Rumen living corynebacterium, microbial agent and application of microbial agent

Abstract

The invention relates to the field of microorganisms, in particular to rumen living corynebacterium, a microbial agent and application of the rumen living corynebacterium and the microbial agent. The invention provides a rumen living corynebacterium, and the preservation number of the rumen living corynebacterium is CGMCC (China General Microbiological Culture Collection Center) No.33059. The rumen living corynebacterium has the advantages that the rumen living corynebacterium has the preservation number of CGMCC No.33059; the invention provides rumen living corynebacterium and application thereof to whey wastewater to research functions of the rumen living corynebacterium and fill the blank of domestic research.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, in particular to rumen-inhabiting Corynebacterium, microbial agents and applications thereof. Background Art

[0002] Corynebacterium refers to Gram-positive rods. It is named because one or both ends of its members are swollen and rod-shaped. It has multiple forms and irregular arrangement, often in a fence or V shape. It is unevenly stained, with darker metachromatic particles at both ends. It has no spores and most strains are non-motile. It is aerobic or facultatively anaerobic, has special nutritional requirements, and the G+C gram molecular content in DNA is 52~68%. The cell wall polysaccharides are mainly arabinose and galactose.

[0003] There are many studies on Corynebacterium, mostly focusing on glutamate, lysine and valine, such as patent (CN201610515320.X) a Corynebacterium glutamicum and its application, patent (CN 202011285971.7) Corynebacterium glutamicum with high glutamate production and its application; patent (CN 201610800601.X) a method for fermentation production of L-lysine and modified Corynebacterium, patent (CN 201911370732.9) a Corynebacterium with high valine production and its construction method and application, etc. However, there are fewer studies on rumen-resident Corynebacterium. Hong Yongcong's research found that rumen-resident Corynebacterium has a good degradation ability for cypermethrin, while Wang Zhihong's research found that rumen-resident Corynebacterium can secrete phytase, and other functions are still unclear. Summary of the invention

[0004] In view of this, the present invention provides rumen-resident Corynebacterium, microbial agent and application thereof. The present invention provides a rumen-resident Corynebacterium and applies it to whey wastewater to study its function, thus filling the gap in domestic research.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides Corynebacterium vitaeruminis, whose deposit number is CGMCC No.33059.

[0007]

[0008] The present invention also provides a microbial agent, comprising: the above-mentioned Corynebacterium vitaeruminis.

[0009] The present invention also provides the use of the above-mentioned Corynebacterium vitaeruminis and / or the above-mentioned microbial agent in treating whey wastewater.

[0010] In some embodiments of the present invention, the Corynebacterium vitaeruminis and / or the microbial agent in the above application reduces the pH value of whey wastewater.

[0011] In some embodiments of the present invention, the Corynebacterium vitaeruminis and / or the microbial agent in the above application increases the suspended matter content of whey wastewater.

[0012] The present invention also provides a method for treating whey wastewater, wherein the rumen-resident Corynebacterium vitaeruminis and / or the microbial agent are inoculated, fermented and cultured, and mixed with the whey wastewater.

[0013] In some embodiments of the present invention, in the above method, the fermentation culture uses a fermentation medium; the fermentation medium includes: YPD medium.

[0014] In some embodiments of the present invention, in the above method, the YPD medium comprises: 2% (m / v) glucose, 1.0% (m / v) yeast powder, 2% (m / v) peptone and 1.8% (m / v) agar powder.

[0015] In some embodiments of the present invention, in the above method, the inoculation amount is 1.0%, and the viable count is 5900×10 4 cfu / mL.

[0016] The rumen-resident Corynebacterium isolated by the invention produces lactic acid, can grow on both LB medium and YPD medium, does not produce protease, has good bacterial sedimentation performance, has flocculation effect, and has an optimum addition amount of more than 7.5%. It can be applied to whey wastewater to increase the content of suspended matter. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0018] Figure 1 Shows the colony morphology of LBSW24-01;

[0019] Figure 2 Shows the morphology of LBSW24-01 colonies under microscope;

[0020] Figure 3 The fermentation liquid caused potassium permanganate to fade;

[0021] Figure 4 Corynebacterium rumenicola was shown to settle for 10 minutes.

