A kind of be raw material production pharmaceutical grade protein peptide powder with salmon fish method
Technical field:
The invention belongs to technical field of bioengineering, be specifically related to a kind of with salmon fish for raw material production medical grade protein peptide powder
Method.
Technical background:
Protein peptide powder is not only fermentation method pharmaceutical industry and is used the important nitrogen nutrition source of culture medium, is also food and health product interpolation
And the important source material of aminoacids complex.Conventional medical grade protein peptide powder mainly uses peptone, with Carnis Bovis seu Bubali cream, and the molten starch of fish
Etc. for main, but at spawn culture in this, as nitrogen nutrition source and produce the index of the aspects such as bacterium rate and all can not meet requirement, and egg
White peptone impurity is many, containing pollutant and heavy metal, affects the medical safe of product.Therefore select pollution-free and content of beary metal is low
Raw material become improve peptone quality principal element.During selecting, owing to marine fishes move among deep-sea,
Unmanned is ecological environmental pollution, and content of beary metal is low, and fish body protein content is high, and sight is gradually locked in ocean by research worker
Fish.Through comparison, it has been found that the fish skin protein content that salmon fish records is more than 80%, and salmon fish fish protein content is high
Reach 17.2%, every 100 grams of flesh of fish ability energy reach 139 kilocalories, the nutritive index of the various Fish of Integrated comparative, preserve difficulty or ease, be subject to
The degree polluted, the harm etc. to human body, we show that deep-sea cold water salmon fish is substantially better than other fingerling such as tuna, cod
Fish, fish, Trichiurus haumela, mackerel, squid, be the preferred raw materials well producing pharmaceutical grade protein peptide powder.
It is related to the report utilizing Fish to carry out extracting fish protein as raw material at present.Such as application number:
201310096815X, a kind of method utilizing compound protease to prepare active collagen, its method used is through pre-
The fish skin mixed liquor processed adds compound protease and carries out enzymolysis, and compound protease selects papain, bromelain, pancreas
In protease, neutral protease, animal proteolytic enzyme, acid protease and pepsin two kinds, the enzymolysis solution obtained is entered
Row is centrifugal, ultrafiltration, concentrate, saltout and obtain.Such as application number: 2012102539328, the preparation method of a kind of fish brain microcapsule, its
After the method used adds trypsin digestion in the equal serosity of fish brain being ultrasonically treated, enzyme denaturing, add pepsin
Enzyme enzymolysis, it is thus achieved that enzymolysis solution, adds other nutritional labeling through spraying, being dried and obtain in enzymolysis solution.Application number:
2015101175690, a kind of tuna fish bone collagen protein sources zinc chelating collagen peptide and its production and use, its method is
Through processing addition pepsin enzymolysis in the tuna bone collagen feed liquid prepared, then adjust pH value, add pancreas
Protease hydrolyzed, obtains enzymatic hydrolysate, by enzymatic hydrolysate ultrafiltration, macroporous resin chromatographic column removing impurities, ethanol elution, lyophilization and
?.Application number: 2004100356188, the preparation method of fish protein biology peptide fertilizer, its method is at fresh fish, fresh fish skin or fresh fish
The leftover bits and pieces of bone adds digestive enzyme trypsin or pepsin is degraded, then filter, concentration, sterilizing.
Although there being the above-mentioned extraction process about fish protein, but use above-mentioned enzymolysis process that salmon fish is extracted, enzyme
Solving result and show that its degree of hydrolysis can only achieve between 25-50%, hydrolysis degree is relatively low, and little peptide molecular weight is more than 10000, egg
White peptide presents bitterness, and for abatement bitterness, the most conventional technique is to use membrane filtration, is separated by the little peptide of less than 10000,
Thereby increase separating difficulty, the difficulty cleaning film and pollution.Meanwhile, the fat content in salmon fish is up to 7.8%, colloid contains
Amount is more than 5%, and enzymolysis solution is not readily separated and is layered.Using a conventional step to be centrifuged the method combined with membrance separation cannot be by enzyme
The fish oil, colloid, fat and the impurity that solve in liquid are completely separated, and also bring the difficulty of cleaning to membrance separation.During additionally enzymolysis obtains thing
The content of free amino acid is too low, less than 20%.Above-mentioned enzymolysis obtains the index of thing and does not reaches far away wanting of pharmaceutical grade protein peptide powder
Ask, it is therefore necessary to the enzymolysis process for salmon fish is further improved.
