CN114540216A - Lactobacillus plantarum for degrading oleuropein and application thereof - Google Patents
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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
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a lactobacillus plantarum strain and application thereof in degradation of oleuropein.
Background
Oleuropein is one of byproducts generated in olive processing, and analysis of the metabolic pathway of oleuropein shows that the oleuropein is broken in the glycosidic bond under the action of glycosidase to generate glucose and oleuropein, and the ester bond of oleuropein is broken under the high-temperature reaction to be degraded into hydroxytyrosol and elemi-nic acid. The activities of resisting cancer, oxidation, aging, osteoporosis and the like of hydroxytyrosol are reported and confirmed in documents to be higher than that of oleuropein, and the current yield of the hydroxytyrosol is low, so that the oleuropein is expensive and not easy to obtain, so that the additional value of products can be improved by researching the degradation of the oleuropein into the hydroxytyrosol, and the problem of insufficient yield of the hydroxytyrosol is solved.
At present, three main types of methods for degrading oleuropein exist. The first is that the chemical method is adopted to carry out acid hydrolysis or alkaline hydrolysis on oleuropein, the yield of the method can reach 7.41 percent and 4.09 percent respectively, but the components of secondary metabolites generated by the method are complex, and secondary reaction is easy to occur under severe reaction conditions, so that the product yield is low, and the environment is easily polluted if the chemical reagent is not properly treated. Secondly, the enzyme method is adopted for degradation, the yield can reach 6.07 percent, but the enzyme is expensive and the reaction conditions are harsh. Thirdly, biological methods are adopted, which are the most popular and promising methods at present. Once the mature fermentation process is mastered, the method can be applied to industrial production in a large scale, greatly improves the production efficiency, reduces the production cost and is environment-friendly. However, the technology for degrading oleuropein by using microorganisms is not mature at present, so that a high-efficiency specific strain is sought, and the high-flux conversion of oleuropein to hydroxytyrosol is of great significance.
At present, a plurality of strains for degrading oleuropein are reported in foreign literatures. Santos finds that the lactobacillus plantarum Lac plant6907 can take oleuropein as a carbon source and degrade the oleuropein into hydroxytyrosol with the degradation rate of 90 percent and the generation rate of the hydroxytyrosol of 30 percent, and intermediate products such as oleuropein aglycone and the like are detected. The Bouzid is prepared by mixing olive oil waste with Aspergillus niger for fermentation, the yield of the hydroxytyrosol is 2.94g/kg, and the purity is 85%. Khoufi uses another Aspergillus niger to ferment olive oil wastewater, and the yield of hydroxytyrosol is 0.8 g/L. At present, no strain and mature fermentation process are found to realize the stable and efficient conversion of oleuropein to hydroxytyrosol.
Disclosure of Invention
The invention mainly aims to solve the problems of low degradation yield, complex components of degradation products, harsh process conditions, high cost, environmental pollution and the like existing in the process of degrading the oleuropein by using a biological method, and provides a novel specific and efficient strain, so that the novel strain can be used for stably and efficiently generating hydroxytyrosol by using the oleuropein as a unique carbon source.
Another object of the present invention is to provide the use of the above mentioned strains for the degradation of oleuropein.
Aiming at the purposes, 1 lactobacillus plantarum strain capable of efficiently degrading oleuropein to generate hydroxytyrosol is screened from traditional fermentation products in different producing areas on the market and named as lactobacillus plantarum (FBEL-07), and the strain is preserved in the common microorganism center of the committee for preservation and management of Chinese microorganisms in 2021, 10, 15 days, and the preservation unit address is as follows: the microbial research institute of China academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, has a preservation number of CGMCC No.23608, and a 16s DNA sequence shown in SEQ ID No. 1.
The bacterial colony of the lactobacillus plantarum (FBEL-07) screened by the invention is purple after being dyed, is gram-positive bacteria, and has short rod-shaped bacteria, blunt circles at two ends, no spores and no flagella.
