CN106119173B - A kind of shrimps low molecular peptide of no bitter taste and the preparation method and application thereof - Google Patents
A kind of shrimps low molecular peptide of no bitter taste and the preparation method and application thereof Download PDFInfo
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Abstract
The present invention discloses a kind of shrimps low molecular peptide and the preparation method and application thereof of no bitter taste, belongs to field of biotechnology.The present invention provides a kind of bacillus subtilis YBPE-4, deposit number: CCTCC NO:M2016359.By shrimps through drying, crushing, neutral protease enzymolysis, isolated enzymolysis liquid.It is configured to fermentation medium using enzymolysis liquid, soluble starch and potassium dihydrogen phosphate, mature bacillus subtilis YBPE-4 culture solution is accessed, is stirred 132~144h of aerobic fementation；Fermentation liquid removes thallus through centrifugation, obtains supernatant, through ultrafiltration membrance filter, obtains the filtrate that molecular weight is 360~5000Da；Filtrate finally obtains the shrimps low molecular peptide product of no bitter taste through drying.The present invention has many advantages, such as that shrimps low molecular peptide bitter taste removal effect is good, efficiency of pcr product is high, and lower production costs are suitable for low value shrimp industrialization production without bitter taste low molecular peptide, and application prospect is good.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of shrimps low molecular peptide of no bitter taste and preparation method thereof with
Using.
Background technique
China is the big country of aquaculture and processing, and only the annual output of shrimps was above 1,000,000 tons in recent years.Wherein, shrimp
Class processing generates a large amount of leftover bits and pieces, and fishing obtains a large amount of unmanageable shrimps, accounts for about 40% or more of shrimps total amount,
It is typically only capable to be used to produce animal feed or production shrimp paste food etc..Shrimps processing fent and low value shrimp are rich in albumen
Matter develops the valuable source of the products such as amino acid, active peptide, flavouring base material as people in the recent period.Low molecular peptide, which has, easily to be inhaled
It receives and the characteristics such as flavor, different flavor depend on peptide chain length, amino acid composition and arrangement etc., is opened in field of food industry
The hot spot of hair and application.But after peptide chain warp enzymolysis, excessive hydrophobic amino acid is exposed to peptide end, easily leading to product is in
Existing bitter taste, serious bitter taste will affect low molecular peptide in the application of field of food.
The method of low molecular peptide bitter taste removal mainly has absorption method, encapsulation method, microencapsulation method, enzymatic isolation method and micro- life at present
Object method etc., wherein enzymatic isolation method and microbial method are the debitterizing methods with great potential, are the hot spots of bitter taste removal research.Enzymatic hydrolysis
Debittering france is mainly directly cut off using hydrophobic amino acid of the aminopeptidase to peptide end, and microorganism debittering france then passes through microorganism point
Aminopeptidase is secreted, and then cuts off the hydrophobic amino acid at peptide end and reaches de- bitter purpose.Aminopeptidase high-yield strains report is few, domestic ammonia
The production of peptide enzyme preparation is still blank, and the flavor protease preparation of only Novozymes Company's production can be applied to low molecular peptide at present
De- hardship.But flavor protease preparation is the mix preparation of endo protease and exoproteinase, it is de- to carry out enzymatic hydrolysis using it
When bitter, low molecular peptide Degree of Enzymatic Hydrolysis is not easy to control, easily causes product molecule too small too low with specific product yield.In addition, ammonia peptide
The cost of enzyme extraction and purification accounts for about 70% or so of enzyme preparation production cost, if directly taken off using aminopeptidase preparation
Hardship, there are also the disadvantages that production cost is excessively high.Therefore, breeding aminopeptidase high-yield strains, directly using microbe fermentation method to low point
Sub- peptide carries out de- hardship, will have the characteristics that efficiency of pcr product is high, production cost is low, is the de- hardship side of the following most application potential
Method.
Summary of the invention
In order to overcome the disadvantages and deficiencies of the prior art, the primary purpose of the present invention is that providing a kind of bacillus subtilis
Bacterium.
Another object of the present invention is to provide a kind of preparation methods of the shrimps low molecular peptide of no bitter taste.
Another object of the present invention is to provide the shrimps low molecular peptides that no bitter taste is prepared in above-mentioned preparation method.
A further object of the present invention is to provide the applications of the shrimps low molecular peptide of above-mentioned no bitter taste.
The purpose of the invention is achieved by the following technical solution:
The present invention provides a kind of bacillus subtilis, and it is from beans that strain, which is named as Bacillus subtilis YBPE-4,
Separation obtains in fermented soya beans, salted or other wise.
The preservation information of described bacillus subtilis (Bacillus subtilis) YBPE-4: depositary institution: Chinese allusion quotation
Type culture collection (CCTCC), preservation date: on June 30th, 2016, preservation address: the Chinese Wuhan Wuhan University is protected
Hiding number: CCTCC NO:M 2016359.
