao Haibo Biotechnology Co., Ltd. Staphylococcus aureus ATCC 6538; Aspergillus niger was purchased from Qingdao Haibo Biotechnology Co., Ltd. Aspergillus niger ATCC 16404; Saccharomyces cerevisiae was purchased from Guangzhou Huankai Biotechnology Co., Ltd. Saccharomyces cerevisiae ATCC 9763.

[0079] Culture Medium

[0080] (1) Beef extract peptone liquid medium: Sodium chloride 5 g / L, beef extract 3 g / L, peptone 10 g / L, prepare liquid medium. Sterilize at 115 °C for 20 min and reserve.

[0081] (2) Beef extract peptone solid medium: Prepared by adding 1.5 - 2.5% agar during the preparation of its liquid medium, sterilize at 115 °C for 20 min and reserve.

[0082] (3) Yeast complete medium YPD: Yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, prepare liquid medium. Sterilize at 115 °C for 20 min and reserve. Adding 1.5 - 2.5% agar powder becomes YPD solid medium.

[0083] (4) Potato dextrose broth (PDB) medium (purchased from Hangzhou Microorganism Reagent Co., Ltd.) and potato dextrose agar (PDA) medium (purchased from Hangzhou Microorganism Reagent Co., Ltd.) are used to culture Aspergillus niger, and the culture temperature is 30 °C.

[0084] Among them, beef extract peptone medium is used to culture Staphylococcus aureus, and the culture temperature is 37 °C; yeast complete medium YPD is used to culture Saccharomyces cerevisiae, and the culture temperature is 30 °C.

[0085] 1.1 Preparation of Antibacterial Agent

[0086] Use ultrapure water to prepare the antibacterial fermentation compositions prepared in Examples 1 - 5 and Comparative Examples 1 - 6 into a sample solution of 200 mg / mL. After autoclaving at 121 °C for 20 min, reserve.

[0087] 1.2 Activation of strains

[0088] Inoculate the cryopreserved strains into the corresponding liquid medium. Staphylococcus aureus is cultured in a shaker at 37°C for 24 h, and Saccharomyces cerevisiae and Aspergillus niger are cultured in a shaker at 30°C for 24 h. Use an inoculation loop to inoculate the activated bacterial liquid onto the corresponding solid medium. After the strains are cultured at the corresponding temperature for 24 h, select single colonies and inoculate them into 10 mL of liquid medium, and culture them in a shaker at the corresponding temperature for 24 h. Measure the absorbance value at 600 nm every 2 hours to determine the logarithmic growth phase of the strains. Determine the bacterial liquid concentration by the plate counting method, and dilute the bacterial liquid concentration to 1.0×108 CFU / mL with the corresponding liquid medium.

[0089] 1.3 Determination of inhibition zone

[0090] Place sterile filter paper with a diameter of 6 mm into the sterilized bacteriostatic agent and soak for 30 min. Pipette 100 μL of the bacterial liquid in the logarithmic growth phase and evenly spread it on the corresponding solid medium. Then place the soaked filter paper in the center of the solid medium and gently press it with sterile forceps to make it stick tightly to the surface of the solid medium. Cover the petri dish and place it in a constant temperature incubator for 24 h. Staphylococcus aureus is cultured at 37°C, and Saccharomyces cerevisiae and Aspergillus niger are cultured at 30°C. After the culture is completed, measure the diameter of the inhibition zone. The diameter of the inhibition zone is measured with a vernier caliper and repeated 3 times, and the average value is taken.

[0091] Table 1 Determination results of the diameter of the inhibition zone

[0092]

[0093] Note: Different letters in the same column represent significant differences, P≤0.05.

[0094] As can be seen from Table 1, obvious inhibition zones appeared in the antibacterial fermentation composition, indicating that the antibacterial fermentation composition has antibacterial effects on Staphylococcus aureus, Saccharomyces cerevisiae and Aspergillus niger. The inhibition zone diameters of Examples 1-5 are large, indicating obvious antibacterial effects on Staphylococcus aureus, Saccharomyces cerevisiae and Aspergillus niger.

[0095] Compared with Example 1, the antibacterial effects of the antibacterial fermentation compositions of Comparative Examples 1-2 on Staphylococcus aureus, Saccharomyces cerevisiae and Aspergillus niger are poor, indicating that the antibacterial effect of a single traditional Chinese medicine is not as good as that of the traditional Chinese medicine fermentation composition.

