阅读说明：本技术 一种用于检测调味品中难培养菌的培养基和检测方法 (Culture medium and detection method for detecting refractory bacteria in seasoning ) 是由 黄寿聪 王洪江 桂军强 于 2020-06-23 设计创作，主要内容包括：本发明公开了一种用于检测调味品中难培养菌的培养基,包含MRS 0.040～0.070g/mL、蔗糖0.005～0.030g/mL、复配生长因子0.0005～0.0020g/mL和待测调味品稀释液200～500mL/L；所述复配生长因子包含以下重量份的组分：L-半胱氨酸30～70份、FeSO<Sub>4</Sub>·7H<Sub>2</Sub>O 30～70份和吐温-80 0～30份；所述待测调味品稀释液中待测调味品的质量浓度为40～70％。本发明所述培养基中添加待测调味品稀释液和复配生长因子,为变质菌的生长提供适宜的微量元素和生长因子,缩短了变质菌的培养时间,提高了检测效率。本发明还公开了一种调味品的质量检测方法。(The invention discloses a culture medium for detecting refractory bacteria in a seasoning, which comprises 0.040-0.070 g/mL of MRS, 0.005-0.030 g/mL of sucrose, 0.0005-0.0020 g/mL of compound growth factor and 200-500 mL/L of seasoning diluent to be detected; the compound growth factor comprises the following components in parts by weight: 30-70 parts of L-cysteine and FeSO 4 ·7H 2 30-70 parts of O and 800-30 parts of Tween-; the mass concentration of the condiment to be detected in the condiment diluent to be detected is 40-70%. The culture medium is added with the condiment diluent to be detected and the compound growth factor, so that appropriate trace elements and growth factors are provided for the growth of spoilage bacteria, the culture time of the spoilage bacteria is shortened, and the detection efficiency is improved. The invention also discloses a quality detection method of the seasoning.)

1. A culture medium for detecting refractory bacteria in a seasoning is characterized by comprising 0.040-0.070 g/mL of MRS broth, 0.005-0.030 g/mL of cane sugar, 0.0005-0.0020 g/mL of compound growth factor and 200-500 mL/L of seasoning diluent to be detected;

the compound growth factor comprises the following components in parts by weight: 30-70 parts of L-cysteine and FeSO₄·7H₂30-70 parts of O and 800-30 parts of Tween-;

the mass concentration of the condiment to be detected in the condiment diluent to be detected is 40-70%.

2. The culture medium for detecting bacteria difficult to culture in a seasoning according to claim 1, wherein the pH of the culture medium is 3.5 to 4.2.

3. The medium according to claim 1, wherein the seasoning to be tested is soy sauce, seasoning wine, vinegar or compound seasoning sauce.

4. The culture medium for detecting the difficult-to-culture bacteria in the seasoning according to claim 1, wherein the preparation method of the culture medium comprises the following steps:

(1) adding MRS broth, sucrose and compound growth factor into tertiary water, boiling for dissolving, and cooling to obtain material A;

(2) adding the filtered diluent of the seasoning to be detected into the material A, and uniformly mixing to obtain a material B;

(3) adjusting the pH value to 3.5-4.2, and sterilizing to obtain the culture medium.

5. A seasoning quality detection method, characterized in that detection is carried out by using the culture medium for detecting the bacteria difficult to culture in the seasoning as claimed in any one of claims 1 to 4.

6. The seasoning quality inspection method of claim 5 wherein the seasoning quality inspection method comprises the steps of:

(a) inoculating the seasonings to be tested into the culture medium, culturing for 2-7 days at 29-31 ℃, and arranging multiple groups of parallel samples;

(b) in the culture period, when at least one of the conditions (i) to (iii) occurs, judging the condiment to be tested to be unqualified; otherwise, judging the seasoning to be tested to be qualified;

(i) bubbles were observed for at least 1 sample;

(ii) stickiness and stringiness were observed for at least 1 sample;

(iii) at least 1 sample showed X₁-X₀≥0.20g/100mL，X₁Denotes the total acid, X, of the medium during the cultivation₀Is the initial total acid of the medium.

7. The seasoning quality inspection method according to claim 6, wherein in the step (a), 1mL of the seasoning to be tested is inoculated into a culture medium, and the culture medium is contained in a Duchen vial.

