CN110463933A - A kind of enzyme-linked preparation method for closing preparation squid nutrients biological product of bacterium - Google Patents
A kind of enzyme-linked preparation method for closing preparation squid nutrients biological product of bacterium Download PDFInfo
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Abstract
The present invention provides a kind of bacterium the enzyme-linked preparation method for closing preparation squid nutrients biological product, belongs to sleeve-fish product technical field, includes the steps that in squid homogenate while neutral proteinase being added and bacillus subtilis bacterium solution is cultivated；Bacillus subtilis bacterium solution used is cultivated 8-12h under conditions of 35-40 DEG C, 100-150r/min and is made, wherein contain 2- aminopyrimidine and guanidine hydrochloride in LB meat soup by the way that bacillus subtilis to be inoculated in LB meat soup.Preparation method of the present invention passes through the bacterium enzyme synergy of bacillus subtilis and neutral proteinase, soluble protein high conversion rate, and squid nutrients biological product obtained contains higher Soluble Protein Contents and unsaturated fatty acid, has good sense organ flavor；Squid nutrients biological product obtained preferably can also inhibit harmful bacteria to grow, and extend the freshness date of squid nutrients biological product.
Description
Technical field
The invention belongs to sleeve-fish product technical fields, and in particular to a kind of enzyme-linked close of bacterium prepares squid nutrients biological product
Preparation method.
Background technique
Squid is called Chinese squid, is a kind of ocean mollusks.Squid can be divided into three big portions on the whole
Point: squid meat, head and foot, squid skin and internal organ, this three parts accounts for 50%, 25%, the 25% of its weight respectively, wherein edible
Part account for the 75% of its weight, be higher by general 20% than other most of fish, it can be seen that, the whole utilization of squid
Rate or relatively high.The protein content of squid is higher, and the amino acid classes for probably having 20%, and containing are relatively abundanter,
In many essential amino acids that needed by human body is wanted all are encompassed within；And the crude fat content of squid is relatively low, wherein phosphatide is its master
Constituent is wanted, the 40%-50% of lipid total amount is probably accounted for.The working process of squid has many by-products and generates, such as
The leftover bits and pieces such as calamary head, eye, epidermis, cartilage and internal organ probably account for the 20%-25% of squid total weight.The processing of these squids
By-product is full of nutrition, not only contains a large amount of protein, lipid, also rich in taurine, vitamin, chondroitin etc., however people
But ignore its higher value application.But the moisture content of squid is bigger, in addition its intracorporal enzyme is very active, causes
Leftover bits and pieces is unable to long term storage, and with the extension of storage time, leftover bits and pieces is also with dense bad smell.For under squid
The processing of heel causes difficulty of processing bigger due to lacking mature processing technology, in turn results in high cost, low profit
Situation, thus many people see that this situation is just hung back.
Biotechnology is the hot spot studied at present, and microbial fermentation is that one kind is novel, in actual production
Arrived application, and be proved to safe and reliable technology, microbial fermentation technology application also achieve good economic benefit with
Applied value, thus it is with wide development space.Probiotics is a kind of feasible microorganism dietary supplements, by its
Effect in enteron aisle valuably influences host.In human research it has been reported that it is more relevant to the intake of probiotics with it is strong
The relevant effect of health, including mitigate lactose intolerance and Immune-enhancing effect, promote food digestion, it is bad to destroy to produce useful product
Microorganism, supplement the digestive ferment missed function (due to miss or the gene of defect), and maintain the pH value of digestive system, etc.
Deng.
Summary of the invention
It is an object of the present invention to provide a kind of enzyme-linked conjunctions of the bacterium of soluble protein conversion ratio to prepare squid nutrients biological
The preparation method of product, squid nutrients biological product obtained contain higher Soluble Protein Contents and unsaturated fatty acid, tool
There is good sense organ flavor；Squid nutrients biological product obtained preferably can also inhibit harmful bacteria to grow, and extend squid battalion
The freshness date of health Tetramune.
The technical solution that the present invention is taken to achieve the above object are as follows:
A kind of enzyme-linked preparation method for closing preparation squid nutrients biological product of bacterium, including neutrality will be added simultaneously in squid homogenate
The step of protease and bacillus subtilis bacterium solution are cultivated.
