Culture medium for detecting microorganisms producing foreign taste in beer, preparation method and application thereof

Abstract

The present invention relates to a culture medium for detecting microorganisms producing foreign taste in beer, a preparation method and an application thereof, and relates to the field of microbial technology. The culture medium comprises a nitrogen source and a carbon source, wherein the nitrogen source is fish meal peptone. The culture medium can rapidly culture microorganisms producing foreign taste in beer in a relatively short period of time, so that an operator can judge whether the beer is contaminated by microorganisms based on the culture results.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a culture medium for detecting microorganisms producing foreign taste in beer, and a preparation method and application thereof. Background Art

[0002] Occasionally, beer may smell sour or contaminated. The main reason for this problem is that the cleaning is not thorough after the production is stopped or the residual materials are corrupted at high temperature to produce metabolic byproducts such as butyric acid. This type of contamination is usually detected by microbial analysis and inspection, and there are both bacterial spores and other harmful bacteria in beer. When different types of bacteria are mixed together, the requirements for the culture medium are more stringent. Bacterial spores and anaerobic microorganisms usually have different growth requirements, and it is challenging to design a culture medium that meets both. Spores require specific nutrients and environmental conditions to germinate, while anaerobic microorganisms require an anaerobic environment. When culturing bacterial spores and anaerobic microorganisms at the same time, it is necessary to balance the growth conditions such as pH, temperature, and oxygen concentration to ensure that both can grow without inhibiting each other. A culture medium with too high a sugar concentration has a certain inhibitory effect on the growth and reproduction of spore bacteria, affecting its growth and reproduction; the pH of the culture medium also has a significant effect on the growth and inhibition of the strain, and has an important effect on the growth and metabolite accumulation of the bacteria; the demand for nitrogen source content varies greatly among different strains, and is affected by many factors. The concentration and type of nitrogen source will affect the growth rate and metabolic pathway of the bacteria. Different microorganisms have different adaptability to nitrogen sources. Some microorganisms can exhibit good growth and metabolic activity under specific nitrogen source conditions, while other microorganisms may not be able to survive or grow slowly under these conditions.

[0003] Since bacterial spores and anaerobic microorganisms may grow at different rates and have different adaptability to the environment, it is more difficult to detect and identify these microorganisms during the culture process. Therefore, it is very important to quickly develop a culture medium that is suitable for both bacterial spores and strict anaerobes to quickly achieve the cultivation of strains.

[0004] At present, beer culture media are basically developed for specialized culture or broad-spectrum culture. For example, plate count agar solid medium (PCA) generally cultures fungi under aerobic conditions, including yeast, mold, spores and aerobic bacteria, while NBB medium is for culturing anaerobic beer harmful bacteria, and UBA is a culture medium for the targeted cultivation of lactic acid bacteria and acetic acid bacteria. The culture time of the above-mentioned culture media generally takes about 7 days, which has a relatively high time cost and cannot quickly block the microbial contamination of the production process. Bacterial spores are divided into aerobic or facultative anaerobic Bacillus (Bacilli) and strictly anaerobic Clostridia (Clostridia). These bacteria usually have a certain symbiotic relationship with anaerobic bacteria. For example, it has been reported in the literature that Bacillus subtilis, which is usually considered to be an aerobic bacterium, can actually grow under strict anaerobic conditions using nitrate as an electron acceptor, indicating that it has facultative anaerobic characteristics. [1] We previously found that conventional liquid culture media such as wort medium and NBB medium cannot achieve the culture of mixed bacteria of Clostridium spores and anaerobic bacteria.

[0005] Therefore, developing a culture medium that can rapidly culture spore-forming bacteria and strict anaerobic bacteria simultaneously so as to achieve subsequent rapid detection is an urgent problem to be solved. Summary of the invention

[0006] In view of the above problems, the present invention provides a culture medium for detecting microorganisms that produce foreign taste in beer. The culture medium can quickly culture the microorganisms that produce foreign taste in beer in a relatively short period of time, so that operators can judge whether the beer is contaminated by microorganisms based on the culture results.

[0007] In order to achieve the above object, the present invention provides a culture medium for detecting foreign taste in beer, the culture medium comprises a nitrogen source and a carbon source, and the nitrogen source is fish meal peptone.