A kind of preparation method of the shrimps low molecular peptide of no bitter taste, comprises the following steps:
(1) shrimps digest: the shrimp through being dried being pulverized and sieved, powder is obtained；Powder is mixed with water, then in addition
Property protease digested, after enzymatic hydrolysis heating enzyme is inactivated, obtain enzymolysis liquid；
(2) preparation of seed liquor: taking enzymolysis liquid made from part steps (1), be added 15~25g/L soluble starch and
1.5~2.5g/L potassium dihydrogen phosphate adjusts pH6.5~7.5, accesses aminopeptidase producing strains strain after sterilization and cooling, then 33~
37 DEG C, 24~28h is cultivated under 160~200rpm, obtain seed liquor；
(3) ferment debitterness of low molecular peptide: taking enzymolysis liquid made from remaining step (1), and 30~40g/L solubility is added and forms sediment
Powder and 1.5~2.0g/L potassium dihydrogen phosphate adjust pH6.5~7.5, are fitted into air agitation fermentor, after sterilization and cooling, access
Seed liquor made from step (2), ferments at 33~37 DEG C, and speed of agitator control is 200~250rpm, according to thallus need
Oxygen content control ventilation ratio is 0.2~1.6vvm (initial volume relative to fermentation liquid), until 132~144h terminates to ferment；
(4) take off it is bitter after low molecular peptide separation and drying: the fermentation liquid after step (3) fermentation is subjected to thallus inactivation, so
Thallus and large particulate matter in centrifugation removal fermentation liquid afterwards, collect supernatant；The ultrafiltration membrane for being 5000Da using molecular cut off
It carries out ultrafiltration and removes macromolecular substances in supernatant, obtain permeate；Recycle the ultrafiltration membrane that molecular cut off is 360Da to saturating
It crosses liquid and is concentrated and is removed small-molecule substance, obtain concentrate；Then, the further vacuum evaporation of liquid will be concentrated by ultrafiltration simultaneously
It is dry, obtain the low molecular peptide of no bitter taste.
In order to which the present invention is better achieved, soluble starch described in step (2) and potassium dihydrogen phosphate are food-grade
Raw material；
Shrimp described in step (1) is preferably shrimp, and shrimp belongs to low value shrimp, just focuses on comprehensive utilization；If it is prawn,
It is eaten after being mainly directly used in edible or simple processing.
Sieving described in step (1) was preferably 80~120 meshes；
The mass ratio (liquid-solid ratio) of water described in step (1) and powder is preferably (15~20): 1；
The dosage of neutral proteinase described in step (1) be every gram of protein (albumen contained by powder) be added 4000~
The neutral proteinase of 5000U；
The condition of enzymatic hydrolysis described in step (1) is preferably 45~55 DEG C of 4~6h of enzymatic hydrolysis；
The condition of inactivation described in step (1) is preferably heated to 80 DEG C~90 DEG C, 10~15min of constant temperature；
The dosage of enzymolysis liquid described in step (2) is 5%~10% of enzymolysis liquid gross mass made from step (1)；
Aminopeptidase producing strains described in step (2) are preferably bacillus subtilis YBPE-4, deposit number CCTCC
NO:M 2016359；
Cell concentration in seed liquor described in step (2) is preferably (1.0~2.5) × 1010A/mL；
The condition of sterilizing described in step (2) and step (3) is preferably 121 DEG C of sterilizing 20min；
Fermentation liquid described in step (3) ferments to 72h, and leucine amino peptidase vigor reaches 3400~4000U/mL；
The condition of inactivation described in step (4) is preferably 121~125 DEG C, keeps the temperature 15~25min, adds to thallus
Heat inactivation；It is centrifuged again after being preferably cooled to 70~75 DEG C after heat inactivation；
The condition of centrifugation described in step (4) is preferably that 5000~6000rpm is centrifuged 15~25min；
The mode that supernatant is collected described in step (4) is preferred are as follows: after fermentation liquid centrifuge separation, preferably with being equivalent to
The deionized water of 20%~30% fermentating liquid volume washs centrifugal sediment, is then centrifuged for separating, washing liquid, collects and merges twice
It is centrifuged resulting supernatant；
Supernatant is filtered using the ultrafiltration membrane that molecular cut off is 5000Da described in step (4), works as concentrate
When volume reaches the 10%~12% of supernatant volume, the deionized water of 2~3 times of volumes is added to concentrate, then carries out second
Secondary ultrafiltration, makes the 10%~12% of final volume of the concentrated liquid fermentating liquid volume, collects and the permeate that merges two times of ultrafiltration；
The volume of concentrate described in step (4) is preferably the 10%~12% of permeate；
The mode of concentration described in step (4) is preferred are as follows: using the ultrafiltration membrane that molecular cut off is 360Da to permeate
It is concentrated, when the volume of the concentrated liquid reaches the 10%~12% of permeate volume, going for 2~3 times of volumes is added to concentrate
Ionized water, then second of ultrafiltration is carried out, make the 10%~12% of final volume of the concentrated liquid permeate volume, collects final
Concentrate；
After ultrafiltration concentration liquid vacuum evaporation described in step (4), 55%~65% moisture is removed；
Drying preferably spray drying described in step (4)；
A kind of shrimps low molecular peptide of no bitter taste, is prepared by above-mentioned preparation method；
The molecular weight of the shrimps low molecular peptide without bitter taste is 360~5000Da, relative to protein in raw material
Yield is 51%~54%；
Application of the shrimps low molecular peptide without bitter taste in food production or food additives production.
