A bacteriostatic agent prepared from soy sauce residue and a preparation method and application thereof

Abstract

The application discloses a method for preparing a bacteriostatic agent by using soy sauce residues as raw materials, and comprises the following steps: (1) after the soy sauce residues are soaked with an ethanol solution, pre-filtration is carried out, the filtrate is desalted by using a nanofiltration device, and the nanofiltrate after desalting is rotary evaporated to remove ethanol to obtain a salt-free crude extract; (2) the salt-free crude extract in step (1) is subjected to fat removal treatment by using petroleum ether, and the raffinate phase is collected by centrifugation; (3) the raffinate phase in step (2) is sequentially extracted by using equal volumes of chloroform and ethyl acetate, the ethyl acetate extraction phase is collected, the ethyl acetate is removed by using a rotary evaporation method, the residue is dissolved with sterile water, and then microfiltration is carried out, so that the bacteriostatic agent is obtained. The method has low raw material cost, and the bacteriostatic agent prepared by using the method has remarkable bacteriostatic effect. The application further discloses the bacteriostatic agent prepared by using the method and application of the bacteriostatic agent in preservation of condiments.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial agent technology, specifically relating to an antibacterial agent prepared from soy sauce residue, its preparation method, and its application. Background Technology

[0002] Growing consumer demand and awareness of the health benefits of soy foods have fueled the expansion of soy products in the food market. The production of soy milk, tofu, or soy sauce generates a significant amount of soybean residue, a byproduct. Asian countries and non-Asian countries with high soybean consumption account for the majority of global soybean residue production (1.4 billion tons). Particularly in China, soy sauce residue is a major byproduct of soy sauce production, generating approximately 0.67 tons of residue for every ton of soy sauce produced. As a high-value-added material, the effective utilization of soy sauce residue warrants exploration. Addressing the waste of soy sauce residue is a significant challenge for the soy sauce processing industry, as it can improve energy efficiency, help food companies maximize economic benefits, and promote sustainable social development.

[0003] Adding food preservatives to condiments is a simple, efficient, and low-cost method for food preservation. With the increasing pursuit of green and healthy lifestyles and in-depth research into natural antibacterial substances, more and more plant-derived active substances have been found to possess antibacterial effects, serving as alternatives to traditional chemical antibacterial agents and showing great potential in the green control of pathogenic microorganisms. The process of realizing the high-value utilization of multiple components in soy sauce residue remains a current research hotspot. Designing a process to fully utilize the active substances in soy sauce residue and develop a new antibacterial agent with high added value and good antibacterial properties would be a truly ingenious invention. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing an antibacterial agent using soy sauce residue as raw material. This method has low raw material costs and the prepared antibacterial agent has a significant antibacterial effect.

[0005] Another objective of this invention is to provide an antibacterial agent prepared using the above method.

[0006] The final objective of this invention is to provide the application of the above-mentioned antibacterial agent in the preservation of condiments.

[0007] The first objective of this invention can be achieved by the following technical solution: a method for preparing an antibacterial agent using soy sauce residue as raw material, comprising the following steps:

[0008] (1) Soak the soy sauce residue in ethanol solution, pre-filter, desalt the filtrate using nanofiltration equipment, and remove the ethanol from the desalted nanofiltrate by rotary evaporation to obtain a salt-free crude extract.

[0009] (2) The salt-free crude extract in step (1) was degreased with petroleum ether and the raffinate was collected by centrifugation;

[0010] (3) Extract the raffinate phase from step (2) sequentially with equal volumes of chloroform and ethyl acetate, collect the ethyl acetate extract phase, remove ethyl acetate by rotary evaporation, dissolve the residue in sterile water, and then filter it through a microporous membrane to obtain the antibacterial agent.

[0011] In the above method for preparing antibacterial agents using soy sauce residue as raw material:

[0012] Optionally, the moisture content of the soy sauce residue in step (1) is 45-55%.

[0013] Optionally, the soaking in step (1) is performed at a temperature of 25-30°C for 24-36 hours.

[0014] Optionally, the ratio of the amount of soy sauce residue to the amount of ethanol solution in step (1) is 1g: 5-8mL, wherein the volume percentage of ethanol in the ethanol solution is 80-95%.

[0015] Optionally, gauze pre-filtration is used in step (1).

[0016] Optionally, in step (1), when using nanofiltration equipment for desalination, a nanofiltration membrane with a molecular weight of 300 Da is used for filtration, the pressure during filtration is 0.2 MPa, the temperature is 25 °C, and the inlet and outlet flow rates are 10 L / h.

