Method for detecting corrosion resistance of oyster sauce

Abstract

The invention relates to the technical field of seasoning preservation, in particular to a method for detecting the corrosion resistance of oyster sauce, which comprises the following steps: preparing a spore suspension by taking aspergillus niger ZE902 and aspergillus oryzae ZA306 as indicator bacteria; inoculating the spore suspension into a to-be-detected oyster sauce sample for culturing; in the culture process, observing whether the surface of the obtained culture is mildewed or not, sampling and detecting the viable count of indicator bacteria in the obtained culture, and judging the corrosion resistance of the oyster sauce sample to be detected according to the obtained observation result and the detected viable count of indicator bacteria. According to the method, the preservative capacity of the oyster sauce in the shelf life can be accurately judged, and the method is more representative than a traditional challenge experiment method.

Description

Technical Field

[0001] The present application relates to the technical field of condiment preservation, and in particular to a method for detecting the antiseptic performance of oyster sauce. Background Art

[0002] At present, oyster sauce contains rich nutrients such as protein, amino acids and sugars. Under the conditions of low salt content, high water activity and suitable temperature, it is very easy for microorganisms to multiply and cause mildew. Especially in the seasons between spring and summer, when the air is humid and the relative humidity is high, mold microorganisms often breed on the surface of oyster sauce and even at the mouth of the bottle, causing mildew and even white film on the mouth of the bottle, which seriously affects storage and storage after opening.

[0003] In order to make a more accurate judgment on the antiseptic properties of oyster sauce products, the USP method of the United States Pharmacopoeia was introduced to conduct antiseptic challenge experiments. However, the USP method uses a single Aspergillus niger for the experiment, which is different from the easily infected mold of oyster sauce itself, and it is easy to make a wrong judgment.

[0004] As far as the anti-corrosion performance is concerned, traditional technologies such as:

[0005] The literature mentions the following antiseptic challenge test methods used in the cosmetics industry: the single inoculation method, which inoculates 1% volume of a single bacterium or a single mold solution into the product to make the concentration of bacteria or fungi in the sample between 10 5 -10 6 cfu / g. Stir with a glass rod or vortex to mix evenly and place in an incubator at 22.5±5℃. Take samples to test the bacterial content on the 0th, 3rd, 7th, 14th, 21st and 28th day after inoculation to determine whether the product has passed the preservation test.

[0006] It is mentioned in the literature that the Cosmetics Association currently uses a three-repetition microbial challenge test: weigh 30g of the test sample, add bacteria once every 2 weeks (the 1st, 3rd and 5th week of the experiment), and add 1×10 mold each time. 5 -1×1O 6 cfu / g. Take samples and analyze the bacterial content on the 0th, 7th and 14th day after adding bacteria (before the last addition of bacteria) to determine whether the product has passed the preservation.

[0007] There is a documented method for detecting the anti-corrosion ability of seasonings, including the following steps: 1) Prepare a bacterial suspension with the prepared test strains; 2) Inoculate the prepared bacterial suspension into the test sample; 3) At the moment of inoculation and at regular intervals, take equal amounts of samples from the inoculated test sample and detect the number of bacteria contained in the samples; 4) Compare the number of bacteria detected at regular intervals with the initial number of bacteria at the moment of inoculation. If the difference ≤ 0, it is determined that the anti-corrosion ability of the test sample is qualified, and the smaller the difference, the stronger the anti-corrosion ability of the test sample is judged. Further, the test strains are selected from standard strains or strains isolated from the product; alternatively, the test strains are selected from both standard strains and strains isolated from the product. The standard strains include at least one of bacteria, molds, and yeasts. When the test strains are selected as standard strains, appropriate strains are selected from the standard strains according to the water activity aw of the test sample: 1) If aw ≥ 0.90, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Aspergillus niger are selected respectively for steps 2) - 4); 2) If 0.85 < aw < 0.90, Staphylococcus aureus, Candida albicans, and Aspergillus niger are selected respectively for steps 2) - 4); 3) If aw ≤ 0.85, Candida albicans and Aspergillus niger are selected respectively for steps 2) - 4).

