A kind of Serratia marcescens and its acid-reducing application

Abstract

The present invention discloses a kind of Serratia marcescens and its acid-reducing application. The strain name is Serratia marcescens (Serratia sp.) JS‑043, and the deposit number is GDMCC NO: 62046. The strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on the 5th floor of Building 59, No. 100, Xianlie Middle Road, Guangzhou on November 9, 2021. The Serratia sp.JS‑043 strain in the present invention has excellent tolerance to high concentrations of citric acid, and can improve its pH value by biotransforming plant acidic substances such as organic acid compounds in citrus juice and citrus residues, reduce or eliminate the inhibitory effect of low pH conditions on the activity of fermentation microorganisms, and effectively improve the utilization efficiency of acidic organic matter such as citrus juice.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and particularly relates to Serratia marcescens and an acid-reducing application thereof. Background Art

[0002] Citrus fruits are mainly used for direct consumption and industrial production, among which the most important processing methods are juicing and making canned citrus segments. Citrus residue is a by-product of the citrus processing industry, accounting for more than 50% of the weight of fresh citrus fruits, mainly including citrus peel (60-65%), seeds (0-10%), residual pulp and citrus pith (30-35%). In addition, due to the expansion of the tangerine peel industry, a large number of citrus industries targeting citrus peels have continued to develop, making tangerine juice gradually become a by-product of tangerine peel. These tangerine juices contain a large amount of organic acids, mainly citric acid, which will cause great pollution to the environment if not properly disposed of. Discharging them into rivers will inhibit the growth of microorganisms in the water and seriously threaten the self-purification system of the water body; abandoning them in farmland without treatment will cause soil acidification and increase the activity of metal ions in the soil, affecting the development of crops and bringing a series of environmental problems.

[0003] Orange juice and other acidic juices of plants contain a large amount of organic matter, nitrogen, phosphorus, potassium and other nutrients necessary for plant growth. They can be used in feed or fertilizer production and soil improvement after processing, and are biomass resources with great potential value. At present, it is a common mode in this field to use microbial fermentation to process orange juice and other resources. Through the metabolism of microorganisms, they are converted into fermented beverages or raw materials, and then further processed into finished products. However, orange juice and other products contain a large amount of organic acid compounds, mainly citric acid. These organic acids will inhibit the activity of fermenting microorganisms, thereby hindering the conversion of biomass during the fermentation process, resulting in a decrease in the quality of the fermented product or fermentation failure, and will produce unpleasant odors, causing environmental pollution and waste of resources. Therefore, how to transform and eliminate the organic acids in orange juice during the fermentation process has become a technical problem that needs to be solved in the resource utilization of orange juice.

[0004] At present, most strategies to solve this problem are to use alkaline substances such as quicklime and calcium carbonate. Although this can alleviate the problem of fermentation failure caused by organic acids to a certain extent, the cost of adding additional chemical reagents is high for large-scale fermentation, and these chemical reagents are prone to secondary pollution to the environment, destroy the nutrition of organic matter, and are not conducive to resource utilization. When used to prepare organic fertilizers, it is easy to cause land salinization. Therefore, it is urgent to find an efficient and environmentally friendly method for reducing acidity of acidic organic matter. Summary of the invention

[0005] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a Serratia marcescens.

[0006] Another object of the present invention is to provide the application of the Serratia marcescens.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A species of Serratia marcescens, named Serratia marcescens ( Serratia sp.) JS-043, with the deposit number GDMCC NO: 62046. The strain was deposited on November 9, 2021 at the Guangdong Provincial Microbiological Culture Collection Center on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0009] The activity of the Serratia marcescens is greater than or equal to 1×10 10 cfu / g.

[0010] The application of the Serratia marcescens in degrading acidic organic matter and / or organic acid compounds.

[0011] The acidic organic matter is a plant acidic substance; preferably fruit juice and / or pomace; more preferably citrus juice and / or citrus pomace; further preferably citrus juice.

[0012] The tangerine juice is the tangerine juice produced by processing tangerine peel, and mainly includes the liquid obtained by squeezing citrus pulp.

[0013] The organic acid compound includes at least one of citric acid, malic acid and acetic acid, preferably citric acid.

[0014] A biological bacterial agent for degrading acidic organic matter and / or organic acid compounds, the active ingredient of which is the above-mentioned Serratia marcescens.

