CN1052511C

CN1052511C - Technology for preparation of gallic acid by using enzyme process

Info

Publication number: CN1052511C
Application number: CN93107994A
Authority: CN
Inventors: 邓厚暲
Original assignee: GUIZHOU PROVINCIAL INST OF CHEMICAL TECHNOLOGY
Current assignee: GUIZHOU PROVINCIAL INST OF CHEMICAL TECHNOLOGY
Priority date: 1993-07-08
Filing date: 1993-07-08
Publication date: 2000-05-17
Anticipated expiration: 2013-07-08
Also published as: CN1083532A

Abstract

The present invention provides a process for producing gallic acid by an enzyme method, which relates to a technology for preparing aromatic ring compound derivatives by the enzyme method. The present invention has the goal of providing the process with the advantages of high product yield, low raw material unit consumption and reasonable resource utilization. In the process of the present invention, fermentation enzyme preparation and hydrolysis acid preparation are correspondingly and respectively carried out in different containers; simultaneously, the reaction time and the conditions such as the optimum temperature, the pH value, the dissolved oxygen quantity, etc. of the two steps of the reaction are respectively controlled, so both of the two steps of the reaction reach the optimum state, and tannin is totally hydrolyzed. When used for producing the gallic acid, the process of the present invention has the advantages of high product yield, low raw material consumption, improved production environment, simple flow path, wide raw material adaptability and large economic benefit improvement amplitude.

Description

The technology of preparation of gallic acid by using enzyme

The present invention relates to use the method for enzyme to produce the technology of aromatic compound derivative, in particular to the technology of preparation of gallic acid by using enzyme.

Gallic acid has another name called doubly acid, gallic acid (gallic acid), and chemical name is 3,4,5-trihydroxybenzoic acid (3,4,5-trihydroxy benzoic acid), and molecular formula is (OH) ₃C ₆H ₂COOHH ₂O, structural formula is:

Molecular weight 188.14, white, needle-shaped crystals is a kind of broad-spectrum fine chemical product.It is raw material that industrial Turkey-galls commonly used (chinese nut galls) or tower draw (tara, Caesalpinia spinosaKuntze), produces gallic acid by chemical hydrolysis or microbe fermentation method.Wherein, chemical hydrolysis is to be catalyzer with strong acid or highly basic, makes the Chinese tannin hydrolysis generate gallic acid.Then, through filtration, concentrate, a series of chemical processes such as decolouring, drying make product.

Use the sulphuric acid hydrolysis method,, in reaction process, can destroy a part of benzene ring structure in the tannin, and side reaction is arranged because the vitriol oil has strong oxidizing property; Therefore, hydrolysis conversion only 85%, total recovery is 69%, and raw material is underused, and the product economy benefit is not high.

Than acid-hydrolysis method height, but must use a large amount of alkali with alkali hydrolysis method (CN1057255A) product yield, then use the sulfuric acid neutralization reaction product again as catalyzer.Make the alkali process (U.S.Patent 2723992.1955) of raw material with Turkey-galls, need to use a large amount of organic solvents carry out extracting and separating, obtain product through processes such as solvent recuperation, decolouring, dryings again.The solvent extration facility investment is big, organic solvent cost height, and solvent extraction and recovery technology complexity should not adopt at home.

The principle of microbe fermentation method (CN87103987) is to utilize microorganism to ferment in containing the aqueous solution of tannin, with the glucose after the Weibull hydrolysis as carbon source, for microorganism growth, breeding, meanwhile microorganism through inducing the generation enzyme, tannin generates gallic acid through the catalytic hydrolysis effect of enzyme again in solution.The production cycle of this method is long, and hydrolysis is incomplete, also has the not hydrolysis conversion of 15%～20% tannin after the fermentation ends.

The purpose of this invention is to provide a kind of process for producing gallic acid that overcomes above-mentioned shortcoming,, reduce raw material consumption, simplify technical process in order to improve product yield, economical, reasonably utilize the vegatable tannin resource.

Secular scientific research and production practice prove already: a variety of moulds can both generate tannase (tannase) with the inductor effect under certain condition, wherein, make with the most use, the most ripely with black-koji mould, and the applicating history in more than 100 year has been arranged.Tannase claims tannin ethanoyl lytic enzyme (tannin acyl enzyme) again, belongs to the outer inducible enzyme (induced enzyme) of spore, is a kind of biological catalyst with hydrolyzation catalysis performance.Under optimum conditions, ester bond, glycosidic link and the phenol key hydrolysis of contracting in single-minded, efficient, the directed cracking tannin of its energy generates gallic acid.