The lactobacillus plantarum FBEL-07 screened by the method can be used for degrading oleuropein to produce hydroxytyrosol, and the specific method comprises the following steps: activating a lactobacillus plantarum FBEL-07 strain, inoculating the strain on an oleuropein solid culture medium, culturing in an incubator at 30-40 ℃, screening out a plate with a single colony, selecting the single colony, inoculating the single colony in an oleuropein liquid culture medium containing 1-3 mmol/L of oleuropein with the pH of 6.0-7.0, fermenting in a shaker at 30-40 ℃ for 72-120 hours, adding an antioxidant 2, 6-di-tert-butyl-4-methylphenol (BHT) into the fermentation liquor after the fermentation is finished, and continuously reacting in the shaker at 40-60 ℃ for 8-36 hours.
The formula of the oleuropein solid culture medium is as follows: 1-3 mmol/L oleuropein, 4-4.5 g/L disodium hydrogen phosphate, 2.5-2.8 g/L potassium dihydrogen phosphate, 0.8-1.2 g/L ammonium nitrate, 0.15-0.3 g/L magnesium sulfate heptahydrate, 0.01-0.03 g/L calcium chloride, 0.01-0.02 g/L ferrous sulfate heptahydrate, 0.002-0.003 g/L manganese sulfate heptahydrate, 15-20 g/L agar and the balance of water.
The formula of the oleuropein liquid culture medium is as follows: 1-3 mmol/L oleuropein, 4-4.5 g/L disodium hydrogen phosphate, 2.5-2.8 g/L potassium dihydrogen phosphate, 0.8-1.2 g/L ammonium nitrate, 0.15-0.3 g/L magnesium sulfate heptahydrate, 0.01-0.03 g/L calcium chloride, 0.01-0.02 g/L ferrous sulfate heptahydrate, 0.002-0.003 g/L manganese sulfate heptahydrate, and the balance of water.
The invention has the following beneficial effects:
1. the novel lactobacillus plantarum strain FBEL-07 screened by the invention can utilize oleuropein as a unique carbon source to efficiently and specifically convert the oleuropein into hydroxytyrosol, and can solve the problems of low yield, low purity, high production cost and long production period of the hydroxytyrosol.
2. The strain can play a role more efficiently and stably under the conditions of the optimal pH, temperature and initial concentration of oleuropein for degrading the fermented oleuropein into hydroxytyrosol, improves the technical method for biologically degrading the oleuropein and further improves the yield of the hydroxytyrosol.
Drawings
FIG. 1 is a microscopic image of the morphological identification of Lactobacillus plantarum strain FBEL-07.
FIG. 2 is a phylogenetic tree.
FIG. 3 is the effect of pH on the fermentation of oleuropein by Lactobacillus plantarum strain FBEL-07.
FIG. 4 is the effect of culture temperature on Oleuropein fermentation by Lactobacillus plantarum strain FBEL-07.
FIG. 5 is the effect of the initial concentration of oleuropein on the fermentation of oleuropein by Lactobacillus plantarum strain FBEL-07.
Detailed Description
The invention is explained in more detail below with reference to exemplary embodiments, to which the scope of protection of the invention is not limited.
Example 1
(1) Screening of strains
First, preliminary screening of the strains
The traditional fermented products collected and selected in different producing areas on the market in a sterile operating platform comprise the Chinese garden old crock pickled vegetables of Sichuan tunnel, the Shumeiwei northeast sauerkraut of Shenyang of Liaoning, the Korean radish pickled vegetables of Xiubi home garden along the Yangling side of Jilin, the northern five-season spicy cabbage of Helongjiang Harbin, the Gansu Tianshui pickled vegetables of Gansu Tianshui, the stinky tofu embryo of Changsha in Hunan and the like. Respectively taking 2mL of samples to inoculate in 100mL of MRS liquid culture medium, shaking at 37 ℃, and shaking at 200rpm/min for 24 h.
Taking 1mL of the culture solution, adding 9mL of sterile physiological saline for diluting by 10 times, and sequentially diluting by 10 times in a gradient manner2、 103、104、105、106、107And (4) taking 30 mu L and 50 mu L of each dilution solution to coat and inoculate the solution in MRS solid culture medium, and culturing the solution in a constant temperature incubator at 37 ℃ for 48 h.