[0017] Optionally, the reduced pressure during rotary evaporation in steps (1) and (3) is 0.08 MPa and the temperature is 40–50 °C.

[0018] Optionally, in step (2), the salt-free crude extract from step (1) is degreased with petroleum ether 4 to 6 times, preferably 6 times.

[0019] Optionally, in step (2), the centrifuge speed is 6000-8000 r / min and the centrifugation time is 6-10 min.

[0020] Optionally, a microporous membrane with a pore size of 0.22 μm is used for filtration in step (3).

[0021] The second objective of the present invention can be achieved by the following technical solution: an antibacterial agent for preserving condiments, prepared by the above method.

[0022] The last objective of the present invention can be achieved by the following technical solution: the application of the above-mentioned antibacterial agent in the preservation of condiments.

[0023] Optionally, the amount of the antibacterial agent added is 0.2% to 1% of the total mass of the seasoning.

[0024] Optionally, the condiment is soy sauce.

[0025] Furthermore, the condiment is low-sodium soy sauce.

[0026] Applying the antibacterial agent of this invention to the preservation of condiments can stabilize the quality of condiments during storage and improve economic benefits.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] (1) This invention extracts soy sauce residue by extracting it with ethanol solution, and by treating it by removing protein and fat, and then extracting it by polarity in sequence. Compared with the simple extraction of soy sauce residue directly with ethyl acetate, this invention obtains a soy sauce residue extract with simpler composition and higher purity.

[0029] (2) The ethyl acetate extract obtained by the method of the present invention contains a variety of natural antioxidants and has antioxidant activity, which can extend the shelf life of condiments in the application of condiment preservation.

[0030] (3) The preparation method of the present invention is relatively simple, safe and reliable, and the extractant used in the extraction process is readily available and easy to use. At the same time, the raw material cost is very low, and the prepared antibacterial agent has a significant antibacterial effect.

[0031] (4) The antibacterial raw materials used in this invention are all permitted in food processing and there are no limits on the amount of addition. They can be adjusted according to the needs of use.

[0032] (5) On the one hand, the present invention obtains antibacterial agents from soy sauce residue, which expands the application range of soy sauce residue and improves the comprehensive economic value of soy sauce residue. On the other hand, it also provides a natural source of antibacterial agents for the preservation of condiments. At the same time, the method is simple to operate, safe and reliable, and has low cost. Whether it is laboratory small-scale preparation or large-scale factory production, it has cost advantages. Attached Figure Description

[0033] Figure 1 The effect of different concentrations of antibacterial agents on the growth curve of Bacillus subtilis within 24 hours in Example 4;

[0034] Figure 2 The HPLC chromatograms of the standard and crude ethyl acetate extract in Example 5 are shown below.

[0035] Figure 3 This is a thin-layer chromatogram of the components of the crude ethyl acetate extract separated by silica gel in Example 5;

[0036] Figure 4 This is a positive electrospray ionization diagram of characteristic peak 5 purified and separated in Example 5;

[0037] Figure 5 This is a positive electrospray ionization diagram of characteristic peak 6 purified and separated in Example 5;

[0038] Figure 6 This is a positive electrospray ionization diagram of characteristic peak 7 purified and separated in Example 5. Detailed Implementation

[0039] To illustrate the present invention in more detail, the following embodiments are provided. It should be emphasized that the following embodiments are for illustrative purposes only and are not intended to limit the scope or content of the present invention.

[0040] In this document, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0041] It should be noted that the following comparative examples are not prior art, but are only set up for comparison with the examples and are not intended to limit the present invention.

[0042] Example 1

[0043] The method for preparing an antibacterial agent using soy sauce residue as raw material provided in this embodiment includes the following steps:

[0044] (1) Soak the soy sauce residue in an ethanol aqueous solution, pre-filter it with gauze, desalt the filtrate with nanofiltration equipment, and remove the ethanol by rotary evaporation to obtain a salt-free crude extract.

[0045] in:

[0046] The volume percentage of ethanol in the aqueous ethanol solution is 80%.

[0047] Soaking is performed at 27°C for 24 hours;

[0048] The moisture content of the soy sauce residue raw material is 53%;

[0049] The ratio of soy sauce residue to ethanol aqueous solution is 1g:5mL;

[0050] The rotary evaporation conditions were 0.08 MPa and 45 °C.

[0051] The desalination conditions are as follows: nanofiltration technology is used, and a nanofiltration membrane with a molecular weight of 300 Da is used for filtration. The filtration conditions are: pressure 0.2 MPa, temperature 25℃, and inlet and outlet flow rates 10 L / h.

