CN103638515B - Anti gram-positive bacterium composition of anglerfish skin collagen peptide - Google Patents
Anti gram-positive bacterium composition of anglerfish skin collagen peptide Download PDFInfo
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Abstract
The invention discloses an anti gram-positive bacterium composition of anglerfish skin collagen peptide, and the anti gram-positive bacterium composition is prepared by compounding an anglerfish skin collagen peptide solution with a concentration of 100-200 mg/ml and a ceftiofur sodium solution with a concentration of 20 mug/mL according to a volume ratio of 3- 4:1. By combination use and collaborative effects of anglerfish skin collagen peptide and antibiotic ceftiofur sodium, the anti gram-positive bacterium composition has a better bacteria resistance and bacteriostasis activity on gram-positive bacterium, reduces the antibiotic dose, reduces drug residues, avoids or reduces the production of drug resistant strains, and improves the economic added value of anglerfish skin.
Description
Technical field
The present invention relates to a kind of bactericidal composition, particularly Yi Zhong angler collagen peptide resisting gram-positive bacteria compositions.
Background technology
In processing of aquatic products process, leftover bits and pieces accounts for 30% ~ 55% of raw material fish, and in the leftover bits and pieces such as fish head, fish skin, fishbone and the residual flesh of fish thereof, collagen content does not reach 20% ~ 50% not etc.Preliminary Results shows; aquatic collagen protein and polypeptide have protection gastric mucosa, antiulcer action, antiallergic, blood pressure lowering, cholesterol reducing, defying age, promotion wound healing, strengthen the multiple physiological active functions (Xu Haiju such as bone strength and prevention of osteoporosis, prevention arthritis, the reparation of promotion corneal epithelium wound and the growth of promotion corneal epithelial cell; Cai Jian; Chen Jiangping. the fundamental characteristics of aquatic collagen protein matter and the application [J] in food processing thereof. Zhejiang Agriculture science; 2010, (2): 324-327).
Angler belongs to cold warm nature demersal fishes, and volt seabed of often dwelling, be distributed in North Western Pacific, northern East China Sea, the Huanghai Sea and the Bohai Sea are originated in China.In the processing of angler, a large amount of fish skin is discarded by as leftover bits and pieces, this not only pollutes environment, and be the waste of resource, add current aquatic collagen protein and polypeptide characteristic inherently with and applied research in medical science, beauty and skin care, food, chemical industry and biomaterial etc. relative less, limit the scope of its application.In order to the economic value added of Ti Gao angler skin, need people constantly to explore its new physiologically active, widen the range of application of collagen peptide energetically.There are some researches show so far, Os Bovis seu Bubali collagen peptide, Silurus asotus fish bone zymolyte, Optimization of Low Value Fish Protein polypeptide-calcium chelate and collagen protein metal complex have antibacterial activity.And the research of antibacterial bacteriostatic activity that up to the present Hai Mei You angler polypeptide and antibiotic are worked in coordination with is reported.
Summary of the invention
The object of the present invention is to provide Yi Zhong angler collagen peptide resisting gram-positive bacteria compositions Tong Guo angler collagen peptide and antibiotic coupling, collaborative performance effect, there is preferably antibacterial bacteriostatic active to gram positive bacteria, reach simultaneously and reduce antibiotic dosage, reduce drug residue, avoid or reduce the object that Resistant strain produces, the economic value added of Ti Gao angler skin.
The technical solution adopted for the present invention to solve the technical problems is:
An angler collagen peptide resisting gram-positive bacteria compositions, to state angler collagen peptide bactericidal composition be 100-200mg/mL angler collagen peptide solution and concentration by concentration be that 20 μ g/mL ceftiofur sodium solutions are according to the composite combination solution formed of the volume ratio of 3-4:1.
