CN114540216A - Lactobacillus plantarum degrading oleuropein and its application - Google Patents

Abstract

The invention discloses lactobacillus plantarum for degrading oleuropein and application thereof, wherein the lactobacillus plantarum is named as lactobacillus plantarum FBEL-07, the strain is preserved in China general microbiological culture Collection center (CGMCC) at 10 months and 15 days 2021, the preservation number is CGMCC No.23608, and the 16s DNA sequence is shown as SEQ ID No. 1. The strain can utilize oleuropein as a unique carbon source, efficiently and specifically converts the oleuropein into hydroxytyrosol, can solve the problems of low yield, low purity, high production cost and long production period of the hydroxytyrosol, can more efficiently and stably play a role under the conditions of optimal pH, temperature and initial concentration of the oleuropein for degrading the fermented oleuropein into the hydroxytyrosol, improves the technical method for biologically degrading the oleuropein, and further improves the yield of the hydroxytyrosol.

Description

Lactobacillus plantarum degrading oleuropein and its application

technical field

The invention belongs to the technical field of microorganisms, and in particular relates to a Lactobacillus plantarum strain and its application in degrading oleuropein.

Background technique

Oleuropein is one of the by-products of olive processing. Analysis of the metabolic pathway of oleuropein shows that the glycosidic bond of oleuropein will be broken under the action of glycosidase to generate glucose and oleuropein. The primary ester bond will be broken and degraded into hydroxytyrosol and elemenic acid. It has been reported and confirmed that the anticancer, antioxidative, antiaging, antiosteoporosis and other activities of hydroxytyrosol are higher than those of oleuropein. At present, the production of hydroxytyrosol is still very low, which makes it expensive and difficult to obtain. Research on the degradation of oleuropein to hydroxytyrosol can improve the added value of products and make up for the problem of insufficient hydroxytyrosol production.

At present, there are three main categories of methods for degrading oleuropein. The first is to use chemical methods for acid hydrolysis or alkali hydrolysis of oleuropein. The yields of this method can reach 7.41% and 4.09%, respectively. However, the secondary metabolites generated by this method have complex components, and under severe reaction conditions, extremely Secondary reactions are prone to occur, resulting in low product yields and environmental pollution if chemical reagents are not handled properly. The second is the use of enzymatic degradation, the yield can reach 6.07%, but the enzyme is expensive and the reaction conditions are harsh. The third is the use of biological methods, which is currently the hottest and most promising method. Because once the mature fermentation process is mastered, it can be applied to industrial production on a large scale, greatly improving production efficiency, reducing production costs, and being environmentally friendly. However, the technology of using microorganisms to degrade oleuropein is not yet mature, so it is of great significance to seek high-efficiency specific strains to convert oleuropein to hydroxytyrosol with high throughput.

At present, foreign literature reports mention many strains that degrade oleuropein. Santos found that a strain of Lactobacillus plantarum Lac plant6907 could use oleuropein as a carbon source and degrade it into hydroxytyrosol with a degradation rate of 90%, but the production rate of hydroxytyrosol was 30%. Intermediates such as oleuropein aglycone were detected. product. Bouzid was fermented with olive oil waste and Aspergillus niger, the yield of hydroxytyrosol was 2.94g/kg, and the purity was 85%. Khoufi used another strain of Aspergillus niger to ferment olive oil wastewater, and the yield of hydroxytyrosol was 0.8 g/L. So far, no single strain and mature fermentation process have been found to achieve stable and efficient conversion of oleuropein to hydroxytyrosol.

SUMMARY OF THE INVENTION

The main purpose of the present invention is to solve the existing problems such as low degradation yield, complex degradation product components, harsh process conditions, high cost, environmental pollution and the like that exist in the degradation of oleuropein by biological methods, and provide a specific and efficient new strain , enabling it to utilize oleuropein as the sole carbon source to stably and efficiently generate hydroxytyrosol.

Another object of the present invention is to provide the application of the above strain for degrading oleuropein.

For the above purpose, the present invention screened out a Lactobacillus plantarum strain capable of efficiently degrading oleuropein to generate hydroxytyrosol from traditional fermented products of different origins in the market, named as Lactobacillus plantarum FBEL-07, The strain has been deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee on October 15, 2021. The address of the depositor is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. The preservation number is CGMCC No. .23608, whose 16s DNA sequence is shown in SEQ ID NO.1.