[0052] (2) The crude extract was degreased with petroleum ether 6 times, and the raffinate was collected by centrifugation;

[0053] (3) The raffinate phase was extracted sequentially with equal volumes of chloroform and ethyl acetate, and the ethyl acetate extract phase was finally collected. Ethyl acetate was removed by rotary evaporation, the residue was dissolved in sterile water, and then filtered through a 0.22 μm microporous membrane to obtain the antibacterial agent. The reduced pressure during rotary evaporation was 0.08 MPa and the temperature was 45 °C.

[0054] Example 2

[0055] Unlike Example 1, the soy sauce residue was soaked in an aqueous ethanol solution with a volume percentage of 90%.

[0056] Example 3

[0057] Unlike Example 1, the soy sauce residue was soaked in an aqueous ethanol solution with a volume percentage of 95%.

[0058] Comparative Example 1

[0059] Soy sauce residue was extracted by soaking in an 80% (by volume) aqueous ethanol solution.

[0060] Comparative Example 2

[0061] The soy sauce residue was extracted by soaking in ethyl acetate at a volume percentage of 80%.

[0062] Example 4

[0063] The antibacterial agents in Examples 1-3 and Comparative Examples 1-2 were tested as follows:

[0064] (I) Antibacterial test

[0065] Common spoilage bacteria found in condiments, such as Bacillus subtilis, Escherichia coli, and Staphylococcus aureus, were selected and activated in suitable culture media using conventional methods until the colony count reached 1 × 10⁻⁶. 7 CFU / mL, refrigerate for later use.

[0066] Take a sterile 96-well plate and add 100 μL of broth culture medium to all wells. Add 100 μL of the antibacterial agents prepared in Examples 1-3 and Comparative Examples 1-2 to the first row. Mix well with a pipette and add 100 μL to the second row. Repeat this operation to dilute by half to the next row until the 7th row is finished.

[0067] The experimental group was added with 100 μL of bacterial suspension, with the broth used as a negative control and the broth used as a positive control. The mixture was incubated at 37°C for 24 hours. The OD value was measured at 600 nm using a microplate reader. The effect of different concentrations of antibacterial agents on the growth curve of Bacillus subtilis within 24 hours in Example 2 is as follows: Figure 1 As shown.

[0068] The specific antibacterial effects are shown in Table 1:

[0069] Table 1. Minimum inhibitory concentrations of Examples 1-3 and Comparative Examples 1-2

[0070]

[0071] As shown in Table 1, the natural antibacterial agents prepared in Examples 1-3 all have good antibacterial ability against the tested bacteria. The antibacterial effect of Example 2 is the best. This is because the soy sauce residue extract contains a variety of antibacterial active substances, which have obvious inhibitory effects on Escherichia coli, Bacillus subtilis and Staphylococcus aureus.

[0072] (2) Antioxidant experiment

[0073] Take 0.15 mL of the antibacterial agents prepared in Examples 1-3 and Comparative Examples 1-2, respectively, and add 0.15 mL of DPPH (2×10⁻⁶) to each. - 4 Mix the DPPH solution (0.15 mL / L) thoroughly and react in the dark for 30 min. Zero the sample solution with anhydrous ethanol and measure the absorbance at 517 nm. Simultaneously, measure the absorbance at 517 nm of a mixture of 0.15 mL of the sample solution (standard solution) and 0.15 mL of ethanol (blank) (A) at 517 nm. Then measure the absorbance at 517 nm of 0.15 mL of DPPH solution and 0.15 mL of ethanol (control) at 517 nm. Repeat the same measurement three times. Substitute the detected absorbance values ​​into the calculation formula to obtain the scavenging rate for each concentration. Then calculate the half-inhibitory concentration (IC50) from the scavenging rate and the corresponding concentration. 50 ).

[0074] Formula for calculating DPPH free radical scavenging rate:

[0075] DPPH free radical scavenging rate (%) = [1 - (Sample A - Blank A) / Control A] × 100%

[0076] A 样品 0.15 mL DPPH solution + 0.15 mL standard / sample solutions of different concentrations

[0077] A 样品 0.15 mL of standard / sample solutions of different concentrations + 0.15 mL of anhydrous ethanol solution

[0078] A 样品 0.15 mL DPPH solution + 0.15 mL anhydrous ethanol solution

[0079] Table 2. Antioxidant performance of Examples 1-3 and Comparative Examples 1-2

[0080] Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 DPPH removal rate 80％ 94％ 93％ 30％ 57％ <![CDATA[IC 50 Value 0.23 mg / mL 0.07 mg / mL 0.10 mg / mL 1.28 mg / mL 0.62 mg / mL

[0081] As shown in Table 2, the natural antibacterial agents prepared in Examples 1-3 all have strong antioxidant capacity. The antioxidant effect of Example 2 is the best. This is because the soy sauce residue extract contains active substances with good antioxidant effects, which have a significant effect on free radical scavenging and can delay the oxidation reaction of oils in condiments, thus achieving a preservation effect.