Preparation method of the present invention passes through the bacterium enzyme synergy of bacillus subtilis and neutral proteinase, can make soluble protein
Conversion ratio reaches 24.38%, obtains rich in nutriments such as amino acid and unsaturated fatty acids.Preparation method of the present invention can press down
Harmful bacteria growth processed, extends the freshness date of squid nutrients biological product, and together can be squid nutrients biological with neutral proteinase
Product assigns good sense organ flavor.The method that preparation method of the present invention uses bioconversion, environmentally protective, the probiotics energy of addition
It is enough to complement each other with nutriment contained by squid itself, while being opened up for conversion technology and marine product by-product utilized
Road.
Preferably, the solid-to-liquid ratio of squid homogenate and distilled water is 1:3-11 in squid homogenate.
Preferably, the additive amount of neutral proteinase is 500-6000u/g.
Preferably, the additive amount of bacillus subtilis bacterium solution is 10-35%.
Preferably, bacillus subtilis bacterium solution is by the way that bacillus subtilis to be inoculated in LB meat soup, in 35-40 DEG C, 100-
8-12h is cultivated under conditions of 150r/min to be made.Bacillus subtilis bacterium solution should select the thallus in the logarithmic growth later period, because
The stage bacterial metabolism is vigorous, and enzymatic productivity is most strong.
It is furthermore preferred that containing 2- aminopyrimidine and guanidine hydrochloride in LB meat soup.Bacillus subtilis producing enzyme system rich in
System, can secrete a large amount of extracellular protease, such as carbohydrase, protease, lipase and amylase etc., wherein most
Extracellular proteinase is secreted by Sec approach；2- aminopyrimidine and guanidine hydrochloride presence in LB meat soup can influence base when DNA replication dna
Normal pairing, cause gene that transversion, conversion, missing and frameshift mutation etc. occurs, it is higher withered to obtain protease production
Careless bacillus liquid, the bacillus subtilis bacterium solution can secrete more albumen during culture in squid homogenate
Enzyme can further increase soluble protein conversion ratio, be higher than 32%.In addition, the bacillus subtilis bacterium solution energy that the culture medium obtains
More protein-based Substances are enough released, the inhibiting effect of harmful bacteria growth are enhanced, so as to prolong
The freshness date of long made biological products.
It still more preferably, is ‰ 2- aminopyrimidine of 0.01-0.1 and 0.03- containing mass concentration in LB meat soup
0.08 ‰ guanidine hydrochlorides.The protease production for the bacillus subtilis that LB meat soup under this condition obtains improves 3 times or more.
Preferably, cultivation temperature is 20-60 DEG C, revolving speed 100-150r/min, time 12-60h.
Compared with prior art, the invention has the benefit that preparation method of the present invention passes through bacillus subtilis in
Property protease bacterium enzyme synergy, soluble protein conversion ratio can be made to reach 24.38%, obtained rich in amino acid and unsaturated lipid
The nutriments such as fat acid.Preparation method of the present invention preferably can inhibit harmful bacteria to grow, and extend the freshness date of biological products, and
With neutral proteinase good sense organ flavor can be assigned for squid nutrients biological product together.Preparation method of the present invention is turned using biology
The method of change, environmentally protective, the probiotics of addition can complement each other with nutriment contained by squid itself, while turn for biology
Change technology and marine product by-product utilized blaze the trail.
Present invention employs above-mentioned technical proposals to provide a kind of bacterium the enzyme-linked preparation side for closing preparation squid nutrients biological product
Method compensates for the deficiencies in the prior art, reasonable design, easy operation.
Detailed description of the invention
Fig. 1 is the measurement result of producing bacillus subtilis enzymatic activity in test example 1 of the present invention；
Fig. 2 is the measurement result of soluble protein conversion ratio in test example 1 of the present invention；
Fig. 3 is the assay result of unsaturated fatty acid in flavor squid nutrients biological product in test example 1 of the present invention.
Specific embodiment
It is next by the following examples that the present invention is furture elucidated.However, it should be understood that the embodiment is merely illustrative
Purpose, be not intended to limit scope and spirit of the present invention.
