CN105861565B - A kind of turbot fish-skin tunning with antioxidant activity - Google Patents
A kind of turbot fish-skin tunning with antioxidant activity Download PDFInfo
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Abstract
The present invention provides a kind of turbot fish-skin tunning with antioxidant activity, is made of aspergillus oryzae fermentation turbot fish-skin homogenate.The advantages of present invention can effectively degrade fish-skin albumen, have simple process, and cost consumption is low, be easy industrialization improves its added value using turbot fish-skin and provides new way to be deep.Meanwhile the low molecular weight fish-skin fermentation liquid that degree of hydrolysis is 30~50% is obtained, product amino acid rich in, wherein glycine, glutamic acid, alanine, proline content are higher, have high trophism, are the replenishers of good amino acid and protein.In vitro in Oxidation Resistance Test, tunning shows high DPPH clearance rate and Fe2+Chelating ability, this illustrates that the substance has the function of chelated microelement, can be used as novel micro element amino acid nutrition addictive and is added in food and health care product.
Description
Technical field
The invention belongs to Natural Antioxidants preparation technical fields, and in particular to a kind of big water chestnut with antioxidant activity
Flounder skin tunning.
Background technique
The cradle that ocean is bred as life, be richly stored with the multiple resources such as biology, chemistry, mineral, the energy, wherein
Protein resource is the important composition ingredient of living marine resources, since ocean is deposited used in many extreme environments, so its biological egg
Bai Ziyuan is far longer than land in type and quantity.In recent years, the demand with people to aquatic products increased, sea fishery
It is grown rapidly, consequently also produces many processing byproducts, such as fish-skin, fish head, fish-bone, internal organ etc., these by-products
The random discarding of object not only results in the wasting of resources and brings great environmental problem.With the development of science and technology more next
It is more research shows that these by-products are good marine protein sources, can be used as the extraction raw material of active peptide and enzyme preparation.
Turbot belongs to Osteichthyes Pleuronectiformes Bothidae, is commonly called as brill, be worldwide flounder flounder master support kind it
One, it is distributed widely in Asia, Europe and South America, from after the China of introducing in 1992, industrial aquaculture is concentrated mainly on Circum-Bohai Sea
Three provinces, one city is littoral, is more than 60000 tons of the output value with annual output, becomes the main culture fishery in China.Turbot is usually used in
The factorial production of steck and fillet can generate a large amount of fish-skin by-product, albumen rich in fish-skin in process
Matter, therefore a kind of conversion technology for being translated into active polypeptide is found, it can not only increase turbot added value and mention
High economic benefit, while environmental pressure can also be mitigated.
Summary of the invention
The object of the present invention is to provide a kind of to prepare the preparation method with anti-oxidation active substance from turbot fish-skin
And application, to make up the deficiency of present technology.
Present invention firstly provides a kind of turbot fish-skin tunning with antioxidant activity, is to use aspergillus oryzae
(Aspergillus oryzae) ferments made of turbot fish-skin homogenate；
The turbot fish-skin homogenate is to carry out boiling after turbot fish skin raw material to be added to water, after being cooled to room temperature
It carries out homogenate and fish-skin fermenation raw liquid is made；
Turbot fish-skin tunning of the invention, specific preparation method comprise the following steps that
1) boiling is carried out after turbot fish skin raw material being added water, homogenate is carried out after being cooled to room temperature, fish-skin proferment is made
Liquid；
2) it uses according to the fish-skin fermenation raw liquid of step 1) method preparation as culture medium, the strain that activation is added is trained
It supports, obtains seed culture medium；
3) seed culture medium prepared in step 2) is inoculated into the fish-skin fermenation raw liquid according to the preparation of step 1) method,
Inoculum concentration is 10~30%, and 5~8d is cultivated under strain optimum temperature；
4) filtering fermentation liquor for obtaining step 3), centrifugation obtain fermentation polypeptide liquid, by what is obtained after the freeze-drying of gained liquid
Yellow powder substance is the anti-oxidant tunning of turbot fish-skin；
The mass ratio of fish skin raw material and water is preferably 1:3 in the step 1)；
Actication of culture condition in the step 2) is: 30 DEG C, 180rmp, incubation time 2d；
The optimal inoculum concentration of the step 3) is 10% and optimal fermentation time is 7d.