The Lactobacillus plantarum FBEL-07 colonies screened out by the present invention are purple after staining, and are Gram-positive bacteria.

The Lactobacillus plantarum (Lactobacillus plantarum) FBEL-07 screened out by the present invention can be used for degrading oleuropein to produce hydroxytyrosol, and the specific method is as follows: after the Lactobacillus plantarum FBEL-07 strain is activated, it is inoculated on oleuropein solid medium Placed in a 30-40 ℃ incubator for cultivation, screened out the plate that grew a single colony, and then picked a single colony and inoculated it into a liquid medium containing 1-3 mmol/L oleuropein with a pH of 6.0-7.0. Fermentation at 30～40℃ for 72～120 hours, after the fermentation is completed, add antioxidant 2,6-di-tert-butyl-4-methylphenol (BHT) to the fermentation broth, and place on a shaker at 40～60℃ to continue The reaction is carried out for 8 to 36 hours.

The formula of above-mentioned oleuropein solid medium is: oleuropein 1～3mmol/L, disodium hydrogen phosphate 4～4.5g/L, potassium dihydrogen phosphate 2.5～2.8g/L, ammonium nitrate 0.8～1.2g/L , magnesium sulfate heptahydrate 0.15～0.3g/L, calcium chloride 0.01～0.03g/L, ferrous sulfate heptahydrate 0.01～0.02g/L, manganese sulfate heptahydrate 0.002～0.003g/L, agar 15～20g/ L, and the rest is water.

The formula of above-mentioned oleuropein liquid culture medium is: oleuropein 1～3mmol/L, disodium hydrogen phosphate 4～4.5g/L, potassium dihydrogen phosphate 2.5～2.8g/L, ammonium nitrate 0.8～1.2g/L , 0.15～0.3g/L of magnesium sulfate heptahydrate, 0.01～0.03g/L of calcium chloride, 0.01～0.02g/L of ferrous sulfate heptahydrate, 0.002～0.003g/L of manganese sulfate heptahydrate, and the rest are water.

The beneficial effects of the present invention are as follows:

1. The present invention screens out a novel Lactobacillus plantarum strain FBEL-07, which can utilize oleuropein as the only carbon source to convert it into hydroxytyrosol efficiently and exclusively, which can solve the problem that the hydroxytyrosol yield is not high, The problems of low purity, high production cost and long production cycle.

2. The strain of the present invention can play a more efficient and stable role under the conditions of the optimum pH, temperature, and initial concentration of oleuropein to degrade oleuropein into hydroxytyrosol, thereby improving the technical method for biological degradation of oleuropein , to further increase the production of hydroxytyrosol.

Description of drawings

Fig. 1 is a micrograph of the morphological identification of Lactobacillus plantarum strain FBEL-07.

Figure 2 is a phylogenetic tree.

Figure 3 is the effect of pH on the fermentation of oleuropein by Lactobacillus plantarum strain FBEL-07.

Figure 4 shows the effect of culture temperature on the fermentation of oleuropein by Lactobacillus plantarum strain FBEL-07.

Figure 5 is the effect of the initial concentration of oleuropein on the fermentation of oleuropein by Lactobacillus plantarum strain FBEL-07.

Detailed ways

The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the protection scope of the present invention is not limited to these embodiments.

Example 1

(1) Screening of strains

①Preliminary screening of strains

The traditional fermented products from different origins in the market were collected on the aseptic operation table, including the sauerkraut from Caixiangyuan Laotan in Suining, Sichuan, the shredded Northeast pickled cabbage from Bimei in Shenyang, Liaoning, and the Korean radish kimchi from Xiubinjiayuan in Yanbian, Jilin. Beiwuji spicy cabbage from Harbin, Heilongjiang, Longpuyuantianshuijiangshui sauerkraut from Tianshui, Gansu, and stinky tofu raw embryos from Changsha, Hunan. 2 mL of samples were taken and inoculated into 100 mL of MRS liquid medium, and incubated at 37°C with a shaker at 200 rpm/min for 24 h.

Take 1 mL of the above-mentioned culture medium, add 9 mL of sterile normal saline to dilute 10 times, and then successively dilute 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , and 10 ⁷ times successively, and draw 30 μL and 50 μL of each dilution solution respectively. The cloth was inoculated in MRS solid medium and cultured in a constant temperature incubator at 37°C for 48h.

