CN1456666A

CN1456666A - Production of pectase by solid mould oxalate fermenting

Info

Publication number: CN1456666A
Application number: CN 03120793
Authority: CN
Inventors: 张洪勋; 白志辉
Original assignee: Research Center for Eco Environmental Sciences of CAS
Current assignee: Research Center for Eco Environmental Sciences of CAS
Priority date: 2003-03-20
Filing date: 2003-03-20
Publication date: 2003-11-19

Abstract

A process for preparing pectinase by solid fermentation of oxalate blue mold includes such steps as providing beet dregs as carbon source and ammonium sulfate as nitrogen source, inoculating oxalate blue mold B2H-2002, solid fermenting at 30 deg.c and 70% of relative humidify for 72 hr, extracting in water, and filtering to obtain filtrate (pectainase liquid). Its advantage is high output rate.

Description

The penicillium oxalicum solid state fermentation is produced polygalacturonase

The invention belongs to the microbial fermentation field.

Polygalacturonase is a kind of industrial enzymes, and its main character is the depolymerized pectin material.This character of polygalacturonase is mainly used in fruit juice processing and brewing fruit wine.In addition, polygalacturonase comes unstuck at the fermentation of tea and coffee, orange excystation clothing, fiber crops material, also there is application in fields such as wastewater treatment, papermaking, oil expression.It is disease-resistant that the patent of invention that the author applies for recently successfully is applied to inducing plant.

The substratum of producing polygalacturonase at present mainly comprises: inductor, carbon source, nitrogenous source and trace element.Wherein inductor mainly is pectin or the biomass that are rich in pectin substance, and carbon source is based on carbohydrate and wheat bran, and nitrogenous source mainly contains peptone, yeast extract paste, corn juice and inorganic ammonia salt.The high-yield strain for pectase penicillium oxalicum BZH-2002 that the present invention screens from environment with the author produces bacterium, with abandoned biomass and inorganic nitrogen-sourced be that raw material carries out solid state fermentation and produces polygalacturonase.This method principal feature is as follows: 1. the abandoned biomass that contains pectin substance is as inductor and carbon source; 2. inorganic nitrogen is made nitrogenous source; 3. tap water is a moisture; 4. inoculation penicillium oxalicum BZH-2002 under about 30 ℃ of temperature, ambient moisture about 70%, ingress of air condition, leaves standstill cultivation and reached product enzyme peak in about 72 hours.5. through flooding, promptly get pectase liquid.6. method for degumming is measured enzyme activity, and the polygalacturonase productive rate reaches 1.2 * 10 ⁵The units/gram beet pulp.It is raw materials used inexpensive that this method prepares polygalacturonase, enzyme productive rate height, and fermentation period is short, and technology is simple, has very high industrialized developing and is worth.

Polygalacturonase preparation method of the present invention thes contents are as follows:

1. the used penicillium oxalicum BZH-2002 of the present invention bacterial strain is to separate, screen acquisition from environment.This bacterial strain is stored in the PDA substratum.

2. it is as follows to produce enzyme substratum proportion of composing (w/w): beet pulp 24%, ammonium sulfate 2%, tap water 74%, carbamate additives for low phosphorus hydrochlorate.

3. the solid state fermentation working method is as follows: be equipped with substratum by 2 described raw material ratio preparations; Substratum is sub-packed in the triangular flask of 500mL, every bottle of 30g, and vapor sterilization is 20 minutes under 0.1Mpa pressure, also can directly use raw material fermentation; Inoculate the bacterial classification of 1 described method preparation, under about 30 ℃ of temperature, ambient moisture about 70%, ingress of air condition, leave standstill and cultivated 72 hours, use flooding, promptly get pectase liquid.

4. polygalacturonase is measured and is adopted method for degumming, measuring method is as follows: get the test tube that 1% pectin solution (with 0.05M citric acid/trisodium citrate damping fluid and the citrus pectin preparation of pH4.8) that 5mL prepared the same day places  25 * 250mm, 0.05M citric acid/trisodium citrate damping fluid the 10mL that adds pH4.8, test tube was in 40 ℃ of pre-thermal equilibrium of water bath with thermostatic control 5 minutes, the enzyme liquid that adds 5mL dilution certain multiple, and pick up counting immediately, react the 1mL that takes a sample after 3 minutes and put into test tube, add 2 1%I immediately ₂/ KI solution shakes up, and adds the 2mL Virahol again, rotates test tube, observes precipitation and whether disappears the accurate recording usually time.The enzyme activity unit definition: under the aforesaid operations condition, the enzyme amount that made 1 milligram of pectin come unstuck required fully in 30 minutes is an enzyme activity unit.

Embodiment 1: the different strain solid state fermentation produces the enzyme experiment

Produce the enzyme substratum with above-mentioned dish slag, tested bacterial classifications such as penicillium oxalicum, aspergillus niger, mucor mucedo, grey mold respectively, the product enzyme situation of each bacterial classification sees Table 1.

Table 1

Bacterial classification	Enzyme productive rate (units/gram dish slag)
Bacterial classification	Enzyme productive rate (units/gram dish slag)	Penicillium oxalicum BZH-2002	????1.2×10 ⁵
Mould sp.	????9.6×10 ³	Penicillium oxalicum BZH-2002	????1.2×10 ⁵
Mould sp.	????9.6×10 ³	Aspergillus niger BH	????8.5×10 ³
Aspergillus niger CGMCC0455	????1.5×10 ⁴	Aspergillus niger BH	????8.5×10 ³
Aspergillus niger CGMCC0455	????1.5×10 ⁴	Mucor mucedo	????7.8×10 ²
Grey mold	????5.3×10 ²	Mucor mucedo	????7.8×10 ²

Embodiment 2: different nitrogen sources is to producing the influence of polygalacturonase

Bacterial classification penicillium oxalicum BZH-2002 is stored in 4 ℃ of PDA substratum in the refrigerator, transfers half a year 1 time.Be transferred to the inclined-plane of new configuration before the use.