② separation and purification of bacterial strain
Selecting a single bacterial colony with a smooth and convex surface, a complete edge and a milky white color, repeatedly separating and purifying for 2-3 times, then selecting a bacterial colony with suspected lactobacillus growth characteristics to 4mL of MRS liquid culture medium, carrying out shake cultivation at 37 ℃ and 200rpm for 24h, screening 352 bacterial strains, and respectively mixing with a 30% glycerol aqueous solution according to a volume ratio of 1: 1, mixing, and storing in a refrigerator at-80 ℃ for later use.
③ rescreening of bacterial strains
Taking out the strain stored in refrigerator at-80 deg.C, placing on ice, thawing, adding 9-containing strain into 100 μ L in clean benchDiluting 10 times in a centrifuge tube of 00 mu L sterile normal saline, and then sequentially diluting 10 times in a gradient way2、 103、104、105、106、107And (4) doubling. And (3) respectively sucking 30 mu L and 50 mu L of each dilution solution, coating and inoculating the solution to an MRS solid culture medium, and culturing the solution in a constant-temperature incubator at 37 ℃ for 48 hours. After the activated strain grows out of a single colony, a sterile forceps is used for clamping a gun head to pick the single colony, the single colony is placed in a sterilized test tube containing 2mL of MRS liquid culture medium, and the shaking table culture is carried out at 37 ℃ and 200rpm/min for 24 h.
Diluting the activated bacterial liquid, coating the diluted bacterial liquid on an oleuropein solid culture medium, and culturing in an incubator at 37 ℃. The plate on which the single colony grew was selected, and the single colony was then picked up in a liquid medium containing 100mL oleuropein and shake-cultured at 37 ℃ and 200 rpm/min. Blank controls were also set: the same 100mL oleuropein broth was not inoculated with the bacteria.
The formula of the MRS liquid culture medium is as follows: 10g/L of peptone, 5g/L of beef powder, 20g/L of glucose, 4g/L of yeast powder, 5g/L of sodium acetate, 2g/L of dipotassium phosphate, 0.2g/L of magnesium sulfate, 2g/L of triammonium citrate, 0.05g/L of magnesium sulfate, 801 mL/L of tween-801, and the balance of water; MRS solid culture medium is additionally added with agar 15 g/L.
The formula of the oleuropein liquid culture medium is as follows: 1mmol/L oleuropein, 4.26g/L disodium hydrogen phosphate, 2.65g/L potassium dihydrogen phosphate, 1g/L ammonium nitrate, 0.2g/L magnesium sulfate heptahydrate, 0.02g/L calcium chloride, 0.01g/L ferrous sulfate heptahydrate, 0.002g/L manganese sulfate heptahydrate, and the balance of water; the oleuropein solid culture medium is added with 15g/L of agar.
Identification of intermediate products
And (5) injecting and detecting according to an HPLC detection method. The specific operation is as follows: 1) preparing a mobile phase: the water phase is prepared from formic acid, methanol and water according to a volume ratio of 3: 50: 947, and the organic phase is prepared from methanol and acetonitrile according to the volume ratio of 1: 1, preparing the medicament. After the preparation, the mixture is filtered by suction to remove impurities, and then is subjected to ultrasonic treatment for 15min to remove bubbles. 2) Preparing a standard solution: weighing oleuropein, oleuropein and hydroxytyrosol, respectively 0.01g, dissolving in 80% methanol water solution, adding into brown volumetric flask to 100mL to obtain 100 μ g/mL mother solution, and storing at 4 deg.C in dark place. When in use, the above standard mother liquor is diluted in gradient to obtain standard solutions with concentrations of 20 μ g/mL, 40 μ g/mL, 60 μ g/mL, 80 μ g/mL and 100 μ g/mL. Respectively sucking 1mL of each standard solution, mixing to obtain a mixed standard solution, and storing at 4 ℃ in a dark place. 3) Preparing a sample: sucking 1mL of the bacterial liquid cultured in the third step in a 1.5mL centrifuge tube, centrifuging at 10000rpm/min for 5min, sucking 100 mu L of supernatant into 900 mu L of 80% methanol aqueous solution, performing vortex oscillation for 30s, sucking by a disposable sterile needle tube, and filtering by a 0.22 mu m filter membrane to a sample injection bottle. 4) Elution was performed by liquid chromatography gradient, with the following procedure: 0-3 min, and 100% of organic phase; 3-27 min, wherein the volume ratio of the water phase to the organic phase is 40%: 60 percent; 27-45 min, setting the temperature of the organic phase at 35 ℃ and the flow rate at 1mL/min, wherein the organic phase is 100%, and the sample injection amount is 10 mu L. Several standard solutions were then injected separately and the peak retention time was recorded under the same conditions. And then injecting a sample into the mixed standard solution, and recording the peak emergence sequence and the relative retention time of various standards. The separation degree of each standard product is high, and the retention time of the peak is stable, so that the method is feasible and can be used for measuring the sample. 5) And (3) sample determination: and comparing the sample chromatogram with the standard chromatogram according to the retention time of different standards, and performing qualitative analysis to preliminarily determine an intermediate product generated by the oleuropein degraded after the lactobacillus fermentation. 6) And (4) analyzing results: analysis of HPLC spectrogram shows that the retention time of the peak of the degradation product is consistent with that of the peak of oleuropein standard product after oleuropein is fermented by lactic acid bacteria.