[0015] A method for degrading acidic organic matter and / or organic acid compounds comprises the following steps:

[0016] (1) adding peanut bran and molasses to acidic organic matter and / or organic acid compounds to prepare a fermentation medium;

[0017] (2) The activated Serratia marcescens is inoculated into the fermentation medium and aerated and fermented to degrade the organic acid compounds in the acidic organic matter.

[0018] The acidic organic matter in step (1) is a plant acidic substance; preferably fruit juice and / or pomace; more preferably citrus juice and / or citrus pomace; and even more preferably citrus juice.

[0019] The added amount of the peanut bran is calculated based on 5-30 g of peanut bran per 100 ml of tangerine juice; preferably, it is calculated based on 18.75 g of peanut bran per 100 ml of tangerine juice.

[0020] The amount of molasses added is calculated based on 1-10 g molasses per 100 ml mandarin juice; preferably, it is calculated based on 6.25 g molasses per 100 ml mandarin juice.

[0021] The organic acid compound in step (1) includes at least one of citric acid, malic acid and acetic acid; preferably citric acid.

[0022] The concentration of the organic acid compound in the fermentation medium is 0-100 g / L, preferably 10-60 g / L.

[0023] The formula of the fermentation medium described in step (1) is preferably: 70-90 ml of citrus juice, 5-25 g of peanut bran, and 5 g of molasses; more preferably: 80 ml of citrus juice, 15 g of peanut bran, and 5 g of molasses.

[0024] The main organic acid component of the citrus juice is more than 90% of citric acid, and the initial pH is about 3.0.

[0025] The activated Serratia marcescens described in step (2) is preferably Serratia marcescens in the logarithmic growth phase or the stable phase; it is preferably prepared by the following steps: inoculating the above-mentioned Serratia marcescens on an activation medium (plate medium) for activation, and then inoculating it in a seed medium for shaking culture to obtain a seed culture solution of Serratia marcescens in the logarithmic growth phase or the stable phase.

[0026] The activated culture medium (plate culture medium) is preferably MRS agar culture medium.

[0027] The activation temperature is 28-34°C, preferably 30°C.

[0028] The activation time is 20 to 30 h, preferably 24 h.

[0029] The seed culture medium is MRS agar culture medium or MRS broth culture medium.

[0030] The rotation speed of the shaking culture is 150-210 rpm, preferably 180 rpm.

[0031] The shaking culture temperature is 28-34°C, preferably 30°C.

[0032] The shaking culture time is 18 to 30 h, preferably 24 h.

[0033] The inoculation amount of Serratia marcescens in step (2) is 5% to 12% by volume, preferably 10% by volume.

[0034] The fermentation aeration rate in step (2) is 0.01-3 L / L·min, preferably 1 L / L·min.

[0035] The fermentation temperature in step (2) is 28-34°C, preferably 30°C.

[0036] The fermentation time in step (2) is 60 to 84 h, preferably 72 h.

[0037] In step (2), the pH value of the organic acid compound after degradation is 5.0-5.3.

[0038] Compared with the prior art, the present invention has the following advantages and effects:

[0039] (1) The present invention screened and obtained a strain of acid-reducing microorganisms Serratia sp. JS-043, this strain can improve the pH of citrus juice by biotransformation of organic acids in citrus juice, reduce or eliminate the inhibitory effect of low pH conditions on the activity of fermentation microorganisms, and effectively improve the utilization efficiency of acidic organic matter such as citrus juice.

[0040] (2) Serratia marcescens obtained by screening according to the present invention Serratia sp. JS-043 can still grow well when the citric acid concentration is as high as 100g / L (the citric acid concentration tolerated by conventional Serratia marcescens is generally less than 20g / L), indicating that it has an excellent ability to tolerate high concentrations of citric acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the colony morphology of the strain Serratia sp. JS-043; where a is the colony morphology; b is the staining result of the strain under a microscope (×100).

[0042] Figure 2 The strain was constructed based on the Neighbor-Joining method ( Serratia sp. JS-043) Phylogenetic tree diagram.

[0043] Figure 3 It is a strain Serratia sp. The adaptability of JS-043 to different types of carbon sources (galactose, xylose, glucose, sucrose, succinic acid, citric acid, malic acid, acetic acid).