It is by above-mentioned turbot the present invention also provides a kind of better turbot fish-skin tunning of antioxidant effect
It after the freeze-drying concentration of skin fermentation liquid, is redissolved with ultrapure water, configuration concentration is the solution of 1g/mL, crosses 0.45 μm of inorganic moisture film, upper G-
15 gel chromatographic columns are separated, applied sample amount 2mL, and using distilled water as eluent, flow velocity 1mL/min, Detection wavelength is
220nm collects the eluent that elution time is 17~23.5min and 24.75~34.3min；
The present invention can effectively degrade fish-skin albumen, have simple process, and cost consumption is low, be easy the excellent of industrialization
Point improves its added value for deep utilization turbot fish-skin and provides new way.Meanwhile obtain degree of hydrolysis be 30~50% it is low
Molecular weight fish-skin fermentation liquid, product amino acid rich in, wherein glycine, glutamic acid, alanine, proline content compared with
Height has high trophism, is the replenishers of good amino acid and protein.In vitro in Oxidation Resistance Test, tunning
Show high DPPH clearance rate and Fe2+Chelating ability, this illustrates that the substance can be used as novel micro with chelated microelement
Element amino acid nutrition addictive is added in food and health care product, to prevent disease (iron deficiency caused by microelement deficiencies
Property anaemia, rickets etc.).In addition, obtained product not only has good color (faint yellow) by the processing of this technology,
Raw material fishy smell can also be removed, fish-skin flavor is effectively improved, improves the consumer acceptability of product, more preferably is applied to eat for it
Product and health products trade are laid a good foundation.
Detailed description of the invention
Fig. 1: turbot leather is for antioxidant process flow chart；
Fig. 2: turbot fish-skin fermentation front and back molecular weight distribution liquid phase figure；
Fig. 3: turbot fish-skin fermentation front and back antioxidation activity in vitro variation diagram；
Fig. 4: G-15 purifying figure after the fermentation of turbot fish-skin；
Fig. 5: turbot fish-skin fermentation front and back flavor variations radar map.
Specific embodiment
Antioxidant of the invention mainly carries out biodegrade to turbot fish-skin using microorganism, by by fish-skin
Middle inactive protein is degraded to small peptide or amino acid hydrolyticsolution obtains corresponding antioxidant activity.The present invention has selected common food
Grade strain obtains anti-oxidation active substance by direct fermentation turbot fish-skin, mainly includes the preparation of fish-skin culture medium, strain training
It supports, the separation of actication of culture, inoculation fermentation, product.As shown in Figure 1, steps are as follows for preparation method of the invention:
One, fish-skin preparation of culture medium:
The condition 1. fish-skin is precooked: solid-liquid ratio is placed in boiling water bath for 1:3~3:2 fish-skin liquid and boils 30min；
After 2. fish-skin liquid of precooking is cooled to room temperature, it is transferred in refiner and is crushed, broken condition are as follows: 4000rmp, 40s；
3. fish-skin homogenate is dispensed into triangular flask, high pressure sterilization, condition are as follows: 121 DEG C, 20min.
Two, actication of culture:
4. fermenting microbe cultivates 2d under respective optimal culture conditions, actication of culture is carried out；
Three, prepared by anti-oxidation active substance:
5. fermentation: 10%~30% seed liquor being inoculated into 25%~60% fish-skin fermentation liquid, ferment 5~8d, fermentation
30 DEG C~37 DEG C of temperature, revolution 180rmp；
7. antioxidant separates；By fermentation liquid through 4 layers of filtered through gauze, filtrate centrifugation, centrifugal condition are as follows: revolution
4 DEG C of 8000rmp, temperature, centrifugation time 15min.It is centrifuged acquired solution, gained pale yellow powder is institute after being freeze-dried 2d
Obtain big squama flounder skin antioxidant.
The present invention is described in detail below with reference to embodiment.
