TWI363801B - Method for producing rhamnolipid and mediumn of the same - Google Patents

Description

1363801

〇 is bound to β-hydroxydecanoic by one or two rhamnose. It can reduce the surface tension of water from 72 dcm to 72~30 dyne/cm, and reduce the interfacial tension between water and n-hexadecane to 1 mN/m. The emulsification ability, after being mixed with oils and hydrocarbons, causes emulsification and decomposes oils and hydrocarbons. It is one of the most effective biological interface and active agents to date. Ο In addition, because rhamnolipids are easily combined with heavy metals in soil or wastewater for Rhamnolipid-Metal Complexation Reaction, they are also currently used for soil pollution control to extract heavy metal compounds from contaminated soil. . The combination of rhamnolipid and metal ions is stable: (Stability Constant, log K) is the order ion (Al3+) > copper ion (Cu2+) > lead ion (Pbi+) > cadmium ion (0 ( 12+)>Zinc ion (Zn2+)>Iron ion 063+)> Mercury ion (1^2+)> Calcium ion (〇&2+)>Cobalt ion (Co2+)> Nickel ion ( Ni2+)>Manganese ion (Mn2+)>Magnesium ion (Mg2+)> Potassium ion (K+). Antibacterial property is another important feature of Rhamnolipid. It is known that rhamnolipid has antibacterial and antifungal effects and can effectively control plants Pythium aphanidermatum, Phytophthora capsicilasmopara and Plasmopara lactucae-radicis as well as harmful The spread of pathogen spores of the seaweed flowers Heterosigma akashivo, Prorocentrum dentatemhe and Gymnodnium sp. In addition, the preparation of liposome (LiP〇s_es) from rhamnosyl ester 6 1363801 kg has excellent biodegradability and stability of storage. It is suitable for delivery vehicles for medicines and cosmetics. In the past year, it has also been found that the addition of rhamnolipid can effectively help the skin cells of burned skin to regenerate, and it is more effective in the high application of high-valued lipophylping and its high value. It is a biosurfactant with deep development potential. However, although rhamnolipid has the above-mentioned advantages, it still faces bottlenecks such as low yield and high production cost in practical use, so that commercial production cannot be effectively implemented. In view of this, it is urgent to propose a kind of fermentation of rhamnolipids to improve the production and concentration of rhamnoic acid and reduce the manufacturing cost. And time. SUMMARY OF THE INVENTION Therefore, the embodiment of the present invention provides a method of providing a buckthorn (four) method comprising at least the following steps: First, a bacterial solution containing a plurality of Pseudomonas aer Uginosa is provided. Then, the bacterial liquid is inoculated into the fermentation medium for vinegar production to produce β. Among them, the fermentation medium contains at least 〇.. (4) oil, money salt, unloading inorganic salt, sodium-containing inorganic salt, iron-containing inorganic salt, Contains in the bow inorganic salt, magnesium and inorganic salts and corn dressing. Another embodiment of the present invention provides a fermentation medium suitable for inoculating a green bacillus for fermenting to produce rhamnose, wherein the fermentation medium comprises at least: glycerin having a weight percentage substantially between 6 and 16. GlyCer〇1); corn syrup (Corn Steep LiqUOr) with a weight percentage between 2 and 5; sulphuric acid ((NH4)2S〇4) with a content between 8 mM and mM in essence; Potassium dihydrogen potassium (KH2P〇4) with a quality between 3〇mM and 5〇; phosphorus 7 1363801 sodium hydrogenate (Na2HP04) with a content between 3〇mM and 5〇mM2; Calcium chloride (CaCl2) between 3μΜ and 5μΜ; sodium Ethylenediamine tetraacetic acid (Na-EDTA) in the range of 介 ΙμΜ to 8 μΜ; the content is substantially 70 μΜ A sulphuric acid heptahydrate lock between 850 μΜ (MgS〇 4. 7 out of 0); and iron sulfate heptahydrate (FeS04 · 7Η20) with a content between 1 and 8 in nature.