The principle of the invention: neutral proteinase is endo type protease, for the hydrolysis of shrimp protein, can be cut off from any position
Peptide chain converts protein to the low molecular peptide of different molecular weight；Bacillus subtilis CCTCC NO:M 2016359 is high yield
The strain of aminopeptidase is grown and is metabolized in the culture medium containing above-mentioned low molecular peptide, aminopeptidase can be largely secreted, from peptide
Chain N-terminal cuts off hydrophobic amino acid, and the bitter taste of low molecular peptide is made to weaken or eliminate；After the de- hardship of microbial fermentation, it is centrifuged, surpasses
Filter membrane separation and concentration, then through drying, finally can get the low molecular peptide without bitter taste.Core of the invention is aminopeptidase Producing Strain
The fermentation technique of kind is eliminated the bitter taste of low molecular peptide, is solved low molecular peptide bitter taste and limit it by the de- bitter effect of microbial fermentation
Using the problem of.
The present invention compared with the existing technology, is had the following advantages and beneficial effects:
(1) present invention carries out ferment debitterness to low molecular peptide using aminopeptidase high-yield strains, with flavor protease debittering france
It compares, not only has many advantages, such as that bitter taste removal effect is good, efficiency of pcr product is high, and enzyme preparation can be saved and isolate and purify cost, have
Conducive to the comprehensive production cost of reduction.
(2) present invention is before de- bitter, without the processing such as enzymatic hydrolysis separating liquid being separated, being dried, directly utilizes dried shrimps powder
Enzymolysis liquid is configured to the culture medium of aminopeptidase high-yield strains, can save the energy consumption cost of intermediate product drying and generation, also favorably
In reduction production cost.
(3) present invention selects low value shrimp for raw material, comprehensive utilization zymolysis technique, microbial fermentation technology and Ultra filtration membrane
Technology etc. produces the low molecular peptide of no bitter taste, and both the comprehensive utilization for low value shrimp provided an effective way, is also other low values
The comprehensive utilization of aquatic products provides valuable reference, may advantageously facilitate the development of culture fishery and processing industry.
(4) present invention is by shrimps through drying, crushing, neutral protease enzymolysis, isolated enzymolysis liquid.Using enzymolysis liquid,
Soluble starch and potassium dihydrogen phosphate are configured to fermentation medium, access mature bacillus subtilis YBPE-4 (CCTCC NO:
M 2016359) culture solution, it is stirred 132~144h of aerobic fementation；Fermentation liquid removes thallus through centrifugation, obtains supernatant, warp
Ultrafiltration membrance filter obtains the filtrate that molecular weight is 360~5000Da；Filtrate finally obtains low point of shrimps of no bitter taste through drying
Sub- peptide product.The present invention has many advantages, such as that shrimps low molecular peptide bitter taste removal effect is good, efficiency of pcr product is high, lower production costs,
It is suitable for low value shrimp industrialization production without bitter taste low molecular peptide, application prospect is good.
Detailed description of the invention
Fig. 1 is the de- bitter flow chart of the microorganism of shrimps polypeptide of the present invention.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Neutral proteinase is purchased from the extensive and profound in meaning star Biotechnology Co., Ltd in Beijing, enzyme activity 6.0 × 104U/g；
The condition for the sterilizing being related in embodiment is 121 DEG C of sterilizing 20min；
The definition of leucine amino peptidase vigor: under conditions of 40 DEG C, pH8.0, it is catalyzed L-Leu -4- nitrobenzene per minute
Enzyme amount needed for amine generates 1 μ g paranitroanilinum is 1 enzyme activity unit；
Embodiment 1
(1) separation and screening of aminopeptidase high-yield strains
Enriched medium: glucose 50g/L, peptone 10g/L, KH2PO42g/L, MgSO4·7H2O0.5g/L, NaCl
5g/L, pH7.0,121 DEG C of sterilizing 20min, it is spare after cooling.
Plate isolation base: glucose 5g/L, casein 15g/L, KH2PO41g/L, NaCl 5g/L, agar 20g/L,
PH7.0,121 DEG C of sterilizing 20min, it is spare after cooling.
Slant medium: glucose 5g/L, peptone 5g/L, yeast extract 5g/L, NaCl 5g/L, agar 20g/L,
PH7.0,121 DEG C of sterilizing 20min, it is spare after cooling.