[0096] Compared with Example 1, the antibacterial effects of the antibacterial compositions of Comparative Examples 3-4 on Staphylococcus aureus, Saccharomyces cerevisiae and Aspergillus niger are poor, indicating that the antibacterial fermentation composition prepared by the preparation method used in the present invention has a good antibacterial effect.

[0097] The antibacterial effect of the antibacterial fermentation composition of Comparative Examples 5-6 is lower than that of Example 1, which indicates that the combined fermenting agent used in the present invention can better release the active ingredients in traditional Chinese medicine and exhibits a stronger antibacterial effect.

[0098] 1.4 Determination of minimum inhibitory concentration (MIC)

[0099] According to the two-fold dilution method, the antibacterial fermentation compositions of Example 1 and Comparative Examples 1-2 were prepared into antibacterial solutions of 200 mg / mL, 100 mg / mL, 50 mg / mL, 25 mg / mL, 12.5 mg / mL, 6.25 mg / mL, and 3.125 mg / mL with sterile water, and after mixing evenly, they were reserved for use. 100 μL of the activated bacterial solution was inoculated into fresh liquid medium, and antibacterial solutions of different concentrations in each group were added to the liquid medium so that the final concentrations were 20 mg / mL, 10 mg / mL, 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, and 0.3125 mg / mL. Staphylococcus aureus was cultured at 37 °C, and Saccharomyces cerevisiae and Aspergillus niger were cultured at 30 °C. After culturing in a shaker for 24 h, the results were observed, and the minimum inhibitory concentration was the one where the culture medium was clear and there was no turbidity. Each experiment was repeated 3 times.

[0100] Table 2 Determination of minimum inhibitory concentration (MIC) (mg / mL)

[0101]

[0102] It can be seen from the MIC determination results in Table 2 that the antibacterial effect of the antibacterial fermentation composition of Example 1 is better than that of Comparative Example 1 and Comparative Example 2, which indicates that the antibacterial effect of the traditional Chinese medicine fermentation composition is better than that of a single traditional Chinese medicine. Among them, the MIC value of the antibacterial fermentation composition of Example 1 against Staphylococcus aureus is 0.625 mg / mL, the MIC value against Saccharomyces cerevisiae is 1.25 mg / mL, and the MIC value against Aspergillus niger is 2.5 mg / mL.

[0103] 1.5 Bactericidal effect of traditional Chinese medicine antibacterial agent

[0104] 1.0 mL of the diluted solution of the activated bacterial solution was taken, and according to the MIC determination results under item "1.4", appropriate concentrations of the traditional Chinese medicine antibacterial solutions of Example 1, Comparative Example 1, and Comparative Example 2 were added respectively so that the final concentration was 1 MIC, and in addition, sterile water was used as the blank control group; Staphylococcus aureus was cultured at 37 °C, and Saccharomyces cerevisiae and Aspergillus niger were cultured at 30 °C.

[0105] 1.5.1 Determination of extracellular proteins and nucleic acids of bacterial cells

[0106] After culturing for 6 h respectively, the bacterial liquid was collected, centrifuged at 10,000 rpm for 5 min using a centrifuge, the supernatant was aspirated, and the absorbance values at 260 nm and 280 nm were measured using a microplate reader. Each experiment was repeated 3 times.

[0107] 1.5.2 Determination of the content of extracellular alkaline phosphatase (AKP) in bacterial cells

[0108] After culturing for 6 h respectively, the bacterial liquid was collected, centrifuged at 10,000 rpm for 5 min using a centrifuge, the supernatant was aspirated, and the AKP activity of the supernatant was determined according to the instructions of the kit. Each experiment was repeated 3 times.

[0109] Table 3 Determination of nucleic acid content

[0110]

[0111] Note: Different letters in the same column represent significant differences, P≤0.05.

[0112] Table 4 Determination of protein content

[0113]

[0114] Note: Different letters in the same column represent significant differences, P≤0.05.

[0115] Table 5 Determination of AKP activity.

[0116]

[0117] Note: Different letters in the same column represent significant differences, P≤0.05.

[0118] Proteins and nucleic acids exist inside cells, and AKP exists between the cell wall and the cell membrane. It cannot be detected extracellularly in normal cells. Only when the cell integrity is damaged, proteins, nucleic acids, and AKP will be released from the cells and can be detected extracellularly. Therefore, the integrity of the cell membrane can be determined by analyzing the amount of leaked proteins, nucleic acids, and AKP. As can be seen from Tables 3, 4, and 5, compared with the blank control group, the contents of proteins, nucleic acids, and AKP in Example 1 increased, indicating that after adding the traditional Chinese medicine bacteriostatic agent of the present invention, the cell membrane of the bacteria was damaged, causing the leakage of proteins, nucleic acids, and AKP in the bacterial cells, thereby playing a bacteriostatic role. Compared with Example 1, the contents of proteins, nucleic acids, and AKP in Comparative Example 1 and Comparative Example 2 decreased, indicating that the bacteriostatic effect of the bacteriostatic agent composed of the combination of honeysuckle and camellia nitidissima is better than that of the single bacteriostatic agent.