[0008] The inventors found in the research process that even if there is microbial contamination in the beer brewing process due to incomplete cleaning during the shutdown, the number of microorganisms is very small (for example, the content of anaerobic bacteria in brewing washing water is less than 10 2 The number of microorganisms in the beer is within the order of magnitude range, so it is difficult for operators to simply and directly determine whether there is microbial contamination from the cleaning water sample. However, if no pre-judgment or treatment is performed in the early stage, the subsequent microorganisms will continue to multiply in the beer, which will not only produce foreign tastes, but also affect product safety. Therefore, in order to be able to simply and accurately determine whether there is microbial contamination that causes foreign tastes in a short period of time, the inventors proposed the above-mentioned culture medium, which can quickly cultivate microorganisms that produce foreign tastes in beer in a relatively short period of time, so that operators can determine whether the beer is contaminated by microorganisms based on the culture results.

[0009] In one embodiment, the culture medium is composed of the following raw materials in mass fractions:

[0010]

[0011] When liquid culture medium needs to be prepared, the amount of coagulant used is 0; when solid culture medium needs to be prepared, the amount of coagulant used is not 0.

[0012] In one embodiment, the carbon source includes at least one of glucose, fructose, syrup, honey, and malt powder.

[0013] In one embodiment, the syrup includes at least one of fructose-glucose syrup, starch syrup, and molasses.

[0014] In one embodiment, the coagulant includes at least one of agar, gelatin, carrageenan, konjac flour, and guar gum.

[0015] In one embodiment, the pH adjuster includes sodium hydroxide solution and a buffer, and the buffer includes at least one of a phosphate buffer system and a Borate buffer system.

[0016] In one embodiment, the phosphate buffer system includes K 2 HPO 4 、NaH 2 PO 4 .

[0017] In one embodiment, the Borate buffer system includes Na 2 B 4 O 7 10H 2 O.

[0018] The present invention also provides a method for preparing the culture medium, comprising the following steps: mixing raw materials, adjusting pH value, sterilizing, and deoxygenating to obtain the culture medium.

[0019] In one embodiment, the pH is 6-8.

[0020] In one embodiment, the sterilization temperature is 118-124°C, and the time is 10-20 minutes; the deoxygenation time is 45-55 hours.

[0021] The present invention also provides a method for detecting foreign taste-producing microorganisms in beer, comprising the following steps: taking a sample to be tested under sterile conditions, inoculating it into the culture medium, adopting aerobic culture or anaerobic culture, and analyzing the culture results.

[0022] When the target microorganism is an aerobic or facultative anaerobic microorganism such as wild yeast or mold, the above culture medium is cultured aerobically.

[0023] In one embodiment, the temperature of the anaerobic culture is 25-39°C.

[0024] In one embodiment, the sample to be tested includes weak wort, a cleaning water sample of a pipe, a pot or a tank.

[0025] In one embodiment, when the amount of bacteria in the sample to be tested is small, the detection method further includes filtering after taking the sample to be tested, and inoculating the residue obtained by filtering into the culture medium.

[0026] In one embodiment, the analysis includes: testing the culture results to determine whether the sample to be tested is contaminated by microorganisms;

[0027] The judgment rule includes: if the turbidity of the culture medium increases and / or a foreign smell appears, it is judged that the sample to be tested is contaminated by microorganisms.

[0028] In one embodiment, the detection includes detecting at least one of bacterial count, smell, GC or GC-MS.

[0029] In one embodiment, the bacterial cell amount is obtained by detecting the turbidity of the culture sample; the foreign smell is detected by smell, GC or GC-MS, and the foreign smell includes sourness and odor; the GC or GC-MS detection determines that there is at least one of acetic acid, butyric acid, isovaleric acid, isopentanol, propionic acid or indole in the culture result, then it is determined that there is a foreign smell.

[0030] In one embodiment, the microorganisms include at least one of aerobic or facultative anaerobic microorganisms such as bacterial spores, anaerobic bacteria, wild yeast, and mold.

[0031] In one embodiment, the cell spores are derived from at least one of Bacillus, Clostridium, Sporolaetobacilbs, and Proteobacteria.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The culture medium for detecting microorganisms producing foreign taste in beer, as well as its preparation method and application, can realize rapid culture of microorganisms producing foreign taste in beer in a relatively short period of time, and the OD600 value can reach about 0.6-0.8 within 3 days, so that operators can judge whether the beer is contaminated by microorganisms based on the culture results. DETAILED DESCRIPTION

[0034] For ease of understanding of the present invention, the present invention will be described more fully below with reference to relevant embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0036] source:

[0037] Unless otherwise specified, the reagents, materials, and equipment used in this example are all commercially available; the experimental methods are all conventional experimental methods in the art unless otherwise specified.

[0038] Example 1

[0039] 1. Culture medium components.