Fermentation medium: soluble starch 40g/L, peptone 20g/L, KH2PO41.5g/L, MgSO4·7H2O 0.5g/
L, pH7.0,121 DEG C of sterilizing 20min, it is spare after cooling.
Fermented soya bean are commercially available material, by the enriched medium of 10g fermented soya bean access 200mL, are placed in the condition of 30 DEG C, 200rpm
Lower shaken cultivation 16h.Culture solution is subjected to gradient dilution, is coated on plate isolation base, be placed at 30 DEG C culture 36~
48h has 56 plants of bacterial strain of obvious transparent circle around picking colony, accesses slant medium, after turning out lawn, is placed in 4 DEG C of ice
Case preservation.
The slant strains of each bacterial strain are respectively connected to the triangular flask equipped with 200mL fermentation medium, every bottle of 2 ring bacterium of access
Tongue fur is placed in 37 DEG C, shaken cultivation 72h under conditions of 200rpm.Culture solution is centrifuged 20min under the conditions of 6000rpm, takes supernatant
Liquid measures aminopeptidase vigor.Aminopeptidase vigour-testing method reference literature (Wu Qingxun, Gu Haixian, Zhao Guangao .N+Inject mutagenesis
Breeding aminopeptidase superior strain and fermentation condition tentatively optimize [J] food and fermentation industries, 2006,32 (1): 15-18).Through surveying
It is fixed, the producing enzyme highest of bacterial strain YBPE-4 (i.e. bacillus subtilis YBPE-4CCTCC NO:M 2016359), the ammonia of fermentation liquid
Peptidase activity reaches 3882U/mL, therefore fermenting microbe preferably of the invention.
(2) identification of strain
Molecular biology identification is carried out using 16S rDNA, first extraction bacterial strain YBPE-4 (i.e. bacillus subtilis YBPE-
4CCTCC NO:M 2016359) 16S rDNA, through PCR amplification, purifying, send to sequencing company carry out Sequence Detection, then exist
Homology is retrieved in NCBI (http://blast.ncbi.nlm.nih.gov).
Through Sequence Detection, the DNA fragmentation overall length of the 1492R/27F PCR product of the strain is 1439bp, i.e. withered grass gemma
The 16S rDNA sequence of bacillus (Bacillus subtilis) YBPE-4 bacterial strain, specific as follows:
TCCTATACATGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTTAGCGGCGGACGGGTGAGT
AACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACC
GCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGG
TAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCA
GACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGAT
GAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTA
CCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTA
TTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGG
AAACTGGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGG
AACACCAGTGGCGAAAGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTA
GATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAA
CGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCG
GTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGAT
AGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAA
GTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAA
ACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAG
AACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTC
GACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACA
CCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTGTAGGAGCCAGCCGCCGAAGTGAC
ATG。
Through homology search, it is found that the strain and the homology of bacillus subtilis (Bacillus subtilis) reach
99%, therefore the strain is accredited as bacillus subtilis, is named as bacillus subtilis (Bacillus subtilis) YBPE-
4。
(3) preservation of strain
The strain is sent to China typical culture collection center and carries out preservation, above-mentioned qualification result obtains Chinese Typical Representative training
The confirmation of object collection is supported, and obtains deposit number, as bacillus subtilis YBPE-4CCTCC NO:M 2016359.
The preservation information of described bacillus subtilis (Bacillus subtilis) YBPE-4: depositary institution: Chinese allusion quotation
Type culture collection (CCTCC), preservation date: on June 30th, 2016, preservation address: the Chinese Wuhan Wuhan University is protected
Hiding number: CCTCC NO:M 2016359.
