CN103740657A - Earthworm biological composite enzyme - Google Patents

Abstract

An earthworm biological composite enzyme is disclosed. A certain amount of live earthworm is cleaned by utilization of an ultrasonic cleaner so that dirt in earthworm viscera can be discharged as much as possible, then fished out, drained to remove excess water and weighed. 900 kg of the earthworm is added into a bioreactor and then 50 kg of enzymatic bacteria and 50 kg of biological assistants are added. The materials are stirred uniformly and subjected to aerobic fermentation for 48 h. Filtrate is subjected to ultrafiltration to concentrate effective components, and fed into a vacuum freeze drier to be dried at a sublimation temperature not higher than 30 DEG C for 4-10 h into dry powder. The dry powder is smashed, sealed and packaged with aluminum plastic film, and stored in a refrigerator.

Description

The biological composite enzyme of earthworm

Technical field:

The invention belongs to food and field of biological pharmacy.

Technical background:

Modern medicine study prove earthworm have heat-clearing, calm, anticonvulsion, relieving asthma, antihistamine, hypotensive, thrombolysis, anticancer effect.Earthworm protein matter content is very high, accounts for 8%～10% of fresh earthworm, 56%～66% of dry weight, and amino acid is in the majority with L-glutamic acid, Methionin, arginic content.Earthworm lyophilized powder and body intraluminal fluid Analysis of Fatty Acid Content show, unsaturated fatty acid content is high, and saturated fatty acid content is low.Particularly have anticancer, hypotensive, prevent that the linoleic acid content of the effect such as arteriosclerosis, beauty treatment is higher, and ten tricarboxylic acid are obviously different from other animal, these meet the requirement of current mankind heath food structure.

Earthworm is rich in the functional factors such as nucleic acid, VA, Vc, VE, and the trace element such as Fe, Mn, Cu, Zn, Se is abundant, they with other Chinese medicinal materialss in organism complexing bring into play larger pharmacological effect, wherein Se can also promote immunization of cell.In addition, earthworm is still containing the main sp act material such as lumbricin (lumbritn), earthworm alkali (lumbrofobrim), terrestro-lumbrolysin (terrestrolumbrolysin) and purine, choline, cholesterol.But what study at present at most is several enzymes (being) of earthworm.

One, earthworm enzyme system and physiological function

1, plasmin (fibrinolytic enzyme)

Earthworm is as the existing history in several thousand of the medicinal material activating blood circulation and dispersing blood clots.The plasmin that fresh earthworm body includes belongs to wherein the serine protease of one group of strong solution fibrin of tool and thrombus effect, can separate and obtain from the earthworm of different genera.

2, Lumbrukinase (lumbrinonase)

Lumbrukinase is a kind of serine protease extracting from earthworm, has the polypeptide chain folded form of typical chymotrypsin protein enzyme serine protease, with the original special avidity of scleroproein, and has Fibrinolytic Activity.Lumbrukinase energy obvious degradation Fibrinogen, reduces platelet aggregation rate and blood viscosity.Now have and much studies have shown that Lumbrukinase has strong fibrinolytic in vitro, thereby make it may become a kind of very promising thrombolysis preparation.

3, anti-tumor protein

Xie Jiangbi etc., take Eisenia foetida as raw material, separate and obtain one group of anti-tumor active protein composition (being called for short earthworm extract).Ancestral sacrifices one's life for etc., and to choose Eisenia foetida be raw material, studied the restraining effect for tumour cell by the isolated sample of the whole bag of tricks.Result shows that a certain protein ingredient after separation and purification can effectively suppress the growth of BEL-7402 cell (human primary liver cancer's clone), HeLa cell (Human cervical cancer cell lines).

4, hemolysin

From coelomic fluid of earthworm, can be separated to the protein with hemolytic activity, wherein indivedual protein is bifunctional protein, has and dissolves and the erythrocytic activity of aggegation.

5, digestive ferment

Earthworm plays an important role in Organic residues conversion and soil nutrient circulation, for the feeding habits of clear and definite different ecological type earthworm and the ability of digesting organic materials thereof, Zhang Baogui etc. have measured table and have occupied the activity that occupies type earthworm William Pheretimatschiliensis (Pheretima guillemi) enteron aisle inner cellulose enzyme, proteolytic enzyme, acid phosphatase and alkaline phosphatase under type earthworm cellulase (Eisenia fetida) and upper food; Also proteolytic enzyme, Phosphoric acid esterase and C02 respiratory intensity and original soil in William Pheretimatschiliensis movement are compared simultaneously, result shows, cellulase enteron aisle inner cellulose enzymic activity is far away higher than William Pheretimatschiliensis, and proteolytic enzyme and acidity and alkaline phosphatase activities are significantly lower than William Pheretimatschiliensis; The difference of two kinds of earthworm enteron aisle digestive ferment activation and cellulase are directly take plant residue as food, and William Pheretimatschiliensis is relevant as eating take the microorganism on half Organic residues decomposing.And draw different types of earthworm life habit difference, and feeding habits difference, in body, composition also there are differences.