People are through for many years production practice and research (Agr.Biol.Chem., Vol.32.No.7, P.803～809,1968), find to generate the top condition of tannase and the top condition of enzymic hydrolysis tannin, comprise temperature, pH value, dissolved oxygen amount, inductor concentration, medium component etc., most inequality.In general fermentative Production technology, the generation of tannase and the reaction of Hydrolysable Tannins all are to carry out in same reactor (fermentor tank).Because two top condition differences that reaction requires, during the fermentation, always attend to one thing and lose sight of another, mutual restriction, the both can not reach the optimum response state.Here it is causes the incomplete major cause of tannin hydrolysis.

For solving this difficult problem in the fermentation method technology, the solution that the contriver proposes is: two reactions of the system of will fermenting enzyme and hydrolysis relieving haperacidity relatively separate with two containers respectively to be carried out, and makes the two all reach optimum regime.Like this, the tannin hydrolysis conversion can reach more than 98%, near or reach Quantitative yield.Mandatory declaration, the two both had any different system enzyme and relieving haperacidity, was related again, complemented each other, respectively give priority to, be not absolute division.In the system enzyme process, contain the enzyme mycelium pellet in a large number to generate, meanwhile also turn out a large amount of healthy and strong aspergillus niger spores; In the relieving haperacidity process,, proceed fermentation simultaneously again, produce a part of enzyme, impel the tannin complete hydrolysis, therefore need to supply with enough dissolved oxygens too based on Hydrolysable Tannins.

In fermentation system enzyme process, the proportioning of every fermentation parameter and substratum is very important, must strict control.Optimal temperature is 27～33 ℃, and optimum temperuture is 30 ℃; Initial pH value is 5～6.5, adds alkali in the reaction process and is adjusted between pH3～5, and be best with pH3.5～4.5; Dissolved oxygen amount is controlled at 3.5～5.0gO ₂Between/the Lh.Adopt aspergillus niger No.316 (aspergillus neiger No.316) as the zymogenic bacteria kind.

In the system enzyme substratum, the C/N ratio is 5～15, and tannin concentration is 1%～8%; The carbon source add-on is 0.3～1 times of tannin amount beyond the tannin.Additional carbon comprises that sucrose, glucose, honeybee sugar etc. all can use; Adding nitrogenous source can all can use as ammonium nitrate, ammonium chloride, ammonium sulfate, ammonium phosphate, SODIUMNITRATE, saltpetre etc. with any inorganic ammonium salt or nitrate.The inorganic salt and the yeast extract paste of a spot of sulphur, phosphorus, potassium, sodium, zinc, magnesium also should be arranged in the substratum or tie up the B compounds of group as nutritional additive.

The tannase solution made from above-mentioned fermentation condition and substratum proportioning can be used for hydrolysis relieving haperacidity, and the relieving haperacidity optimal temperature is 30～37 ℃, and optimum temperuture is 34～35 ℃; Adding ammoniacal liquor and regulate pH between 4～7 in reaction process, is best with pH5.5～6.5; Dissolved oxygen amount is 3.5～5.0gO ₂/ Lh.With Turkey-galls is raw material, and tannin concentration is 6%～10% (W/V); Drawing powder with tower is raw material, and add-on is 12%～20% (W/V).

Use the technology of preparation of gallic acid by using enzyme of the present invention that following advantage is arranged: (1) reaction times is shorter than fermentation method, product yield height, and raw material consumption is low.With containing Turkey-galls that tannin do not wait and Ta La respectively as raw material, the product yield is respectively 47%～52% and 28%～33% of raw material, and total recovery can reach 80%～90%.Compare with acid-hydrolysis method, produce with technology of the present invention is annual that 1 ton of product can be saved 0.3～0.4 ton of Turkey-galls or 0.6～0.9 ton of tower draws.(2) improved production environment.Technological reaction condition of the present invention relaxes, no high temperature, high top pressure operation, and " three wastes " pollute little, administer easily; The consumption of acid, alkali and gac is than the obvious reduction of chemical hydrolysis.(3) adaptability to raw material is wide.Both can use Turkey-galls or tower to draw and be raw material; The Turkey-galls that also available tannin content is low and pectin substance is high (claiming doubly flower again) is as raw material.(4) technical process is simple.Compare " hydrolysis " preceding lixiviate workshop section that saves with existing production method; " hydrolysis " back is economized to decrystallize and decolour and is operated each once.

Accompanying drawing is a process flow diagram of the present invention.Through the bacterial classification seed pre-culture, changing fermentation system enzyme in culture medium after sterilization solution then over to, after measured enzymic activity qualified after, promptly be used for hydrolysis relieving haperacidity.After hydrolysis reaction finishes, add sulfuric acid or hydrochloric acid and regulate pH, remove by filter filter residue, then filtrate is concentrated, obtain coarse crystallization after the cooling.Coarse-grain is separated, water-soluble again, add activated carbon decolorizing, recrystallization.And then separate, reclaiming mother liquor, last drying and pulverizing can obtain the gallic acid product.