Embodiment 2
(1) shrimps digest
Shrimp through being dried is crushed and is sieved with 100 mesh sieve, powder is obtained；Weighing 1200g powder, (protein content is
56.2%) 18L water, is added, mixes；Then 56.2g neutral proteinase is added, 55 DEG C of enzymatic hydrolysis 4h are then heated to 85 DEG C of constant temperature
12min inactivates enzyme, is cooled to room temperature, and obtains enzymolysis liquid；
(2) preparation of seed liquor
900mL enzymolysis liquid is taken, 18g soluble starch and 1.8g potassium dihydrogen phosphate is added, pH7.0 is adjusted, is averagely distributed into 5
A 1000mL triangular flask accesses the inclined-plane bacterium of bacillus subtilis YBPE-4 (CCTCC NO:M 2016359) after sterilization and cooling
Kind, every bottle of 2 ring lawns of access are cultivated for 24 hours under 37 DEG C, 160rpm, and obtaining cell concentration is 1.08 × 1010The seed of a/mL
Liquid；
(3) ferment debitterness of low molecular peptide
Remaining enzymolysis liquid 17.1L is taken, 684g soluble starch and 29.93g potassium dihydrogen phosphate is added, is mixed, is adjusted
PH7.0 is fitted into the air agitation fermentor of 30L, after sterilization and cooling, seed liquor made from access 900mL step (2), starting
Stir and be passed through filtrated air to ferment: during the fermentation, fermentation temperature control is 37 DEG C, and speed of agitator control is
220rpm；Ferment 0~60h, and as biomass increases, ventilatory capacity is incrementally increased by 3.6L/min to 28.8L/min；Fermentation 61~
72h, ventilatory capacity control are 28.8L/min；Ferment 73~108h, and ventilatory capacity control is 21.6L/min；Ferment 109~120h, leads to
Tolerance control is 18L/min；Ferment 121~144h, and ventilatory capacity control is 14.4L/min；Fermentation terminates fermentation to 144h；Its
In, it ferments to 72h, the leucine amino peptidase vigor of fermentation liquid reaches 3426U/mL；
(4) take off it is bitter after low molecular peptide separation and drying
With the fermentation liquid after steam heating stepses (3) fermentation to 123 DEG C, keep the temperature 20min, be cooled to 70 DEG C, 5500rpm from
Heart 20min washs centrifugal sediment with 5.4L deionized water, then carries out second of centrifugation, collects and merges centrifugation gained twice
Supernatant, obtain 18.6L supernatant；Ultrafiltration is carried out to supernatant using the ultrafiltration membrane that molecular cut off is 5000Da, works as hair
When zymotic fluid is concentrated into 2.23L, 6.69L deionized water is added, mixes, carries out second of ultrafiltration, is again concentrated into dope
2.23L, collect with the permeate that merges two times of ultrafiltration, obtain permeate 23L；Then the ultrafiltration membrane for being 360Da with molecular cut off
Ultrafiltration is carried out to permeate, when dope volume reaches 2.76L, 8.28L deionized water is added, mixes, carries out second of ultrafiltration,
Dope is concentrated into 2.76L, the low molecular peptide solution that collection concentrate, as molecular weight are 360~5000Da again；In vacuum
It is evaporated in vacuo under conditions of degree -0.08MPa, temperature 60 C, concentrate is further concentrated into 1.24L, then be sprayed
Dry, control inlet air temperature is 180 DEG C, and leaving air temp is 75 DEG C, finally obtains 365g powder product, as low point without bitter taste
Sub- peptide.It through detection and calculates, low molecular peptide content is 94.8% in powder product, and low molecular peptide is relative to the yield of protein
51.3%.The subjective appreciation of bitter taste is carried out using quinine sulfate solution as standard control object, products obtained therefrom does not have bitter taste.
Embodiment 3
(1) shrimps digest
Shrimp through being dried be crushed into 80 meshes, obtain powder；Weighing 900g powder, (protein content is
56.2%) 18L water, is added, mixes；Then 33.72g neutral proteinase is added, 50 DEG C of enzymatic hydrolysis 5h are then heated to 80 DEG C, perseverance
Warm 15min, inactivates enzyme, is cooled to room temperature, and obtains enzymolysis liquid；
(2) preparation of seed liquor
1800mL enzymolysis liquid is taken, 27g soluble starch and 2.7g potassium dihydrogen phosphate is added, pH6.5 is adjusted, is averagely distributed into
10 1000mL triangular flasks access the inclined-plane of bacillus subtilis YBPE-4 (CCTCC NO:M 2016359) after sterilization and cooling
Strain, every bottle of 2 ring lawns of access, cultivates 28h under 33 DEG C, 200rpm, and obtaining cell concentration is 2.49 × 1010The kind of a/mL
Sub- liquid；
(3) ferment debitterness of low molecular peptide:
Remaining enzymolysis liquid 16.2L is taken, 567g soluble starch and 32.4g potassium dihydrogen phosphate is added, is mixed, is adjusted
PH6.5 is fitted into the air agitation fermentor of 30L, after sterilization and cooling, seed liquor made from access 1800mL step (2), starting
Stir and be passed through filtrated air to ferment: during the fermentation, fermentation temperature control is 33 DEG C, and speed of agitator control is
250rpm；Ferment 0~54h, and as biomass increases, ventilatory capacity is incrementally increased by 3.6L/min to 28.8L/min；Fermentation 55~
64h, ventilatory capacity control are 28.8L/min；Ferment 65~72h, and ventilatory capacity control is 25.2L/min；Ferment 73~84h, ventilation
Amount control is 21.6L/min；Ferment 85~102h, and ventilatory capacity control is 18L/min；Ferment 103~132h, and ventilatory capacity control is
14.4L/min；Fermentation terminates fermentation to 132h；Wherein, to 72h, the leucine amino peptidase vigor of fermentation liquid reaches for fermentation
3992U/mL；
(4) take off it is bitter after low molecular peptide separation and drying
With the fermentation liquid after steam heating stepses (3) fermentation to 121 DEG C, keep the temperature 25min, be cooled to 72 DEG C, 5000rpm from
Heart 25min washs centrifugal sediment with 3.6L deionized water, then carries out second of centrifugation, collects and merges centrifugation gained twice
Supernatant, obtain 18.4L supernatant；Ultrafiltration is carried out to supernatant using the ultrafiltration membrane that molecular cut off is 5000Da, when upper
When clear liquid is concentrated into 1.84L, 3.68L deionized water is added, mixes, carries out second of ultrafiltration, is again concentrated into dope
1.84L, collect with the permeate that merges two times of ultrafiltration, obtain permeate 20.2L；Then the ultrafiltration for being 360Da with molecular cut off
Film carries out ultrafiltration to permeate, and when dope volume reaches 2.02L, 4.04L deionized water is added, mixes, and carries out super for the second time
Dope is concentrated into 2.02L, the low molecular peptide solution that collection concentrate, as molecular weight are 360~5000Da again by filter；?
