Method for collecting microorganisms and application thereof

Abstract

The invention discloses a method for collecting microorganisms, which comprises the following steps: 1) taking microbial fermentation liquor, centrifuging, and discarding supernatant to obtain thallus precipitate; (2) adding a sodium chloride solution which is 0.5-2 times of the original volume of the fermentation liquor into the thallus precipitate, and resuspending thalli to obtain a resuspension solution; and (3) centrifuging for 5-25 minutes at the speed of 7000-12500 rpm, and discarding the supernatant to obtain the thalli. The invention also discloses an application of the method in preparation of a high-activity bacterial agent. It is proved that compared with a traditional method for cleaning and collecting microorganisms with sterile water, when the method is adopted for collecting thalli, the loss amount is smaller, the prepared microbial agent is higher in viable count, and the good application prospect is achieved.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a method for collecting microorganisms and its application. Background Art

[0002] With the improvement of people's lives and the increasing emphasis on physical health, traditional medicines such as Chinese herbal medicine and Western medicine can no longer meet consumers' requirements for health care functions. On the contrary, they are more interested in the intestinal flora that accompanies us throughout our lives, because there have been a large number of reports showing that these microorganisms are key environmental factors that directly or indirectly have beneficial or harmful effects on the human body. Some of these microorganisms, such as probiotics, have been widely studied due to their beneficial properties to the host, and have been developed into probiotic preparations for direct ingestion to regulate intestinal flora or used as starters for related fermented products. However, the efficacy of probiotics is dose-dependent, and they are almost ineffective under low-dose conditions, so this requires producers to cultivate and collect the above-mentioned probiotics in large quantities to meet the dosage value required for the function to be exerted.

[0003] At present, the collection of probiotics is mainly completed by centrifugal collection, among which the centrifugal rate and cleaning agent are important factors affecting the number and purity of live bacteria contained in the final bacterial agent. It is well known that the collection efficiency is proportional to the centrifugal rate. However, too high a centrifugal speed has high requirements on the equipment on the one hand, and high shear force will also cause certain damage to the bacteria on the other hand. Therefore, the rate of centrifugal collection is relatively limited for industrial production enterprises. The purpose of the cleaning agent is to replace the fermentation liquid and metabolites remaining in the bacterial sediment, thereby eliminating the influence of these impurities on subsequent fermentation. This step is particularly important in the process of preparing the fermentation agent. For most probiotics, using sterile water as a cleaning agent is sufficient to achieve the purpose of efficient collection and cleaning, but for some microorganisms that produce extracellular polysaccharides, the resuspension after washing the bacteria with sterile water cannot be separated by centrifugation at all. This is because the extracellular polysaccharides of these microorganisms have excellent dispersibility in pure water, so that the microbial cells in the resuspension system are stably and evenly dispersed therein, and are not easy to be collected by centrifugation. Therefore, it is urgent to find a method to improve the efficiency of microorganism collection so that the above-mentioned easily dispersed exopolysaccharide-producing microorganisms can be more effectively collected by centrifugation. The above-mentioned problems are urgent to be solved by those skilled in the art. Summary of the invention

[0004] In order to solve the above technical problems, the present application provides a method for collecting microorganisms, comprising the following steps: 1) taking microbial fermentation liquid, centrifuging, discarding the supernatant, and obtaining a bacterial precipitate; 2) adding a sodium chloride solution with a volume of 0.5 to 2 times the original volume of the fermentation liquid to the bacterial precipitate, resuspending the bacteria to obtain a resuspended liquid; 3) centrifuging at 7000 to 12500 rpm for 5 to 25 minutes, discarding the supernatant, and obtaining the bacteria.

[0005] The present application also provides the application of the above method in collecting bacteria or preparing bacterial agents.