[0008] There is also a document that records a method for testing and evaluating the ability of a cosmetic preservative system. The implementation steps are as follows: (1) Use a sterilized dilution tube containing 10 ml of saline as a base tube, use an inoculation loop to take 1 loop of each pure strain of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger, and Salmonella to be verified, and add it to the base tube. Oscillate it on an oscillator for 1-2 minutes to prepare a uniform test sample solution; (2) Directly sample 45 ml of liquid products; or 45 g of solids and semi-solids. If the sample is water-soluble, add sterile saline to prepare a 45 ml uniform solution; if the sample is oil-soluble, add liquid paraffin and sterile saline in a ratio of 3:7 to prepare a 45 ml uniform solution; (3) Hold the inoculation loop and place it in a sterile saline tube. (4) At the same time, take 1 ml of pure bacterial strain solution and add it to a sterilized test tube containing 9 ml of physiological saline. The pipette cannot touch the liquid surface. Mix well and prepare a 1:10 dilution solution. Then dilute it successively to 1:100, 1:1000, and dilute it successively to 10-6 dilution. Replace one pipette for each dilution. (5) After dilution, select 1:10 according to the content of the sample bacteria. 1:1000, 1:10000, 1:100000 diluted bacterial solution were added to two sterile plates at 1 ml / plate. The tip of the pipette should not touch the plate wall. Then 15 ml of nutrient agar medium melted and cooled to 45-50 °C was poured in for blank counting. After the culture medium solidified, it was turned over and placed in an incubator for 2 days of incubation. The numbers were counted separately for use. (6) The test data was calculated as the pure bacterial strain content according to GB standard. This base number is the initial data of the pure bacterial strain. (7) On the 1st, 7th, 14th and 28th day, 1 ml of sample was taken and diluted to 1:10, 1:100, 1:1000, and then diluted to 10-6 dilution in sequence. For bacteria tested on the 1st day and fungi tested on the 7th day, the dilutions were 1:1000, 1:10 000 inoculation sample, then select 1:10 and 1:100 as inoculation samples, select 2 dilutions for inoculation and culture respectively, and calculate the culture data as the colony count of the sample liquid; (8) Calculate the test data as the colony count of microorganisms in each verification time period in the same way as GB; (9) Divide the colony count obtained in step (8) by the colony count obtained in step (6), and take the logarithm of the reciprocal of the result with the base ten as the antiseptic ability index for each test time period; (10) When the antiseptic ability index of bacteria on the first day is greater than 1, and the antiseptic ability index on the 7th day and above is greater than 3, and the antiseptic ability index of fungi including Candida albicans and Aspergillus niger is greater than 1 on the 7th day, and the antiseptic ability index on the 14th day and above is greater than 3, it proves that the antiseptic effect is significant and the product has strong anti-pollution ability.

[0009] However, using traditional anti-corrosion performance testing methods, the test results are difficult to accurately reflect the actual shelf life anti-corrosion performance of oyster sauce. Therefore, it is urgent to develop a shelf life anti-corrosion test method for oyster sauce products that can accurately reflect the anti-corrosion performance of oyster sauce in the usage scenario. Summary of the invention

[0010] Based on this, one or more embodiments of the present application provide a method for detecting the antiseptic properties of oyster sauce, including the following technical solutions:

[0011] One or more embodiments of the present application provide a method for detecting the antiseptic properties of oyster sauce, the method comprising the following steps:

[0012] Using Aspergillus niger ZE902 and Aspergillus oryzae ZA306 as indicator bacteria, spore suspension was prepared;

[0013] Inoculating the spore suspension into the oyster sauce sample to be tested for cultivation;

[0014] During the culturing process, observe whether there are signs of mold on the surface of the culture, and take samples to detect the number of live indicator bacteria in the culture. The antiseptic performance of the oyster sauce sample to be tested is determined based on the observation results and the number of live indicator bacteria detected.

[0015] In some embodiments of the present application, the detection method comprises the following steps:

[0016] Inoculating the spore suspension into the oyster sauce sample to be tested for the first time to carry out the first stage of culture, and inoculating the spore suspension into the oyster sauce sample for the second time to carry out the second stage of culture;

[0017] During the first stage of cultivation and the second stage of cultivation, the surface of the culture is observed at a preset time point to see if there are signs of mold, and samples are taken to detect the number of live indicator bacteria in the culture. The antiseptic properties of the oyster sauce sample to be tested are determined based on the observation results and the number of live indicator bacteria.

[0018] In some embodiments of the present application, the first inoculation day is counted as day 0, the first stage of culture lasts from day 0 to day 14, and the second stage of culture begins after the second inoculation on day 14, and the second stage of culture lasts until day 28.