Embodiment 1:
A kind of enzyme-linked preparation method for closing preparation squid nutrients biological product of bacterium, includes the following steps:
S1: being crushed homogenate after Squid By-products are cleaned, is placed in high-pressure steam sterilizing pan 120 DEG C, after the 30min that sterilizes, be placed in-
20 DEG C of freezer storages are spare；
S2: bacillus subtilis is inoculated in LB meat soup conical flask, the shaking table culture 10h under conditions of 37 DEG C, 120r/min
Bacillus subtilis bacterium solution is obtained, for use；
S3: taking squid homogenate 2g in 50ml centrifuge tube, 10g distilled water is added according to solid-to-liquid ratio 1:5, liquid-transfering gun draws withered grass bud
Neutral proteinase is added in centrifuge tube, and according to 2000u/g in spore bacillus liquid 10% (200ul), in 40 DEG C, pH 7.0,120r/
48h is cultivated under conditions of min, and then culture solution is freeze-dried to get biological products.
Embodiment 2:
A kind of enzyme-linked preparation method for closing preparation squid nutrients biological product of bacterium, includes the following steps:
S1: being crushed homogenate after Squid By-products are cleaned, is placed in high-pressure steam sterilizing pan 120 DEG C, after the 30min that sterilizes, be placed in-
20 DEG C of freezer storages are spare；
S2: bacillus subtilis is inoculated in LB meat soup conical flask, the shaking table culture 10h under conditions of 37 DEG C, 120r/min
Bacillus subtilis bacterium solution is obtained, for use；
S3: taking squid homogenate 2g in 50ml centrifuge tube, 18g distilled water is added according to solid-to-liquid ratio 1:9, liquid-transfering gun draws withered grass bud
Neutral proteinase is added in centrifuge tube, and according to 500u/g in spore bacillus liquid 30% (600ul), in 40 DEG C, pH 7.0,120r/
48h is cultivated under conditions of min, and then culture solution is freeze-dried to get biological products.
Embodiment 3:
A kind of enzyme-linked preparation method for closing preparation squid nutrients biological product of bacterium, includes the following steps:
S1: being crushed homogenate after Squid By-products are cleaned, is placed in high-pressure steam sterilizing pan 120 DEG C, after the 30min that sterilizes, be placed in-
20 DEG C of freezer storages are spare；
S2: bacillus subtilis is inoculated in LB meat soup conical flask, phonetic for 0.1 ‰ 2- amino containing mass concentration in LB meat soup
Pyridine and 0.08 ‰ guanidine hydrochlorides, shaking table culture 10h obtains bacillus subtilis bacterium solution under conditions of 37 DEG C, 120r/min, for use；
S3: taking squid homogenate 2g in 50ml centrifuge tube, 18g distilled water is added according to solid-to-liquid ratio 1:9, liquid-transfering gun draws withered grass bud
Neutral proteinase is added in centrifuge tube, and according to 500u/g in spore bacillus liquid 30% (600ul), in 40 DEG C, pH 7.0,120r/
48h is cultivated under conditions of min, and then culture solution is freeze-dried to get biological products.
Embodiment 4:
A kind of enzyme-linked preparation method for closing preparation squid nutrients biological product of bacterium, includes the following steps:
S1: being crushed homogenate after Squid By-products are cleaned, is placed in high-pressure steam sterilizing pan 120 DEG C, after the 30min that sterilizes, be placed in-
20 DEG C of freezer storages are spare；
S2: bacillus subtilis is inoculated in LB meat soup conical flask, and it is 0.01 ‰ 2- amino that mass concentration is contained in LB meat soup
Pyrimidine and 0.03 ‰ guanidine hydrochlorides, shaking table culture 10h obtains bacillus subtilis bacterium solution under conditions of 37 DEG C, 120r/min, for use；
S3: taking squid homogenate 2g in 50ml centrifuge tube, 18g distilled water is added according to solid-to-liquid ratio 1:9, liquid-transfering gun draws withered grass bud
Neutral proteinase is added in centrifuge tube, and according to 500u/g in spore bacillus liquid 30% (600ul), in 40 DEG C, pH 7.0,120r/
48h is cultivated under conditions of min, and then culture solution is freeze-dried to get biological products.