It is evaporated in vacuo under conditions of vacuum degree -0.08MPa, temperature 60 C, concentrate is further concentrated into 0.707L, then carry out
Spray drying, control inlet air temperature are 180 DEG C, and leaving air temp is 75 DEG C, 285g powder product are finally obtained, as without bitter taste
Low molecular peptide.It through detection and calculates, low molecular peptide content is 95.3% in powder product, and low molecular peptide is obtained relative to protein
Rate is 53.7%.The subjective appreciation of bitter taste is carried out using quinine sulfate solution as standard control object, products obtained therefrom does not have bitter taste.
Embodiment 4
(1) shrimps digest
Shrimp through being dried be crushed into 120 meshes, obtain powder；Weighing 1100g powder, (protein content is
56.2%) 19.25L water, is added, mixes, 46.37g neutral proteinase is added, in 45 DEG C of enzymatic hydrolysis 6h, is then heated to 90 DEG C, perseverance
Warm 10min, inactivates enzyme, is cooled to room temperature, and obtains enzymolysis liquid；
(2) preparation of seed liquor
1540mL enzymolysis liquid is taken, 38.5g soluble starch and 3.85g potassium dihydrogen phosphate is added, adjusts pH7.5, average mark
8 1000mL triangular flasks are packed into, 121 DEG C of sterilizing 20min after cooling, access bacillus subtilis YBPE-4 (CCTCC NO:M
2016359) slant strains, every bottle of 2 ring lawns of access, cultivate 26h under 35 DEG C, 180rpm, and obtaining cell concentration is 1.77
×1010The seed liquor of a/mL；
(3) ferment debitterness of low molecular peptide:
Remaining enzymolysis liquid 17.71L is taken, 531g soluble starch and 26.57g potassium dihydrogen phosphate is added, is mixed, is adjusted
PH7.5 is fitted into the air agitation fermentor of 30L, after sterilization and cooling, seed liquor made from access 1540mL step (2), starting
Filtrated air is stirred and is passed through to ferment.During the fermentation, fermentation temperature control is 35 DEG C, and speed of agitator control is
200rpm；Ferment 0~60h, and as biomass increases, ventilatory capacity is incrementally increased by 3.85L/min to 30.8L/min；Fermentation 61
~72h, ventilatory capacity control are 30.8L/min；Ferment 73~84h, and ventilatory capacity control is 23L/min；Ferment 85~108h, ventilation
Amount control is 19.25L/min；Ferment 109~140h, and ventilatory capacity control is 15.4L/min；Fermentation terminates fermentation to 140h；Its
In, it ferments to 72h, the leucine amino peptidase vigor of fermentation liquid reaches 3764U/mL；
(4) take off it is bitter after low molecular peptide separation and drying
With the fermentation liquid after steam heating stepses (3) fermentation to 125 DEG C, keep the temperature 15min, be cooled to 75 DEG C, 6000rpm from
Heart 15min washs centrifugal sediment with 4.8L deionized water, then carries out second of centrifugation, collects and merges centrifugation gained twice
Supernatant, obtain 20.2L supernatant；Ultrafiltration is carried out to supernatant using the ultrafiltration membrane that molecular cut off is 5000Da, when upper
When clear liquid is concentrated into 2.22L, 5.55L deionized water is added, mixes, carries out second of ultrafiltration, is again concentrated into dope
2.22L, collect with the permeate that merges two times of ultrafiltration, obtain permeate 23.5L；Then the ultrafiltration for being 360Da with molecular cut off
Film carries out ultrafiltration to permeate, and when dope volume reaches 2.59L, 6.48L deionized water is added, mixes, and carries out super for the second time
Dope is concentrated into 2.59L, the low molecular peptide solution that collection concentrate, as molecular weight are 360~5000Da again by filter；?
