Natto food antibacterial preservative, preparation method and application

Abstract

The invention provides a natto food antibacterial preservative, a preparation method and application, and belongs to the technical field of food preservation, the preservative is mainly prepared from the following raw materials: mixed beans, protease, bacillus natto and enterococcus faecium; wherein the mixed beans consist of the following components in parts by mass: 30-50 parts of soybeans, 40-50 parts of peas, 5-10 parts of oats and 10-20 parts of wheat; the use amount of the protease is 0.1-0.5% of the total mass of the mixed beans; the use amount of the bacillus natto is 1%-3% of the total mass of the mixed beans; the dosage of the enterococcus faecium is 1%-3% of the total mass of the mixed beans. Through scientific compatibility, the antibacterial and preservative effects of the natto paste are improved, and the technical blank of organic natto preservatives in the field of long-acting bacteriostasis of food bodies is filled.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food preservation, and particularly relates to a natto food antibacterial preservative, a preparation method and application thereof. BACKGROUND

[0002] The chemical preservatives (such as sodium benzoate and potassium sorbate) widely used in the current food industry have significant limitations. Long-term intake of chemical preservatives may cause safety hazards, and natural preservatives such as Nisin are only effective against gram-positive bacteria and have poor stability and are easily inactivated in acidic environments. In addition, inorganic antibacterial agents (such as silver ions and titanium dioxide) have surface antibacterial properties, but have problems such as short action time, easy migration and secondary pollution, and are difficult to achieve long-term inhibition in food bodies. With the increasing demand of consumers for green and safe food, the development of natural preservatives with broad-spectrum, high efficiency and non-toxicity has become an urgent need in the industry.

[0003] Natto bacteria (Bacillus subtilis natto) is a subspecies of Bacillus subtilis, which has been certified by the US FDA as a safe microorganism (GRAS). The antibacterial substances secreted by the natto bacteria include bacteriocins, antibacterial peptides, natto kinase and polysaccharides, which have significant inhibition and even killing effects on a variety of foodborne pathogens (such as Staphylococcus aureus, Escherichia coli and Shigella). Studies have shown that natto fermentation supernatant is effective against gram-positive bacteria (Staphylococcus aureus) and gram-negative bacteria (Escherichia coli), with a minimum inhibitory dilution factor of 26-27 times; the antibacterial protein is stable at pH 4-12 and a temperature of ≤80℃, and is resistant to enzymatic hydrolysis, which is superior to most natural preservatives; the antibacterial components can be decomposed with food in nature, without residual toxicity.

[0004] However, the industrial application of existing natto antibacterial agents still faces three major bottlenecks: (1) low extraction efficiency of active ingredients: the traditional fermentation broth is directly dried or prepared by simple centrifugation, and the active bacteria rate of the prepared bacterial agent is low (usually <50%), and the antibacterial protein is easily inactivated during the extraction process; (2) lack of synergy in the complex system: single natto extract has weak inhibitory effect on molds and yeasts, and needs to be compounded with other natural ingredients (such as perilla essential oil) to broaden the antibacterial spectrum, but the existing compounding technology lacks quantitative optimization of synergistic effect; (3) poor long-term stability: conventional natto bacterial agents can only be stored at room temperature for 2-7 days, and the cold storage shelf life is less than 1 month, which is difficult to meet the needs of food processing and circulation.

[0005] Therefore, the present application is proposed. SUMMARY

[0006] The application aims to provide a natto food antibacterial preservative, a preparation method and application thereof.

[0007] To achieve the above-mentioned purpose of the application, the following technical solutions are adopted:

[0008] A natto food antibacterial preservative is prepared from the following raw materials: mixed beans, protease, natto Bacillus and Enterococcus faecium.

[0009] The mixed beans are composed of 30-50 parts of soybeans, 40-50 parts of peas, 5-10 parts of oats and 10-20 parts of wheat by mass fraction.

[0010] The amount of the protease is 0.1%-0.5% of the total mass of the mixed beans.

[0011] The amount of the natto Bacillus is 1%-3% of the total mass of the mixed beans.

[0012] The amount of the Enterococcus faecium is 1%-3% of the total mass of the mixed beans.

[0013] Preferably, the amount of the natto Bacillus is 2% of the total mass of the mixed beans.