Embodiment 5:
A kind of enzyme-linked preparation method for closing preparation squid nutrients biological product of bacterium, includes the following steps:
S1: being crushed homogenate after Squid By-products are cleaned, is placed in high-pressure steam sterilizing pan 120 DEG C, after the 30min that sterilizes, be placed in-
20 DEG C of freezer storages are spare；
S2: bacillus subtilis is inoculated in LB meat soup conical flask, and it is 0.03 ‰ 2- amino that mass concentration is contained in LB meat soup
Pyrimidine and 0.05 ‰ guanidine hydrochlorides, shaking table culture 10h obtains bacillus subtilis bacterium solution under conditions of 37 DEG C, 120r/min, for use；
S3: taking squid homogenate 2g in 50ml centrifuge tube, 18g distilled water is added according to solid-to-liquid ratio 1:9, liquid-transfering gun draws withered grass bud
Neutral proteinase is added in centrifuge tube, and according to 500u/g in spore bacillus liquid 30% (600ul), in 40 DEG C, pH 7.0,120r/
48h is cultivated under conditions of min, and then culture solution is freeze-dried to get biological products.
Comparative example 1:
A kind of enzyme-linked preparation method for closing preparation squid nutrients biological product of bacterium, includes the following steps:
S1: being crushed homogenate after Squid By-products are cleaned, is placed in high-pressure steam sterilizing pan 120 DEG C, after the 30min that sterilizes, be placed in-
20 DEG C of freezer storages are spare；
S2: bacillus subtilis is inoculated in LB meat soup conical flask, the shaking table culture 10h under conditions of 37 DEG C, 120r/min
Bacillus subtilis bacterium solution is obtained, for use；
S3: taking squid homogenate 2g in 50ml centrifuge tube, 18g distilled water is added according to solid-to-liquid ratio 1:9, liquid-transfering gun draws withered grass bud
Spore bacillus liquid 30% (600ul) cultivates 48h, 90 DEG C of inactivations in centrifuge tube under conditions of 40 DEG C, pH 7.0,120r/min
Neutral proteinase is added according to 500u/g after 30min, 48h is digested at 40 DEG C, then culture solution is freeze-dried to get biology
Product.
Comparative example 2:
A kind of enzyme-linked preparation method for closing preparation squid nutrients biological product of bacterium, includes the following steps:
S1: being crushed homogenate after Squid By-products are cleaned, is placed in high-pressure steam sterilizing pan 120 DEG C, after the 30min that sterilizes, be placed in-
20 DEG C of freezer storages are spare；
S2: bacillus subtilis is inoculated in LB meat soup conical flask, the shaking table culture 10h under conditions of 37 DEG C, 120r/min
Bacillus subtilis bacterium solution is obtained, for use；
S3: taking squid homogenate 2g in 50ml centrifuge tube, 18g distilled water be added according to solid-to-liquid ratio 1:9, is added according to 500u/g
Property protease, digests 48h, then 90 DEG C of inactivation 30min at 40 DEG C, adds bacillus subtilis bacterium solution 30% (600ul), in
40 DEG C, pH 7.0, cultivate 48h under conditions of 120r/min, then culture solution is freeze-dried to get biological products.
Comparative example 3:
A kind of enzyme-linked preparation method for closing preparation squid nutrients biological product of bacterium, includes the following steps:
S1: being crushed homogenate after Squid By-products are cleaned, is placed in high-pressure steam sterilizing pan 120 DEG C, after the 30min that sterilizes, be placed in-
20 DEG C of freezer storages are spare；
S2: bacillus subtilis is inoculated in LB meat soup conical flask, the shaking table culture 10h under conditions of 37 DEG C, 120r/min
Bacillus subtilis bacterium solution is obtained, for use；
S3: taking squid homogenate 2g in 50ml centrifuge tube, 18g distilled water is added according to solid-to-liquid ratio 1:9, liquid-transfering gun draws withered grass bud
Spore bacillus liquid 30% (600ul) cultivates 48h under conditions of 40 DEG C, pH 7.0,120r/min in centrifuge tube, then will training
Nutrient solution is freeze-dried to get biological products.