[0040] Fish meal peptone (peptone from fish, Sinopharm Chemical Reagent Co., Ltd.) 1.2% (mass fraction, the same below), glucose 1.5%.

[0041] 2. Preparation of culture medium.

[0042] Weigh 12g of fish meal peptone and 15g of glucose, and prepare 1000g of liquid culture medium with ultrapure water. Adjust the pH with sodium hydroxide solution first, and then adjust it with a buffer, including phosphate buffer system K 2 HPO 4 +NaH 2 PO 4 、Borate buffer system Na 2 B 4 O 7 10H 2 O, in this embodiment, a phosphate buffer system is selected so that the pH of the culture medium is 6-8 when used.

[0043] 3. Sterilize and deoxygenate the culture medium.

[0044] The solution obtained in step 2 of this example was sterilized at 121 degrees for 15 minutes.

[0045] The sterilized liquid culture medium is divided into small test tubes and deoxygenated in an anaerobic chamber or with a deoxygenating bag for 48 hours (a deoxygenation indicator is placed in the air part at the upper end of the small test tube to detect the deoxygenation. If the deoxygenation is complete, the test paper will not change color).

[0046] 4. Inoculate the sample to be tested.

[0047] Aseptically take a sample of the weak wort produced in large quantities, take 100 ml of the weak wort sample and filter it with 0.45 μm filter paper, put the residue obtained after the filtration together with the filter paper into a small test tube of the deoxygenated liquid culture medium obtained in step 3 of this embodiment for inoculation.

[0048] 5. Anaerobic culture.

[0049] The inoculated sample test tubes were cultured anaerobically at 37 degrees, and the anaerobic conditions in the anaerobic chamber were monitored with deoxygenated test paper.

[0050] After 3 days of cultivation, the culture results were obtained, the OD600 value was measured by turbidimetry, and the culture results were smelled to describe the taste characteristics.

[0051] Comparative Example 1

[0052] The method is basically the same as Example 1, except that conventional wort medium is used to replace fish meal peptone to prepare the culture medium.

[0053] The preparation method of the conventional wort culture medium comprises the following steps: taking a large production wort at 13 degrees, sterilizing it at 121 degrees for 15 minutes, and deoxygenating it. The specific operation of deoxygenation is the same as that in Example 1.

[0054] Comparative Example 2

[0055] The culture medium was prepared according to Example 1, but no bacteria were inoculated into the culture medium. Other aspects were the same as those in Example 1.

[0056] Comparative Example 3

[0057] The method is basically the same as Example 1, except that the fish meal peptone in the culture medium components is replaced by yeast extract (purchased from Beijing Aoboxing Biotechnology Co., Ltd.), and the other methods are the same as Example 1.

[0058] Comparative Example 4

[0059] The method is basically the same as Example 1, except that the fish meal peptone in the culture medium components is replaced by beef extract powder (purchased from Beijing Luqiao Technology Co., Ltd.), and the other methods are the same as Example 1.

[0060] Experimental example

[0061] 1. The ratio of nitrogen sources in the culture medium of each embodiment and comparative example.

[0062] Table 1 Ratio of nitrogen sources in the culture medium of each embodiment and comparative example

[0063] Nitrogen source Amino nitrogen (mass percentage, %) Total nitrogen (mass percentage, %) Example 1 / ≥12.0％ Comparative Example 3 ＞3.0％ ＞12.0％ Comparative Example 4 ≥3.0％ ≥9.0％

[0064] 2. The bacterial concentration and flavor characteristics of the culture medium in each example and comparative example were tested.

[0065] 1. The culture results of the culture medium of each embodiment and comparative example were tested for bacterial concentration by turbidimetry: 200 μL of the cultured sample of each embodiment and comparative example was taken, and the bacterial concentration was measured by turbidimetry at a wavelength of 600 nm to obtain the OD600 value.

[0066] At the same time, some samples were taken out for sniffing, and the sniffing results were recorded. The results are shown in the following table.

[0067] Table 2 Bacterial concentration and olfactory results of the culture results of each embodiment and comparative example

[0068] Serial number OD600 Smell Example 1 0.83 Unpleasant cheese taste, sour smell Comparative Example 1 0.10 The taste of wort Comparative Example 2 0.06 Normal trypsin medium taste Comparative Example 3 0.62 Slightly sour and unpleasant cheese smell Comparative Example 4 0.56 Slightly sour and unpleasant cheese smell

[0069] From the OD600 results, it can be seen that compared with Comparative Examples 1 and 2, the bacteria in Example 1 are in the logarithmic growth phase, the culture medium has an obvious culture effect, and the sample has a typical butyric acid odor.