The advantage that angler collagen peptide has is: be easy to absorb, the not easily generation of inducible resistance bacterial strain, also has stable, good water solubility, almost harmless to higher mammal normal cell, the feature such as noresidue, also has higher biological value and nutritive value simultaneously in vivo.Inventor studies discovery, Dan Chun angler collagen peptide does not have antibacterial bacteriostatic activity to gram positive bacteria, but it is composite with specific antibiotic ceftiofur sodium, performance effect can be worked in coordination with, remarkable enhancing ceftiofur sodium is active to the antibacterial bacteriostatic of gram positive bacteria, reaches and reduces antibiotic dosage, reduces drug residue, avoid or reduce the object that Resistant strain produces, the economic value added of Ti Gao angler skin.In addition, because ceftiofur sodium belongs to extensive pedigree antibiotic, it is active that itself also has antibacterial bacteriostatic definitely for gram negative bacteria, therefore, product of the present invention is mainly used in the antibacterial bacteriostatic of gram positive bacteria, and the antibacterial bacteriostatic simultaneously also had for gram negative bacteria is active.The proportioning of Kong angler collagen peptide and ceftiofur sodium, ensureing that the prerequisite of antibacterial activity exists, can play synergistic function to greatest extent.
As preferably, the molecular weight of Suo Shu angler collagen peptide is at below 2000D.The molecular weight of angler collagen peptide, at below 2000D, is not only easy to be absorbed by the body, and the activity of itself also can be stronger, especially the activity of scavenging hydroxyl, coordinate with ceftiofur sodium, synergistic function is good, and the antibacterial bacteriostatic activity of product is good.
As preferably, the preparation method step of Suo Shu angler collagen peptide is as follows:
(1) Xuan Qu angler skin cleaning drain after, shred to obtain fish skin fragment.
(2) fish skin fragment washes with water after being placed in dilute alkaline soln and soaking 24-30 h, then adds butanol solution degrease, is washed with water to pH and is neutrality and drains after stirring 20-28 h.
(3) the fish skin fragment bruisher drained is smashed to pieces, be placed in dilute acid soln, be heated to 45-90 DEG C, and constantly stir extracting 6-9 h, after filtration, obtain collagen protein crude extract.
(4) in collagen protein crude extract, add pepsin, pepsin consumption is the 1%-2% of angler tare weight amount, under ultrasonic assistant, and enzymolysis 2-6 h at 30-40 DEG C of temperature; Then add flavor protease, flavor protease consumption is the 0.1%-0.2% of angler tare weight amount, and tune pH is 6.5-7.5, under ultrasonic assistant, and enzymolysis 5-7 h at 40-55 DEG C of temperature.
Adopt two enzyme enzymolysis, the cleavage site that pepsin and flavor protease act on peptide bond is different, both can the peptide bond that formed of synergism carboxyl that can cut off aromatic amino acid again can inscribe and circumscribed remove bitterness and to ensure to discharge oligopeptide, its height discharged
fthe relative molecular weight of value collagen peptide is less, more can be absorbed by the body.Ultrasound wave can play Emulsification effect, can make two mixing fully and contacting equably between enzyme and collagen protein, can improve the extraction ratio of enzymolysis speed and collagen peptide.
(5) enzymolysis solution enzyme denaturing, adds after active carbon stirs and filters, after vacuum concentration, and spraying dry get angler collagen peptide.Carry out debitterizing and decoloring with activated carbon adsorption, not only simple to operate, cost is lower, and effective.
By the product that the preparation method of above-mentioned You Xuan angler collagen peptide obtains, there is the feature of high F value, free from extraneous odour, color and luster be homogeneous, be easy to absorption of human body, there is higher biological value and nutritive value.Coordinate with ceftiofur sodium, synergistic function is good.
High
fvalue oligopeptide be a class by not higher than 9 amino acid residues form, the ratio of branched-chain amino acid (Val, Leu, Ile) and the amount of substance of aromatic amino acid (Tyr, Phe, Trp) content namely
fthe mixing oligomerization small peptide polymer of value>=20.From modern nutriology angle, compared with free amino acid, biological value and the nutritive value specific ionization aminoacid of small peptide are higher.Studies have reported that height
fvalue oligopeptide product has physiological functions such as improving hepatic encephalopathy, relieving alcoholic intoxication, hepatoprotective, and physiological function, also can be used for the protein nutritional status and the post exercise supplementary that improve Post operation and bed patient.
As preferably, the dilute alkaline soln described in step (2) is the NaOH solution of 0.25%-0.45%, and Mei Ke angler skin adds the dilute alkaline soln described in 25-45 mL.