②Isolation and purification of strains

Pick a single colony with a smooth surface, convex round, complete edge, and milky white. After repeated isolation and purification for 2 to 3 times, pick a colony with suspected growth characteristics of lactic acid bacteria into 4 mL of MRS liquid medium, and culture at 37 °C and 200 rpm in a shaker. 24h, 352 strains were screened out, respectively mixed with 30% glycerol aqueous solution by volume ratio of 1:1, and stored in a -80°C refrigerator for later use.

③Rescreening of strains

Take out the strains stored in the -80°C refrigerator and place them on ice. After thawing, take 100 μL of the strains from the ultra-clean workbench and add them to a centrifuge tube containing 900 μL of sterile normal saline to dilute 10 times, and then serially dilute 10 ² and 10 ³ in sequence. , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ times. Pipette 30 μL and 50 μL of each dilution solution, respectively, to spread and inoculate on MRS solid medium, and cultivate in a constant temperature incubator at 37 °C for 48 h. After the activated strain grows a single colony, use sterile tweezers to pick up a single colony with a pipette tip and place it in a sterilized test tube containing 2 mL of MRS liquid medium.

The activated bacterial solution was diluted and spread on oleuropein solid medium, and cultured in a 37°C incubator. The plates with single colonies were screened out, and then single colonies were picked and cultured in a liquid medium containing 100 mL of oleuropein and shaken at 37°C and 200 rpm/min. At the same time, a blank control was set: no bacteria were inserted into the same 100 mL oleuropein liquid medium.

The formula of the above-mentioned MRS liquid medium is: peptone 10g/L, beef powder 5g/L, glucose 20g/L, yeast powder 4g/L, sodium acetate 5g/L, dipotassium hydrogen phosphate 2g/L, magnesium sulfate 0.2g/L L, triammonium citrate 2g/L, magnesium sulfate 0.05g/L, Tween-80 1mL/L, and the rest are water; MRS solid medium is additionally added with agar 15g/L.

The formula of above-mentioned oleuropein liquid culture medium is: oleuropein 1mmol/L, disodium hydrogen phosphate 4.26g/L, potassium dihydrogen phosphate 2.65g/L, ammonium nitrate 1g/L, magnesium sulfate heptahydrate 0.2g/L , calcium chloride 0.02g/L, ferrous sulfate heptahydrate 0.01g/L, manganese sulfate heptahydrate 0.002g/L, and the rest are water; oleuropein solid medium is added with agar 15g/L.

④Identification of intermediate products

The samples were injected and detected according to the HPLC detection method. The specific operations are as follows: 1) Preparation of mobile phase: the aqueous phase is prepared from formic acid, methanol, and water in a volume ratio of 3:50:947, and the organic phase is prepared from methanol and acetonitrile in a volume ratio of 1:1. After the preparation is completed, suction filtration is performed to remove impurities, and then ultrasonication is performed for 15 min to remove air bubbles. 2) Preparation of standard solution: Weigh 0.01 g of each of the standard products of oleuropein, oleuropein and hydroxytyrosol, dissolve them in methanol aqueous solution with a volume concentration of 80%, and then use a brown volumetric flask to dilute to 100 mL , to obtain 100 μg/mL standard stock solution, and store at 4°C in the dark. When using, the above standard product mother solutions were respectively diluted in gradient to prepare standard solutions with concentrations of 20 μg/mL, 40 μg/mL, 60 μg/mL, 80 μg/mL and 100 μg/mL. Pipette 1 mL of each standard solution and mix to obtain a mixed standard solution, which is stored at 4°C in the dark. 3) Sample preparation: suck 1 mL of the bacterial solution after the above step (3) into a 1.5 mL centrifuge tube, centrifuge at 10,000 rpm/min for 5 min, suck 100 μL of the supernatant into 900 μL of methanol aqueous solution with a concentration of 80% by volume, and vortex for 30 s , suck it with a disposable sterile syringe and pass it through a 0.22 μm filter membrane to the injection bottle. 4) Gradient elution by liquid chromatography, the elution procedure is: 0～3min, the organic phase is 100%; 3～27min, the volume ratio of the aqueous phase to the organic phase is 40%: 60%; 27～45min, the organic phase is 100%, The temperature was set to 35 °C, the injection volume was 10 μL, and the flow rate was 1 mL/min. Several standard solutions were then injected separately, and the retention times of the peaks under the same conditions were recorded. Then, the mixed standard solution was injected, and the peak sequence and relative retention time of each standard were recorded. If the resolution of each standard is high and the retention time of the peak is stable, the method is feasible and can be used for the determination of samples. 5) Sample determination: According to the retention time of different standard substances, the sample chromatogram is compared with the standard substance chromatogram, and qualitative analysis is carried out to preliminarily determine the intermediate product generated by the degradation of oleuropein after lactic acid bacteria fermentation. 6) Analysis of results: Analysis of the HPLC spectrum showed that after oleuropein was fermented by lactic acid bacteria, the retention time of the peak of the degradation product was consistent with the retention time of the peak of the oleuropein standard product.