[0014] Preferably, the amount of the Enterococcus faecium is 2% of the total mass of the mixed beans.

[0015] Preferably, the protease is neutral protease.

[0016] Preferably, the mixed beans are composed of 45 parts of soybeans, 30 parts of peas, 5 parts of oats and 20 parts of wheat by mass fraction.

[0017] Preferably, the amount of the protease is 0.3% of the total mass of the mixed beans.

[0018] Preferably, the natto Bacillus is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO:M 2021018; and the Enterococcus faecium is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO:M 2021017.

[0019] The application further provides a preparation method of the natto food antibacterial preservative, which comprises the following steps:

[0020] S1, soybean, peas, oats and wheat are expanded, crushed, soaked, and then added with protease for enzymolysis to obtain an enzymolysis solution;

[0021] S2, Bacillus natto is then added to the enzymolysis solution for anaerobic fermentation by standing;

[0022] S3, the temperature is adjusted, Enterococcus faecium is added, and aerobic fermentation is performed by stirring and rotating.

[0023] Preferably, the soaking in S1 is specifically as follows: using warm water at 48-52°C, soaking for 3-5 hours at a water-material mass ratio of 1:3.

[0024] Preferably, the enzymolysis in S1 is performed at 30-40°C and pH 7.

[0025] Preferably, the anaerobic fermentation by standing in S2 is performed at 36-38°C for 22-26 hours.

[0026] Preferably, the temperature is adjusted to 40-44°C in S3, and fermentation is performed for 3-5 days.

[0027] Preferably, the enzymolysis in S1 is performed at 37°C and pH 7.5.

[0028] Preferably, mixed bacteria 1 is added to the enzymolysis solution in S2, and anaerobic fermentation by standing is performed at 37°C for 24 hours.

[0029] Preferably, the temperature is adjusted to 42°C in S3, mixed bacteria 2 is added, aerobic fermentation is performed by stirring and rotating for 5 days.

[0030] Preferably, the stirring and rotating rate is 120 rpm.

[0031] Preferably, the specific steps of the expansion treatment are as follows: microwave expansion treatment is performed for 60 seconds using a 900-watt microwave.

[0032] Preferably, the crushing in the crushing step needs to be performed to 50-100 μm.

[0033] The application also provides application of the natto food antibacterial preservative prepared by the preparation method to food preservation.

[0034] Compared with the prior art, the application has the following beneficial effects:

[0035] The natto paste prepared by the specific raw material ratio, combined with the microwave puffing pretreatment and the two-stage composite bacteria fermentation process can cover gram-positive bacteria and negative bacteria at the same time, has broad-spectrum antibacterial property, overcomes the short storage period problem of traditional natto agent, has long-term stability, uses the natural natto bacteria and lactic acid bacteria composite system, the antibacterial components can be naturally decomposed, has no chemical residue risk, and meets the green food preservation trend. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 : finished natto paste;

[0037] Figure 2 : the antibacterial test results of the natto paste on E. coli K88, wherein 5-2 are the natto paste original sample and the distilled water diluted 50 times, 100 times and 200 times samples respectively, and 1 is the distilled water control;

[0038] Figure 3 : the antibacterial test results of the natto paste on Staphylococcus aureus, wherein 5-2 are the natto paste original sample and the distilled water diluted 50 times, 100 times and 200 times samples respectively, and 1 is the distilled water control. DETAILED DESCRIPTION

[0039] The technical solutions of the present application will be described clearly and completely in combination with specific embodiments, but those skilled in the art will understand that the following described embodiments are part of the embodiments of the present application, not all the embodiments, and are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application. The specific conditions are not specified in the embodiments, and are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, and are conventional products that can be purchased on the market.

[0040] The protease used in the embodiments of the present application is neutral protease (50,000 U / g, product number: 9068-59-1, purchased from Beijing Solabio Technology Co., Ltd.). The preservation number of Bacillus subtilis DK-03 is CCTCC NO: M 2021018, which was preserved in the China Center for Type Culture Collection (address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province) on January 5, 2021; the preservation number of Enterococcus faecium DK-01 is CCTCC NO: M 2021017, which was preserved in the China Center for Type Culture Collection (address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province) on January 5, 2021.