[0019] In some embodiments of the present application, the preset time points include day 0, day 7, day 14, day 21 and day 28.

[0020] In some embodiments of the present application, judging the antiseptic performance of the oyster sauce sample to be tested according to the obtained observation results and the number of viable indicator bacteria includes:

[0021] (1) No signs of mold, m1-14≤m1-0×10 -2 , m1-0>m1-7>m1-14, m2-28≤m2-14×10 -2 , m2-14>m2-21>m2-28, then the anti-corrosion ability is excellent;

[0022] (2) No signs of mold, m1-7≤m1-0×10 -1 ,m2-21≤m2-14×10 -1 , if the indicator bacteria content in each culture stage decreases, the antiseptic ability is passed;

[0023] (3) If mold is observed in the culture, the antiseptic performance fails;

[0024] The live counts of the indicator bacteria detected on the 0th day, the 7th day, the 21st day and the 28th day were recorded as m1-0, m1-7, m2-21 and m2-28, respectively. The live counts of the indicator bacteria detected before and after the second inoculation on the 14th day were recorded as m1-14 and m2-14, respectively.

[0025] In some embodiments of the present application, in the spore suspension, the ratio of conidia of Aspergillus niger ZE902 to conidia of Aspergillus oryzae ZA306 is 1:(0.8-1.2).

[0026] In some embodiments of the present application, the culture conditions include: temperature of 28°C-32°C, oyster sauce mass of 50g-105g, the amount of oyster sauce not exceeding 1 / 2 of the total volume of the container, sealed, and time of 28d-30d.

[0027] In some embodiments of the present application, 10 4 CFU-10 5 The concentration of CFU spores was used for inoculation.

[0028] In some embodiments of the present application, the mass of the spore suspension is 0.5%-1% of the mass of the oyster sauce sample to be tested.

[0029] In some embodiments of the present application, the method for detecting the number of viable indicator bacteria includes using a plate colony counting method.

[0030] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. DETAILED DESCRIPTION

[0031] Below in conjunction with embodiment and example, the application is further described in detail.It should be understood that these embodiments and examples are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing implementation modes and embodiments and are not intended to limit this application.

[0033] the term

[0034] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0035] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0036] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0037] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0038] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0039] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of this application.

[0040] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0041] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0042] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0043] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0044] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0045] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0046] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0047] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0048] In order to more accurately judge the anti-corrosion ability level of oyster sauce products during their shelf life and the mold and thinning situation during the shelf life, the traditional shelf life verification at room temperature was abandoned, and a fast, simple and accurate method for shelf life anti-corrosion verification of oyster sauce was developed. This method can quickly judge the anti-corrosion ability of oyster sauce during its shelf life, and is more accurate than the results of traditional anti-corrosion challenge experiments.

[0049] This application has developed a new type of oyster sauce shelf life preservation challenge test method, which can accurately determine the shelf life preservation ability of oyster sauce and is more representative than the traditional challenge test method. This application optimizes the traditional preservation challenge test method, making the operation simpler, the periodicity shorter, and more in line with the actual use scenario of consumers. Specifically, the process of consumers opening the oyster sauce packaging cap multiple times to use the oyster sauce is a continuous and repeated contamination process. Correspondingly, this application adopts secondary inoculation, which is closer to the consumer's use process than the single inoculation.

[0050] The present application provides a method for detecting the antiseptic performance of oyster sauce, the method comprising the following steps:

[0051] Using Aspergillus niger ZE902 and Aspergillus oryzae ZA306 as indicator bacteria, spore suspension was prepared;

[0052] Inoculating the spore suspension into the oyster sauce sample to be tested for cultivation;

[0053] During the culturing process, observe whether there are signs of mold on the surface of the culture, and take samples to detect the number of live indicator bacteria in the culture. The antiseptic performance of the oyster sauce sample to be tested is determined based on the observation results and the number of live indicator bacteria detected.

[0054] In some examples of the present application, the detection method comprises the following steps:

[0055] Inoculating the spore suspension into the oyster sauce sample to be tested for the first time to carry out the first stage of culture, and inoculating the spore suspension into the oyster sauce sample for the second time to carry out the second stage of culture;

[0056] During the first stage of cultivation and the second stage of cultivation, the surface of the culture is observed at a preset time point to see if there are signs of mold, and samples are taken to detect the number of live indicator bacteria in the culture. The antiseptic properties of the oyster sauce sample to be tested are determined based on the observation results and the number of live indicator bacteria.