Solid state fermentation produces the enzyme experiment: add various inorganic nitrogen-sourced and organic nitrogen source preparation product enzyme substratum respectively, the proportioning of each substratum all is beet pulp 24% (w/w), nitrogen 0.55% (w/w), and about moisture 74% (w/w), the carbamate additives for low phosphorus hydrochlorate.Inoculation penicillium oxalicum BZH-2002 carries out solid state fermentation, the results are shown in Table 2.：

Table 2

Nitrogenous source	Enzyme productive rate (units/gram dish slag)
Nitrogenous source	Enzyme productive rate (units/gram dish slag)	????(NH ₄) ₂SO ₄	????1.2×10 ⁵
????NH ₄Cl	????4.3×10 ⁴	????(NH ₄) ₂SO ₄	????1.2×10 ⁵
????NH ₄Cl	????4.3×10 ⁴	????NH ₄NO ₃	????9.1×10 ³
????NaNO ₃	????8.5×10 ³	????NH ₄NO ₃	????9.1×10 ³
????NaNO ₃	????8.5×10 ³	Trisodium citrate	????3.5×10 ⁴
Urea	????8.4×10 ³	Trisodium citrate	????3.5×10 ⁴
Urea	????8.4×10 ³	Msg powder type	????8.4×10 ³
Yeast powder	????2.8×10 ⁴	Msg powder type	????8.4×10 ³
Yeast powder	????2.8×10 ⁴	Wheat bran	????4.0×10 ⁴
Soybean cake powder	????6.0×10 ⁴	Wheat bran	????4.0×10 ⁴
Soybean cake powder	????6.0×10 ⁴	Do not add nitrogenous source	????9.0×10 ³

Embodiment 3: the productive rate of different fag-end solid-state fermentation polygalacturonases

Being carbon source with Receptaculum Helianthi, beet pulp, apple residue, orange peel, Pericarpium Musae respectively, is that the enzyme substratum is produced in the nitrogenous source preparation with ammonium sulfate, and the proportioning of each substratum (w/w) all is a waste residue 24%, ammonium sulfate 2%, water 78%, carbamate additives for low phosphorus hydrochlorate.Inoculation penicillium oxalicum BZH-2002 carries out solid state fermentation, the results are shown in Table 3.

Table 3

Waste residue	Enzyme productive rate (units/gram waste residue)
Waste residue	Enzyme productive rate (units/gram waste residue)	Receptaculum Helianthi	????1.2×10 ⁵
Beet pulp	????1.2×10 ⁵	Receptaculum Helianthi	????1.2×10 ⁵
Beet pulp	????1.2×10 ⁵	Apple residue	????2.6×10 ⁴
Orange peel	????3.1×10 ⁴	Apple residue	????2.6×10 ⁴
Orange peel	????3.1×10 ⁴	Pericarpium Musae	????8.5×10 ³

Biological material specimens of the present invention is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, the address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica, 100080; Preservation date: on March 14th, 2003; Deposit number: CGMCC No.0907; Classification name: penicillium oxalicum (Penicillium oxalicum).

Claims

With the abandoned biomass that contains pectin substance as carbon source, as nitrogenous source, add 3 times of water with inorganic nitrogen, inoculation penicillium oxalicum BZH-2002 bacterial strain carries out solid state fermentation, the tunning flooding under certain temperature, humidity, ventilation condition, filter, filtrate is pectase liquid.
2. in 1 described method of claim the, penicillium oxalicum BZH-2002 bacterial strain is for producing unique bacterial strain of polygalacturonase, and this bacterial strain separates, screens acquisition from environment, and its enzymatic production efficient is because of the raw material difference, control condition difference and changing.
3. in 1 described method of claim the, the carbon source of preparation polygalacturonase comprises that beet pulp, Receptaculum Helianthi and pomace etc. are rich in the abandoned biomass of pectin substance.Described beet pulp is meant the waste residue (claiming the beet pulp or the particle dregs of rice again) that produces in the beet sugar production process, is processed into moisture below 12%; Receptaculum Helianthi is that Sunflower Receptacle is removed the Semen Helianthi post-treatment and becomes moisture graininess below 12%; Pomace is meant pericarp and the waste residue that the processing fruits process produces, and is processed into moisture graininess below 12%.In fermention medium, carbon source accounts for about 25% (w/w) of substratum gross weight.
4. in 1 described method of claim the, nitrogenous source can be with the waste water (as gourmet powder waste water) of higher ammonia-nitrogen content, also available inorganic ammonium salt (as ammonium sulfate, ammonium chloride, ammonium nitrate etc.).In fermention medium, ammonia nitrogen content accounts for about 0.5% (w/w) of substratum gross weight.
5. in 1 described method of claim the, the temperature of solid state fermentation is about 30 ℃, and ambient relative humidity is about 70%, and the scope of application is bigger, need not strict control.
6. in 1 described method of claim the, pectase liquid can be according to service requirements, and concentrating under reduced pressure becomes high density polygalacturonase solution or lyophilize to become pulvis; Also can by saltout, separation purification process such as ultrafiltration, column chromatography obtains the higher zymin of purity.