[0006] The beneficial effects of the present invention include but are not limited to: (1) The activity of the bacterial agent prepared by the microbial collection method of the present invention is greater than 2×10 11 CFU / g; (2) when the microorganism collection method of the present application is used to collect bacteria, the loss is small; (3) the microorganism collection method of the present application has the advantages of low cost and simple operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The present application will be further described in the form of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not restrictive, and include:

[0008] Figure 1 This is a comparison of the centrifugation results after resuspension in the cleaning solution in Scheme 1 and Example 3. DETAILED DESCRIPTION

[0009] In order to more clearly illustrate the technical solutions of the embodiments of this specification, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some examples or embodiments of this specification. For ordinary technicians in this field, this specification can also be applied to other similar scenarios based on these drawings without creative work. Unless it is obvious from the language environment or otherwise explained, the same reference numerals in the figures represent the same structure or operation.

[0010] As shown in this specification and claims, unless the context clearly indicates an exception, the words "a", "an", "an" and / or "the" do not refer to the singular and may also include the plural. Generally speaking, the terms "comprise" and "include" only indicate the inclusion of the steps and elements that have been clearly identified, and these steps and elements do not constitute an exclusive list. The method or device may also include other steps or elements.

[0011] Flowcharts are used in this specification to illustrate the operations performed by the system according to the embodiments of this specification. It should be understood that the preceding or following operations are not necessarily performed precisely in order. Instead, the steps may be processed in reverse order or simultaneously. At the same time, other operations may also be added to these processes, or one or more operations may be removed from these processes.

[0012] The present application provides a method for collecting microorganisms, comprising the following steps: 1) taking microbial fermentation liquid, centrifuging, discarding the supernatant, and obtaining bacterial precipitate; 2) adding a sodium chloride solution with a volume of 0.5 to 2 times the original volume of the fermentation liquid to the bacterial precipitate, resuspending the bacteria to obtain a resuspended liquid; 3) centrifuging at 7000 to 12500 rpm for 5 to 25 minutes, discarding the supernatant, and obtaining the bacteria.

[0013] In some embodiments, in the step 1), the centrifugation condition can be 8000-12000rpm, 10-20min. For example, the centrifugation condition can be about 8200, 8400, 8600, 8800, 9000, 9200, 9400, 9600, 9800, 10000, 10200, 10400, 10600, 10800, 11000, 11200, 11400, 11600, 11800 or 12000rpm. It also includes any range characterized by a combination of the above end values, which will not be repeated here. For example, the centrifugation time can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20min. It also includes any range characterized by a combination of the above end values, which will not be repeated here.

[0014] In some embodiments, preferably, in step 1), the centrifugation condition can be 10000 rpm, 15 min.

[0015] In some embodiments, in step 2), a sodium chloride solution of 0.75 to 1.5 times the original volume of the fermentation liquid is added to the bacterial precipitate. For example, a sodium chloride solution of about 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45 or 1.5 times the original volume of the fermentation liquid is added to the bacterial precipitate. Any range characterized by a combination of the above end values ​​is also included, which will not be repeated here.

[0016] In some embodiments, in step 2), a sodium chloride solution with an amount 1 times the original volume of the fermentation liquid is added to the bacterial precipitate.

[0017] In some embodiments, in the step 3), the centrifugal condition can be 8750-11250rpm, 10-20min. For example, the centrifugal condition can be about 8750, 8850, 8950, 9050, 9150, 9250, 9350, 9450, 9550, 9650, 9750, 9850, 9950, 10050, 10150, 10250, 10350, 10450, 10550, 10650, 10750, 10850, 10950, 11050, 11150 or 11250rpm. It also includes any range characterized by a combination of the above end values, which will not be repeated here. For example, the centrifugal time can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20min. Any range characterized by a combination of the above-mentioned end values ​​is also included and will not be repeated here.

[0018] In some embodiments, preferably, in step 3), the centrifugation condition can be 10000 rpm, 15 min.