[0057] In some examples of the present application, the first inoculation day is counted as day 0, the first stage of culture lasts from day 0 to day 14, and the second stage of culture begins after the second inoculation on day 14, and the second stage of culture lasts until day 28.

[0058] In some examples of the present application, the preset time points include day 0, day 7, day 14, day 21 and day 28.

[0059] In some examples of the present application, judging the antiseptic performance of the oyster sauce sample to be tested according to the obtained observation results and the number of viable indicator bacteria includes:

[0060] (1) No signs of mold, m1-14≤m1-0×10 -2, m1-0>m1-7>m1-14, m2-28≤m2-14×10 -2 , m2-14>m2-21>m2-28, then the anti-corrosion ability is excellent;

[0061] (2) No signs of mold, m1-7≤m1-0×10 -1 ,m2-21≤m2-14×10 -1 , if the indicator bacteria content in each culture stage decreases, the antiseptic ability is passed;

[0062] (3) If mold is observed in the culture, the antiseptic performance fails;

[0063] The live counts of the indicator bacteria detected on the 0th day, the 7th day, the 21st day and the 28th day were recorded as m1-0, m1-7, m2-21 and m2-28, respectively. The live counts of the indicator bacteria detected before and after the second inoculation on the 14th day were recorded as m1-14 and m2-14, respectively.

[0064] In some examples of the present application, in the spore suspension, the ratio of conidia of Aspergillus niger ZE902 to conidia of Aspergillus oryzae ZA306 is 1:(0.8-1.2), for example, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2.

[0065] In some examples of the present application, the culture conditions include: a temperature of 28°C-32°C (for example, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32°C), an oyster sauce mass of 50g-105g (for example, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105g), the amount of oyster sauce does not exceed 1 / 2 of the total volume of the container, sealed, and the time is 28d-30d (for example, 28, 28.5, 29, 29.5, 30d).

[0066] In some examples of the present application, 10 4 CFU-10 5 The concentration of CFU spores was used for inoculation.

[0067] In some examples of the present application, the mass of the spore suspension is 0.5%-1% (for example, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%) of the mass of the oyster sauce sample to be tested.

[0068] In some examples of the present application, the method for detecting the number of viable indicator bacteria includes using a plate colony counting method.

[0069] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0070] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0071] 1. Experimental Procedure

[0072] 1.1 Selection of indicator microorganisms In the long-term shelf life research of oyster sauce, the inventors found that using Aspergillus niger ZE902 and Aspergillus oryzae ZA306 as indicator bacteria for preservation testing can more accurately reflect the actual situation of the shelf life of oyster sauce.

[0073] Aspergillus niger ZE902 (hereinafter referred to as specific mold M1) was deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 28, 2023, with the deposit number GDMCCNO: 63690. Relevant information is recorded in the Chinese invention patent application CN117821261A.

[0074] Aspergillus oryzae ZA306 (hereinafter referred to as specific mold M2) was deposited in the Guangdong Provincial Microbiological Culture Collection Center on May 13, 2024, with the deposit number GDMCC No: 64621. The relevant information is recorded in the Chinese invention patent application CN118516244A.

[0075] 1.2 Detection Method

[0076] 1.2.1 Testing methods for this application

[0077] (1) Preparation of mixed spore suspension

[0078] Inoculate the fresh culture of specific mold M1 into PDA medium, culture in a 30℃ constant temperature incubator for 3-5 days, add 3-5mL 0.85wt% sterile saline, elute the mold spores, and prepare M1 spore suspension. Prepare the spore suspension of specific mold M2 by the same operation, then mix the mold spore suspensions of M1 and M2 evenly with a vortex shaker, aspirate the spore suspension into a sterile test tube by an appropriate method, add an appropriate amount of 0.85wt% sterile saline, and use the hemocytometer to count 10 spores per 1mL. 6 -10 7CFU of mold mixed spore suspension (in the present application example, the blood cell count unit is cfu / mL, and the oyster sauce system bacterial content unit is cfu / g). At the same time, the plate method is used to determine the bacterial count of 1 mL of mixed spore suspension.