High temperature reaction
The oleuropein is determined to generate oleuropein element after being degraded by glycosidase according to the metabolic pathway of the oleuropein, and ester bonds of the oleuropein element are broken at high temperature to generate the target product hydroxytyrosol. Adding antioxidant BHT 0.01% of the total volume of the fermentation liquid into the fermentation liquid, placing the fermentation liquid in a constant temperature incubator at 50 ℃ for high temperature reaction, and sampling for HPLC determination at 8h, 12h and 24h respectively. Finally, 10 strains with better capability of degrading oleuropein into hydroxytyrosol are screened out.
(2) Identification of strains
The 10 screened strains with better ability of degrading oleuropein into hydroxytyrosol are respectively subjected to morphological identification, the physiological and biochemical characteristics and biological identification are determined according to the following methods, and the species and the characteristics of the strains are determined.
(ii) morphological identification
And respectively picking single bacterial colonies with smooth surface, convex round shape, complete edge and milky color in the culture medium for gram staining, observing and taking pictures under an oil mirror, and identifying the morphological characteristics of the single bacterial colonies.
② measurement of physiological and biochemical characteristics
The screened 10 strains with better ability of degrading oleuropein into hydroxytyrosol are respectively subjected to a catalase test, an oxidase test, a starch hydrolysis test, a gelatin liquefaction test, a V-P reaction and a methyl red test, and the physiological and biochemical characteristics of the strains are measured.
③ identification of biology
And (3) carrying out 16s DNA identification on the screened 10 strains with better capability of degrading oleuropein into hydroxytyrosol, extracting the DNA of the strains by using a DNA extraction kit according to a kit method, and sending the extracted DNA to Shanghai biological engineering company Limited for sequencing. The sequencing results were aligned to known sequences in NCBI, and phylogenetic trees were constructed using MEGA-X software.
Through morphological identification, physiological and biochemical characteristic identification and biological identification, a strain screened from the northern five-season spicy cabbage of Harbin of Heilongjiang is comprehensively determined to be a strain with the optimal capability of degrading oleuropein into hydroxytyrosol. The stained strains are purple, gram-positive bacteria, short rod-shaped bacteria, round ends, no spores, no flagella, and the results are shown in FIG. 1. According to phylogenetic tree analysis, the bacterium is determined to be a lactobacillus plantarum strain, and the result is shown in fig. 2.
The lactobacillus plantarum strain with the best ability to degrade oleuropein into hydroxytyrosol, which is determined by the results, is named as lactobacillus plantarum (FBEL-07), is preserved in the China general microbiological culture Collection center (CGMCC) in 2021, 10 months and 15 days, and the preservation unit address is as follows: the microbial research institute of China academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, has a preservation number of CGMCC No.23608, and a 16s DNA sequence shown in SEQ ID No. 1.