Comparative example 4:
A kind of enzyme-linked preparation method for closing preparation squid nutrients biological product of bacterium, includes the following steps:
S1: being crushed homogenate after Squid By-products are cleaned, is placed in high-pressure steam sterilizing pan 120 DEG C, after the 30min that sterilizes, be placed in-
20 DEG C of freezer storages are spare；
S2: bacillus subtilis is inoculated in LB meat soup conical flask, the shaking table culture 10h under conditions of 37 DEG C, 120r/min
Bacillus subtilis bacterium solution is obtained, for use；
S3: it takes squid homogenate 2g in 50ml centrifuge tube, 18g distilled water is added according to solid-to-liquid ratio 1:9, then adds according to 500u/g
Enter neutral proteinase, 48h is digested at 40 DEG C, then enzymolysis liquid is freeze-dried to get biological products.
Comparative example 5:
A kind of enzyme-linked preparation method for closing preparation squid nutrients biological product of bacterium, includes the following steps:
S1: being crushed homogenate after Squid By-products are cleaned, is placed in high-pressure steam sterilizing pan 120 DEG C, after the 30min that sterilizes, be placed in-
20 DEG C of freezer storages are spare；
S2: bacillus subtilis is inoculated in LB meat soup conical flask, and it is 0.03 ‰ 2- amino that mass concentration is contained in LB meat soup
Pyrimidine, shaking table culture 10h obtains bacillus subtilis bacterium solution under conditions of 37 DEG C, 120r/min, for use；
S3: taking squid homogenate 2g in 50ml centrifuge tube, 18g distilled water is added according to solid-to-liquid ratio 1:9, liquid-transfering gun draws withered grass bud
Neutral proteinase is added in centrifuge tube, and according to 500u/g in spore bacillus liquid 30% (600ul), in 40 DEG C, pH 7.0,120r/
48h is cultivated under conditions of min, and then culture solution is freeze-dried to get biological products.
Comparative example 6:
A kind of enzyme-linked preparation method for closing preparation squid nutrients biological product of bacterium, includes the following steps:
S1: being crushed homogenate after Squid By-products are cleaned, is placed in high-pressure steam sterilizing pan 120 DEG C, after the 30min that sterilizes, be placed in-
20 DEG C of freezer storages are spare；
S2: bacillus subtilis is inoculated in LB meat soup conical flask, and it is 0.05 ‰ guanidine hydrochlorides that mass concentration is contained in LB meat soup,
Shaking table culture 10h obtains bacillus subtilis bacterium solution under conditions of 37 DEG C, 120r/min, for use；
S3: taking squid homogenate 2g in 50ml centrifuge tube, 18g distilled water is added according to solid-to-liquid ratio 1:9, liquid-transfering gun draws withered grass bud
Neutral proteinase is added in centrifuge tube, and according to 500u/g in spore bacillus liquid 30% (600ul), in 40 DEG C, pH 7.0,120r/
48h is cultivated under conditions of min, and then culture solution is freeze-dried to get biological products.
Test example 1:
1. the measurement of producing bacillus subtilis enzymatic activity
The measurement of 1.1 producing bacillus subtilis proteinase activities
1.1.1 enzyme activity defines: for 1mL liquid enzymes under the conditions of 40 DEG C, pH=7.5, it is one that 1min hydrolyzed casein, which generates 1 μ g tyrosine,
A enzyme activity unit, is indicated with U/mL.
1.1.2 the drafting of standard curve: take the tyrosine that 1.00mL concentration is 0,10,20,30,40,50 μ g/mL molten respectively
Liquid is in test tube, number 0,1,2,3,4,5, and the sodium carbonate liquor that 5.00mL concentration is 0.4mol/L is added, and after mixing, is added
After 1.00mL Folin reagent mixes, after being placed in 40 DEG C of water-baths the 20min that develops the color, take out in right amount with spectrophotometer with blank tube 0
Pipe is zeroing pipe, measures absorbance A of the 1-5 pipe at wavelength 680nm.Using the concentration of tyrosine as abscissa, each solution is inhaled
Shading value is ordinate, draws tyrosine standard curve and calculates regression equation simultaneously.Calculate the tyrosine when OD value is 1
It measures (μ g), as extinction constant K, K value should be within the scope of 95-100.This test need to do parallel test.