[0079] (2) Specific microbial inoculation

[0080] Take the intact oyster sauce sample and divide it into 50-100g in a sterile glass bottle. Spray the prepared specific mold mixed spore suspension evenly so that the total mold spore inoculation amount in 1g oyster sauce sample is 2×10 4 CFU, where the concentration of M1 spores in the oyster sauce sample was 1×10 4 cfu / g, M2 spore concentration is 1×10 4 cfu / g, the inoculation ratio of M1 and M2 is 1:1, the mass of the inoculated mixed spore suspension shall not exceed 0.5%-1% of the total mass of oyster sauce, and the above-mentioned specific mold mixed spore suspension shall be inoculated in an ultra-clean workbench, wherein the volume of oyster sauce shall not exceed 1 / 2 of the volume of the glass bottle. After inoculation, the lid shall be closed and sealed for storage, and placed in an incubator at 30°C for accelerated culture, and the total colony count of the oyster sauce system shall be tested on the 0th day (the day of inoculation), the 7th day, and the 14th day, respectively, and each test shall be repeated 3 times.

[0081] After the test was completed on the 14th day, a second spraying inoculation of the specific mold mixed spore suspension prepared in step (1) was performed, wherein the spore concentration of M1 in the oyster sauce sample was 1×10 4 cfu / g, the spore concentration of M2 is 1×10 4 cfu / g, the inoculation ratio of M1 and M2 was 1:1. The operation steps were as above. Then, the total number of colonies in the oyster sauce system was detected on the 21st and 28th days. Each test was repeated three times.

[0082] (3) Determination of surviving bacteria

[0083] On the 0th, 7th, 14th, 21st and 28th days after inoculation of the mixed spore suspension, 10 g (mL) of the oyster sauce sample was taken from each of the above glass bottles and diluted with 0.85 wt% sterile water (fungus) to a dilution ratio of 1:10, 1:10 and 2:10. 2 , 1:10 3 Equal dilution level.

[0084] The plate colony counting method was used to determine the number of bacteria in each sample. Bengal red agar and PDA medium were used for the determination of fungi.

[0085] (4) Judgment basis of anti-corrosion challenge

[0086] The viable counts of the indicator bacteria detected on the 0th day, the 7th day, the 21st day and the 28th day are recorded as m1-0, m1-7, m2-21 and m2-28 respectively. The viable counts of the indicator bacteria detected before and after the second inoculation on the 14th day are recorded as m1-14 and m2-14 respectively. The judgment basis includes:

[0087] ①Excellent anti-corrosion effect: no signs of mold m1-14≤m1-0×10 -2 , m1-0>m1-7>m1-14, m2-28≤m2-14×10 -2 , m2-14>m2-21>m2-28, it indicates that the anti-corrosion effect is excellent.

[0088] ② Anticorrosion test passed: After two inoculation tests, the oyster sauce system did not show any mold during the 28-day anticorrosion test, and the bacterial content in the system showed a significant downward trend, meeting the requirement of m1-7≤m1-0×10 -1 ,m2-21≤m2-14×10 -1 If the indicator bacteria content in each culture stage decreases, the preservation is judged to have passed.

[0089] ③ Failure of antiseptic challenge test: After two inoculation tests, if mold appears in the oyster sauce system after any inoculation test, the total colony count will no longer be performed and the antiseptic test will be directly judged as failed.

[0090] 1.2.2 Control Method (Traditional USP Preservative Challenge Test Procedure)

[0091] (1) Dispense 50-100 g of the sample to be tested into a clean glass bottle;

[0092] (2) Inoculate a single bacterial suspension of the special mold (Aspergillus niger) for antiseptic challenge. Mix the sample and the bacterial suspension evenly. The volume of the inoculated bacterial suspension shall not exceed 0.5%-1% of the total volume of the sample. The inoculation system concentration of Aspergillus niger is 1×10 4 cfu / g.

[0093] (3) Observe the moldy surface of oyster sauce on days 0, 7, 14, 21, and 28 after inoculation and test and record the total number of live bacteria in the system.