[0119] 1.6 Application of traditional Chinese medicine bacteriostatic agent in fruit juice

[0120] Experimental grouping: experimental group (bacteriostatic fermentation composition of Example 1), positive control group (chemical preservative: sodium benzoate), blank control group (without preservative), with 5 bottles in each group.

[0121] Experimental method:

[0122] After sterilizing the bacteriostatic agents in the experimental group and the positive control group, add them to 10 mL of fruit juice under aseptic conditions so that the concentration of the bacteriostatic agent is 0.625 mg / mL. Inoculate 1.0×103 CFU / mL of the bacterial liquid in the logarithmic growth phase into the fruit juice, mix well and seal. Place the fruit juices of the experimental group, the positive control group and the blank control group in an aging box for observation. When the blank control group deteriorates, detect the number of microorganisms in the beverage to evaluate the effect of the bacteriostatic agent on extending the shelf life of the beverage.

[0123] Plate counting method: Take 1 mL of fruit juice from each group and dilute it 10-fold into samples with different concentration gradients. Select a 1000-fold dilution for each sample. Take 200 μL of the diluted solution and evenly coat it on the corresponding solid medium. Cover the petri dish and incubate it in an incubator for 24 h. Staphylococcus aureus is cultured at 37 °C, and Saccharomyces cerevisiae and Aspergillus niger are cultured at 30 °C. Measure and record the number of colonies.

[0124] Table 6 Effects of bacteriostatic agents on the number of colonies (CFU / mL)

[0125]

[0126] The results show that: as can be seen from Table 6, compared with the blank control group, the total number of colonies using sodium benzoate and the bacteriostatic agent provided by the present invention decreases, indicating that both the bacteriostatic agent of the present invention and sodium benzoate can well control the growth of Staphylococcus aureus, Saccharomyces cerevisiae and Aspergillus niger, thus playing a bacteriostatic role.

[0127] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than a limitation on the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A fermentation composition for inhibiting foodborne harmful bacteria, characterized in that, It is mainly prepared from traditional Chinese medicine and a starter, wherein the traditional Chinese medicine includes honeysuckle and golden camellia.

2. The fermentation composition according to claim 1, wherein The traditional Chinese medicine, by weight, includes 30 - 50 parts of honeysuckle and 40 - 180 parts of golden camellia.

3. The fermentation composition according to claim 1, wherein The weight ratio of the honeysuckle to the golden camellia is 1:(1.3 - 4).

4. The fermentation composition according to claim 1, wherein The weight ratio of the traditional Chinese medicine to the starter is (625 - 800):

1.

5. The fermentation composition according to claim 1, wherein The starter includes 0.08 - 0.2 parts of Lactiplantibacillus plantarum and 0.02 - 0.1 parts of Lactobacillus acidophilus.

6. A method for preparing the fermentation composition according to any one of claims 1-5, characterized in that, It includes the following steps: (1) Soak the traditional Chinese medicine in water and then decoct and extract to obtain a decoction; (2) Sterilize the decoction to obtain a sterilized solution; (3) Add the starter to the sterilized solution for fermentation to obtain a fermentation broth; (4) Perform solid-liquid separation on the fermentation broth, and concentrate, spray-dry the clear fermentation liquid to obtain the product.

7. The preparation method according to claim 6, characterized in that, In the step (1), the weight ratio of water to the traditional Chinese medicine is (8 - 12):1; the soaking time of the traditional Chinese medicine in step (1) is 15 - 30 min; the decocting temperature is 80 - 110 °C, and the decocting time is 30 - 80 min.

8. The preparation method according to claim 6, characterized in that, In the step (3), the fermentation temperature is 20 - 35 °C, and the fermentation time is 12 - 24 h.

9. Use of the antibacterial composition according to any one of claims 1 - 5 or the antibacterial composition obtained by the preparation method according to any one of claims 6 - 8 in the preparation of antibacterial products and as an antibacterial agent in the food field.

10. A product for inhibiting foodborne harmful bacteria, characterized in that, It includes the composition according to claim 1 and other pharmaceutically or food-acceptable excipients.

Images