1.1.3 it the measurement of sample: with 5% inoculum concentration by bacillus subtilis bacterium solution, is cultivated in 37 DEG C, 120r/min
After 48h, 5mL culture solution is taken, is centrifuged 5min in 12000rpm/min, supernatant is crude enzyme liquid.Take appropriate diluted enzyme solution 1.0mL
2min is preheated in 40 DEG C of water-baths.4 test tubes are taken, respectively number 0,1,2,3.It is suitably dilute that 1mL is separately added into four test tubes
Crude enzyme liquid after releasing, the casein solution 1.0mL being then added after preheating at 1,2, No. 3 shake up, while being added 2mL's in No. 0 pipe
Trichloroacetic acid solution.After keeping the temperature l0min at 40 ± 0.2 DEG C, add 0.4mol/L trichloroacetic acid 2.0mL respectively toward 1,2, No. 3 pipe
Reaction is terminated, the casein solution of 1mL is added toward No. 0 pipe.After standing l0min, filtered with filter paper.1.0mL filtrate is taken, is added
0.4mol/L NaCO3Solution 5.0mL adds Folin reagent 1.0mL, and the 20min that develops the color at 40 ± 0.2 DEG C.In 680nm wavelength,
10mm cuvette measures absorbance OD.
1.1.4 the active unit of enzyme is calculated according to following formula:
Vigor=A × K × 4/10 × nU/mL of protease；
In formula, the mean OD value of A- sample parallel test；
K- extinction constant；
The total volume of 4- reaction reagent；
The 10- enzyme digestion reaction time；
N- enzyme solution dilutes general times.
The measurement of 1.2 producing bacillus subtilis lipase actives
With 5% inoculum concentration by bacillus subtilis bacterium solution, after 37 DEG C, 120r/min culture 48h, 500 μ L culture solutions, In are taken
12000r/min is centrifuged 5min, takes supernatant, dilutes 1000 times with the Tris-HCl buffer of 50mmoL/L, pH 8.5, takes 500
Reaction system is added in μ L dilution, and reaction control systems then use the dilution of 500 μ L Tris-HCl buffers substitution fermentation liquid.
Reaction carries out 10min in 37 DEG C of shaking baths, and the ice-cold dehydrated alcohol of 5mL is added and terminates reaction.Use control reaction solution as
Reference measures the absorbance A of reaction solution at 406nm.The lipase active of every mL fermentation liquid is calculated according to following equation:
The definition (U) of unit of lipase activity: under the conditions of 37 DEG C, pH 8.5, hydrolysis 4- nitrobenzophenone caprylate is produced in 1min
Enzyme amount required for the p-nitrophenol of raw 1 μm of oL.
The measurement result of producing bacillus subtilis enzymatic activity such as Fig. 1, embodiment 2 obtain the production protease activity of bacillus subtilis
Property be 14.78U/mL, yielding lipase activity be 8.32U/mL；Embodiment 3-5 obtains the protease production point of bacillus subtilis
Not Wei 45.26U/mL, 46.12U/mL, 48.01U/mL, yielding lipase activity be respectively 27.58U/mL, 26.89U/mL,
28.33U/mL；The protease production that comparative example 4-5 obtains bacillus subtilis is respectively 13.53U/mL, 15.24U/mL, produces rouge
Fat enzymatic activity is respectively 9.22U/mL, 9.05U/mL.These results suggest that the producing enzyme that embodiment 3-5 obtains bacillus subtilis is living
Property be better than embodiment 2, comparative example 4-5, this shows 2- aminopyrimidine in LB meat soup and guanidine hydrochloride in the presence of when can influence DNA replication dna
The normal pairing of base causes gene that transversion, conversion, missing and frameshift mutation etc. occurs, and it is higher withered to obtain inulinase-producing activity
Careless bacillus liquid.
2. the measurement of soluble protein conversion ratio
Soluble protein conversion ratio is calculated according to the following formula:
Soluble protein conversion ratio (%)=(M-N)/P × 100%；
Wherein,
M is soluble protein content (being measured using Forint phenol method) in biological products；
N is soluble protein content (being measured using Forint phenol method) in Squid By-products；
P is Squid By-products total protein content (being measured using Kjeldahl's method).