8. The seasoning quality detection method of claim 7 wherein the culture medium in the Duchen vial is bubble free.

9. Use of the culture medium for detecting bacteria difficult to culture in seasoning according to any one of claims 1 to 4 for detecting bacteria difficult to culture in seasoning.

Technical Field

The invention relates to a detection method of microorganisms, in particular to a culture medium and a detection method for detecting refractory bacteria in a seasoning.

Background

The seasoning is an auxiliary food which can increase the color, the fragrance and the taste of dishes, promote appetite and benefit human health. Its main function is to improve the quality of the dish and meet the sensory requirements of consumers, thus stimulating appetite and improving human health. If the seasoning is not sterilized properly in the production process, part of thermophilic acid-resistant bacteria can cause the seasoning to deteriorate in shelf life. For the microbial detection of the difficult-to-culture type of the seasoning, the currently related technologies mainly have the following aspects:

patent numbers: 201811113012.X culture medium and detection method for aerogenic bacteria in cooking wine, which discloses a culture medium and detection method for aerogenic bacteria in cooking wine, comprising the following steps: diluting a cooking wine sample to be detected, inoculating the diluted cooking wine sample into a fermentation tube containing a culture medium, culturing the diluted cooking wine sample at the temperature of 30 +/-1 ℃ for 48 +/-2 hours, and detecting aerogenic bacteria according to whether the fermentation tube produces the gas or not; the culture medium consists of the following raw materials in parts by weight: 10-30 parts of sucrose, 3-6 parts of yeast extract, 4-10 parts of peptone, 3-10 parts of sodium chloride, 2-8 parts of yellow wine, 801-2 parts of tween-801, 0.05-1 part of sodium thioglycolate, 0.2-0.6 part of magnesium sulfate heptahydrate, 0.01-0.1 part of methionine, 0.01-0.2 part of cysteine and 1000 parts of distilled water. The detection method can detect the culture-refractory bacteria of the cooking wine which are difficult to grow on the conventional culture medium, has good correlation between the detection result of the detection method and the deterioration and gas production of the food, and provides a basis for improving the production process, controlling the product quality and improving the product quality; the method has the advantages of high detection efficiency, simplicity, low cost, easiness in popularization and the like, and is suitable for industrial production application. The method mainly monitors the bacteria which are difficult to culture and produce gas by means of the deterioration and the gas production of the cooking wine product, but does not monitor the bacteria which are difficult to culture and produce acid but not produce gas.

Patent No. CN201410579443.0 culture medium and detection method for detecting aerogenic bacteria and membranogen bacteria in vinegar discloses a culture medium and detection method for detecting aerogenic bacteria and membranogen bacteria in vinegar, wherein the culture medium is a liquid culture medium prepared from potatoes, glucose, yeast extract, beef extract, ammonium sulfate and the like serving as raw materials, the detection method is to inoculate a vinegar sample to be detected in a test tube provided with a Du's small tube and then culture the vinegar sample for 72 hours for observation, and if bubbles and mycoderm appear in the test tube, the vinegar is unqualified and cannot be directly delivered from a factory. The method mainly determines a culture medium and a detection method of aerogenic and membrane-producing bacteria in vinegar, determines whether vinegar products deteriorate or not through bubbles and mycoderm appearance in a test tube, but does not disclose monitoring of non-aerogenic bacteria but acid-producing bacteria which are difficult to culture.

Therefore, the components of the existing various culture media have obvious differences, and the simultaneous culture and rapid detection of all bacteria difficult to culture cannot be realized.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a culture medium and a detection method for detecting bacteria difficult to culture in a seasoning.

In order to achieve the purpose, the invention adopts the technical scheme that: a culture medium for detecting refractory bacteria in a seasoning comprises 0.040-0.070 g/mL of MRS broth, 0.005-0.030 g/mL of sucrose, 0.0005-0.0020 g/mL of compound growth factor and 200-500 mL/L of seasoning diluent to be detected;

the compound growth factor comprises the following components in parts by weight: 30-70 parts of L-cysteine and FeSO₄·7H₂30-70 parts of O and 800-30 parts of Tween-;

the mass concentration of the condiment to be detected in the condiment diluent to be detected is 40-70%.