[0019] In some embodiments, the microorganism may be a bacterium that secretes exopolysaccharides. In some embodiments, preferably, the microorganism may be Lactobacillus rhamnosus. In some embodiments, more preferably, the microorganism may be Lactobacillus rhamnosus CGMCC No.13310.

[0020] In some embodiments, the sodium chloride solution may be an aqueous solution of sodium chloride. In some embodiments, preferably, the solute concentration in the sodium chloride solution may be 0.7% to 1.1%, and the pH may be 7.0 to 7.4. For example, the solute concentration in the sodium chloride solution may be approximately 0.7%, 0.8%, 0.9%, 1.0% or 1.1%. Any range characterized by a combination of the above end values ​​is also included, which will not be repeated here. For example, the pH of the sodium chloride solution may be approximately 7.0, 7.1, 7.2, 7.3 or 7.4. Any range characterized by a combination of the above end values ​​is also included, which will not be repeated here.

[0021] In some embodiments, more preferably, the sodium chloride solution can be physiological saline.

[0022] The present application also provides the application of the above method in collecting bacteria or preparing bacterial agents.

[0023] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0024] Example 1

[0025] 1. Materials and methods

[0026] Preparation of Lactobacillus rhamnosus fermented liquid: freeze-dried powder of Lactobacillus rhamnosus CGMCC No.13310 (provided by Bright Dairy Co., Ltd.) was dissolved with a small amount of sterile distilled water, a ring was streaked on MRS solid medium (purchased from Merck Co., Germany) with an inoculating loop, and taken out after anaerobic culture at 37°C for 24 h, a single colony was picked out with an inoculating loop and put into 10 mL MRS liquid medium (purchased from Merck Co., Germany), a vortex oscillator was used to evenly disperse the colony in the liquid medium, anaerobic culture was taken out at 37°C for 24 h, and the inoculum was inoculated into MRS liquid medium with 2% (v / v), and after anaerobic culture at 37°C for 24 h, a Lactobacillus rhamnosus fermented liquid was obtained, and the viable bacteria concentration of Lactobacillus rhamnosus in the fermented liquid was determined to be 2.3 × 10 9 CFU / mL.

[0027] The centrifuge of the present application (BECKMAN COULTER, Avanti JXN-30) is equipped with a rotor (JA-25.50).

[0028] The deposit information of the strain in this application is as follows:

[0029] Strain name: Lactobacillus rhamnosus

[0030] The accession number is: CGMCC No.13310;

[0031] Deposit date: November 15, 2016;

[0032] Name of depository: General Microbiology Center of China Microbiological Culture Collection Administration;

[0033] Abbreviation of depository unit: CGMCC;

[0034] Address of the preservation unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0035] 2. Collection of Lactobacillus rhamnosus

[0036] Take 20 mL of Lactobacillus rhamnosus fermentation broth and add it to a 50 mL centrifuge tube. Centrifuge at 10000 rpm for 15 min, discard the supernatant to obtain a bacterial precipitate, add physiological saline twice the original volume of the fermentation broth to the bacterial precipitate to resuspend the bacteria, centrifuge again (7000 rpm, 25 min), discard the supernatant, and obtain bacterial A.

[0037] Example 2

[0038] 1. Materials and methods

[0039] Same as Example 1.

[0040] 2. Collection of Lactobacillus rhamnosus

[0041] Take 20 mL of Lactobacillus rhamnosus fermentation broth and add it to a 50 mL centrifuge tube. Centrifuge at 10000 rpm for 15 min, discard the supernatant to obtain the bacterial precipitate, add 3 / 2 times the original volume of the fermentation broth of normal saline to the bacterial precipitate to resuspend the bacteria, centrifuge again (8750 rpm, 20 min), discard the supernatant, and obtain bacterial B.

[0042] Example 3

[0043] 1. Materials and methods

[0044] Same as Example 1.