[0041] Example 1

[0042] Preparation of Natto Paste: 45g soybean, 30g pea, 5g oat, 20g wheat were used for raw material pretreatment: after 900w microwave puffing treatment for 60 seconds, ultrafine grinding to 50-100 micron particle size, using 50℃ warm water according to the material water ratio 1:3 soak 3-5 hours. Next, add 0.3% neutral protease (50,000 U / g, item number: 9068-59-1, purchased from Beijing Solabio Technology Co., Ltd.), enzymolysis at 37℃, pH 7.5 for 1h.

[0043] Subsequently, the strain was added for 2-stage fermentation treatment, the first stage 37℃, adding 2% of the total mass of beans Enterococcus faecalis, static anaerobic fermentation for 24 hours; the second stage temperature adjustment to 42℃, adding 2% of the total mass of beans Enterococcus faecalis, stirring speed 120 rpm, aerobic fermentation for 3-5 days; stop fermentation when the original soybean flavor disappears, put into the freezer for cold storage. The prepared natto paste is shown in Figure 1

[0044] Experimental Example 1

[0045] Preparation of bacterial solution: single colonies of Staphylococcus aureus (ATCC 25923) and Escherichia coli K88 were inoculated into 5ml broth and cultured at 35℃ for 4-6 hours (logarithmic growth phase). The bacterial solution was adjusted to 0.5 McFarland turbidity (≈1.5×10 8 CFU / ml) with normal saline.

[0046] Plate inoculation: a sterile cotton swab was soaked in the bacterial solution and the excess liquid was squeezed off the tube wall. The MHA and LB plates were rotated and smeared in three directions (ensure uniformity and no gaps, Staphylococcus aureus was smeared on MHA plates and Escherichia coli was smeared on LB plates), and the surface was allowed to dry for 5 minutes.

[0047] Natto paste sample: 200μl of the natto paste sample, 50-fold, 100-fold and 200-fold dilutions of distilled water were spotted onto the pathogenic bacteria plates (Oxford cups can be used), with a distance of ≥24mm (to avoid overlapping interference), and then transferred to a 37℃ incubator for culture within 15 minutes. The experiment was repeated 3 times, and after 16-18 hours of inverted culture in a 37℃ constant temperature incubator, the diameter of the inhibition zone was measured with a vernier caliper (including the diameter of the Oxford cup, accurate to 0.1mm).

[0048] The results of the natto paste sample inhibition are shown in Table 1, Figures 2-3 The original natto paste sample had strong inhibition activity against Escherichia coli and Staphylococcus aureus, the 50-fold diluted sample had moderate inhibition activity, the 100-fold diluted sample could still inhibit the growth of Escherichia coli and Staphylococcus aureus, and the 200-fold diluted sample did not detect inhibition activity.

[0049] ​Table 1: Natto paste bacteriostatic circle determination results (average ± standard error) of pathogenic bacteria

[0050]

[0051]

[0052] Common gram-negative pathogenic bacteria Escherichia coli and common gram-positive pathogenic bacteria Staphylococcus aureus were selected as indicator bacteria to evaluate the bacteriostatic effect of natto paste. The results showed that the natto paste had obvious bacteriostatic activity against Escherichia coli and Staphylococcus aureus. After 50-fold dilution, the bacteriostatic activity was moderate, and after 100-fold dilution, the growth of Escherichia coli and Staphylococcus aureus could still be inhibited (if 1% natto paste is added, the growth of Escherichia coli and Staphylococcus aureus can still be inhibited, which can achieve good preservation and bacteriostatic effect).

[0053] Experimental Example 2

[0054] Bread was prepared as follows: flour, yeast, salt and water were mixed, and 2% by mass of natto paste was added, and kneaded into a smooth dough. The dough was placed in a warm place to ferment until the volume increased by about 2 times, and after fermentation was completed, the bubbles were gently discharged, and the dough was divided into small pieces. The dough was placed in a preheated oven and baked at 180℃ for 15 minutes to complete the bread making, and the shelf life of the natto bread was tested, and the test basis was GB / T 20981-2021 "General Quality of Bread". The product was stored at 45℃±2℃ (temperature / ℃) for accelerated destructive test, and the changes of product quality and food safety related indexes were evaluated by node test results, so as to infer the shelf life range of the product. The detection scheme is shown in Table 2, and the test results are shown in Table 3.