Embodiment 1: the screening of turbot fish-skin fermenting microbe
Lactic acid bacteria is respectively as turbot fish-skin fermenting microbe, actication of culture condition；37 DEG C, anaerobism stationary culture, training
Supporting the time is 2d.Activated strain is accessed in turbot fish-skin culture medium, inoculum concentration 10%, solid-liquid ratio 40%, hair
The ferment time is 5d.Fermentation liquid obtains tunning freeze-drying through 4 layers of filtered through gauze, filtrate centrifugation, and measurement result shows: the product water
Xie Du is that 5.19%, DPPH clearance rate is 10.83%.
Embodiment 2: the screening of turbot fish-skin fermenting microbe
Saccharomycete is respectively as turbot fish-skin fermenting microbe, actication of culture condition；30 DEG C, 180rmp, incubation time
For 2d.Activated strain is accessed in turbot fish-skin culture medium, inoculum concentration 10%, solid-liquid ratio 40%, fermentation time
For 5d.Fermentation liquid obtains tunning freeze-drying through 4 layers of filtered through gauze, filtrate centrifugation, and measurement result shows: degree of hydrolysis 3.44%,
DPPH clearance rate is 38.89%.
Embodiment 3: the screening of turbot fish-skin fermenting microbe
Aspergillus oryzae is respectively as turbot fish-skin fermenting microbe, actication of culture condition；30 DEG C, 180rmp, incubation time
For 2d.Activated strain is accessed in turbot fish-skin culture medium, inoculum concentration 10%, solid-liquid ratio 40%, fermentation time
For 5d.Fermentation liquid obtains tunning freeze-drying through 4 layers of filtered through gauze, filtrate centrifugation, and measurement result shows: degree of hydrolysis is
37.43%, DPPH clearance rate are 58.35%.
It can be seen that under identical fermentation condition from embodiment 1-3, aspergillus oryzae has best degradation energy to turbot
The product of power, acquisition has high degree of hydrolysis and DPPH Scavenging activity.
The preparation of 4 turbot fish-skin antioxidant of embodiment
Fish-skin is shredded, weighs 25g, is added 75mL distilled water, after the 30min that precooks, is homogenized high pressure sterilization, access has activated
The aspergillus oryzae of 2d ferments, inoculum concentration 10%, and fermentation temperature is 30 DEG C, fermentation time 6d, and the fermentation liquid of acquisition is lyophilized,
Measuring DPPH clearance rate is 67.5%.
The preparation of 5 turbot fish-skin antioxidant of embodiment
Fish-skin is shredded, weighs 40g, is added 60mL distilled water, after the 30min that precooks, is homogenized high pressure sterilization, access has activated
The aspergillus oryzae of 2d ferments, inoculum concentration 10%, and fermentation temperature is 30 DEG C, fermentation time 7d, and the fermentation liquid of acquisition is lyophilized,
Measuring DPPH clearance rate is 69.18%.
The preparation of 6 turbot fish-skin antioxidant of embodiment
Fish-skin is shredded, weighs 60g, is added 40mL distilled water, after the 30min that precooks, is homogenized high pressure sterilization, access has activated
The aspergillus oryzae of 2d ferments, inoculum concentration 10%, and fermentation temperature is 30 DEG C, fermentation time 8d, and the fermentation liquid of acquisition is lyophilized,
Measuring DPPH clearance rate is 50.12%.
The preparation of 7 turbot fish-skin antioxidant of embodiment
Fish-skin is shredded, weighs 40g, is added 60mL distilled water, after the 30min that precooks, is homogenized high pressure sterilization, access has activated
The aspergillus oryzae of 2d ferments, inoculum concentration 20%, and fermentation temperature is 30 DEG C, fermentation time 6d, and the fermentation liquid of acquisition is lyophilized,
Measuring DPPH clearance rate is 68.67%.
The preparation of 8 turbot fish-skin antioxidant of embodiment
Fish-skin is shredded, weighs 60g, is added 40mL distilled water, after the 30min that precooks, is homogenized high pressure sterilization, access has activated
The aspergillus oryzae of 2d ferments, inoculum concentration 20%, and fermentation temperature is 30 DEG C, fermentation time 7d, and the fermentation liquid of acquisition is lyophilized,
Measuring DPPH clearance rate is 41.38%.