[0022] Biological Deposit Description

[0023] Biological material: LBSW24-01; classification name: Corynebacterium vitaeruminis; deposited in the General Microbiology Center of China Culture Collection Administration on December 13, 2024; address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit number: CGMCCNo.33059. DETAILED DESCRIPTION

[0024] The invention discloses rumen-inhabiting Corynebacterium, a microbial agent and applications thereof.

[0025] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0026] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0027] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0028] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0029] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0030] The present invention independently screened and isolated a strain of Corynebacterium, which was identified as rumen-resident Corynebacterium vitaeruminis and deposited in the General Microbiology Center of China Microbiological Culture Collection Administration. The strain name is LBSW24-01, the deposit number is CGMCC No.33059, and the deposit date is December 13, 2024.

[0031] The ruminal bacillus LBSW24-01 with the deposit number of CGMCC No.33059 has oval cells under a microscope, no spores, no flagella, and is a Gram-positive bacterium; the strain is cultured in a 37°C constant temperature incubator for 24 hours and can form milky white colonies with smooth surfaces, protrusions and neat edges on LB solid culture medium.

[0032] The rumen-inhabiting Corynebacterium LBSW24-01 contains the gene sequence shown in SEQ ID NO:1.

[0033] The rumen-inhabiting Corynebacterium can grow on both YPD medium and LB medium, but YPD medium is superior to LB medium.

[0034] The rumen-resident Corynebacterium produces lactic acid by fermentation.

[0035] The rumen-inhabiting Corynebacterium has good sedimentation performance and flocculation effect.

[0036] The rumen-inhabiting Corynebacterium has no protease activity.

[0037] Applying the rumen bacteria to whey wastewater at an addition rate of 1.0% (v / v) can significantly increase the suspended solids content in the effluent by more than 25%.

[0038] In Examples 1 to 4 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0039] The present invention will be further described below in conjunction with embodiments:

[0040] Example 1 Strain Isolation

[0041] It was separated from whey wastewater of a sewage treatment plant in Boxing County, Binzhou City, Shandong Province. 10 mL of wastewater was added to 90 mL of sterile water with glass beads, shaken in a shaker for 30 minutes, and then diluted to 10^4 in a clean bench. 100 μL was spread on a YPD plate and cultured in a constant temperature incubator at 37°C for 24 hours. Monoclones of different morphology and size were picked out in the clean bench and added to 15 mL of fresh YPD liquid culture medium, and cultured in a constant temperature incubator at 35°C for 24 hours. Each strain of bacteria was stored in 3 glycerol tubes, and the remaining bacterial liquid was used for standby use.

[0042] Functional screening: pH of the obtained bacterial solution was tested by pH electrode method, YPD medium was used as negative control, and strains with pH lower than the negative control were selected as candidate strains for the next test. Three acid-producing strains were isolated, among which JM4 could grow on both YPD medium and LB medium. The screened strain JM4 was streaked on LB plate and cultured in a 37℃ constant temperature incubator for 24 hours. A milky white colony with smooth surface, protrusions and neat edges was formed. The colony morphology was as follows: Figure 1 As shown, the cells are oval in shape, without spores or flagella, and their microscopic morphology is as follows Figure 2 As shown, it was sent to BGI (Hubei, Wuhan) for sequencing, and the sequence is shown in the gene sequence table SEQ ID NO: 1. After BLAST comparison, the sequence was 99.93% homologous to Corynebacterium vitaeruminis DSM 20294, belonging to Corynebacterium vitaeruminis, and was named LBSW24-01.

[0043] Example 2 Fermentation medium screening

[0044] The LB medium formula is: sodium chloride 10 g / L, yeast powder 5 g / L, peptone 10 g / L, agar powder 18 g / L (solid), distilled water as the balance, pH 6.8~7.0, sterilized at 121℃ for 20 min.

[0045] The formula of YPD medium is: glucose 20 g / L, yeast powder 10 g / L, peptone 20 g / L, agar powder 18 g / L (solid), and the balance is distilled water. Sterilize at 121℃ for 20 min.

[0046] Activation of bacterial strains: In a clean bench, pick the bacterial strain with the deposit number of CGMCC No.33059 from the slant and put it into 200 mL LB liquid culture medium, and culture it in a shaking incubator at 37°C and 150 rpm for 24 h;

[0047] Inoculation: The prepared liquid seeds were inoculated into 200 mL LB liquid medium and 200 mL YPD liquid medium at a 1% (v / v) inoculation rate, respectively, and cultured in a shaking incubator at 37°C and 150 rpm for 12 h.