[0082] (3) Application test:

[0083] In this invention, an antibacterial agent was added at a mass percentage of 0.2% and mixed with seasonings. Each batch was packaged in 50g portions, with a blank sample included. After packaging, the mixture was sterilized at 115℃ for 40 minutes. After cooling to room temperature, the mixture was stored at room temperature and continuously observed. The total bacterial count of each group of seasonings was tested according to the national standard testing methods. The results are shown in Table 3.

[0084] Table 3. Changes in total bacterial count in condiments (unit: CFU / g)

[0085] Days 0 7 14 21 30 Example 1 <10 70 260 800 <![CDATA[1.3*10 3 ]]> Example 2 <10 <10 <10 30 30 Example 3 <10 <10 90 200 390 Comparative Example 1 <10 200 730 <![CDATA[2.6×10 3 ]]> <![CDATA[6.7×10 4 ]]> Comparative Example 2 <10 800 <![CDATA[1.5×10 3 ]]> <![CDATA[9.4×10 3 ]]> <![CDATA[4.2×10 5 ]]> blank <10 <![CDATA[3.4×10 4 ]]> <![CDATA[6.1×10 8 ]]> Severe mold growth on the surface Rotten and stinking

[0086] As shown in Table 3, a large number of colonies grew in the control group as early as day 7, with a total colony count reaching 3.4 × 10⁻⁶. 4 The total bacterial count was CFU / g, accompanied by a putrid odor. The total bacterial count in other comparative examples also increased significantly with the extension of the enhanced culture time. However, in Example 2, no significant increase in the total bacterial count was observed within 30 days, and the condiments remained fresh and odorless.

[0087] At the same time, sensory evaluations were conducted on each group at the beginning. Ten evaluators were trained in accordance with the evaluation standards, which are shown in Table 4. Then, they were smelled and tasted. The average value was taken after scoring. The sensory evaluation results are shown in Table 5.

[0088] Table 4 Sensory Evaluation Criteria

[0089] Evaluation Project Evaluation criteria Color The closer it is to a reddish-brown color with an oily sheen, the higher the score (0-20 points). fragrance The stronger the aroma of fermented soybean esters, the higher the score; any off-odors will result in a deduction of points (0-20 points). taste The stronger the aroma of fermented soybean esters, the higher the score; any off-odors will result in a deduction of points (0-30 points). Organizational Form The better the overall sensory evaluation of oil content, viscosity, and the number of fermented soybean pieces, the higher the score (0-30 points).

[0090] Table 5 Sensory evaluation results

[0091] Color fragrance taste Organizational Form Example 1 13 15 22 25 Example 2 16 18 25 26 Example 3 15 14 19 27 Comparative Example 1 13 12 20 26 Comparative Example 2 12 16 20 24 blank 15 16 25 25

[0092] According to the sensory evaluation results in Table 5, the sensory evaluation of the seasoning in Example 2 was the highest in all indicators. The results of the other examples were not much different from the blank control. This indicates that the reasonable addition of antibacterial agents did not significantly change the flavor and taste of the seasoning. However, the comparative examples were not as good as the blank control in all aspects, and the color and flavor scores were slightly lower.

[0093] Example 5

[0094] The antibacterial agent in Example 2 was separated from the sample solution by optimizing the silica gel chromatography column conditions, and the components were preliminarily identified by HPLC, thin-layer chromatography, and LC-MS.

[0095] The HPLC identification process and results were as follows: The crude ethyl acetate extract of soy sauce residue (component S, the ethyl acetate extract phase collected in Example 2) was analyzed by HPLC. Solvent A consisted of 0.1% (v / v) formic acid, and the solvent was chromatographically pure water; solvent B consisted of chromatographically pure acetonitrile. The elution program was as follows: 5% B phase elution for 3 min, followed by 15% B phase elution for 10 min, then 32.5% B phase elution for 12 min, then 100% B phase elution for 13 min, then 100% B phase elution for 15 min, and finally 5% B phase elution for 22 min. The flow rate was 1 mL / min. The HPLC chromatograms of the standard and the crude ethyl acetate extract are shown below. Figure 2 .