[0094] 2. Verify the results

[0095] 2.1 Verification Example 1

[0096] The antiseptic performance test of commercially available oyster sauce sample A includes the following steps:

[0097] 1. Open the oyster sauce sample A, take 100g and pack it into sterilized glass bottles for later use;

[0098] 2. Prepare a mixed spore suspension of oyster sauce-specific molds M1 and M2 according to the method in 1.2.1. The concentration of the suspension is 5.5×10 6 cfu / g;

[0099] 3. Use an alcohol spray bottle to evenly spray the specific mold mixed spore suspension on the surface of the oyster sauce sample so that the surface of the oyster sauce sample is evenly covered with the mixed spore suspension. The spraying amount is 0.5 mL;

[0100] 4. Place the oyster sauce sample A inoculated with the specific mixed spore suspension in a 30°C constant temperature incubator and screw on the lid. Observe the moldy signs on the surface on the 0th, 7th and 14th day after inoculation and test and record the total number of colonies in the system;

[0101] 5. After testing the total colony count on the 14th day, repeat step 3 and inoculate an equal amount of specific mixed spore suspension for the second time. Observe the signs of mold on the surface of the oyster sauce on the 21st and 28th days and test and record the total colony count of the system.

[0102] 6. Perform the preservation test of oyster sauce A according to the traditional United States Pharmacopoeia challenge test method in Item 1.2.2, and observe and record the signs of mold and the total number of colonies during the experiment.

[0103] 2.2 Verification Example 2

[0104] The antiseptic performance of commercially available oyster sauce sample B is tested, including the following steps:

[0105] 1. Open the sealed oyster sauce sample B, take 80g and pack it into sterilized glass bottles for later use;

[0106] 2. Prepare a mixed spore suspension of M1 and M2 according to the method in 1.2.1. The concentration of the suspension is 3×10 6 cfu / g;

[0107] 3. Use an alcohol spray bottle to evenly spray the specific mold mixed spore suspension on the surface of the oyster sauce sample so that the surface of the oyster sauce sample is evenly covered with the mixed spore suspension. The spraying amount is 0.8 mL;

[0108] 4. Place the oyster sauce sample A inoculated with the specific mixed spore suspension in a 30°C constant temperature incubator and screw on the lid. Observe the moldy signs on the surface on the 0th, 7th and 14th day after inoculation and test and record the total number of colonies in the system;

[0109] 5. After testing the total colony count on the 14th day, repeat step 3 and inoculate an equal amount of specific mixed spore suspension for the second time. Observe the signs of mold on the surface of the oyster sauce on the 21st and 28th days and test and record the total colony count of the system.

[0110] 6. Perform the preservation test of Oyster Sauce B according to the traditional USP challenge test method in 1.2.2, and observe and record the signs of mold and total colony count during the experiment.

[0111] 2.3 Verification Example 3

[0112] The antiseptic performance test of commercially available oyster sauce sample C includes the following steps:

[0113] 1. Open the oyster sauce sample A and dispense 50g of it into sterilized glass bottles for later use;

[0114] 2. Prepare a mixed spore suspension of M1 and M2 according to the method in 1.2.1. The concentration of the suspension is 1×10 6 cfu / g;

[0115] 3. Use an alcohol spray bottle to evenly spray the specific mold mixed spore suspension on the surface of the oyster sauce sample so that the surface of the oyster sauce sample is evenly covered with the mixed spore suspension. The spraying volume is 1 mL;

[0116] 4. Place the oyster sauce sample A inoculated with the specific mixed spore suspension in a 30°C constant temperature incubator and screw on the lid. Observe the moldy signs on the surface on the 0th, 7th and 14th day after inoculation and test and record the total number of colonies in the system;

[0117] 5. After testing the total colony count on the 14th day, repeat step 3 and inoculate an equal amount of the mixed spore suspension for the second time. Observe the signs of mold on the surface of the oyster sauce on the 21st and 28th days and test and record the total colony count of the system.

[0118] 6. Perform the preservation test of Oyster Sauce C according to the traditional United States Pharmacopoeia challenge test method in 1.2.2, and observe and record the signs of mold and the total number of colonies during the experiment.

[0119] 2.4 Verification Example 4

[0120] The antiseptic performance test of commercially available oyster sauce sample D includes the following steps:

[0121] 1. Open the oyster sauce sample D and dispense it into sterilized glass bottles in 60g portions for later use;

[0122] 2. Prepare a mixed spore suspension of M1 and M2 according to the method in 1.2.1. The concentration of the suspension is 4×10 6 cfu / g;

[0123] 3. Use an alcohol spray bottle to evenly spray the mixed spore suspension on the surface of the oyster sauce sample so that the surface of the oyster sauce sample is evenly covered with the mixed spore suspension. The spraying amount is 0.6 mL;

[0124] 4. Place the oyster sauce sample A inoculated with the mixed spore suspension in a 30°C constant temperature incubator and screw on the lid. Observe the moldy signs on the surface on the 0th, 7th and 14th day after inoculation and test and record the total number of colonies in the system.