[0055] Table 2: Detection scheme

[0056]

[0057] As shown in Table 3, the sample was stored at a temperature of 45℃±2℃ for 24 days, and the tested items met the requirements of GB / T 20981-2021 "General Quality of Bread" and GB 7099-2015 "National Food Safety Standard for Cakes and Bread". According to the formula calculation, it is inferred that the shelf life of the product at room temperature (25℃) can reach the expected 3 months. According to the test results, the natto paste preservative prepared by the technical scheme of the application has excellent antibacterial and preservative effect.

[0058] Table 3: Test results:

[0059]

[0060]

[0061] Comparative Example

[0062] Commercial preservative and fungicide: sodium benzoate (purchased from Shanghai Yuan Ye Biotechnology Co., Ltd., item number: S30127), potassium sorbate (purchased from Shanghai Yuan Ye Biotechnology Co., Ltd., item number: T25295).

[0063] Bread was prepared according to the method in Example 2, with 2% of the commercial preservative and fungicide sodium benzoate added.

[0064] Bread was prepared according to the method in Example 2, with 2% of the commercial preservative and fungicide potassium sorbate added.

[0065] The bacteriostatic activity of the prepared fermented natto paste preservative and sodium benzoate and potassium sorbate was determined by dilution plate count method. The results were as follows: the fermented natto paste preservative had a bacteriostatic rate of 97.3% on Staphylococcus aureus and 99.8% on Escherichia coli; sodium benzoate had a bacteriostatic rate of 90.5% on Staphylococcus aureus and 92.1% on Escherichia coli; and potassium sorbate had a bacteriostatic rate of 89.6% on Staphylococcus aureus and 87.3% on Escherichia coli.

[0066] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the same; although the present application has been described in detail with reference to the foregoing examples, those of ordinary skill in the art will understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for some or all of the technical features; and such modifications or substitutions do not cause the essence of the corresponding technical solution to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. An antibacterial and preservative agent for natto food, characterized in that, It is mainly made from the following raw materials: mixed legumes, protease, Bacillus natto, and Enterococcus faecalis; The mixed legumes, by weight, consist of 30-50 parts soybeans, 40-50 parts peas, 5-10 parts oats, and 10-20 parts wheat. The amount of the protease used is 0.1%-0.5% of the total mass of the mixed legumes; The amount of Bacillus natto used is 1%-3% of the total mass of the mixed beans; The amount of Enterococcus faecalis used is 1%-3% of the total mass of the mixed legumes.

2. The antibacterial and preservative agent for natto food according to claim 1, characterized in that, The amount of Bacillus natto used is 2% of the total mass of the mixed beans.

3. The antibacterial and preservative agent for natto food according to claim 1, characterized in that, The amount of Enterococcus faecalis used is 2% of the total mass of the mixed legumes.

4. The antibacterial and preservative agent for natto food according to claim 1, characterized in that, The mixed legumes, by weight, consist of 45 parts soybeans, 30 parts peas, 5 parts oats, and 20 parts wheat; The amount of protease used is 0.3% of the total mass of the mixed legumes.

5. The antibacterial and preservative agent for natto food according to claim 1, characterized in that, The Bacillus natto is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 2021018; the Enterococcus faecalis is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M2021017.

6. A method for preparing an antibacterial and preservative agent for natto food as described in any one of claims 1-5, characterized in that, Includes the following steps: S1. Soybeans, peas, oats and wheat are puffed, crushed and soaked, and then protease is added to carry out enzymatic hydrolysis to obtain enzymatic hydrolysate; S2. Then Bacillus natto is added to the enzymatic hydrolysate and allowed to ferment anaerobically. S3. Adjust the temperature, add Enterococcus faecalis, stir and rotate for aerobic fermentation.

7. The method for preparing the antibacterial and preservative agent for natto food according to claim 6, characterized in that, The soaking process in S1 involves soaking the material in warm water at 48℃-52℃ for 3-5 hours at a water-to-material mass ratio of 1:

3.

8. The method for preparing the antibacterial and preservative agent for natto food according to claim 7, characterized in that, In the pulverizing step, the soybeans, peas, oats, and wheat need to be pulverized to 50-100μm.

9. The application of an antibacterial preservative for natto prepared by the preparation method according to any one of claims 7-8 in food preservation.

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