As a result as shown in fig. 2, it can be seen that soluble protein conversion ratio is high respectively in 2 preparation method of embodiment 1 and embodiment
Up to 24.04% and 24.38%, in comparative example 1-4 preparation method soluble protein conversion ratio be respectively 22.69%, 18.63%,
11.83% and 23.99%, so, for soluble protein high conversion rate in comparative example 1-4, this illustrates this hair in 1 preparation method of embodiment
Bright preparation method passes through the bacterium enzyme synergy (bacterium enzyme acts on simultaneously) of bacillus subtilis and neutral proteinase, and can be improved can
Molten albumen conversion ratio；In addition, soluble protein conversion ratio is lower than embodiment 3-5 in embodiment 1-2 and comparative example 5-6 preparation method,
This illustrates the mistake that embodiment 3-5 is higher with the protease production of bacillus subtilis bacterium solution, is cultivated in squid homogenate
More protease can be secreted in journey, can further increase soluble protein conversion ratio, be higher than 32%.
3. the assay of unsaturated fatty acid in flavor squid nutrients biological product
Referring to the measurement of fat, saturated fat (acid), unsaturated fat (acid) total in GB/T22223-2008 food, using hydrolysis
Extraction-gas chromatography.Through gas chromatographic detection, its fatty acid composition is analyzed, as a result fig. 3, it is shown that embodiment
1 and embodiment 2 made from flavor squid nutrients biological product the content of unsaturated fatty acid be higher than comparative example 1-4；This explanation
Preparation method of the present invention passes through the bacterium enzyme synergy (bacterium enzyme acts on simultaneously) of bacillus subtilis and neutral proteinase, Neng Gouti
The content of unsaturated fatty acid in high flavor squid nutrients biological product；In addition, wind made from embodiment 1-2 and comparative example 5-6
The content of unsaturated fatty acid is lower than embodiment 3-5 in taste squid nutrients biological product, this illustrates embodiment 3-5 withered grass gemma
The yielding lipase activity of bacillus liquid is higher, and more lipase can be secreted during culture in squid homogenate,
To further increase the content of unsaturated fatty acid in flavor squid nutrients biological product, it is higher than 78%.
Test example 2:
This test examples of Antibacterial Activity of biological products chooses four kinds of common spoilage organisms Escherichia coli, salmonella, golden yellow
Color staphylococcus and Bacillus cercus carry out antibacterial work to Squid By-products fermentation liquid using Odontothrips loti as indicator bacteria
Property measurement.
The preparation of indicator bacteria bacteria suspension: first weighing in proportion and configure 50ml for MRS broth bouillon, adds in water-bath
Heat makes it completely dissolved, and is then sealed respectively toward 6ml is dispensed in the test tube of 4 18ml with gauze and brown paper equipped with broth cultivation
The test tube mouth for supporting base, is put into 121 DEG C of sterilizing 20min of high-pressure sterilizing pot for test tube and rubber stopper.After sterilizing, by 4 kinds of test tubes
The indicator bacteria of inclined-plane culture is inoculated into 4 sterilized broth bouillon test tubes respectively under super-clean bench aseptic condition with oese
In, rubber stopper is covered, 4 kinds of different indicator bacterias are marked with marking pen.4 test tubes are shaken under 37 DEG C, 150r/min
For 24 hours, the concentration of indicator bacteria is 10 at this time for bed culture7-109cfu/ml。
Odontothrips loti bacteriostatic test: indicator bacteria is placed in culture dish, with sterile spreader by its even spread, then again
3 sterilized Oxford cup aseptic nippers, are equably placed on solid medium, toward in the detection plate prepared
Oxford cup is moved plus 100 μ L mass concentrations are the biological products solution (sterile water dissolution) of 1.5g/L, is put in 3 Oxford cups identical
Liquid, i.e., the same detection plate done 3 it is parallel；After adding fermentation liquid, it will test plate and put 4 DEG C of refrigerator 12h into, then
It will test plate again and be placed in 37 DEG C of constant incubator and cultivate 16h, carefully take out culture dish, use calliper to measure Oxford cup at once
Antibacterial circle diameter.The results are shown in Table 1, it can be seen that embodiment 1-5 and comparative example 2-3,5-6 obtain biological products solution pair
Escherichia coli, salmonella, staphylococcus aureus and Bacillus cercus have bacteriostasis, and comparative example 1 and 4 must be raw
Tetramune solution on E. coli, salmonella, staphylococcus aureus and Bacillus cercus are then without bacteriostasis；Together
When, embodiment 4 and embodiment 5 obtain biological products solution on E. coli, salmonella, staphylococcus aureus and wax-like bud
The fungistatic effect of spore bacillus is best, this illustrates 2- aminopyrimidine in LB meat soup and guanidine hydrochloride in the presence of can influence alkali when DNA replication dna
The normal pairing of base causes gene that transversion, conversion, missing and frameshift mutation etc., the bacillus subtilis bacterium solution tool of acquisition occurs
There is the corrupt mushroom harmful bacteria growth of stronger inhibition, so as to extend the freshness date of made biological products.