[0045] 2. Collection of Lactobacillus rhamnosus

[0046] Take 20 mL of Lactobacillus rhamnosus fermentation broth and add it to a 50 mL centrifuge tube. Centrifuge at 10,000 rpm for 15 min, discard the supernatant to obtain a bacterial pellet, add 1 times the original volume of the fermentation broth with physiological saline to the bacterial pellet to resuspend the bacteria, centrifuge again (10,000 rpm, 15 min), discard the supernatant, and obtain bacterial cell C.

[0047] Example 4

[0048] 1. Materials and methods

[0049] Same as Example 1.

[0050] 2. Collection of Lactobacillus rhamnosus

[0051] Take 20 mL of Lactobacillus rhamnosus fermentation broth and add it to a 50 mL centrifuge tube. Centrifuge at 10000 rpm for 15 min, discard the supernatant to obtain bacterial pellet, add 3 / 4 times the original volume of the fermentation broth with physiological saline to the bacterial pellet to resuspend the bacteria, centrifuge again (11250 rpm, 10 min), discard the supernatant, and obtain bacterial D.

[0052] Example 5

[0053] 1. Materials and methods

[0054] Same as Example 1.

[0055] 2. Collection of Lactobacillus rhamnosus

[0056] Take 20 mL of Lactobacillus rhamnosus fermentation broth and add it to a 50 mL centrifuge tube. Centrifuge at 10000 rpm for 15 min, discard the supernatant to obtain the bacterial precipitate, add 1 / 2 times the original volume of the fermentation broth with physiological saline to the bacterial precipitate to resuspend the bacteria, centrifuge again (12500 rpm, 5 min), discard the supernatant, and obtain bacterial E.

[0057] Effect Example 1 - The proportion of viable bacteria in the supernatant obtained by centrifugation after resuspension to the total number of viable bacteria

[0058] The supernatant after the cells were resuspended in physiological saline in Examples 1 to 5 was taken for viable bacteria counting and volume measurement, and the fermentation broth volume and its viable bacteria concentration were substituted into the following formula to calculate the proportion of viable bacteria in the supernatant to the total viable bacteria (P). The results are as follows:

[0059] As shown in Table 1.

[0060]

[0061] Table 1 The proportion of viable bacteria in the supernatant obtained by resuspension and centrifugation in the total number of viable bacteria in Examples 1 to 5

[0062] Supernatant source Example 1 Example 2 Example 3 Example 4 Example 5 Proportion (%) 4.32 3.05 0.01 0.04 0.03

[0063] It can be seen from the above table that when physiological saline is used as the medium for resuspending and washing the bacteria, the number of live Lactobacillus rhamnosus in the supernatant after re-centrifugation accounts for less than 5% of the total number of live bacteria, which indicates that the loss rate caused by this method of washing the bacteria will be less than 5%.

[0064] Effect Example 2--Preparation of Lactobacillus rhamnosus Agent

[0065] The bacterial cells A, B, C, D and E prepared in the above Examples 1 to 5 were resuspended in 2 mL of pre-sterilized skim milk (10%, w / v), freeze-dried and then counted. The results are shown in Table 2 below.

[0066] Table 2 Activity of the bacterial agent prepared from the bacterial cells prepared in Examples 1 to 5

[0067]

[0068] As shown in the table above, when physiological saline is used as the medium for resuspending and washing the bacteria, the loss caused by re-centrifugation is low (as shown in Example 1), so that the activity of the final freeze-dried bacterial preparation is stably maintained at 2×10 11 CFU / g and above.

[0069] Comparative Example 1

[0070] The type of cleaning solution, the amount of cleaning solution added, the speed and time of centrifugation in Example 3 were adjusted one by one, and the following group of different methods for collecting Lactobacillus rhamnosus cells were obtained. Referring to the methods described in Effect Examples 1 and 2, the proportion of the number of viable bacteria in the supernatant obtained after resuspension and centrifugation in the total number of viable bacteria in each group of methods, as well as the activity of the prepared bacterial agent, were calculated, and the results are shown in Table 3 below.