As preferably, in the butanol solution described in step (2), the volumn concentration of n-butyl alcohol is the butanol solution that 7-10%, Mei Ke angler skin adds described in 25-45 mL.
As preferably, the mass fraction of the dilute acid soln described in step (3) is person's citric acid solution of 0.2-0.4%, and Mei Ke angler skin adds the dilute acid soln described in 55-75 mL.
As preferably, in step (4), ultrasound condition is: ultrasonic frequency is 25-40 kHz, and power is 100-400 W.
As preferably, add activated carbon in step (5) and stir 1-3 h at 40-60 DEG C.
The invention has the beneficial effects as follows:
1, Tong Guo angler collagen peptide and the coupling of antibiotic ceftiofur sodium, collaborative performance effect, there is preferably antibacterial bacteriostatic active to gram positive bacteria, reach simultaneously and reduce antibiotic dosage, reduce drug residue, avoid or reduce the object that Resistant strain produces, the economic value added of Ti Gao angler skin.
2, because ceftiofur sodium belongs to extensive pedigree antibiotic, it is active that itself also has antibacterial bacteriostatic definitely for gram negative bacteria, therefore, product of the present invention is mainly used in the antibacterial bacteriostatic of gram positive bacteria, and the antibacterial bacteriostatic simultaneously also had for gram negative bacteria is active.
3, angler collagen peptide have be easy to absorb, the not easily generation of inducible resistance bacterial strain, also has stable, good water solubility, almost harmless to higher mammal normal cell, the feature such as noresidue, also has higher biological value and nutritive value simultaneously in vivo.
Accompanying drawing explanation
The antibacterial effect of the compositional liquor that figure 1 Shi angler collagen peptide and collaborative antibiotic are prepared in different ratio mode compares.
Fig. 2 is that staphylococcus aureus normal growth curve and compositional liquor suppress curve comparison.
Detailed description of the invention
Below by specific embodiment, and by reference to the accompanying drawings, technical scheme of the present invention is described in further detail.
In the present invention, if not refer in particular to, the raw material adopted and equipment etc. all can be buied from market or this area is conventional.Method in following embodiment, if no special instructions, is the conventional method of this area.
Embodiment 1:
An angler collagen peptide resisting gram-positive bacteria compositions, to state angler collagen peptide bactericidal composition be 100mg/mL angler collagen peptide solution and concentration by concentration be that 20 μ g/mL ceftiofur sodium solutions are according to the composite combination solution formed of the volume ratio of 3:1.
Embodiment 2:
An angler collagen peptide resisting gram-positive bacteria compositions, to state angler collagen peptide bactericidal composition be 200mg/mL angler collagen peptide solution and concentration by concentration be that 20 μ g/mL ceftiofur sodium solutions are according to the composite combination solution formed of the volume ratio of 4:1.The molecular weight of angler collagen peptide is at below 2000D.
the preparation method of angler collagen peptide is as follows:
method 1:
Get clean Hou angler skin 1000 g scissor cut into about length and width be 2 cm × 2 cm fragments (fish skin can fully contact solution in turn ensure that fragment not easily too small cause run off) after.
Adding 25L mass fraction in angler skin fragment is 0.45% NaOH solution, pH is washed with water to for neutral after soaking 24 h, adding 35 L volumn concentrations is again the butanol solution of 9%, is washed with water to pH and is neutrality and drains (quality that improve collagen protein) after stirring 28 h.
Smash to pieces draining angler skin fragment (smash fish skin fragment to pieces and fully can be dissolved in extracting solution to make collagen protein, improve the extraction ratio of fish skin), adding 55 L mass fractions is the citric acid solution of 0.4%, is heated to 55 DEG C, and constantly stir extracting 6 h, obtain collagen protein crude extract after filtration.
In collagen protein crude extract, add pepsin, pepsin addition Wei angler tare weight amount 1%, be 40 kHz in ultrasonic frequency, power is 100 W, and temperature is carry out enzymolysis 4 h at 35 DEG C; Adjust pH to be 6.5, add flavor protease Liang Wei angler tare weight amount 0.15%, under ultrasonic assistant, (ultrasonic frequency is 40 kHz, and power is 100 W) set temperature is 50 DEG C of enzymolysis 6 h.