⑤ High temperature reaction

According to the metabolic pathway of oleuropein, it was determined that oleuropein was degraded by glycosidase to generate oleuropein, and the ester bond of oleuropein would be broken at high temperature to generate the target product hydroxytyrosol. 0.01% antioxidant BHT of the total volume of the fermentation broth was added to the fermentation broth, and the fermentation broth was placed in a 50°C constant temperature incubator for high temperature reaction, and samples were taken at 8h, 12h and 24h for HPLC determination. Finally, 10 strains with better ability to degrade oleuropein to hydroxytyrosol were screened out.

(2) Identification of strains

Morphological identification of 10 strains with better ability to degrade oleuropein to hydroxytyrosol was carried out respectively, and their physiological and biochemical characteristics were determined according to the following methods, biological identification, and the species and characteristics of the strains were determined.

① Morphological identification

The single colony with smooth surface, convex round, complete edge and milky white color in the medium was picked for Gram staining, observed and photographed under an oil microscope, and its morphological characteristics were identified.

② Determination of physiological and biochemical properties

Catalase test, oxidase test, starch hydrolysis test, gelatin liquefaction test, V-P reaction and methyl red test were performed on the 10 strains screened out with better ability to degrade oleuropein to hydroxytyrosol. biochemical properties.

③ Biological identification

The 10 strains screened out with better ability to degrade oleuropein to hydroxytyrosol were identified by 16s DNA, DNA extraction kit was used, strain DNA was extracted according to the kit method, and the extracted DNA was sent to Shanghai Sangon Bioengineering Ltd. for sequencing. The sequencing results were compared with the known sequences in NCBI, and then a phylogenetic tree was constructed using MEGA-X software.

Through morphological identification, physiological and biochemical characteristics identification, and biological identification, it was comprehensively determined that a strain selected from the northern five seasons spicy cabbage in Harbin, Heilongjiang was the strain with the best ability to degrade oleuropein to hydroxytyrosol. The strains were all purple after staining, and were Gram-positive bacteria. According to phylogenetic tree analysis, it was determined that the bacteria was a Lactobacillus plantarum, and the results are shown in Figure 2.

The Lactobacillus plantarum strain with the best ability to degrade oleuropein to hydroxytyrosol determined by the above results was named Lactobacillus plantarum FBEL-07, and was preserved in the General Committee for the Collection of Microorganisms of China on October 15, 2021. Microbiology Center, depository address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing.

Adopt the above-mentioned Lactobacillus plantarum FBEL-07 to ferment oleuropein, investigate the influence of different conditions on the degradation ability of the bacterial strain, and the specific experiments are as follows:

(1) The effect of pH on the fermentation reaction

After Lactobacillus plantarum FBEL-07 was activated, it was inoculated on oleuropein solid medium for cultivation, and placed in a 37°C incubator for cultivation. The plates with single colonies were screened out, and then single colonies were picked and inoculated in 100 mL of oleuropein liquid medium containing 3 mmol/L pH 5.0, 5.5, 6.0, 6.5, 7.0, and 7.5, respectively, at 37 ° C, 200 rpm /min shaker for 5 days. At the same time, a blank control was set: no bacteria were inserted into the same 100 mL oleuropein liquid medium. The growth of bacteria, the degradation rate of oleuropein and the production rate of hydroxytyrosol were determined. The experiment was repeated 3 times with 3 parallels each time. The experimental results in Figure 3 show that other factors are constant, the fermentation effect is better when the pH is 6.0-7.0, and the fermentation effect is the best when the pH is 6.5, the degradation rate of oleuropein is 88%, and the production of hydroxytyrosol is 47%.