[0125] 5. After testing the total colony count on the 14th day, repeat step 3 and inoculate an equal amount of the mixed spore suspension for the second time. Observe the signs of mold on the surface of the oyster sauce on the 21st and 28th days and test and record the total colony count of the system.

[0126] 6. Perform the preservation test of oyster sauce D according to the traditional USP challenge test method in Item 1.2.2, and observe and record the signs of mold and the total number of colonies during the experiment.

[0127] The statistical analysis of the anti-corrosion performance test results of the above methods is as follows:

[0128] Table 1. Verification results of oyster sauce shelf life antiseptic challenge experiment

[0129] sample Oyster Sauce A Oyster sauce B Oyster Sauce C Oyster sauce D Concentration of mixed spore suspension cfu / g <![CDATA[5.5×10 7 ]]> <![CDATA[3×10 7 ]]> <![CDATA[1×10 7 ]]> <![CDATA[4×10 6 ]]> Detection of cfu / g on day 0 <![CDATA[5.5×10 5 ]]> <![CDATA[3×10 5 ]]> <![CDATA[1×10 5 ]]> <![CDATA[4×10 4 ]]> Detection of cfu / g on the 7th day <![CDATA[2.5×10 3 ]]> <![CDATA[5×10 3 ]]> <![CDATA[2.5×10 4 ]]> <![CDATA[3.5×10 3 ]]> Detection of cfu / g on the 14th day <![CDATA[1.2×10 1 ]]> <![CDATA[2.6×10 2 ]]> <![CDATA[9.5×10 2 ]]> get mouldy Detection concentration after secondary inoculation cfu / g <![CDATA[4.6×10 6 ]]> <![CDATA[1.5×10 6 ]]> <![CDATA[2.5×10 6 ]]> get mouldy Detection of cfu / g on the 21st day <![CDATA[5.2×10 3 ]]> <![CDATA[4.6×10 3 ]]> <![CDATA[9.5×10 3 ]]> get mouldy Detection of cfu / g on the 28th day ＜10 <![CDATA[2.8×10 3 ]]> get mouldy get mouldy Anticorrosion results excellent pass Not passed Not passed Oyster sauce molds when left exposed at room temperature No mold No mold get mouldy get mouldy

[0130] Table 2. Results of traditional USP antiseptic challenge test

[0131] sample Oyster Sauce A Oyster sauce B Oyster Sauce C Oyster sauce D Bacterial suspension concentration cfu / g <![CDATA[5.5×10 7 ]]> <![CDATA[3×10 7 ]]> <![CDATA[1×10 7 ]]> <![CDATA[4×10 6 ]]> Detection of cfu / g on day 0 <![CDATA[5.5×10 5 ]]> <![CDATA[3×10 5 ]]> <![CDATA[1×10 5 ]]> <![CDATA[4×10 4 ]]> Detection of cfu / g on the 7th day <![CDATA[2.0×10 3 ]]> <![CDATA[6.3×10 4 ]]> <![CDATA[2.5×10 4 ]]> <![CDATA[3.5×10 3 ]]> Detection of cfu / g on the 14th day <![CDATA[1.5×10 2 ]]> <![CDATA[3.2×10 2 ]]> <![CDATA[5.6×10 3 ]]> <![CDATA[1.6×10 3 ]]> Detection of cfu / g on the 21st day ＜10 ＜10 <![CDATA[4.6×10 2 ]]> get mouldy Detection of cfu / g on the 28th day ＜10 ＜10 <![CDATA[2.5×10 2 ]]> get mouldy Anticorrosion results excellent excellent pass Not passed Oyster sauce molds when left exposed at room temperature No mold No mold get mouldy get mouldy

[0132] As can be seen from the above table, compared with the results of the room temperature open placement experiment, the test results of the oyster sauce preservative performance test method newly developed in this application are highly consistent with the actual mold situation, while the United States Pharmacopoeia USP test method has certain limitations in the oyster sauce system and fails to distinguish oyster sauce products with intermediate preservative power. Compared with the single standard indicator bacteria-Aspergillus niger, the two strains of mold selected in this application are more representative of oyster sauce products and can make accurate judgments on the actual preservative power of oyster sauce products.