The lactobacillus plantarum FBEL-07 is adopted to ferment oleuropein, the influence of different conditions on the degradation capability of the strain is examined, and the specific experiment is as follows:
(1) effect of pH on the fermentation reaction
After the lactobacillus plantarum FBEL-07 is activated, the lactobacillus plantarum FBEL-07 is inoculated on an oleuropein solid culture medium for culture and is placed in an incubator at 37 ℃ for culture. The plate on which the single colony grew was selected, and the single colony was inoculated into 100mL of oleuropein liquid medium containing 3mmol/L of oleuropein at pH 5.0, 5.5, 6.0, 6.5, 7.0, and 7.5, respectively, and shake-cultured at 37 ℃ and 200rpm/min for 5 days. Blank control was also set: the same 100mL oleuropein broth was not inoculated with the bacteria. The growth amount of the cells, the oleuropein degradation rate and the hydroxytyrosol production rate were measured. The experiment was repeated 3 times, 3 replicates each time. The experimental result of figure 3 shows that the fermentation effect is better when the pH value is 6.0-7.0, and the fermentation effect is the best when the pH value is 6.5, the degradation rate of oleuropein is 88%, and the yield of hydroxytyrosol is 47%.
(2) Effect of culture temperature on fermentation reaction
After the lactobacillus plantarum FBEL-07 is activated, the lactobacillus plantarum FBEL-07 is inoculated on an oleuropein solid culture medium for culture and is placed in an incubator at 37 ℃ for culture. The plate on which the single colony grew was selected, and the single colony was inoculated into 100mL of an oleuropein liquid medium containing 3mmol/L of pH 6.5, and cultured in a shaker at 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, at 200rpm/min for 5 days. Blank control was also set: the same 100mL oleuropein broth was not inoculated with the bacteria. The growth amount of the cells, the oleuropein degradation rate and the hydroxytyrosol production rate were measured. The experiment was repeated 3 times, 3 replicates each time. The experimental result of figure 4 shows that the fermentation effect is better when the temperature is 30-40 ℃ and is the best when the temperature is 35 ℃, the degradation rate of oleuropein is 90% and the yield of hydroxytyrosol is 55% under certain other factors.
(3) Effect of Oleuropein initial concentration on fermentation reaction
After the lactobacillus plantarum FBEL-07 is activated, the lactobacillus plantarum FBEL-07 is inoculated on an oleuropein solid culture medium for culture and is placed in an incubator at 37 ℃ for culture. The plate on which the single colony grew was selected, and the single colony was inoculated into 100mL of an oleuropein liquid medium containing an initial concentration of 1mmol/L, 2mmol/L, 3mmol/L, 4mmol/L, 5mmol/L and a pH of 6.5, respectively, and cultured in a shaker at 37 ℃ at 200rpm/min for 5 days. Blank control was also set: the same 100ml liquid medium was not inoculated with bacteria. The growth amount of the cells, the oleuropein degradation rate and the hydroxytyrosol production rate were measured. The experiment was repeated 3 times, 3 replicates each time. The experimental result of figure 5 shows that the fermentation effect is better when the initial concentration of the oleuropein is 1-3 mmol/L, the fermentation effect is the best when the initial concentration of the oleuropein is 2mmol/L, the degradation rate of the oleuropein is 93%, and the generation amount of hydroxytyrosol is 60%.