[0044] Figure 4 It is a strain Serratia sp. The degradation capacity of JS-043 for different initial concentrations of citric acid (unit: g / L); where A is the strain density; B is the system pH value; and C is the citric acid removal rate. DETAILED DESCRIPTION

[0045] The present invention will be described in further detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0046] Example 1

[0047] 1. Screening of strains

[0048] (1) Enrichment: Wash the surface of Xiaoqinggan (Xiaoqinggan fruits collected from orchards in Jiangmen City, Guangdong Province) with ultrapure water, cut into small pieces, put into enrichment medium, and shake (180 rpm) and static culture for 3 to 5 days;

[0049] (2) Screening: Starting from the third day, take an appropriate amount of enriched bacterial liquid and spread it on the rescreening medium every other day;

[0050] (3) Rescreening: Pick colonies of different morphologies from the rescreening medium and inoculate them into the rescreening medium (by observing the pH changes, further screen the strains that can degrade citric acid and thus increase the pH), shake or statically culture them, and measure the changes in pH every 2 days;

[0051] (4) Separation and purification: The bacterial liquid obtained by repeated screening that can increase the pH value (based on the initial pH of the culture medium) is separated and purified multiple times by the spread plate method. The purified strains are then inoculated into repeated screening culture medium with citric acid (10 g / L) as the sole carbon source for cultivation. The strain with the strongest ability to degrade citric acid is then selected as the target strain.

[0052] The composition of the enrichment medium is as follows: 4 g yeast extract, 5 g tryptone, 0.55 g potassium dihydrogen phosphate, 0.425 g potassium chloride, 0.125 g calcium chloride, 0.125 g magnesium sulfate, 0.0025 g ferric chloride, 0.0025 g manganese sulfate, 12 g citric acid, pH 6.5, dissolved in 1 L distilled water;

[0053] Rescreening medium: 10 g trisodium citrate, 0.1 g ferrous sulfate, 0.5 g ammonium sulfate, 0.4 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate, 0.2 g manganese sulfate, pH 4.0, dissolved in 1 L distilled water.

[0054] , strain morphological identification

[0055] The strains screened above were inoculated into MRS solid medium and activated at 30°C for 24 h, and then a few colonies were picked for Gram staining:

[0056] 1) Smear fixation.

[0057] 2) Stain with ammonium oxalate crystal violet for 1 minute.

[0058] 3) Rinse with distilled water.

[0059] 4) Add iodine solution and cover the surface with dye for about 1 minute.

[0060] 5) Wash with water and absorb the moisture with absorbent paper.

[0061] 6) Add a few drops of 95% (v / v) alcohol and shake gently to decolorize. After 20 seconds, wash with water and absorb the water.

[0062] 7) Stain with dilute safranin solution for 1 minute and then rinse with distilled water.

[0063] 8) Dry and observe under a microscope.

[0064] The strain morphology and staining results are as follows Figure 1 shown.

[0065] 3. Molecular Biology Identification

[0066] The strain was cultured in MRS medium until the logarithmic phase of growth, and then the bacterial genomic DNA extraction kit (Shanghai Sangon Biotechnology) was used to extract the DNA of the test strain, and PCR amplification was performed using bacterial universal primers 27F (5'-AGAGTTTGATCATGGCTCAG-3') and 1492R (5'-TAGGGTTACCTTGTTACGACTT-3').

[0067] PCR reaction system: 0.5 µL bacterial genomic DNA, with Mg 2+ 2.5 µL of 10× Buffer, 1 µL of dNTP, 0.2 µL of DNA polymerase, 0.5 µL of 10 µmol / L upstream and downstream primers, and then add double distilled water to 25 µL.

[0068] PCR reaction conditions: pre-denaturation at 94 °C for 4 min, denaturation at 94 °C for 45 s, annealing at 55 °C for 45 s, extension at 72 °C for 1 min, for a total of 30 cycles; repair and extension at 72 °C for 10 min, and termination of the reaction at 4 °C.

[0069] The PCR amplification product was sent to Meiji Biopharmaceutical Technology Co., Ltd. (Shanghai) for sequencing (SEQ ID NO.1). The sequencing results were compared on NCBI, and then the Neighbor-Joining method on the software MEGA 6 was used to construct the phylogenetic tree of the strain. Figure 2 shown.

[0070] According to the colony morphology and molecular biological identification results of the strain, the strain was named Serratia Serratia sp. JS-043, this strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC for short, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou) on November 9, 2021, with the deposit number GDMCC NO: 62046.

[0071] , the strain's broad-spectrum adaptability to different carbon sources and its ability to reduce acidity in citrus juice

[0072] (1) Activate the strain Serratia sp. JS-043 was inoculated into a culture medium containing 10 g / L of different carbon sources (carbon sources: galactose, xylose, glucose, sucrose, succinic acid, citric acid, malic acid, acetic acid), and cultured at 180 rpm for 24 h with three replicates to investigate the strain's adaptability to different carbon sources.