[0048] The colony count and suspended matter changes were detected respectively. The detection methods referred to GB4789.2-2016 Determination of total colony count in food microbiology examination, GB11901-1989 Gravimetric method for determination of suspended matter in water quality and GB / T 6432-2018 Kjeldahl method for determination of crude protein in feed.

[0049] Table 1 Culture medium optimization

[0050]

[0051] After testing, it was found that the colony count and suspended matter content of rumen-inhabiting Corynebacterium on YPD medium were much higher than those on LB medium. Therefore, the optimal fermentation medium for rumen-inhabiting Corynebacterium is YPD medium.

[0052] Example 3 Functional identification of rumen-inhabiting Corynebacterium

[0053] 3.1 Determination of amino acid content

[0054] Previous studies have shown that Corynebacterium produces amino acids or small molecule organic acids. In order to verify the function of this strain, the amino acid content and pH changes of the fermentation broth were detected. The detection steps are as follows: take an appropriate amount of fermentation broth, filter it with filter paper, and use the filtrate for amino acid content and pH detection. YPD is used as a control. The amino acid content detection method refers to GB / T 8314-2013 Determination of the total amount of free amino acids in tea, and the pH is determined using the pH electrode method.

[0055] Table 2 Determination of amino acid content

[0056]

[0057] After testing, the results are shown in Table 2. The free amino acid content of YPD is higher than that of the fermentation broth, indicating that the fermentation process consumes amino acids. The pH of the fermentation broth is lower than that of YPD, which also indicates that H is produced during the fermentation process. + At the same time, the fermentation liquid can decolorize the potassium permanganate solution (such as Figure 3 ), indicating that lactic acid was produced during the fermentation process. The lactic acid content was tested to be 976 mg / L.

[0058] 3.2 Flocculation analysis

[0059] 3.2.1 Flocculation rate detection

[0060] In Example 2, it was found that the sedimentation effect of rumen bacteria was better (such as Figure 4As shown), it is speculated that the rumen bacteria may have a flocculation effect, so the flocculation rate of the obtained bacterial solution is tested. The determination method is as follows: 5 g / L kaolin suspension is prepared with kaolin (analytical grade) as a simulated suspended solid polluted water body. 100 mL of simulated water sample is added to a 250 mL conical flask, and 2 mL of bacterial solution and 0.1 mL of 5% (w / v) CaCl2 solution as a coagulant are added respectively. The pH is adjusted to 6.8-7.0 with NaOH and HCl, and mixed at 500 rpm on a magnetic stirrer for 5 minutes. At the same time, a simulated water sample with YPD culture medium added is used as a blank control under the same conditions. After standing for 10 minutes, 10 mL of supernatant is aspirated. The absorbance of the supernatant of the sample to be tested and the absorbance of the supernatant of the blank control solution are measured at 550 nm using an ultraviolet spectrophotometer, and the flocculation rate is calculated using the following formula:

[0061] Flocculation rate (%) = (BA) × 100 / B

[0062] In the above formula,

[0063] A-measure the absorbance of the sample supernatant;

[0064] B- absorbance of blank solution supernatant.

[0065] Table 3 Flocculation test of rumen bacteria

[0066]

[0067] After testing and analysis, it was found that rumen bacteria have certain flocculation properties, and the amount of addition needs to be studied.

[0068] 3.2.2 Effect of bacterial addition amount on flocculation

[0069] Add 100 mL of simulated water sample to each of 7 250 mL conical flasks, add 0 mL, 2 mL, 5 mL, 7.5 mL, 10 mL, 15 mL and 20 mL of fermentation broth of Ruminobacterium LBSW24-01, add 20 mL, 18 mL, 15 mL, 12.5 mL, 10 mL, 5 mL and 0 mL of YPD medium respectively, add 0.12 mL of 5% (w / v) CaCl2 solution as coagulant to each flask, adjust the pH to 6.8-7.0 with NaOH and HCl, mix at 500 rpm on a magnetic stirrer for 5 minutes, let stand for 10 minutes, and take 10 mL of supernatant. Use ultraviolet spectrophotometer to measure the absorbance of the supernatant of the sample to be tested at 550 nm, and calculate the flocculation rate using the following formula:

[0070] Flocculation rate (%) = (BA) × 100 / B

[0071] In the above formula,

[0072] A-measure the absorbance of the sample supernatant;

[0073] B- absorbance of blank solution supernatant.