6, antibacterial peptide

Zhang Xichun etc. adopt ammonium sulfate precipitation, ultrafiltration, cationic exchange separates and anti-phase fast protein liquid chromatogram (FPLC) is analyzed, two kinds of new earthworm antimicrobial peptide F-1 and F-2 have been obtained, through electric spray ion source mass spectrum (ESI-MS), measure, its relative molecular mass is 535.27 and 519.27, tandem mass spectrum (MS/MS) data show that the peptide sequence of F-1 is 190Ac-Ala-Met-Val-Ser-Ser, the peptide order of F-2 is Ac-Ala-Met-Val-Gly-Thr, minimal inhibitory concentration (MIC) experiment shows, F-1 and F-2 are to Enterococcus gallinarum (Enterococcus gallinarum), Pseudomonas aeruginosa (Pseudomonas pyocyanea), Boydii is lever (Acinetobacterbaumanii) not, the minimal inhibitory concentration of Klebsiella terrigena (Klebsiella terrigena) is respectively 11.4mg/L and 12.85mg/L, minimal inhibitory concentration to enterococcus faecalis (Enterococcus faecalis) is respectively 22.8mg/L and 25.68mg/L, fungi white chain strain bacterium (Candidaalbicans) is not shown as to restraining effect completely.

7, calcium binding protein

Zhang Yanhong etc. are take Eisenia foetida as material, and by DEAE-FastFlow ion exchange chromatography, CaM-Sepharose affinity chromatography, separation and purification obtains CaM binding Proteins from Earthworm (CaMBPs).Wang Caiqin etc. take Eisenia foetida as material separation purifying calmodulin, and obtain a kind of new calcium binding protein.The quantitative assay of Wang Libo etc. application infrared spectrum technology secondary structure and the variation of its secondary structure under the effect of different ratios Ca2+, Tb3+ ion of the new calcium binding protein of earthworm (NCBP) in hydration shell.Result shows, in NCBP, α-helix content is higher compared with content low and β-sheet, and with the adding of different ratios overall situation ion, the variation at β-sheet peak, 1629cm-1 place is more obvious.Find, NCBP has the SDS-PAGE phenomenon of calcium binding protein family feature simultaneously, but does not have the activity that activates PDE target enzyme, tentatively thinks, NCBP contains the concentration dependent special structure and function with Ca2+.In addition, the intermolecular polymerization of the combination of Tb3+ initiation NCBP under high density.Prompting wants prudent to the use of rare earth element.

8, catalase

Xu Weihong etc. with Eisenia foetida (Eisenia foetida) tissue homogenate through sulfuric acid amine salt analyse, dialyse concentrated after, obtain two catalases with huge pillar preparation and Purified in electrophoresis, be glycoprotein, molecular weight is respectively 39kD and 40kD, the activity of the equal inhibitory enzymes of metal ion such as KCN and Cu2+, Cd2+, Zn2+, Ni2+, Pb2+, Fe2+ have activation.In Zhili Pheretimatschiliensis (Pheretima tschiliensis) body, in experiment, Zn2+ significantly improves catalatic total activity, and Cd2+, Ni2+ reduced before this, improved subsequently enzyme total activity.

9, Pseudocholinesterase

Ding Shihua etc. to the TritonX-100 extract of Eisenia foetida after the processing of PEG2000-potassium phosphate buffer, ion exchange chromatography and gel-filtration, obtain electrophoretically pure Pseudocholinesterase, this enzyme is through being accredited as glycoprotein, gel filtration method and SDS-PAGE method record its Mt and are respectively 59000 and 58000, acidic amino acid content accounts for 24%, basic aminoacids content accounts for 12%, maximal ultraviolet absorption λ=272nm; Enzyme effect optimum temperuture T=39 ℃, thermostability is poor; Optimal pH is 7.8, more stable at pH7.0-8.0 enzyme, to the Ka value of sulfur iodide vagusstoff, is 88 μ mol/L, has excessive substrate inhibition phenomenon .Mg2+, Ca2+ and Mn2+ less on the impact of enzymic activity.DEP has strongly inhibited effect to this enzyme.