It is evaporated in vacuo under conditions of vacuum degree -0.08MPa, temperature 60 C, concentrate is further concentrated into 1.04L, then carry out
Spray drying, control inlet air temperature are 180 DEG C, and leaving air temp is 75 DEG C, 348g powder product are finally obtained, as without bitter taste
Low molecular peptide.It through detection and calculates, low molecular peptide content is 94.9% in powder product, and low molecular peptide is obtained relative to protein
Rate is 53.4%.The subjective appreciation of bitter taste is carried out using quinine sulfate solution as standard control object, products obtained therefrom does not have bitter taste.
Comparative example 1
(1) shrimps digest
Shrimp through being dried is crushed and is sieved with 100 mesh sieve, powder is obtained；Weighing 900g powder, (protein content is
56.2%) 18L water, is added, mixes, 33.72g neutral proteinase is added in 50 DEG C of enzymatic hydrolysis 5h and is then heated to 80 DEG C, constant temperature
10min inactivates enzyme, is cooled to room temperature, and obtains enzymolysis liquid；
(2) debittering by enzymatic hydrolysis of flavor protease
In the enzymolysis liquid made from step (1), addition 40.5g flavor protease (Novozymes Company, enzyme activity 1.5 ×
104U/g), in 50 DEG C of enzymatic hydrolysis 2h, 80 DEG C are then heated to, constant temperature 15min inactivates enzyme, is cooled to room temperature spare；
(3) take off it is bitter after low molecular peptide separation and drying
By it is de- it is bitter after enzymolysis liquid be centrifuged 20min in 5000rpm, wash centrifugal sediment with 5.4L deionized water, then into
Row second is centrifuged, and is collected and is centrifuged resulting supernatant twice with merging, obtains 21.5L supernatant；It is using molecular cut off
The ultrafiltration membrane of 5000Da carries out ultrafiltration to supernatant, and when the volume of the concentrated liquid is 2.15L, 6.45L deionized water is added, mixes,
Carry out second of ultrafiltration, dope be concentrated into 2.15L again, collect with the permeate that merges two times of ultrafiltration, obtain permeate
25.8L；Then, ultrafiltration is carried out to permeate with the ultrafiltration membrane that molecular cut off is 360Da, when dope volume reaches 2.58L,
5.16L deionized water is added, mixes, carries out second of ultrafiltration, dope is concentrated into 2.58L again, collects concentrate, as divides
The low molecular peptide solution that son amount is 360~5000Da；Vacuum steaming is carried out under conditions of vacuum degree -0.08MPa, temperature 60 C
Hair, is further concentrated into 0.92L for concentrate, then be spray-dried, and control inlet air temperature is 180 DEG C, leaving air temp 75
DEG C, finally obtain 218g powder product.It through detection and calculates, low molecular peptide content is 94.3% in powder product, low molecular peptide
Yield relative to protein is 40.6%.Subjective appreciation (the reference of bitter taste is carried out using quinine sulfate solution as standard control object
Document: the partial purification and property research [J] food and fermentation industries of Pan Jinquan Mucor AS3.2778 proline aminopeptidase,
2011,37 (4): 26-31), slight bitter is presented in products obtained therefrom.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. a kind of bacillus subtilis, it is characterised in that: entitled bacillus subtilis (Bacillus subtilis) YBPE-
4, it is preserved in the China typical culture collection center of Wuhan University, Wuhan, China city on June 30th, 2016, deposit number:
CCTCC NO:M 2016359。
2. a kind of preparation method of the shrimps low molecular peptide of no bitter taste, characterized by comprising the steps of:
(1) shrimps digest: the shrimp through being dried being pulverized and sieved, powder is obtained；Powder is mixed with water, and neutral egg is then added
White enzyme is digested, and heating inactivates enzyme after enzymatic hydrolysis, obtains enzymolysis liquid；
(2) preparation of seed liquor: taking enzymolysis liquid made from part steps (1), be added 15~25g/L soluble starch and 1.5~
2.5g/L potassium dihydrogen phosphate adjusts pH6.5~7.5, after sterilization and cooling access aminopeptidase producing strains strain, then 33~37 DEG C,
24~28h is cultivated under 160~200rpm, obtains seed liquor；
(3) ferment debitterness of low molecular peptide: taking enzymolysis liquid made from remaining step (1), be added 30~40g/L soluble starch and
1.5~2.0g/L potassium dihydrogen phosphate adjusts pH6.5~7.5, is fitted into air agitation fermentor, after sterilization and cooling, accesses step
(2) seed liquor made from is fermented at 33~37 DEG C, and speed of agitator control is 200~250rpm, according to thallus oxygen demand
Control ventilation ratio is 0.2~1.6vvm, until 132~144h terminates to ferment；
(4) take off it is bitter after low molecular peptide separation and drying: the fermentation liquid after step (3) fermentation is subjected to thallus inactivation, then from
The heart removes thallus and large particulate matter in fermentation liquid, collects supernatant；It is carried out using the ultrafiltration membrane that molecular cut off is 5000Da
Ultrafiltration removes macromolecular substances in supernatant, obtains permeate；Recycle the ultrafiltration membrane that molecular cut off is 360Da to permeate
It is concentrated and is removed small-molecule substance, obtains concentrate；Then, the further vacuum evaporation of liquid will be concentrated by ultrafiltration and do
It is dry, obtain the low molecular peptide of no bitter taste；
Aminopeptidase producing strains described in step (2) are bacillus subtilis described in claim 1.