[0074] Table 4 Relationship between bacterial solution addition amount and flocculation rate

[0075]

[0076] It can be seen from the table that in order to achieve the best flocculation effect of rumen bacteria, the minimum amount of bacterial solution added is 7.5%.

[0077] 3.3 Protease activity detection

[0078] In order to verify whether Corynebacterium rumensis has the characteristics of protease production, the fermentation broth of Corynebacterium rumensis was subjected to protease activity detection-Folin phenol method, and the detection steps were as follows: 1) The control group (YPD) and the experimental group (LBSW24-01 fermentation broth) were diluted 5 times respectively, and 1 mL of the dilution was taken and preheated at 40℃ for 2 minutes; 2) 2 mL of trichloroacetic acid was added to the control group, and 1 mL of casein solution was added to the experimental group, and the mixture was shaken and kept at 40℃ for 10 minutes; 3) 1 mL of casein solution was added to the control group, and 2 mL of trichloroacetic acid was added to the experimental group, and the mixture was shaken and kept at 40℃ for 10 minutes, and filtered; 4) 1 mL of the filtrate was taken, 5 mL of sodium carbonate solution was added, and then 1 mL of Folin phenol solution was added, and the mixture was placed in a 40℃ water bath for color development for 20 min; 5) The absorbance was measured at a wavelength of 680 nm using a spectrophotometer, and the amino acid content was found from the standard curve; water was used instead of the fermentation broth to perform operations 4) and 5) as the reference solution.

[0079] The protease activity of the fermentation broth of Bacillus subtilis LBSW24-01 was detected to be 0 μ / mL, so this Bacillus subtilis did not produce protease.

[0080] Table 5

[0081]

[0082] Example 4 Application of Ruminobacterium spp. in whey wastewater

[0083] Take two 500 mL Erlenmeyer flasks and add 200 mL of whey wastewater; add 2 mL of distilled water and 2 mL of LBSW24-01 fermentation broth respectively, seal with plastic wrap and place in a shaker, shake and culture at 150 rpm and 37℃ for 24 hours. After the fermentation is completed, detect the pH change, take 50 mL for suspended matter content determination, and the filtrate is used to detect COD. The remaining solution is centrifuged at 5000 rpm for 5 minutes, the supernatant is discarded, and the precipitate is placed in a 105℃ oven and dried for 8 hours for use in the determination of crude protein content.

[0084] Table 6 Application of rumen bacteria in whey wastewater

[0085]

[0086] The results are shown in Table 6. Adding Corynebacterium rumenii can reduce the pH of whey wastewater and increase the suspended solids content, with the suspended solids increasing by 25.29%.

[0087] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Corynebacterium vitaeruminis, characterized in that Its deposit number is: CGMCC No.33059.

2. A microbial agent, characterized in that: include: The rumen-inhabiting Corynebacterium vitaeruminis according to claim 1.

3. Use of the Corynebacterium vitaeruminis according to claim 1 and / or the microbial agent according to claim 2 in treating whey wastewater.

4. The use according to claim 3, characterized in that The Corynebacterium vitaeruminis and / or the microbial agent reduce the pH value of the whey wastewater.

5. The use according to claim 3 or 4, characterized in that: The Corynebacterium vitaeruminis and / or the microbial agent increase the suspended matter content of the whey wastewater.

6. A method for treating whey wastewater, characterized in that: The rumen-resident Corynebacterium vitaeruminis and / or the microbial agent are inoculated, fermented and cultured, and mixed with the whey wastewater.

7. The method according to claim 6, characterized in that The fermentation culture adopts a fermentation medium; the fermentation medium includes: YPD medium.

8. The method according to claim 7, characterized in that The YPD culture medium comprises: 2% (m / v) glucose, 1.0% (m / v) yeast powder, 2% (m / v) peptone and 1.8% (m / v) agar powder.

9. The method according to any one of claims 6 to 8, characterized in that The inoculation volume was 1.0% and the viable count was 5.9×10 7 cfu / mL.

Images