Summary of the invention
For solving utilizing salmon fish to carry out the technical problem in the presence of protein peptide powder extracts purification process as raw material,
The present invention provides a kind of method being raw material production pharmaceutical grade protein peptide powder with salmon fish.
For realizing above-mentioned technical purpose, the technical solution used in the present invention is: a kind of with salmon fish for raw material production medicine
The method of level protein peptide powder, it is characterised in that the method contains the following step:
S1, raw material prepare
Select remaining by-product after the fresh or salmon fish of freezing or its processing, as salmon fish minced meat, fish fillet and fish skin are made
For raw material, it is desirable to the total volatile basic nitrogen of the raw materials such as salmon fish and processing residue by-product thereof, less than 40, is broken for 0.5-1.5cm
Fragment, raw material is put in enzymatic hydrolysis reaction still, add raw material weight 3-4 times soft water;
S2, first step enzymolysis
Select papain, pepsin and trypsin as restriction endonuclease, wherein papain, pepsin and pancreas egg
The ratio of white enzyme is: 1:2:1, and three kinds of enzymes add the 0.2-0.6% that total amount is substrate weight;Hydrolysis temperature 60-65 DEG C, PH5-
8.5, enzymolysis time 3-6 hour, temperature is promoted to 85-95 DEG C, enzyme denaturing 30-40 minute, obtain enzymolysis solution;
S3, the first step separate
It is sent to enzymolysis solution in solid-liquid centrifugation machine separate, centrifuge speed 2500-3500 rev/min, centrifugal difference 0.85-
0.95, the fish pomace after enzymolysis is separated with enzymolysis solution, obtains the initial filter enzymolysis solution containing fish oil, colloid and fat;
S4, second step enzymolysis
Select aminopeptidase or carboxypeptidase as the 0.1-that addition is initial filter enzymolysis solution weight of excision enzyme, aminopeptidase or carboxypeptidase
0.5%, hydrolysis temperature 45-55 DEG C, enzymolysis time is 2-5 hour, and being heated up by enzymolysis solution is heated to 85-90 DEG C, within 25-30 minute, enters
Row enzyme denaturing processes;
S5, second step separate
The enzymolysis solution obtained by S4 step is sent into high velocity liquid liquid centrifuge, and centrifuge speed 8000-10000 rev/min, by enzyme
The fish oil & fat solved in liquid is separated, and obtains suspended enzymolysis solution;
S6, enzymolysis solution stand
Adding Alumen in suspended enzymolysis solution, addition is the 0.02-0.05% of substrate weight, stands 3-6 hour, obtains sedimentation
Enzymolysis solution;
S7, the 3rd step separate
By the enzymolysis solution of sedimentation, send into flat centrifugal, flocculate and bulky grain levitated object are separated, obtains clarification
Enzymolysis solution, flat board is centrifuged the mesh number 600-1000 mesh selecting filter bag, and centrifuge revolution is that 800-1200 turns;
S8, protein peptide powder extract
By clarification enzymolysis solution through ion exchange resin column desalination and heavy metal, then the molecular weight through microfiltration controlled enzymatic hydrolysis liquid
After, dehydration, it is dried, obtains protein peptide powder.
Further, enzymolysis or standing in step S2, S4 and S6 are to carry out in sealed states, while enzymolysis,
Nitrogen it is filled with or by vacuum state in enzymatic hydrolysis reaction still, it is ensured that zymolyte and air exclusion in enzymatic hydrolysis reaction still.
The present invention is by the enzyme used by selected enzymolysis, and by rationally determining the addition of enzyme, controlled enzymatic hydrolysis condition makes institute
The quality of protein peptide powder extracted is greatly improved, and the degree of hydrolysis in hydrolytic process can reach 90%, albumen earning rate up to
More than 70%, the protein peptide powder protein content obtained reaches more than 97%, 18 kinds of amino acid contents more than 85%.Simultaneously by two step enzymes
The employing of solution method, uses restriction endonuclease enzymolysis for the first time separately to carry out with excision enzyme second time enzymolysis, enzymolysis in enzymolysis process
Free amino acid in liquid and little peptide content are more than 50%, and greatly reduce bitter peptides.Three extracted by above two method
Literary composition fish protein Gly-His-Lys indices can fully meet the requirement of pharmaceutical grade protein peptide powder.