95 DEG C are heated to the enzymolysis solution of gained, and keep 7 min enzyme denaturing, activated carbon debitterizing and decoloring is added in enzyme denaturing gained enzymolysis solution, and excessively filter activated carbon after stirring 1 h at 40 DEG C, again the filtrate after debitterizing and decoloring is placed in vacuum rotary evaporator and carries out vacuum concentration, last spraying dry, the angler skin obtained is high
fthe value weight of collagen peptide, purity and
fbe worth as shown in table 1.
control methods 1:preparation condition and method 1 completely the same, difference is, under Ultrasonic Conditions, does not carry out enzymolysis, the weight of the angler collagen peptide obtained, purity and
fbe worth as shown in table 1.
method 2:
Get after clean Hou angler skin 1000 g scissor cut is 2 cm × 2 cm fragments into about length and width.
Adding 45 L mass fractions in angler skin fragment is 0.25% NaOH solution, is washed with water to pH for neutral, then adds the butanol solution that 45L volumn concentration is 7%, be washed with water to pH and be neutrality and drain after stirring 20 h after soaking 30 h.
Smashed to pieces by the angler skin fragment drained, adding 75 L mass fractions is the citric acid solution of 0.2%, is heated to 90 DEG C, and constantly stirs extracting 9 h, obtains collagen protein crude extract after filtration.
In collagen protein crude extract, add pepsin, pepsin addition Wei angler tare weight amount 1.5%, be 25 kHz in ultrasonic frequency, power is 400 W, and temperature is carry out enzymolysis 2 h at 40 DEG C; Adjust pH to be 7, add flavor protease Liang Wei angler tare weight amount 0.2%, under ultrasonic assistant, (ultrasonic frequency is 25 kHz, and power is 400 W) set temperature is 55 DEG C of enzymolysis 5 h.
95 DEG C are heated to the enzymolysis solution of gained, and keep 7 min enzyme denaturing, activated carbon debitterizing and decoloring is added in enzyme denaturing gained enzymolysis solution, and excessively filter activated carbon after stirring 2 h at 60 DEG C, again the filtrate after debitterizing and decoloring is placed in vacuum rotary evaporator and carries out vacuum concentration, last spraying dry, the angler skin obtained is high
fthe value weight of collagen peptide, purity and
fbe worth as shown in table 1.
control methods 2:preparation condition and method 2 completely the same, difference is, under Ultrasonic Conditions, does not carry out enzymolysis, the weight of the angler collagen peptide obtained, purity and
fvalue is as shown in table 1.
method 3:
Get clean Hou angler skin 1000 g scissor cut into about being cut into after length and width are 2 cm × 2 cm fragments.
Adding 35 L mass fractions in angler skin fragment is 0.35% NaOH solution, is washed with water to pH for neutral, then adds the butanol solution that 25L volumn concentration is 10%, be washed with water to pH and be neutrality and drain after stirring 26 h after soaking 30 h.
The angler skin fragment drained is smashed to pieces, in add 60 L mass fractions be the citric acid solution of 0.3%, be heated to 45 DEG C, and constantly stir extracting 7h, after filtration, obtain collagen protein crude extract.
In collagen protein crude extract, add pepsin, pepsin addition Wei angler tare weight amount 2%, be 40kHz in ultrasonic frequency, power is 100 W, and temperature is carry out enzymolysis 6 h at 30 DEG C; Adjust pH to be 7.5, add flavor protease Liang Wei angler tare weight amount 0.1%, under ultrasonic assistant, (ultrasonic frequency is 40 kHz, and power is 100 W) set temperature is 40 DEG C of enzymolysis 7h.
95 DEG C are heated to the enzymolysis solution of gained, and keep 7 min enzyme denaturing, activated carbon debitterizing and decoloring is added in enzyme denaturing gained enzymolysis solution, and excessively filter activated carbon after stirring 3 h at 50 DEG C, again the filtrate after debitterizing and decoloring is placed in vacuum rotary evaporator and carries out vacuum concentration, last spraying dry, Huo get angler skin is high
fthe value weight of collagen peptide, purity and
fbe worth as shown in table 1.
control methods 3:preparation condition and method example 3 completely the same, difference is, under Ultrasonic Conditions, does not carry out enzymolysis, the weight of the angler collagen peptide obtained, purity and
fvalue is as shown in table 1.
antibacterial experiment
1 materials and methods
1.1 experiment materials and reagent
Angler collagen peptide molecular weight is at below 2000D.Ceftiofur sodium, be purchased from Shanghai biological engineering company limited, other reagent is analytical pure.Staphylococcus aureus (gram positive bacteria) is purchased from Guangdong Province's microorganism germ plasma resources bank.Nutrient broth medium, MH culture medium, be purchased from Beijing overpass Technology Co., Ltd..