Part of microorganisms are difficult to culture through a conventional culture medium, the conventional method cannot detect the microorganisms, the microorganisms cannot be effectively killed in advance, turbidity, gas production, no gas production but acid production occur in the shelf life process, and the microorganisms have unpleasant odor and seriously affect the product quality. The culture medium is added with the condiment diluent to be detected and the compound growth factor, so that appropriate trace elements and growth factors are provided for the growth of spoilage bacteria, the culture time of the spoilage bacteria is shortened, and the detection efficiency is improved.

Preferably, the pH of the culture medium is 3.5-4.2.

Preferably, the condiment to be detected is soy sauce, seasoning cooking wine, vinegar or compound seasoning sauce (including tomato sauce, pork rib sauce, garlic chili sauce and the like). The culture medium has a wide detection range, can be used for detecting spoilage bacteria such as gas generation, sticky wire drawing and total acid increase in seasonings such as soy sauce, seasoning wine or compound seasoning sauce, and is more comprehensive in monitoring.

Preferably, the preparation method of the culture medium comprises the following steps:

(1) adding MRS broth, sucrose and compound growth factor into tertiary water, boiling for dissolving, and cooling to obtain material A;

(2) adding the filtered diluent of the seasoning to be detected into the material A, and uniformly mixing to obtain a material B;

(3) adjusting the pH value to 3.5-4.2, and sterilizing to obtain the culture medium.

The invention also aims to provide a quality detection method of the seasoning, which adopts the culture medium for detecting the bacteria difficult to culture in the seasoning for detection.

The detection of the seasoning by adopting the culture medium is short in detection time, and the difficult-to-detect bacteria can be detected in 2 days at the shortest time; for bacteria which are difficult to detect, if the bacteria have no metamorphic bacteria in 7 days, the bacteria can be determined. Compared with the traditional MRS lactic acid bacteria culture medium, the detection time is greatly shortened, and products with unqualified quality can be effectively found in time.

Preferably, the seasoning quality detection method comprises the following steps:

(a) inoculating the seasonings to be tested into the culture medium, culturing for 2-7 days at 29-31 ℃, and arranging multiple groups of parallel samples;

(b) in the culture period, when at least one of the conditions (i) to (iii) occurs, judging the condiment to be tested to be unqualified; otherwise, judging the seasoning to be tested to be qualified;

(i) bubbles were observed for at least 1 sample;

(ii) stickiness and stringiness were observed for at least 1 sample;

(iii) at least 1 sample showed X₁-X₀≥0.20g/100mL，X₁Denotes the total acid, X, of the medium during the cultivation₀Is the initial total acid of the medium.

The spoilage bacteria growing in the seasoning are a general name of microorganisms causing the characteristics of gas production, sticky wiredrawing, total acid increase and the like. For non-aerogenic refractory bacteria, whether the seasoning product is deteriorated or not is determined in all directions through total acid amplitude rise, turbidity return, wiredrawing stickiness and the like, the quality is controlled in advance, and the quality of subsequent products is ensured.

Preferably, in step (a), 1mL of the seasoning to be tested is inoculated into a culture medium contained in a small Duchenne tube.

Preferably, the medium in the duvet tubule is bubble free. Means that no air bubble in the culture medium in the Duchen small tube is selected to inoculate the condiment to be tested so as to reduce the error of observing the air bubble in the subsequent culture period.

The invention also aims to provide the application of the culture medium for detecting the bacteria which are difficult to culture in the seasoning in detecting the bacteria which are difficult to culture in the seasoning.

The invention has the beneficial effects that: the invention provides a culture medium for detecting bacteria difficult to culture in seasonings, which is added with seasoning diluent and compound growth factors, so that appropriate trace elements and growth factors are provided for the growth of spoilage bacteria, the culture time of the spoilage bacteria is shortened, and the detection efficiency is improved. The invention also provides a quality detection method of the seasoning, the seasoning is detected by adopting the culture medium, the detection time is short, and the detection of bacteria which are difficult to detect can be carried out in 2 days at the shortest time; for bacteria which are difficult to detect, if the bacteria have no metamorphic bacteria in 7 days, the bacteria can be determined. Compared with the traditional MRS lactic acid bacteria culture medium, the detection time is greatly shortened, and products with unqualified quality can be effectively found in time. The invention also provides application of the culture medium for detecting the refractory bacteria in the seasoning in detecting the refractory bacteria in the seasoning.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.