Detailed description of the invention
Below in conjunction with embodiment, method provided by the present invention is described further.
Embodiment 1
A kind of be raw material production pharmaceutical grade protein peptide powder with salmon fish method, it is characterised in that the method contains the following step:
S1, raw material prepare
High-quality salmon fish is selected to freeze minced meat, fish fillet and 1500 kilograms of fish skin as raw material, it is desirable to the volatile salts of salmon fish raw material
Base nitrogen is less than 40, by raw material pulverizing to 0.5~1.5cm fragment, puts into 10m3Hydrolysis kettle in, supplement fresh soft water to raw material weight
4 times of amount.During above-mentioned raw materials prepares, raw material can also select fresh salmon fish to oppress, fishbone, soft water in hydrolysis kettle
Addition control as between 3-4 times of solid, concrete numerical value is controlled flexibly according to the water content of raw material;
S2, first step enzyme hydrolysis
Interpolation restriction endonuclease in reactor, the preferred papain of restriction endonuclease, pepsin and the compound enzyme of trypsin composition,
Papain, pepsin and tryptic interpolation weight ratio 1:2:1, three kinds of enzyme overall controls are enzymolysis substrate weight
0.5%, utilize steam that the water in hydrolysis kettle and solid goods and materials are heated to 60 DEG C, adjust pH value to 7.5, hydrolysis time 5 hours,
Enzymolysis solution;
In this step, it is desirable to enzymolysis process, zymolyte completely cuts off with outside air, and specific practice is to be pumped into very by hydrolytic reaction pot
Sky, or in hydrolysis kettle, it is filled with nitrogen, to avoid growing of antibacterial.
Additionally in enzymolysis process, the enzymatic hydrolysis condition that enzymatic hydrolysis condition is not provided by the present embodiment is limited, according in raw material
The flesh of fish, fish skin, internal organs, the respective content Reasonable adjustment enzymatic hydrolysis condition of fishbone, general enzymatic hydrolysis condition is: the interpolation total amount of three kinds of enzymes
Control between the 0.3-0.6% for substrate weight, hydrolysis temperature control 60-65 DEG C, pH value 5-8.5, hydrolysis time 3-6 hour.
In this step, the main purpose utilizing restriction endonuclease is to be cut off by protein peptide chain, is amino by proteolysis
Acid polypeptide;
S3, first step enzymolysis solution separate
In the present embodiment, the solid-liquid separation of enzymolysis solution is first carried out.The method is first to use high-speed horizontal screw settling centrifuge
Separate residue heavy phase, obtain residue and feed liquid.Centrifuge revolution is that 2500-3000 turns, and centrifugal difference selects 0.85-0.95, at this
The solid phase of 100% can be separated by separation, obtain containing suspension (secondary heavy phase), colloid and fat (secondary light phase), salmon fish oil
The initial filter enzymolysis solution of (light liquid phase).
S4, second step enzyme hydrolysis
Owing to papain, pepsin and trypsin belong to restriction endonuclease, it is only capable of cutting off the peptide chain of protein, obtains ammonia
Base acid polypeptide and less than 20% free amino acid and little peptide, reach total amino acids content to obtain free aminoacid content
40-60%, little peptide content reaches the aminoacid enzymolysis solution of 80%, it is necessary to carry out the secondary enzyme as enzyme preparation with aminopeptidase or carboxypeptidase
Solve.
In the implementation case, selecting aminopeptidase or carboxypeptidase is excision enzyme, and addition is the 0.3% of concentration of substrate, enzymolysis temperature
Degree is 50 DEG C, and enzymolysis time is 4.5 hours.Additionally in enzymolysis process, the enzyme that enzymatic hydrolysis condition is not provided by the implementation case
Solution condition limits, and enzymatic hydrolysis condition can the different of actual feed liquid be selected: the 0.1-0.5% being added to substrate weight of enzyme preparation, enzyme
Solving temperature 45-55 DEG C, enzymolysis time is 2-5 hour.Then, enzymolysis solution is heated up and is heated to 85-90 DEG C, within 25-30 minute, carry out
Enzyme denaturing processes.