1.2 instrument and equipment
Micct-Q type Superpure water machine: Japanese Milli-por LIMITED company; AL-104 electronic analytical balance: prunus mume (sieb.) sieb.et zucc. Teller-Tuo benefit Instrument Ltd.; UV-2102 C type ultraviolet spectrophotometer: Shanghai You Nike Instrument Ltd.; The single clean work station of SN-CS-2D: Suzhou Decontamination Equipment Plant; HZQ-X100A type constant-temperature shaking incubator: Shanghai Yiheng Scientific Instruments Co., Ltd; HVE-50 autoclave sterilization pot: Japanese HIRAYAMA, Haier's refrigerator: Qingdao HaiEr Co., Ltd;
1.3 experimental technique
1.3.1 strains tested is cultivated and sample configuration
(1) access in corresponding test tube slant culture medium by the first generation bacterium for test, staphylococcus aureus kind is put in 37 DEG C of calorstats and is all cultivated 24 h, obtains secondary bacterial strain, is placed in 4 DEG C of refrigerator and cooled and hides for subsequent use.
(2) Yi angler collagen peptide mg/mL+ antibiotic (ceftiofur sodium) μ g/mL are with the different compositional liquor of the proportional arrangement of volume ratio 3:1: in table 2, wherein, 0 μ g/mL antibiotic, 0 mg/mL peptide liquid ultra-pure water replace, and carry out antibacterial effect research.
Table 2 test sample allocation list
| Sample sequence number | Peptide liquid concentration | Antibiotic concentration | Peptide liquid: antibiotic (v:v) |
| 1# | 100 mg/mL | 0 μg/mL | 3:1 |
| 2# | 200 mg/mL | 0 μg/mL | 3:1 |
| 3# | 100 mg/mL | 20 μg/mL | 3:1 |
| 4# | 200 mg/mL | 20 μg/mL | 3:1 |
| 5# | 0 mg/mL | 20 μg/mL | 3:1 |
1.3.2 inhibition zone measures
Adopting Odontothrips loti, take ceftiofur sodium as positive control.Staphylococcus aureus adopts Solid nutritional agar plate, and tested bacterium (OD value is about 1.0) the 150 μ L of access exponential phase, spreading rod uniform application, after bacterium liquid fully absorbs, adds each 100 μ L of 1# ~ 5# compositional liquor respectively.
Staphylococcus aureus is placed in 37 DEG C of incubators and cultivates 24 h, each antibacterial circle diameter of vernier caliper measurement (antibacterial circle diameter mm in), Taking Pictures recording.
1.3.3 the mensuration of Bactericidal curves
Phase tested bacterium bacterium liquid (OD is 1.0) 20 mL take the logarithm respectively in culture bottle, and often kind of tested strain gets 5 culture bottles, adds each 10 mL of 1# ~ 5# compositional liquor respectively, arrange normal control, cultivate 26 h in each culture bottle.With the culture medium zeroing not adding bacterium liquid and sample, sampled every two hours and measure absorbance in 600 nm places.
2 results and analysis
fungistatic effect worked in coordination with by 2.1 angler collagen peptides and antibiotic
Grind the antibacterial effect studying carefully the compositional liquor that angler collagen peptide and collaborative antibiotic are prepared in different ratio mode, measure the diameter of inhibition zone and take pictures, seeing shown in Fig. 1 and table 3.
From Fig. 1 and table 3, angler collagen peptide liquid does not have inhibitory action to tested bacterium; When collaborative antibiotic acts on tested bacterium, 3#, 4# compositional liquor is 25.1 mm, 22.5 mm to the inhibition zone of staphylococcus aureus respectively, has obvious inhibitory action.With positive controls ratio, the inhibitory action of 4# compositional liquor to staphylococcus aureus significantly improves.