[0070] 2. Detection of characteristic flavor substances.

[0071] (1) Detection method: Butyric acid and isovaleric acid were detected using a method developed in this experiment based on headspace solid phase microextraction-gas chromatography (HS-SPME-GC) technology, and the external standard method was used for quantification.

[0072] Instruments: GC-7890B gas chromatograph; 50 / 30μm DVB / CAR / PDMS three-phase extraction head; DB-FFAP capillary column (60.00m×0.25mm×0.25μm), all provided by Agilent, USA;

[0073] The extraction conditions of SPME were as follows: extraction time 35 min, extraction temperature 60 °C, and desorption time 7 min.

[0074] Chromatographic conditions: Temperature program: initial temperature 50℃, hold for 1 min, increase to 150℃ at a rate of 10℃ / min, hold for 2 min, then increase to 240℃ at a rate of 15℃ / min, hold for 10 min. Inlet temperature 240℃, detector (FID) temperature 250℃, equilibrium time: 0.1 min. Split ratio: 15:1. Carrier gas: high purity N 2 , purity>99.99%, flow rate: 1mL / min. 2Flow rate: 45 mL / min, air flow rate: 450 mL / min. Injection method: Use solid phase microextraction manual injector for injection.

[0075] Acetic acid was detected by ion chromatography.

[0076] (2) Test results: as shown in the following table.

[0077] Table 3 Detection results of characteristic flavor substances

[0078] Serial number Butyric acid mg / L Isovalerate mg / L Acetic acid mg / L Example 1 1841.98 1.47 1336.84 Comparative Example 1 0.10 0 0.15 Comparative Example 2 0.02 0 0 Comparative Example 3 32.42 0.96 65.24 Comparative Example 4 30.68 0.87 110.24

[0079] From the results of flavor substances, the contents of butyric acid and isovaleric acid, the main flavor substances detected in the sample of Example 1, are significantly higher than those in Comparative Examples 1, 3, and 4.

[0080] 3. High-throughput gene sequencing.

[0081] High-throughput gene sequencing was performed on the culture results of Example 1 and the weak wort stock solution used for sampling in Example 1. The results are shown in the following table.

[0082] Table 4 High-throughput gene sequencing results

[0083]

[0084] It can be seen from the high-throughput gene sequencing results that the bacteria contained in Example 1 are mainly Bacillus and Clostridium.

[0085] In summary, the culture medium developed by this patent can achieve the rapid cultivation of microorganisms that cause beer odor. The microorganisms can reach the logarithmic growth period after 3 days of cultivation. The olfactory result shows a typical contaminated odor, and the content of major flavor substances is also significantly higher than that of the control sample.

[0086] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments 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.

[0087] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A culture medium for detecting microorganisms producing foreign taste in beer, characterized in that: The culture medium comprises a nitrogen source and a carbon source, wherein the nitrogen source is fish meal peptone.

2. The culture medium according to claim 1, characterized in that The culture medium is composed of the following raw materials in mass fractions:

3. The culture medium according to claim 2, characterized in that The carbon source includes at least one of glucose, fructose, syrup, honey and malt powder.

4. The culture medium according to claim 2, characterized in that The coagulant includes at least one of agar, gelatin, carrageenan, konjac flour and guar gum.

5. The culture medium according to claim 2, characterized in that The pH adjuster comprises a sodium hydroxide solution and a buffer, and the buffer comprises at least one of a phosphate buffer system and a Borate buffer system.

6. The method for preparing the culture medium according to any one of claims 1 to 5, characterized in that: The following steps are involved: The raw materials are mixed, the pH value is adjusted, sterilized, and deoxygenated to obtain a culture medium.

7. A method for detecting microorganisms producing foreign taste in beer, characterized in that: The method comprises the following steps: taking a sample to be tested under sterile conditions, inoculating it into the culture medium according to any one of claims 1 to 5, adopting aerobic culture or anaerobic culture, and analyzing the culture results.

8. The detection method according to claim 7, characterized in that: The temperature of the anaerobic culture is 25-39°C.

9. The detection method according to claim 7, characterized in that: The analysis includes: testing the culture results to determine whether the sample to be tested is contaminated by microorganisms; The judgment rule includes: if the turbidity of the culture medium increases and / or a foreign smell appears, it is judged that the sample to be tested is contaminated by microorganisms.

10. The detection method according to claim 9, characterized in that: The microorganisms include at least one of aerobic or facultative anaerobic microorganisms such as bacterial spores, anaerobic bacteria, wild yeast, and mold.

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