Select aminopeptidase or carboxypeptidase to belong to expeptidase as excision enzyme, aminopeptidase or carboxypeptidase, aminoacid can be made
One by one dissociate out from the N-terminal order of polypeptide chain, thus obtain free aminoacid content and reach the enzymolysis solution of 40-60%, keep away
Exempt from the bitterness of protein peptide powder.
In the implementation case, due to diminishing of amino acid molecular amount, amino acid whose ionization degree changes, oil-in-water and oil
The phenomenon of Bao Shui is eliminated, and feed liquid is easily separated into fish oil, colloid and fat (light phase) and aminoacids solution and float (weight
Phase), thus carry out adequate preparation for obtaining the enzymolysis solution of clarification.
S5, the separation of second step enzyme hydrolyzate
Mixed enzymolysis liquid through second step enzymolysis is centrifuged through high speed dish type, and centrifuge revolution is that 8000-10000 turns, by fish
Oil, colloid and fat (light phase) and aminoacids solution and float (heavy phase) separate.In present case, enzymolysis process is same for the second time
Sample is that hydrolysis kettle is evacuated or is filled with nitrogen, to avoid growing of antibacterial in enzymolysis solution.
S6, the 3rd step enzymolysis solution stand
After step S5 separating step, in feed liquid, some suspend, and the protein of not thoroughly hydrolysis and amino acid polypeptide are still
So staying in centrifugal liquid, testing result shows, suspension content is the 2-5% of feed liquid weight, it is therefore desirable to this float carried out
Get rid of.
The present embodiment employed technical scheme comprise that the Alumen adding 0.02% in feed liquid, stands 4 hours, utilizes float
Potential difference, precipitated, can be by whole float precipitations or be adsorbed as oarse-grained solid.In the process, use
In absorption and the Alumen of precipitation purposes, using different additions according to the content difference of float in feed liquid, addition is outstanding
The 0.02-0.05% of float content, stands 3-6 hour.
In present case, standing process is that hydrolysis kettle is evacuated or is filled with nitrogen equally, to avoid antibacterial in enzymolysis solution
Grow.
S7, the 4th step enzymolysis solution separate
In present case, passing through a flat centrifuge through the feed liquid stood, filter bag mesh number selects 800 mesh, centrifuge revolution
Select 1000 turns, obtain the aminoacid enzymolysis feed liquid of clarification.In the process, flat centrifugal technological parameter need to be according to precipitation
With the difference of bulky grain thing, selecting the filter bag of 600-1000 mesh, centrifuge revolution selects 800-3000 to turn.
S8, by enzymolysis clear liquor through ion exchange resin stake desalination and heavy metal, then after ultrafiltration, thickening,
It is dried, obtains protein peptide powder.
In present case, thickening is all carried out with being dried under big flow negative-pressure vacuum, and vacuum reaches-0.08, albumen
Fishy smell in powder reduces by 80%.
The present embodiment selected three steps separate and stood the method combined, the purifying technique of enzymolysis solution has been done one
The improvement of step.The method is pure physical method, be different from conventional one step separate, use activated carbon decolorizing to enzymolysis clear liquid, it is to avoid
The secondary pollution of enzymolysis solution.
By the component content such as following table of the production method gained protein peptide powder that the present embodiment is provided:
Salmon fish protein peptide powder quality index
Purity (conversion is albumen %) |
97.5 |
Ignition residue (%) |
2.5 |
Nitrogen content (in terms of nitrogen) (%) |
15.6 |
Moisture (%) |
2.5 |
18 kinds of amino acid contents (%) |
85 |
Fat (%) |
0.1 |
Amino-acid nitrogen (%) |
7.5 |
95% egg albumen powder molecular weight (dalton) |
< 3000 |
Sanitary index |
Reach medicine national standard requirement |
Heavy metal arsenic (%) |
0.3mg/1000g |
By parameter listed by upper table, this protein peptide powder fully meets the index request of pharmaceutical grade protein peptide powder, can be as medical
Level peptone, health care grade fish protein powder and the raw material of Aminoacids complex powder.