[0071] Table 3 The proportion of viable bacteria in the supernatant obtained after resuspension and centrifugation in the total number of viable bacteria and the activity of the prepared bacterial agents in different methods

[0072]

[0073] From the results shown in the above table, it can be seen that when the type of cleaning solution, the amount of cleaning solution added, the speed and time of centrifugation in the method for improving the efficiency of microbial collection are adjusted outside the preferred range, the proportion of the number of viable bacteria in the supernatant obtained by centrifugation after resuspension in each group in the total number of viable bacteria is significantly increased (>10%), indicating that the implementation of the above comparative scheme will cause more bacterial loss, thereby reducing the activity of the subsequent prepared bacterial agent to a certain extent (<2×10 11 CFU / g).

[0074] It is particularly important to note that the change in the type of cleaning solution has the greatest impact. Specifically, in Comparative Scheme 1, which uses sterile water as the cleaning solution, the number of viable bacteria in the supernatant obtained after resuspension and centrifugation accounts for nearly 90% (86.96%) of the total number of viable bacteria. Such a huge cleaning loss makes the activity of the corresponding bacterial agent prepared subsequently only 1.85×10 10 CFU / g. In Example 3, under the same other conditions, using physiological saline as the cleaning solution can reduce the above proportion to 0.01%, minimizing the loss of bacteria caused by cleaning, thereby ensuring high activity of the bacterial agent (2.21×10 11 CFU / g). (Comparison of the results of centrifugation after resuspension in the cleaning solution in the above two methods is shown in the attached Figure 1 )

[0075] Comparative Example 2

[0076] Referring to the method described in Example 3, the cleaning solution was replaced with NaH2PO4 (0.05M) of pH 5.0 and pH 6.0 to resuspend the bacteria and centrifuge them. Then, referring to the method described in Example 1, the number of live bacteria in the supernatant obtained after centrifugation after treatment with the above-mentioned cleaning solutions of different pH values ​​was calculated as a percentage of the total number of live bacteria (using the method described in Example 3 as a control). The results are shown in Table 4 below.

[0077] Table 4 The proportion of viable bacteria in the supernatant obtained by centrifugation after treatment with different pH cleaning solutions in the total number of viable bacteria

[0078]

[0079] From the results shown in the above table, it can be seen that after the type of cleaning solution in the method for improving the efficiency of microbial collection is adjusted to cleaning solutions of different pH values, the number of viable bacteria in the supernatant obtained after resuspension and centrifugation in each group accounted for a significantly higher proportion of the total number of viable bacteria than that in the control group (>10%), indicating that the pH adjustment scheme cannot achieve the effect of efficiently collecting bacteria.

[0080] Comparative Example 3

[0081] The fermentation broth of Lactobacillus rhamnosus CGMCC No.13310 and Lactobacillus fermentum CGMCC No.1.2133 (purchased from CGMCC, China) was prepared according to the method described in Example 3. The viable bacteria concentration in the fermentation broth was determined to be 2.3×10 9 CFU / mL (Lactobacillus rhamnosus) and 1.3×10 9 CFU / mL (Lactobacillus fermentum). The fermentation liquid was centrifuged according to the method described in Example 3, and the precipitate was collected. Physiological saline with a volume of 1 times the original volume of the fermentation liquid was added to the bacterial precipitate to resuspend the bacterial cells, and the cells were centrifuged again to collect the supernatant.

[0082] The number of viable bacteria in the supernatant obtained by centrifugation after resuspension in the above-mentioned physiological saline according to the method described in Example 1 accounted for the total number of viable bacteria. The results are shown in Table 5 below.