10, lumbricin

Chen Kefu etc. show by experiment, lumbricin 9203 to mouse without analgesia, sedative effect; To rat original position intestines unrestraint effect, rat electrocardio, blood pressure, respiratory rate and depth of respiration are all had no significant effect.There are abnormal conditions in indivedual rats, may be impure relevant with lumbricin 9203; Isolated ileum segments in guinea pigs is had to certain restraining effect, in clinical application, likely produce the side effect of constipation later, remind clinician to pay close attention to.In general, lumbricin 9203 gets a good eye prospect as thrombolysis new drug.

Two, earthworm enzyme conventional extraction process at present

1, the extraction process that used is used

Conventionally the extracting method of earthworm enzyme is to saltout, then by means such as multiple chromatography and HPLC, FPLC and even affinity chromatography or ion-exchange, molecular sieve, gel electrophoresises, from the earthworm of different lines, obtain the simple component of multiple earthworm enzyme, but troublesome poeration, long flow path, and enzyme activity loss is very large, is not suitable for again suitability for industrialized production.

2, recent new extraction process

New process for extracting one:

Extraction process is described: get a certain amount of earthworm alive, washed several times with water, as much as possible make the dirt in its internal organ discharge, then pull out and inhale its excessive moisture with filter paper as far as possible, weigh, the damping fluid that adds 2 times of volumes homogenate 5 minutes in homogenizer, in 4 ℃ of refrigerators after standing 4 hours, high speed centrifugation, discard precipitation, get supernatant liquor reinforcing body ammonium sulfate and reach certain saturation degree, with 4000r/min, centrifugal 10min, collecting supernatant liquor adds solid ammonium sulfate to reach higher saturation ratio again, hold over night, high speed centrifugation, collecting precipitation, then with damping fluid, dissolve this throw out, with dialysis tubing dialysis desalt or concentrated with hyperfiltration process after, use freeze drier freeze-drying.Being now light brown powder shape material, is the raw product of enzyme.

New ten thousand methods two of extracting:

Extraction process is described: select pure compatriot to like that victory (flat No. two greatly) becomes earthworm, screens out the various impurity of rinsing, and drains sewage.Clean earthworm adds finite concentration salt solution, in the homogenate of tissue homogenizer high speed after 5 minutes, and stand at low temperature salt precipitation, high speed centrifugation after saltouing, collects supernatant liquor.Precipitation is used salts solution washed twice, more centrifugal, collects supernatant liquor, merges supernatant liquor, carries out ultrafiltration for the first time, with the water-fast macromole of filtering.By filtrate ultrafiltration again, concentrated effective ingredient.When concentrated solution reaches after 5%-10% concentration, enter vacuum freeze drier, sublimation temperature not higher than 30 ℃ of conditions under, dry about 5-20 hour dry powder, after pulverizing, aluminum plastic film packs, and is placed in refrigerator and preserves.

Summary of the invention:

The biological composite enzyme of earthworm is the whole ferment of earthworm that utilize biotechnology to extract.

One. major equipment:

Ultrasonic Cleaners, earthworm bioreactor, ultrafiltration apparatus, vacuum freeze drier

Two, technical process

Enzymatic microorganism+auxiliary agent

Earthworm---Ultrasonic Cleaners cleaning---earthworm bioreactor---filtrate---ultrafiltration apparatus---vacuum freeze drier----crushing packing alive

Three, produce and describe:

Get a certain amount of earthworm alive, with Ultrasonic Cleaners cleaning, as much as possible make the dirt in its internal organ discharge, then pull elimination excessive moisture out, weigh.Get 900 kilograms of earthworms and add bio-reactor, then add 50 kilograms of enzymatic microorganism, 50 kilograms of biological assistants, stir, and aerobic fermentation 48 hours, carries out ultrafiltration by filtrate, concentrated effective ingredient.Enter vacuum freeze drier, sublimation temperature not higher than 30 ℃ of conditions under, dry about 4-10 hour dry powder, after pulverizing, aluminum plastic film packs, and is placed in refrigerator and preserves.

Four, product and process characteristic

1, use ferment bacterium biological technology, to accelerate earthworm and dissolve, ferment output significantly promotes.

2, processing unit is simple.

3, do not use the chemical assistants such as ammonium sulfate, salt

4, except cleaning, not water, makes concentrate drying easier.

Claims (1)

1. the biological composite enzyme of earthworm is the whole ferment of earthworm that utilize biotechnology to extract.

Get 900 kilograms of earthworms and add bio-reactor, then add 50 kilograms of enzymatic microorganism, 50 kilograms of biological assistants, stir, aerobic fermentation 48 hours.