(2) Influence of culture temperature on fermentation reaction

After Lactobacillus plantarum FBEL-07 was activated, it was inoculated on oleuropein solid medium for cultivation, and placed in a 37°C incubator for cultivation. Screen out the plates that grow a single colony, and then pick a single colony and inoculate it in 100 mL of oleuropein liquid medium containing 3 mmol/L pH 6.5, respectively, at 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃. Incubate for 5 days in a shaker at 200 rpm/min. At the same time, a blank control was set: no bacteria were inserted into the same 100 mL oleuropein liquid medium. The growth of bacteria, the degradation rate of oleuropein and the production rate of hydroxytyrosol were determined. The experiment was repeated 3 times with 3 parallels each time. The experimental results in Figure 4 show that the fermentation effect is better when the temperature is 30-40 °C, and the fermentation effect is the best when the temperature is 35 °C, the degradation rate of oleuropein is 90%, and the production of hydroxytyrosol is 55%. %.

(3) Effect of initial concentration of oleuropein on fermentation reaction

After Lactobacillus plantarum FBEL-07 was activated, it was inoculated on oleuropein solid medium for cultivation, and placed in a 37°C incubator for cultivation. Screen out the plate that grows a single colony, and then pick a single colony and inoculate it in 100 mL of oleuropein liquid with an initial concentration of 1 mmol/L, 2 mmol/L, 3 mmol/L, 4 mmol/L, 5 mmol/L, and a pH of 6.5, respectively. In the medium, the cells were cultured for 5 days in a shaker at 37°C at 200 rpm/min. At the same time, a blank control was set: no bacteria were inserted into the same 100ml liquid medium. The growth of bacteria, the degradation rate of oleuropein and the production rate of hydroxytyrosol were determined. The experiment was repeated 3 times with 3 parallels each time. The experimental results in Fig. 5 show that the fermentation effect is better when the initial concentration of oleuropein is 1-3 mmol/L, and the fermentation effect is the best when the initial concentration of oleuropein is 2 mmol/L, and the degradation rate of oleuropein is the best. 93%, and the production of hydroxytyrosol was 60%.

sequence listing

<110> Shaanxi Normal University

<120> Lactobacillus plantarum degrading oleuropein and its application

<141> 2021-12-20

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Claims (5)

1. A lactobacillus plantarum FBEL-07, the deposit number is CGMCC No. 23608, the 16s DNA sequence of which is shown in SEQ ID NO.1. 2. The purposes of the described Lactobacillus plantarum FBEL-07 of claim 1 in degrading oleuropein. 3. the purposes of Lactobacillus plantarum FBEL-07 in degrading oleuropein according to claim 2, is characterized in that: after the activation of Lactobacillus plantarum FBEL-07 bacterial classification, inoculate on the oleuropein solid medium, Placed in a 30-40 ℃ incubator for cultivation, screened out a plate with a single colony, and then picked a single colony and inoculated it into a liquid medium containing 1-3 mmol/L oleuropein with a pH of 6.0-7.0, at 30-40 Fermentation is carried out at ℃ for 72-120 hours on a shaking table. After the fermentation is completed, the antioxidant 2,6-di-tert-butyl-4-methylphenol is added to the fermentation broth, and the reaction is continued for 8-36 hours on a shaking table at 40-60 ℃. 4. the purposes of Lactobacillus plantarum FBEL-07 according to claim 3 in degrading oleuropein, the formula that it is characterized in that described oleuropein solid medium is: oleuropein 1～3mmol/L, hydrogen phosphate Disodium 4～4.5g/L, Potassium dihydrogen phosphate 2.5～2.8g/L, Ammonium nitrate 0.8～1.2g/L, Magnesium sulfate heptahydrate 0.15～0.3g/L, Calcium chloride 0.01～0.03g/L, Ferrous sulfate heptahydrate 0.01～0.02g/L, manganese sulfate heptahydrate 0.002～0.003g/L, agar 15～20g/L, and the rest are water. 5. the purposes of Lactobacillus plantarum FBEL-07 according to claim 3 in degrading oleuropein, the formula that it is characterized in that described oleuropein liquid medium is: oleuropein 1～3mmol/L, hydrogen phosphate Disodium 4～4.5g/L, Potassium Dihydrogen Phosphate 2.5～2.8g/L, Ammonium Nitrate 0.8～1.2g/L, Magnesium Sulfate Heptahydrate 0.15～0.3g/L, Calcium Chloride 0.01～0.03g/L, Ferrous sulfate heptahydrate 0.01～0.02g/L, manganese sulfate heptahydrate 0.002～0.003g/L, and the rest is water.

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