[0133] On the whole, this application develops a repeated inoculation challenge experiment, and screens oyster sauce-specific spoilage molds for challenge experiments. Repeating the inoculation twice during the shelf life can more accurately judge the preservative power of oyster sauce during the shelf life, and is more in line with actual consumer usage scenarios. The verification results are more accurate than the traditional United States Pharmacopoeia (USP) method.

[0134] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0135] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A method for detecting the antiseptic properties of oyster sauce, characterized in that: The detection method comprises the following steps: Using Aspergillus niger ZE902 and Aspergillus oryzae ZA306 as indicator bacteria, spore suspension was prepared; Inoculating the spore suspension into the oyster sauce sample to be tested for cultivation; During the culturing process, observe whether there are signs of mold on the surface of the culture, and take samples to detect the number of live indicator bacteria in the culture. The antiseptic performance of the oyster sauce sample to be tested is determined based on the observation results and the number of live indicator bacteria detected.

2. The method for detecting the antiseptic properties of oyster sauce according to claim 1, characterized in that: The detection method comprises the following steps: Inoculating the spore suspension into the oyster sauce sample to be tested for the first time to carry out the first stage of culture, and inoculating the spore suspension into the oyster sauce sample for the second time to carry out the second stage of culture; During the first stage of cultivation and the second stage of cultivation, the surface of the culture is observed at a preset time point to see if there are signs of mold, and samples are taken to detect the number of live indicator bacteria in the culture. The antiseptic properties of the oyster sauce sample to be tested are determined based on the observation results and the number of live indicator bacteria.

3. The method for detecting the antiseptic properties of oyster sauce according to claim 2, characterized in that: Taking the day of the first vaccination as day 0, the first stage of culture lasts from day 0 to day 14. After the second vaccination on day 14, the second stage of culture begins and lasts until day 28.

4. The method for detecting the antiseptic properties of oyster sauce according to claim 3, characterized in that: The preset time points include day 0, day 7, day 14, day 21 and day 28.

5. The method for detecting the antiseptic properties of oyster sauce according to claim 4, characterized in that: The antiseptic properties of the oyster sauce sample to be tested are judged according to the obtained observation results and the number of viable indicator bacteria, including: (1) No signs of mold, m1-14≤m1-0×10 -2 , m1-0>m1-7>m1-14, m2-28≤m2-14×10 -2 , m2-14>m2-21>m2-28, then the anti-corrosion ability is excellent; (2) No signs of mold, m1-7≤m1-0×10 -1 ,m2-21≤m2-14×10 -1 , if the indicator bacteria content in each culture stage decreases, the antiseptic ability is passed; (3) If mold is observed in the culture, the antiseptic performance fails; The live counts of the indicator bacteria detected on the 0th day, the 7th day, the 21st day and the 28th day were recorded as m1-0, m1-7, m2-21 and m2-28, respectively. The live counts of the indicator bacteria detected before and after the second inoculation on the 14th day were recorded as m1-14 and m2-14, respectively.

6. The method for detecting the antiseptic properties of oyster sauce according to any one of claims 1 to 5, characterized in that: In the spore suspension, the ratio of conidia of Aspergillus niger ZE902 to conidia of Aspergillus oryzae ZA306 is 1:(0.8-1.2).

7. The method for detecting the antiseptic properties of oyster sauce according to any one of claims 1 to 5, characterized in that: The culture conditions include: temperature of 28°C-32°C, oyster sauce mass of 50g-105g, oyster sauce volume not exceeding 1 / 2 of the total volume of the container, sealed, and culture time of 28d-30d.

8. The method for detecting the antiseptic properties of oyster sauce according to any one of claims 1 to 5, characterized in that: Each 1g of the oyster sauce sample to be tested contains 10 4 CFU-10 5 The concentration of CFU spores was used for inoculation.

9. The method for detecting the antiseptic properties of oyster sauce according to any one of claims 1 to 5, characterized in that: The mass of the spore suspension is 0.5%-1% of the mass of the oyster sauce sample to be tested.

10. The method for detecting the antiseptic properties of oyster sauce according to any one of claims 1 to 5, characterized in that: The method for detecting the number of viable indicator bacteria includes adopting a plate colony counting method.