The preparation of 9 turbot fish-skin antioxidant of embodiment
Fish-skin is shredded, weighs 25g, is added 75mL distilled water, after the 30min that precooks, is homogenized high pressure sterilization, access has activated
The aspergillus oryzae of 2d ferments, inoculum concentration 20%, and fermentation temperature is 30 DEG C, fermentation time 8d, and the fermentation liquid of acquisition is lyophilized,
Measuring DPPH clearance rate is 64.78%.
The preparation of 10 turbot fish-skin antioxidant of embodiment
Fish-skin is shredded, weighs 60g, is added 40mL distilled water, after the 30min that precooks, is homogenized high pressure sterilization, access has activated
The aspergillus oryzae of 2d ferments, inoculum concentration 30%, and fermentation temperature is 30 DEG C, fermentation time 6d, and the fermentation liquid of acquisition is lyophilized,
Measuring DPPH clearance rate is 47.5%.
The preparation of 11 turbot fish-skin antioxidant of embodiment
Fish-skin is shredded, weighs 25g, is added 75mL distilled water, after the 30min that precooks, is homogenized high pressure sterilization, access has activated
The aspergillus oryzae of 2d ferments, inoculum concentration 30%, and fermentation temperature is 30 DEG C, fermentation time 7d, and the fermentation liquid of acquisition is lyophilized,
Measuring DPPH clearance rate is 51.1%.
The preparation of 12 turbot fish-skin antioxidant of embodiment
Fish-skin is shredded, weighs 40g, is added 60mL distilled water, after the 30min that precooks, is homogenized high pressure sterilization, access has activated
The aspergillus oryzae of 2d ferments, inoculum concentration 30%, and fermentation temperature is 30 DEG C, fermentation time 8d, and the fermentation liquid of acquisition is lyophilized,
Measuring DPPH clearance rate is 63.5%.
The preparation of 13 turbot fish-skin antioxidant of embodiment
Fish-skin is shredded, weighs 25g, is added 75mL distilled water, after the 30min that precooks, is homogenized high pressure sterilization, access has activated
The aspergillus oryzae of 2d ferments, inoculum concentration 10%, and fermentation temperature is 30 DEG C, fermentation time 7d, and the fermentation liquid of acquisition is lyophilized,
Measuring DPPH clearance rate is 77.1%.
The basic index of 14 turbot antioxidant of embodiment is analyzed
The fermentation liquid as obtained by embodiment 4-13 all has certain antioxidant activity, wherein product in embodiment 13
Antioxidant activity it is best, clearance rate is analyzed up to 77.1%, therefore to the basic index of the embodiment product, main to wrap
It includes:
1) degree of hydrolysis measures: formol titration and Kjeldahl nitrogen determination free ammonical nitrogen content and total nitrogen is respectively adopted
Content.Degree of hydrolysis measurement result shows that turbot fish-skin degree of hydrolysis after inventive technique degradation can reach 50.45%, this explanation
The process can efficiently use the albumen in fish-skin, to improve its added value.
2) it molecular weight distribution: is measured using efficient liquid phase exclusion chromatography.After sample is diluted 10 times, through 0.22 μm of filter
Filtering, takes 10 μ L sample liquids to be analyzed (table 1).
1 turbot fish-skin stoste of table and antioxidant molecular weight distribution
From this table it can be seen that large protein of the turbot fish-skin mainly by molecular weight greater than 10kDa forms, molecular weight is small
Polypeptide (< 1kDa) only account for 8.37%, after the method for the present invention is handled, molecular weight is greatly reduced, finally, small-molecular peptides content
Up to 84.68% (Fig. 2).
3) hydrolysis amino acid measures: a certain amount of 6mol/L hydrochloric acid hydrolyzate is added in sample, after being filled with nitrogen 30s, hydrolyzes 22h
After take out, be cooled to room temperature.Hydrolyzation sample is concentrated on 50 DEG C of nitrogen evaporator, 1mL0.02mol/L hydrochloric acid liquid is added,
It is filtered through 0.22 μm of filter, filtrate is prepare liquid.20 μ L of prepare liquid is taken to be analyzed with amino-acid analyzer.Amino acid analysis
The results show that turbot fish-skin is good protein resource, amino acid rich in has high nutritive value, wherein
Glycine, alanine, glutamic acid, hydroxyproline, arginine content are higher.The anti-oxidation active substance that the present invention obtains contains more
Amino acids, wherein essential amino acid improves 30.86%, and total amino acid improves 11.76%, in addition, before and after fermentation and anti-
Oxidation activity amino acid content also increases, and has been increased to 45.15% from fermentation preceding 41.18%.This illustrates system of the invention
The antioxidant activity of fish-skin not only can be improved in Preparation Method, while can convert soluble protein for insoluble protein in fish-skin, from
And improve fish-skin albumen availability and trophic function (table 2).