A further embodiment of the present invention provides a fermentation medium suitable for inoculating Pseudomonas aeruginosa for fermentation to produce rhamnolipid, wherein the vinegar medium comprises at least: glycerin having a substantial weight of u 3 ; Corn syrup having a weight percentage of substantially 3.7; ammonium sulfate having a content of substantially 89 mM; potassium dihydrogen phosphate having a content of substantially 40 mM; sodium hydrogen phosphate having a content of substantially 4 〇〇 1 river; calcium chloride having a content of substantially 6 μ ;; The content is substantially 577 μΜ of ethylene = magnesium tetraacetate having a sodium content of substantially 4 μM; and an iron sulfate heptahydrate containing 4 μM. It can be seen from the above that the technical feature of the present invention is to provide a fermentation efficiency including glycerin, sulfuric acid and corn (4) without (four) bacilli, and it is recommended to retreat, and to obtain a large number of ventral sheep in a shorter process time. & glycolipids effectively implement the business of the servant "A - 仟大里的乳李-步摩 w / the obtained buckthorn can be better to enter the fat, ^, see the biological rehabilitation treatment, in the way of autumn The purpose of environmental protection. The purpose of the present invention is to expose the silk e+ 徂 a method of fermenting the lyophile, which is a new type of + 〃 〃 〃 猎 猎 k k k ^ Enhance the acidity of the green pus broth. Xuan - _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ In green

I st. Among them, a carbon source is used in the fermentation process, and a composite nutrient is added by using ammonium sulfate as a nitrogen source. Preparation of buckthorn bacterium fermentation medium by cultivating pus bacillus - glycerol as inorganic salt fermentation medium' and additional

C. The most suitable side of the inorganic hydrochloric acid fermentation medium, and the most important addition of the nursing nutrients* are designed by the experiment: (Response Surface Methodology, RSM) > it # ^ ^ ^ The experimental analysis was carried out at the time of the sister's effort, and then the optimized semi-synthetic acid fermentation medium (Mod&d Seim-synthehc Medium; hereinafter referred to as MSS fermentation medium) was prepared. I. Fermentation bacteria In the application of the fermentation method of the rhamnolipid of the present invention, first, a green bacillus is provided to produce a glycolipid biosurfactant. In a preferred embodiment of the invention, the Pseudomonas aeruginosa LMG i 242 strain is preferred. : The invented P. aeruginosa LMG1242 strain was screened from diesel-contaminated soil. 'For detailed screening methods, please refer to the inventor's "Biotechnol Prog.", April 27, 2007. No. 23, pp. 661 to 666, entitled "Improved Production 〇f Biosurfactant with Newly Isolated P5e «Heartworthy aer «Poor//105α §2" is hereby incorporated by reference. However, the Pseudomonas aeruginosa which was previously selected by the present invention was named as the Pseudomonas aeruginosa S2 strain, and the complex sequence analysis software, such as the National Biotechnology Information Order (Nati〇nal)

Center for Biotechnolcjgy Information ; NCBI) 9 1363801

The Bl〇Edlt〇r sequence analysis software, after aligning the sequence of 16S rRNA, the resulting affinity analysis tree shows that it should be the Pseudomonas aeruginosa LMG1242 strain, as shown in Figure 1. In more detail, please refer to Fig. 1, which is a tree diagram of a microbial affinity analysis according to an embodiment of the present invention. It can be seen from Fig. 1 that the similarity of the Pseudomonas aeruginosa S2 g strain and the green pus g LMG丨M2 g strain to the 16S rRNA sequence is as high as about 1%. At the same time, the physiological activity and metabolic reaction of Pseudomonas aeruginosa S2 strain and Pseudomonas aeruginosa LMG1242 strain are also very similar (not shown), so the selected strain of P. aeruginosa should be Pseudomonas aeruginosa LMG1242 strain. As for the green pus strain, it has been deposited with the Belgian Collection of Microorganisms (Belgian c〇〇rdinated).