[0073] The results are as follows Figure 3 Shown: From Figure 3 It can be seen that the strain can use common organic acids such as citric acid, malic acid, and acetic acid as the sole carbon source. Therefore, the strain may have a good application prospect in the field of degradation of acidic organic matter.

[0074] (2) Peel the Xinhui small green oranges and squeeze the juice to obtain orange juice (the main organic acid component is more than 90% citric acid, and the initial pH is about 3.0). Then add the orange juice, peanut bran (commercially available) and molasses into a 5 L plastic bottle at a ratio of 80%, 15% and 5% (w / v), respectively. After mixing, add 10% (v / v) activated (OD 600 ≥1.0) Serratia sp. JS-043, then, the fermentation was carried out at 30 °C under aeration (1 L / L·min). After 72 h, the pH value and citric acid content of the test system were taken out, and the citric acid removal rate was calculated. This was repeated three times.

[0075] The results showed that the initial pH of the fermentation was 4.21, and the citric acid content was 1.84 g / L. After 72 h of biological acid reduction, the pH rose to 5.30, and the citric acid content dropped to 0.2 g / L. This indicates that this method has a good acid reduction effect on acidic organic matter, especially on acidic organic matter rich in citric acid.

[0076] (3) Activate the strain Serratia sp.JS-043 was inoculated into rescreening medium containing different concentrations (5, 10, 20, 30, 40, 60, 80 and 100 g / L) of citric acid (the citric acid was supplemented or reduced to the target concentration in the rescreening medium in step 1 above, and the pH was adjusted with 1% (v / v) sulfuric acid or 40% (w / v) sodium hydroxide), and cultured with shaking at 180 rpm for 156 h. Samples were taken every 6 h in the early stage and every 12 h in the middle and late stages. Sampling was stopped when the pH was greater than 8 for two consecutive times. The pH value and citric acid content of the system were tested, and the citric acid removal rate was calculated. Three replicates were set.

[0077] The results are as follows Figure 4 Shown: From Figure 4 As can be seen in A, the strain Serratia sp. JS-043 can still grow well when the citric acid concentration is as high as 100 g / L, indicating that it has excellent ability to tolerate high concentrations of citric acid. Figure 4 B and Figure 4 The test results of C showed that at a citric acid concentration of 40 g / L, the strain could still degrade more than 80% of the citric acid and make the pH of the entire system reach above 8, demonstrating excellent tolerance and degradation capabilities.

[0078] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A Serratia marcescens, characterized in that: Serratia marcescens Serratia sp.) JS-043, with the deposit number GDMCC NO: 62046, was deposited on November 9, 2021 at the Guangdong Provincial Microbiological Culture Collection Center on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. Use of the Serratia marcescens described in claim 1 in degrading citric acid.

3. A biological agent for degrading citric acid, characterized in that: The active ingredient is the Serratia marcescens described in claim 1.

4. A method for degrading citric acid in citrus juice, characterized in that: The steps include: (1) adding peanut bran and molasses into orange juice to prepare a fermentation medium; (2) inoculating the activated Serratia marcescens described in claim 1 into the fermentation medium prepared in step (1), and performing aeration fermentation to degrade citric acid in the citrus juice; The concentration of citric acid in the fermentation medium in step (1) is 0 to 40 g / L.

5. The method according to claim 4, characterized in that: The amount of peanut bran added in step (1) is calculated based on 5-30 g of peanut bran per 100 ml of orange juice; The amount of molasses added in step (1) is calculated based on 1-10 g molasses per 100 ml citrus juice.

6. The method according to claim 4, characterized in that: The inoculation amount of Serratia marcescens described in step (2) is 5% to 12% by volume; The fermentation aeration rate in step (2) is 0.01-3 L / L·min; The fermentation temperature in step (2) is 28-34°C; The fermentation time in step (2) is 60 to 84 hours.

7. The method according to claim 4, characterized in that: The activated Serratia marcescens in step (2) is prepared by the following steps: inoculating the Serratia marcescens on an activation medium for activation, and then inoculating the Serratia marcescens on a seed medium for shaking culture to obtain a Serratia marcescens seed culture solution in a logarithmic growth phase or a stationary phase; The activated culture medium is MRS agar culture medium; The activation temperature is 28-34°C; The activation time is 20 to 30 h; The seed culture medium is MRS broth culture medium; The rotation speed of the shaking culture is 150-210 rpm; The shaking culture temperature is 28-34°C; The shaking culture time is 18 to 30 hours.

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