Table 2: turbot fish-skin stoste and the analysis of antioxidant hydrolysis amino acid
4) free amino acid forms: sample pre-treatments: taking 2mL sample, 8mL dehydrated alcohol is added, mixes well, in 4 DEG C
Refrigerator stands 30min, and 6000 × g is centrifuged 5min, takes supernatant 2mL, and 40 DEG C of nitrogen evaporator dryings add 2mL sample diluting liquid, sufficiently
After dissolution, filtered through 0.22 μm of filter, sample detection.The result shows that free ammonia base unit weight acid is practically free of in fish-skin stoste, through sending out
Free aminoacid content dramatically increases after ferment technique processing.Free amino acid before fermentation only has 40mg/g, free ammonia after fermentation
Base acid content is increased to 2190mg/g；Essential amino acids content is 440mg/g, wherein methionine, proline, threonine content compared with
It is high；Anti-oxidant amino acid (account for total free amino acid amount 44.29%) rich in, especially glutamic acid, proline simultaneously
And methionine；Free amino acid is an important factor for constituting flavouring peculiar flavour, and delicious amino acid is also richer in this product
Richness, Fresh ear field and sweet taste amino acid account for the 13.7% and 41.6% of total free amino acid content respectively.Above data is all said
The bright technique can effectively degrade fish-skin, and the product of acquisition is good amino acid nutrient replenishers, while the product also has
Good flavor characteristics (table 3).
3 turbot fish-skin stoste of table and antioxidant free amino acid analysis
The analysis of the big squama flounder antioxidant antioxidant activity of embodiment 15
The diversity of antioxidant active mechanism, it is desirable that evaluation substance antioxidant activity liquid needs various considerations, therefore
Select DPPH, Fe2+Chelating ability and reducing power carry out the evaluation of antioxidant activity to the product that the embodiment of the present invention 13 obtains.
1) DPPH clearance rate: it is 0.06mmol/L DPPH ethanol solution that 3mL concentration is added in the sample of various concentration, is mixed
After even, it is protected from light 30min, light absorption value at 517nm is measured, replaces sample to do blank control with distilled water, positive control is
BHA.
2)Fe2+Chelating ability: after dilution obtains the sample of various concentration, it is separately added into 2mmol/L FeCl2And 0.1mL
5mmol/L phenanthrene alloxazine after reacting 10min, measures light absorption value at 562nm, and distilled water replaces sample to do blank control, positive control
For BHA.
3) 2.5mL PBS (0.2mol/L, pH 6.6) and 1% iron of 2.5mL reducing power: are added in the sample of various concentration
Potassium cyanide solution will be added 10% trifluoroacetic acid of 2.5mL, be stored at room temperature 10min, then take after 50 DEG C of water-bath 20min of mixture
2.5mL distilled water and 0.5mL 0.1%FeCl is added in 2.5mL reaction solution3, 10min is reacted, light absorption value at 700nm, distillation are measured
Water replaces sample to do blank control, and positive control is citric acid.
Measurement result shows that tunning antioxidant activity is significantly larger than fish-skin stoste, and wherein DPPH clearance rate is by 1.2%
It is increased to 67%, chelating ability is increased to 67.62 by 0.32%, and reducing power changes minimum light absorption value and is increased to 0.69 (figure by 0.05
2)。
4 antioxidant activity of table divides result
16 fermentation liquid G-15 gel chromatography initial gross separation of embodiment
After the turbot fermentation liquid freeze-drying concentration that embodiment 13 is obtained, redissolved with ultrapure water, configuration concentration is 1g/mL's
Solution crosses 0.45 μm of inorganic moisture film, and upper G-15 gel chromatographic columns are separated, applied sample amount 2mL, is elution with distilled water
Liquid, flow velocity 1mL/min, Detection wavelength 220nm collect different component and measure sequestering activity.