3. the preparation method of the shrimps low molecular peptide of no bitter taste according to claim 2, it is characterised in that:
Sieving described in step (1) was 80~120 meshes；
The mass ratio of water described in step (1) and powder is (15~20): 1；
The dosage of neutral proteinase described in step (1) is the neutral proteinase that 4000~5000U is added in every gram of protein；
The condition of enzymatic hydrolysis described in step (1) is 45~55 DEG C of 4~6h of enzymatic hydrolysis；
The condition of inactivation described in step (1) is to be heated to 80 DEG C~90 DEG C, 10~15min of constant temperature.
4. the preparation method of the shrimps low molecular peptide of no bitter taste according to claim 2, it is characterised in that:
The dosage of enzymolysis liquid described in step (2) is 5%~10% of enzymolysis liquid gross mass made from step (1)；
Cell concentration in seed liquor described in step (2) is (1.0~2.5) × 1010A/mL.
5. the preparation method of the shrimps low molecular peptide of no bitter taste according to claim 2, it is characterised in that: in step (3)
The fermentation liquid ferments to 72h, and leucine amino peptidase vigor reaches 3400~4000U/mL.
6. the preparation method of the shrimps low molecular peptide of no bitter taste according to claim 2, it is characterised in that:
The condition of inactivation described in step (4) is 121~125 DEG C, keeps the temperature 15~25min, carries out heat inactivation to thallus；Add
It is centrifuged again after being cooled to 70~75 DEG C after heat inactivation；
The condition of centrifugation described in step (4) is that 5000~6000rpm is centrifuged 15~25min.
7. the preparation method of the shrimps low molecular peptide of no bitter taste according to claim 2, it is characterised in that:
The mode of supernatant is collected described in step (4) are as follows: after fermentation liquid centrifuge separation, fermented with being equivalent to 20%~30%
The deionized water of liquid product washs centrifugal sediment, is then centrifuged for separating, washing liquid, collects merging and is centrifuged resulting supernatant twice
Liquid；
Supernatant is filtered using the ultrafiltration membrane that molecular cut off is 5000Da described in step (4), works as the volume of the concentrated liquid
When reaching the 10%~12% of supernatant volume, the deionized water of 2~3 times of volumes is added to concentrate, then carries out second and surpasses
Filter, makes the 10%~12% of final volume of the concentrated liquid fermentating liquid volume, collection and the permeate for merging two times of ultrafiltration.
8. the preparation method of the shrimps low molecular peptide of no bitter taste according to claim 2, it is characterised in that:
The volume of concentrate described in step (4) is the 10%~12% of permeate；
The mode of concentration described in step (4) are as follows: permeate is carried out using the ultrafiltration membrane that molecular cut off is 360Da dense
The deionized water of 2~3 times of volumes is added to concentrate when the volume of the concentrated liquid reaches the 10%~12% of permeate volume for contracting,
Second of ultrafiltration is carried out again, is made the 10%~12% of final volume of the concentrated liquid permeate volume, is collected final concentrate；
After ultrafiltration concentration liquid vacuum evaporation described in step (4), 55%~65% moisture is removed.
9. a kind of shrimps low molecular peptide of no bitter taste, it is characterised in that pass through the described in any item preparation methods of claim 2~8
It is prepared.
10. application of the shrimps low molecular peptide of no bitter taste as claimed in claim 9 in food production or food additives production.
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| CN107400666B (en) * | 2017-09-11 | 2019-11-22 | 广东轻工职业技术学院 | A kind of aminopeptidase and its encoding gene and application |
| CN107811162B (en) * | 2017-12-08 | 2021-03-02 | 玉环市五丰蒸干脱脂鱼粉厂 | Method for preparing easily-preserved whole shrimp pulp by debitterizing and enzymolysis of marine mysid shrimps and application |
| CN110338228B (en) * | 2019-07-26 | 2022-10-14 | 广东轻工职业技术学院 | Soybean milk powder without abdominal distension and preparation method thereof |
| CN110447880A (en) * | 2019-09-17 | 2019-11-15 | 天津春发生物科技集团有限公司 | A kind of preparation method of delicate flavour fermented flavoring material |
| CN110923283A (en) * | 2020-01-14 | 2020-03-27 | 肽晟堂生物科技(常州)有限公司 | Method for preparing functional polypeptide by fermenting eel bones with microorganisms |
| CN112852909B (en) * | 2021-01-25 | 2023-05-09 | 韩山师范学院 | Method for preparing ACE (angiotensin converting enzyme) inhibitory peptide by solid-state fermentation of shrimp heads by mixed strains |
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