[0083] Table 5 The proportion of viable bacteria in the supernatant obtained by centrifugation of bacterial sediments of different strains treated with physiological saline in the total number of viable bacteria

[0084]

[0085] As shown in the above table, the method for improving the microorganism collection efficiency of the present invention is only effective for Lactobacillus rhamnosus CGMCC No.13310 or strains with similar characteristics. If the method is applied to other strains (such as Lactobacillus fermentum CGMCC No.1.2133, etc.), the collection efficiency is obviously not as high as that of Lactobacillus rhamnosus CGMCC No.13310.

[0086] The basic concepts have been described above. Obviously, for those skilled in the art, the above detailed disclosure is only for example and does not constitute a limitation of this specification. Although not explicitly stated here, those skilled in the art may make various modifications, improvements and corrections to this specification. Such modifications, improvements and corrections are suggested in this specification, so such modifications, improvements and corrections still belong to the spirit and scope of the exemplary embodiments of this specification.

[0087] At the same time, this specification uses specific words to describe the embodiments of this specification. For example, "one embodiment", "an embodiment", and / or "some embodiments" refer to a certain feature, structure or characteristic related to at least one embodiment of this specification. Therefore, it should be emphasized and noted that "one embodiment" or "an embodiment" or "an alternative embodiment" mentioned twice or more in different positions in this specification does not necessarily refer to the same embodiment. In addition, certain features, structures or characteristics in one or more embodiments of this specification can be appropriately combined.

[0088] In some embodiments, numbers describing the number of components and attributes are used. It should be understood that such numbers used in the description of the embodiments are modified by the modifiers "about", "approximately" or "substantially" in some examples. Unless otherwise specified, "about", "approximately" or "substantially" indicate that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may change according to the required features of individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and adopt the general method of retaining digits. Although the numerical domains and parameters used to confirm the breadth of their range in some embodiments of this specification are approximate values, in specific embodiments, the setting of such numerical values ​​is as accurate as possible within the feasible range.

[0089] Finally, it should be understood that the embodiments described in this specification are only used to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, as an example and not a limitation, alternative configurations of the embodiments of this specification may be considered consistent with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments explicitly introduced and described in this specification.

Claims

1. A method for collecting microorganisms, characterized in that: The following steps are involved: 1) Take the microbial fermentation liquid, centrifuge it, discard the supernatant, and obtain the bacterial precipitate; 2) adding a sodium chloride solution of 0.5 to 2 times the original volume of the fermentation liquid to the bacterial precipitate, resuspending the bacterial cells to obtain a resuspended liquid; 3) Centrifuge at 7000-12500 rpm for 5-25 min, discard the supernatant, and the bacteria are obtained.

2. The method according to claim 1, characterized in that In the step 1), the centrifugation condition is 8000-12000 rpm for 10-20 min.

3. The method according to claim 1, characterized in that In the step 1), the centrifugation condition is 10000 rpm for 15 min.

4. The method according to claim 1, characterized in that In the step 2), a sodium chloride solution in an amount of 0.75 to 1.5 times the original volume of the fermentation liquid is added to the bacterial precipitate.

5. The method according to claim 1, characterized in that In the step 2), a sodium chloride solution with an amount of 1 times the original volume of the fermentation liquid is added to the bacterial precipitate.

6. The method according to claim 1, characterized in that In the step 3), the centrifugation condition is 8750-11250 rpm for 10-20 min.

7. The method according to claim 1, characterized in that In the step 3), the centrifugation condition is 10000 rpm for 15 min.

8. The method according to claim 1, characterized in that The microorganism is a bacterium that secretes extracellular polysaccharides. Preferably, the microorganism is Lactobacillus rhamnosus.

9. The method according to claim 1, characterized in that The sodium chloride solution is an aqueous solution of sodium chloride. Preferably, the solute concentration in the sodium chloride solution is 0.7% to 1.1%, and the pH is 7.0 to 7.

4. More preferably, the sodium chloride solution is physiological saline.

10. Use of the method according to any one of claims 1 to 9 in collecting bacteria or preparing bacterial agents.

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