This shows, angler collagen peptide can work in coordination with ceftiofur sodium effect suppress staphylococcus aureus, 3#, 4# compositional liquor to the inhibitory action bacterium of staphylococcus aureus apparently higher than positive control.
2.2 compositional liquor Bactericidal curves measure
take incubation time as abscissa, the OD value of different incubation time bacterial suspensions under 600 nm is vertical coordinate, with the compositional liquor of the normal growth curve of the tested bacterium of turbidimetry for Determination (staphylococcus aureus) and different preparation to the Bactericidal curves of tested bacterium, as shown in Figure 2.
As shown in Figure 2, the growth curve of tested bacterium all embodies period of delay, logarithmic (log) phase, stable phase and 4 stages of decline phase, and the Bactericidal curves that with the addition of the compositional liquor of different preparation is different according to the difference of the strain of 3 kinds of tested bacterium and compositional liquor configuration.In Fig. 2, the antibacterial growth curve of 1# and 2# compositional liquor is close with normal growth curve of bacteria shape, the obvious normal growth curve lower than antibacterial in various degree of Bactericidal curves of 3#, 4# and 5# compositional liquor, the Bactericidal curves of 3# and 4# compositional liquor is starkly lower than 5# compositional liquor.Provable thus: 3# and 4# compositional liquor has obvious bacteriostasis to staphylococcus aureus, and bacteriostasis is apparently higher than antibiotics alone.
Above-described embodiment is one of the present invention preferably scheme, not does any pro forma restriction to the present invention, also has other variant and remodeling under the prerequisite not exceeding the technical scheme described in claim.
Claims (6)
1. angler collagen peptide bactericidal composition is in the application preparing anti-Staphylococcus aureus medicine, it is characterized in that: to state angler collagen peptide bactericidal composition be 100-200mg/mL angler collagen peptide solution and concentration by concentration be that 20 μ g/mL ceftiofur sodium solutions are according to the composite combination solution formed of the volume ratio of 3-4:1;
The molecular weight of Suo Shu angler collagen peptide is at below 2000D;
The preparation method step of Suo Shu angler collagen peptide is as follows:
(1) Xuan Qu angler skin cleaning drain after, shred to obtain fish skin fragment;
(2) fish skin fragment washes with water after being placed in dilute alkaline soln and soaking 24-30 h, then adds butanol solution degrease, is washed with water to pH and is neutrality and drains after stirring 20-28 h;
(3) the fish skin fragment bruisher drained is smashed to pieces, be placed in dilute acid soln, be heated to 45-90 DEG C, and constantly stir extracting 6-9 h, after filtration, obtain collagen protein crude extract;
(4) in collagen protein crude extract, add pepsin, pepsin consumption is the 1%-2% of angler tare weight amount, under ultrasonic assistant, and enzymolysis 2-6 h at 30-40 DEG C of temperature; Then add flavor protease, flavor protease consumption is the 0.1%-0.2% of angler tare weight amount, and tune pH is 6.5-7.5, under ultrasonic assistant, and enzymolysis 5-7 h at 40-55 DEG C of temperature;
(5) enzymolysis solution enzyme denaturing, adds after active carbon stirs and filters, after vacuum concentration, and spraying dry get angler collagen peptide.
2. application according to claim 1, is characterized in that: the dilute alkaline soln described in step (2) is the NaOH solution of 0.25%-0.45%, and Mei Ke angler skin adds the dilute alkaline soln described in 25-45 mL.
3. application according to claim 1, is characterized in that: in the butanol solution described in step (2), the volumn concentration of n-butyl alcohol is the butanol solution that 7-10%, Mei Ke angler skin adds described in 25-45 mL.
4. application according to claim 1, is characterized in that: the dilute acid soln described in step (3) is the citric acid solution of mass fraction 0.2-0.4%, and Mei Ke angler skin adds the dilute acid soln described in 55-75 mL.
5. application according to claim 1, is characterized in that: in step (4), ultrasound condition is: ultrasonic frequency is 25-40 kHz, and power is 100-400 W.
6. application according to claim 1, is characterized in that: add active carbon in step (5) and stir 1-3 h at 40-60 DEG C.
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