Collections of Micr〇〇rganisms · ' BCCM), this depository is also a foreign biological material storage agency approved by the Ministry of Economic Affairs' Intellectual Property Office. First, choose a better compound nutrient source

C In an embodiment of the invention, the Pseudomonas aeruginosa LMGI242 strain is first appropriately thawed, unsealed and activated. The stored strain was inoculated on Luria-Bertan KLB) agar plate (Agar Piate) fermentation medium by dilution and sputum method, and cultured at 37±0.21 to activate and isolate a single colony (Single Colony), wherein LB agar was agar The composition of the plate fermentation medium can be, for example, shown in Table 1: Table 1

The composition of gasified sodium (NaCl) 10 1363801 y I is not poor; the inorganic salt is exemplified as the third table: Ο _ component glycerol - acid touch ammonium sulphate dihydrogen potassium sodium hydrogen phosphate gasification calcium seven Hydrated magnesium sulfate ethylene diamine tetraacetate sodium sulfate heptahydrate third table concentration

6%~16% 80mM~98 mM 30 mM~50 mM

H.3 〇/〇89 mM 4〇mM 40 mM 3.5 M 577 M 4 M 4 M 30 mM~50 mM 3 Μ~5 Μ 70 mM- ^ B50mM 1 Μ~8 Μ 1 Μ~8 Μ and select soybean eggs g (SQyt()ne), protein gland (PeptQne), beef extract (Be&f), deuterated protein, brain heart extract (Brain heart Ο, malt extract, corn syrup and yeast) The extract is added as a compound nutrient source to the fermentation medium. w Large ugly protein, peptone, beef extract, trypsin, heart-extracted, malt extract and yeast extract Commercially used fermentation medium, so its fine composition and manufacturing method are not described here. As for the corn syrup used in this month, a by-product of the production of corn starch. In the 42 ° C ~ 5 (TC water in the eight-synthesis 're-use mechanically peeled corn kernels, and placed sulfuric acid 12

Claims (1)