Following examples can be illustrated more clearly in the present invention:

System enzyme embodiment 1 presses following proportioning components preparation substratum:

Tannin 10～100g/L

Sucrose 14g/L

NH ₄Cl 3g/L

KH ₂PO ₄ 1g/L

MgSO ₄·7H ₂O 0.5g/L

ZnSO ₄·7H ₂O 0.3g/L

Yeast extract paste 0.5g/L

Prepared culture medium is divided in several 500ml vibration flasks sterilization.For avoiding the tannin pyrohydrolysis, tannin adds after sterilization, is 5～6.5 with the initial pH value of 5mol NaOH regulator solution, then inserts black-koji mould No.316 bacterial classification immediately, is placed on the THZ-82A constant temperature oscillator and reacts.30 ℃ of design temperatures, the adjusting rotating speed is 130～140r/min, makes that dissolved oxygen remains on 3.5～5.0O in the reaction solution ₂G/L.h regulates for several times with 5mol NaOH, and the pH value of reaction solution is remained between 3.5～4.5, and successive reaction till the activity of measuring enzyme reaches maximum value, promptly was used for relieving haperacidity more than 40 hours.

Make enzyme embodiment 2 and prepare substratum in following composition ratio:

Tannin 10～100g/L

Glucose 16g/L

NH ₄NO ₃ 2.2g/L

KH ₂PO ₄ 1g/L

MgSO ₄·7H ₂O 0.5g/L

ZnSO ₄·7H ₂O 0.3g/L

Compound dimension B 10mg

Prepared culture medium is respectively charged in the vibration flask, and the sterilization back adds tannin.Remaining step is undertaken by system enzyme embodiment 1.

Relieving haperacidity embodiment 1 packs gallnut extract into and has in the special reaction flask of semi-automatic ammonifying device and external acidometer, dissolved oxygen meter, adds an amount of hot water dissolving, and making solution tannin concentration is 8% (W/V).Add the enzyme solution that 10%～20% (V/V) newly makes after being cooled to room temperature.Reaction flask is placed on the THZ-82A constant temperature oscillator reacts, 34 ℃ of design temperatures, speed adjustment makes the reaction solution dissolved oxygen amount remain on 4.5～5.0gO at 140～150r/min ₂/ Lh.Add 10mol ammoniacal liquor in reaction process continuously, keeping pH value of solution is 5.5.Successive reaction is more than 35 hours, till the whole hydrolysis of tannin.Reaction product is adjusted to pH value 1～2 with hydrochloric acid or sulfuric acid, through obtaining product behind coarse crystallization, decolouring, the recrystallization.Raw material contains tannin 64.8%, and the product yield is 47.7% of a raw material, purity 98%.

Relieving haperacidity embodiment 2 draws the powder reaction flask (identical with relieving haperacidity embodiment 1) of packing into tower, adds an amount of hot water dissolving, makes that material quantity is 15% (W/V) in the solution, and the cooling back adds the enzyme solution that 20% (V/V) newly makes, and is put on the constant temperature oscillator and reacts.35 ℃ of design temperatures, rotating speed are 140～150r/min, dissolved oxygen amount 4.5～5gO ₂/ Lh.Adding ammoniacal liquor in the reaction process continuously, to make reacting liquid pH value be 6.0.Reaction times is more than 40 hours, till the tannin hydrolysis fully.Reaction product obtains product through accent pH, coarse crystallization, decolouring, the recrystallization process with relieving haperacidity embodiment 1.Raw material contains tannin 41.13%, and the product yield is 27% of a raw material, and purity is 98.5%.

Claims

1 usefulness microorganism makes tannase in substratum, make the tannin hydrolysis in drawing of Turkey-galls or tower, more after filtration, concentrate, technology that crystallization, decolouring, drying make gallic acid, feature of the present invention comprises:

(1) used bacterial classification is black-koji mould № 316;

(2) the C/N ratio of used substratum is 5～15; Tannin concentration is 1%～8%, and the carbon source add-on beyond the tannin is 0.3～1 times of tannin amount;

(3) fermentation system enzyme separates with two containers respectively with hydrolysis relieving haperacidity two-step reaction and carries out.

2 according to the described technology of claim 1, and the temperature of controlling in the system enzyme process that it is characterized in that fermenting is 27～33 ℃; Initial pH value is controlled to be 5～6.5, adds alkali then and transfers to pH3～5; Dissolved oxygen amount is controlled at 3.5～5.0gO ₂Between/the L.h.

3 according to the described technology of claim 1, it is characterized in that in the hydrolysis relieving haperacidity process, temperature of reaction is controlled at 30～37 ℃; The pH value transfers to 4～7 with ammoniacal liquor, and dissolved oxygen amount is at 3.5～5.0gO ₂/ L.h; Chinese tannin concentration is 6%～10% (W/V) during hydrolysis, and it is 12%～20% that tower draws the powder consumption.