Inhibition zone (mm) of the 1 Squid By-products fermentation liquid of table to four kinds of pathogenic bacteria
Note: inhibition zone is not observed in "-" expression
The prior art of routine techniques dawn known to those skilled in the art in above-described embodiment, thus it is no longer superfluous in detail herein
It states.
The above embodiments are only used to illustrate the present invention, and not limitation of the present invention, the ordinary skill people of this field
Member can also make a variety of changes and modification without departing from the spirit and scope of the present invention.Therefore, all equivalent
Technical solution also belong to scope of the invention, scope of patent protection of the invention should be defined by the claims.
Claims (8)
1. a kind of enzyme-linked preparation method for closing preparation squid nutrients biological product of bacterium, it is characterised in that: including by squid homogenate
In be added neutral proteinase simultaneously and the step of bacillus subtilis bacterium solution is cultivated.
2. a kind of enzyme-linked preparation method for closing preparation squid nutrients biological product of bacterium according to claim 1, feature exist
In: the solid-to-liquid ratio of squid homogenate and distilled water is 1:3-11 in the squid homogenate.
3. a kind of enzyme-linked preparation method for closing preparation squid nutrients biological product of bacterium according to claim 1, feature exist
In: the additive amount of the neutral proteinase is 500-6000u/g.
4. a kind of enzyme-linked preparation method for closing preparation squid nutrients biological product of bacterium according to claim 1 or 3, feature
Be: the additive amount of the bacillus subtilis bacterium solution is 10-35%.
5. a kind of enzyme-linked preparation method for closing preparation squid nutrients biological product of bacterium according to claim 1 or 3, feature
Be: the bacillus subtilis bacterium solution is by the way that bacillus subtilis to be inoculated in LB meat soup, in 35-40 DEG C, 100-150r/
8-12h is cultivated under conditions of min to be made.
6. a kind of enzyme-linked preparation method for closing preparation squid nutrients biological product of bacterium according to claim 5, feature exist
In: contain 2- aminopyrimidine and guanidine hydrochloride in the LB meat soup.
7. a kind of enzyme-linked preparation method for closing preparation squid nutrients biological product of bacterium according to claim 6, feature exist
In: containing mass concentration in the LB meat soup is ‰ 2- aminopyrimidine of 0.01-0.1 and ‰ guanidine hydrochloride of 0.03-0.08.
8. a kind of enzyme-linked preparation method for closing preparation squid nutrients biological product of bacterium according to claim 1, feature exist
In: the cultivation temperature is 20-60 DEG C, revolving speed 100-150r/min, time 12-60h.
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| CN105603031A (en) * | 2016-03-09 | 2016-05-25 | 广东海洋大学 | Aquatic protein active peptide preparing method without adopting high-temperature treatment |
| CN106561999A (en) * | 2016-11-01 | 2017-04-19 | 浙江海洋大学 | Method for preparing feeding peptide by using enzymolysis tuna dark meat |
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| CN105603031A (en) * | 2016-03-09 | 2016-05-25 | 广东海洋大学 | Aquatic protein active peptide preparing method without adopting high-temperature treatment |
| CN106561999A (en) * | 2016-11-01 | 2017-04-19 | 浙江海洋大学 | Method for preparing feeding peptide by using enzymolysis tuna dark meat |
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Application publication date: 20191119 |