Sequence listing
<110> university of Shanxi university
<120> lactobacillus plantarum for degrading oleuropein and application thereof
<141> 2021-12-20
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1201
<212> DNA
<213> Lactobacillus plantarum (Lactobacillus plantarum)
<400> 1
gggcggggcg ggtgctatac atgcagtcga acgaactctg gtattgattg gtgcttgcat 60
catgatttac atttgagtga gtggcgaact ggtgagtaac acgtgggaaa cctgcccaga 120
agcgggggat aacacctgga aacagatgct aataccgcat aacaacttgg accgcatggt 180
ccgagtttga aagatggctt cggctatcac ttttggatgg tcccgcggcg tattagctag 240
atggtggggt aacggctcac catggcaatg atacgtagcc gacctgagag ggtaatcggc 300
cacattggga ctgagacacg gcccaaactc ctacgggagg cagcagtagg gaatcttcca 360
caatggacga aagtctgatg gagcaacgcc gcgtgagtga agaagggttt cggctcgtaa 420
aactctgttg ttaaagaaga acatatctga gagtaactgt tcaggtattg acggtattta 480
accagaaagc cacggctaac tacgtgccag cagccgcggt aatacgtagg tggcaagcgt 540
tgtccggatt tattgggcgt aaagcgagcg caggcggttt tttaagtctg atgtgaaagc 600
cttcggctca accgaagaag tgcatcggaa actgggaaac ttgagtgcag aagaggacag 660
tggaactcca tgtgtagcgg tgaaatgcgt agatatatgg aagaacacca gtggcgaaag 720
cggctgtctg gtctgtaact gacgctgagg ctcgaaagta tgggtagcaa acaggattag 780
ataccctggt agtccatacc gtaaacgatg aatgctaagt gttggagggt ttccgccctt 840
cagtgctgca gctaacgcat taagcattcc gcctggggag tacggccgca aggctgaaac 900
tcaaaggaat tgacggggcc cgcacaagcg tggagcatgt ggtttaattc gaagctacgc 960
gaagaacctt accaggtctt gacatactat gcaaatctaa gaagattaga cgttcccttc 1020
gggacatgga taacaggtgg tgcatgtgtc gtcagctcgt gttcgtgaga tgtttgggta 1080
agtccgcacg aggcgcaccc ttattattca gttgcagcat aagtgggcac tctggtgaga 1140
ctgccgtgac aaaccggaga ggtgggatga cgttcaatca tcatgcctta tgactgggct 1200
g 1201
Claims (5)
1. Lactobacillus plantarum FBEL-07 with preservation number of CGMCC No.23608 and 16s DNA sequence shown in SEQ ID NO. 1.
2. Use of lactobacillus plantarum FBEL-07 according to claim 1 for the degradation of oleuropein.
3. Use of lactobacillus plantarum FBEL-07 according to claim 2 for the degradation of oleuropein, characterized in that: activating a lactobacillus plantarum FBEL-07 strain, inoculating the strain to an oleuropein solid culture medium, culturing in an incubator at 30-40 ℃, screening out a plate with a single colony, selecting the single colony, inoculating the single colony to an oleuropein liquid culture medium containing 1-3 mmol/LpH of 6.0-7.0, fermenting in a shaker at 30-40 ℃ for 72-120 hours, adding an antioxidant 2, 6-di-tert-butyl-4-methylphenol into fermentation liquor after the fermentation is finished, and continuously reacting in the shaker at 40-60 ℃ for 8-36 hours.
4. Use of lactobacillus plantarum FBEL-07 according to claim 3 for the degradation of oleuropein, characterized in that the formulation of the oleuropein solid medium is: 1-3 mmol/L oleuropein, 4-4.5 g/L disodium hydrogen phosphate, 2.5-2.8 g/L potassium dihydrogen phosphate, 0.8-1.2 g/L ammonium nitrate, 0.15-0.3 g/L magnesium sulfate heptahydrate, 0.01-0.03 g/L calcium chloride, 0.01-0.02 g/L ferrous sulfate heptahydrate, 0.002-0.003 g/L manganese sulfate heptahydrate, 15-20 g/L agar and the balance of water.
5. Use of Lactobacillus plantarum FBEL-07 according to claim 3 for the degradation of Oleuropein, characterized by the formula of the Oleuropein broth: 1-3 mmol/L oleuropein, 4-4.5 g/L disodium hydrogen phosphate, 2.5-2.8 g/L potassium dihydrogen phosphate, 0.8-1.2 g/L ammonium nitrate, 0.15-0.3 g/L magnesium sulfate heptahydrate, 0.01-0.03 g/L calcium chloride, 0.01-0.02 g/L ferrous sulfate heptahydrate, 0.002-0.003 g/L manganese sulfate heptahydrate, and the balance of water.
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| EP2901867A1 (en) * | 2013-12-11 | 2015-08-05 | Consiglio Nazionale Delle Ricerche | Method for production of fermented table olives |
| CN111343992A (en) * | 2017-11-08 | 2020-06-26 | 雀巢产品有限公司 | Biotransformation of Oleuropein |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2901867A1 (en) * | 2013-12-11 | 2015-08-05 | Consiglio Nazionale Delle Ricerche | Method for production of fermented table olives |
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