As shown in figure 4, be divided into four eluting peaks by sample after G-15 gel chromatography column, elution time is respectively 8.5~
12min (peak P1), 12.5~16.5min (peak P2), 17~23.5min (peak P3), 24.75~34.3min (peak P4), to four
The sequestering activity at peak is measured, the results showed that the higher Fe of P3 and P4 activity2+Chelating vigor, respectively 65.57% He
70.12%, and P1, P2 have low sequestering activity, respectively 2.25% and 33.38%.It is measured using efficient liquid phase gel column
P3, P4 molecular weight, distribution are concentrated mainly on 400D or less.
The analysis of the big squama flounder antioxidant flavor of embodiment 17
Smell analysis is carried out to antioxidant and fish-skin liquid using electronic nose.Electronic nose is mainly respectively represented not by ten
The metal oxide sensor of commaterial sensibility forms, and primary operational process is as follows: 1mL sample to be tested being taken to be packed into Head-space sampling
In bottle, then electronic nose sample introduction needle is inserted into Head-space sampling bottle to start to measure and is determined, determination condition is sensor self-cleaning time
200s, sensor are zeroed 10s, sampling time 60s, and each sample is repeated 3 times.It is well known that distinctive in fish and its product
Fishy smell is always the main reason for reducing the consumer acceptability of aquatic products, and this flavor is mainly derived from aquaculture mistake
The processes such as microorganism, enzyme and fat oxidation in journey during accumulation in vivo and saving and processing, greatly limit aquatic products depth
Therefore the development of secondary industry seeks the minimizing technology of bad flavor in aquatic products, become and carry out research hotspot this year.From radar
As can be seen that significant changes have occurred in fish-skin liquid flavor in figure peak area, wherein 1,2,3,5,6,8 six sensor response is aobvious
Writing reduces, this explanation effectively improves fish-skin liquid flavor, so that every smell is more evenly distributed, flavor after this technology converts
It is softer, improve consumer acceptance (Fig. 5).It is added in flavouring like sauce as flavor substance, in addition to energy
Outside the organoleptic attribute for enough increasing product, healthcare function can be also brought into, increase added value of product.
5 electronic nose sensor performance result table of table
Claims (7)
1. a kind of turbot fish-skin tunning, which is characterized in that the tunning the preparation method is as follows:
1) boiling is carried out after turbot fish skin raw material being added water, homogenate is carried out after being cooled to room temperature, fish-skin fermenation raw liquid is made；
2) it uses according to the fish-skin fermenation raw liquid of step 1) method preparation as culture medium, the aspergillus oryzae of activation is added
(Aspergillus oryzae) strain is cultivated, and seed culture medium is obtained；
3) seed culture medium prepared in step 2) is inoculated into the fish-skin fermenation raw liquid according to the preparation of step 1) method and is carried out
Fermentation；
4) filtering fermentation liquor for obtaining step 3), centrifugation obtain fermentation polypeptide liquid, the yellow that will be obtained after the freeze-drying of gained liquid
Powdered substance；
5) yellow powder substance is redissolved with ultrapure water, is separated after filtering with G-15 gel chromatographic columns, be with distilled water
Eluent, flow velocity 1mL/min, Detection wavelength 220nm, collection elution time be 17~23.5min and 24.75~
34.3min eluent.
2. tunning as described in claim 1, which is characterized in that the mass ratio of fish skin raw material and water in the step 1)
For 1:3.
3. tunning as described in claim 1, which is characterized in that the actication of culture condition in the step 2) is: 30
DEG C, 180rmp, incubation time 2d.
4. tunning as described in claim 1, which is characterized in that fermentation inoculum concentration is 10% in the step 3).
5. tunning as described in claim 1, which is characterized in that fermentation temperature is 30 DEG C in the step 3), fermentation
Time 7d.
6. tunning as described in claim 1, which is characterized in that the filtering was 0.45 μm of inorganic moisture film.
7. turbot fish-skin tunning described in claim 1 is preparing the application in antioxidant.
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