1363801 Preparation I (pi. month 713⁄4h, patent application scope • Amendment October 21, 2011 amendment replacement page 1. A method for the fermentation of rhamnose ester, including at least: ^ for Pseudomonas aeruginosa (pseud〇monas) a bacterial solution of aeruginosa), wherein the Pseudomonas aeruginosa is a strain of Pseudomonas aeruginosa LMG 1242; and inoculating the bacterial solution into an acid fermentation medium for performing an acid fermentation, wherein the fermentation medium comprises at least one glycerol (Glycer〇) l), a monoammonium salt, a potassium-containing inorganic salt, a sodium-containing inorganic salt, an iron-containing inorganic salt, a calcium-containing inorganic salt, a magnesium-containing inorganic salt, and a corn syrup (c〇rnSteepLiqu〇r). The method for fermenting a rhamnose ester according to claim 1, wherein the biosurfactant is produced at 37±〇21>c, agitation rate of 250±5 rpm, and pH value of 6 3. The method of fermenting the rhamnose ester according to claim 1, wherein the weight percentage of the glycerin is substantially between 6 and 丨6. 4) The rhamnose (four) acid yeast as described in item 1 of the patent scope The method 'in which the weight percentage of the corn syrup is substantially between 2 and 5. The method of squirting the target is not round, and the stagnation is delayed; the sulphuric acid ester of the sulphuric acid ester' includes the sulphuric acid record ((ΝΗ4 ) 2^4), and the content is practically between 98 mM. "6. The fermented yeast of rhamnolipid as described in claim 1 of the patent scope 21 80 1363801 (October 21, 2011, shirt) The page j method is being replaced, wherein the potassium-containing inorganic salt comprises potassium dihydrogen phosphate (ΚΗ2Ρ〇4), and the content is substantially between 30 mM and 50 mM. 7. The buckthorn as described in claim 1 The method for fermenting a sugar ester, wherein the sodium-containing inorganic salt comprises: sodium hydrogen phosphate (Na2HP〇4)' content substantially between 3〇 mM and 50 mM; and sodium Ethylenediamine tetraacetic acid (Sodium Ethylenediamine tetraacetic acid; Na-EDTA) 'the content is substantially between 8 μΜ. 8. The method for fermenting a rhamnose ester according to claim 1, wherein the magnesium-containing inorganic salt comprises magnesium sulfate heptahydrate ( MgS〇4 · 7H2〇), and the content is substantially between 70 μΜ and 850 μΜ. 9_If the scope of patent application is 丨The method for fermenting a rhamnose ester according to the invention, wherein the iron-containing inorganic salt comprises iron sulfate heptahydrate (FeS〇4. 7H2〇), and the content is substantially between 1 μΜ and 8 μΜ. The method for fermenting a rhamnose ester according to the first aspect of the patent, wherein the calcium-containing inorganic salt comprises calcium chloride (Cac丨2), and the content is substantially between 3 μΜ and 5 μΜ. The invention provides the rhamnose of the rhamnose according to the first aspect of the invention, wherein the supply of the corn syrup comprises: immersing a plurality of corn kernels in water from 42 to 50 tons; 22 1363801 October 21, 2011 The rash is peeled off the corn kernels and immersed in a sulfurous acid solution; the corn kernels are removed' and (iv) the sulfurous acid solution; and the sulfurous acid solution is distilled off. iZ. The seedling culture medium is over-extracted with Pseudomonas aeruginosa to produce rhamnoside vinegar, wherein the Pseudomonas aeruginosa is a strain of M. grisea 1 mg (4), and the fermentation medium contains at least: glycerol, one salt, one Containing unloading, machine salt, sodium-containing inorganic salt, iron-containing inorganic salt, - (iv) inorganic salt, - magnesium-containing inorganic salt, and a corn poly-π Γ '丨地 & preparatory yeast culture medium, 1J· «F The weight percentage of the glycerin is substantially between 6 and 16. From the yeast fermentation medium as described in claim 12, the weight percentage of the corn syrup is substantially between 2 and 5. 15. For the medium described in paragraph 12 of the Patent (4), the ammonium salt includes ammonium sulfate ((NH4)2S〇4), and the content is eight ampere " at 8 mM to 16. As claimed in the patent scope The potassium-containing inorganic salt described in the 12th item includes potassium dihydrogen phosphate (KH2P〇4), which is between 30 mM and 50 mM in human soil. 3 The quantity is substantially between 17. For example, please refer to the pseudo-fermentation medium described in Item 1, 135, which contains 1,630,100. The sodium-containing inorganic salt includes: sodium hydrogen phosphate (Na2HP04), and the content is substantially between 3 mM and 5 〇. Between mM; and sodium Ethylenediamine tetraacetic acid (Na-EDTA) 'content is substantially between ιμΜ to 8 μΜ. 18. The fermenting medium according to claim 12, wherein the lock-containing inorganic salt comprises sulphuric acid heptahydrate and the content of enamel is between Chuanxin and 850 μΜ. 19. The fermentation medium of claim 12, wherein the iron-containing inorganic salt comprises iron sulfate heptahydrate and the content is substantially between _ and 8 μΜ. 2〇. For example, the acid fermentation medium described in claim 12, wherein the mother contains inorganic chlorine, and the content is between Between the city and the genus. The glutinous rice U Μ 辑 辑 ' 杂 杂 杂 杂 杂 杂 杂 杂 杂 杂 杂 杂The fermentation is carried out to: produce = sweet and sour, wherein the Pseudomonas aeruginosa is a Pseudomonas aeruginosa I·· strain, and the fermentation medium contains at least: glycerol '% by weight substantially between 6 and 16; = water slurry, The weight percentage is substantially between...··Ammonium: and the content is substantially between 8〇_ and 98%; and the content is substantially between ~~-······················ 24 1363801 | 2 October 21st, gasification of calcium, the content is between 3μΜ and 5μΜ; sodium ethylenediaminetetraacetate, the content is substantially between ΙμΜ to 8μΜ; magnesium sulfate heptahydrate And the content is substantially between 70 μΜ and 850 μΜ; and the ferric sulfate heptahydrate is between 1 μΜ and 8 μΜ. 22· a fermentation medium suitable for inoculating Pseudomonas aeruginosa for fermentation Production of rhamnose ^ day' wherein the Pseudomonas aeruginosa is a green pus sputum (1) 242 strain, and the fermentation Yang group comprising at least:, 11.3 weight percent of the substance; The iron sulfate heptahydrate has a sodium content of 4 μΜ; 6 and a substance content of 577 μΜ; and the content is substantially 4 μΜ. 25