[0096] Five common soy isoflavone standards from soy products, including daidzein, daidzein, genistein, genistein, and daidzein, were compared with component S by high performance liquid chromatography. The results showed that many substances in component S did not correspond one-to-one with the characteristic peaks of the five common soy isoflavones. Furthermore, the characteristic peaks of the main components were not found in the existing technology, and the elution times of the remaining minor components did not correspond to those of the standards. Therefore, further separation and purification by column chromatography and identification by LC-MS are required.

[0097] Silica gel chromatography: The sample prepared above (the ethyl acetate extract collected in Example 2) was uniformly added to the top of a silica gel column, and eluted with gradients of v(dichloromethane):v(ethyl acetate) = 4:1 and 4:3. Thin-layer chromatography (TLC) was used for preliminary analysis of the crude ethyl acetate extract, with a developing solvent of v(dichloromethane):v(ethyl acetate) = 4:3. After development, the spots were detected at a UV wavelength of 254 nm after drying. Silica gel column separation mainly yielded three components with high S content that did not correspond to the characteristic peaks of the standard. The TLC results of the crude ethyl acetate extract components separated by silica gel are shown below. Figure 3 .

[0098] The LC-MS analysis process and results are as follows: Three components with high S content that did not correspond to the characteristic peaks of the standard were separated and identified. However, it is undeniable that other trace components may also have antibacterial effects, but current technology makes it difficult to separate and identify these trace components. The purified three components were dissolved in methanol and identified by LC-MS. The results are as follows: Figure 4 , 5As shown in Figures 6 and 7, through comparative analysis, the relative molecular masses of the three main compounds in the sample are 254, 256, and 272, respectively. These three compounds have not yet been found in the study of active substances in soy products, which is consistent with the theory.

[0099] The specific embodiments of the invention have been described in detail above, but these are merely examples, and the invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications or substitutions to the invention are also within the scope of this invention. Therefore, all equivalent transformations, modifications, and improvements made without departing from the spirit and principles of this invention should be included within the scope of this invention.

Claims

1. A method for preparing an antibacterial agent using soy sauce residue as raw material, characterized in that... Includes the following steps: (1) Soak the soy sauce residue in ethanol solution, pre-filter it, desalt the filtrate using nanofiltration equipment, and remove the ethanol from the desalted nanofiltrate by rotary evaporation to obtain a salt-free crude extract. (2) The salt-free crude extract from step (1) was degreased with petroleum ether and the raffinate was collected by centrifugation; (3) Extract the raffinate phase in step (2) with equal volumes of chloroform and ethyl acetate in sequence, collect the ethyl acetate extract phase, remove ethyl acetate by rotary evaporation, dissolve the residue in sterile water, and then filter it with a microporous membrane to obtain the antibacterial agent. The soy sauce residue in step (1) has a water content of 45-55%, and the ratio of the amount of soy sauce residue to the amount of ethanol solution is 1g: 5-8mL, wherein the volume percentage of ethanol in the ethanol solution is 80%-95%. In step (1), when desalting with nanofiltration equipment, a nanofiltration membrane with a molecular weight of 300 Da is used for filtration. The pressure during filtration is 0.2 MPa, the temperature is 25℃, and the inlet and outlet flow rates are 10 L / h.

2. The method for preparing an antibacterial agent using soy sauce residue as raw material according to claim 1, characterized in that: The pressure reduction during rotary evaporation in steps (1) and (3) is 0.08 MPa and the temperature is 40~50℃.

3. The method for preparing an antibacterial agent using soy sauce residue as raw material according to claim 1, characterized in that: In step (2), the salt-free crude extract from step (1) is degreased 4 to 6 times with petroleum ether.

4. The method for preparing an antibacterial agent using soy sauce residue as raw material according to claim 1, characterized in that: In step (2), the centrifuge speed is 6000~8000 r / min and the centrifugation time is 6~10 min.

5. The method for preparing an antibacterial agent using soy sauce residue as raw material according to claim 1, characterized in that: In step (3), a microporous membrane with a pore size of 0.22 μm is used for filtration.

6. A preservative agent for seasonings, characterized in that: It is prepared by the method described in any one of claims 1-5.

7. The application of the antibacterial agent according to claim 6 in the preservation of condiments.

8. The application according to claim 7, characterized in that: The amount of the antibacterial agent added is 0.2-1% of the total mass of the seasoning.

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