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Crocodylus Porusus Peptide



Cure AIDS by feeding patients to crocodiles !






https://groups.yahoo.com/neo/groups/CrocodylusPorusus/info

Crocodylus Porusus
    
Group Description

The purpose of this group is to confirm, or not, the validity of the information on the Web Site "Biologicalmiracle.com" about "The Antidote", as well as to seek and explore other affordable and non toxic treatments and cures, such as described at web sites: www.robertogiraldo.com - www.aliveandwell.org. The most important part is to get to The Truth about the whole matter: http://www.virusmyth.net/aids/

"The common cold is a thing of the past, even serious infectious diseases such as Cancer, AIDS, SARS and many other life threatening diseases can be helped by the miracle healing powers of the Antidote.

The Antidote does not require a prescription or medical consultation before being taken as it is a natural alternative to Antibiotics or drugs which have failed to kill virulent viruses or bacteria that have developed an immunity to current antibiotics and drugs produced by modern medicine.

The Antidote is a unique Anti-Microbial Peptide offering the widest range of healing power on the market today. It kills all known deadly VIRUSES and BACTERIA in the body. The initial research was carried out over several years ago by the BBC.

The Antidote acts as an additive for your body's immune system. It will fight and protect your body from all virus and bacteria activated infections. The Antidote may be taken safely by children and adults even if on current medication.

The Peptide is a Protein Extract from Crocodylus Porusus, the largest living crocodile in existence and the most ancient living species on earth (over 20 million years old). A Peptide is a Natural Protein made up of Amino Acids strung together that destroy powerful viruses and bacteria by penetrating their membranes.

Check out the facts, links & testimonials pages, which provide an extensive amount of evidence of the Antidote’s healing effects. Take a look for yourself." http://biologicalmiracle.com



http://biologicalmiracle.com/

SHACRO

SHACRO is a very special product combination of 2 synthesized proteins emulsified by a special catalyst that enhances and increases the multiplication ratio of the protein properties. Australian Salt Water Crocodiles and Great White Sharks have an innate immune system as the first line of defence that kicks in as soon as an organism faces any threat-a puncture of the skin, bacteria in the stomach, something nasty in the lungs. Besides the cells, such as macrophages and neutrophils, gobbling up bacteria in this first line of defence, these anti-microbial peptides bash holes in the bacteria’s membrane, which weaken it to the point that it instantly dissipates

SHACRO is a food additive for your body's immune system. It will fight and protect your body from all virus and bacteria activated infections. SHACRO may be taken safely by children and adults even if on current medication...

 On May 31st, 2000, a documentary aired on BBC entitled The Secret Life of Crocodiles, which was the origin for the discovery of a unique anti-microbial peptide in crocodiles. Jill Fullerton-Smith, a senior producer in the Science Department at BBC, decided to investigate, however, she couldn't find any scientists in the world working on the immune response of the crocodile and was on the verge of abandoning the idea. She then saw a newspaper article about a biologist who noticed that a frog in his lab has lost a limb and yet within in few days had healed. The biologist now owned a multi-million dollar research company developing the antibiotic they had found in the frog. Jill rang him, and on his advice decided to hire an American microbiologist to look for a particular particle in the blood of the crocodile. Nobody had ever looked for these peptides in the reptile before. Michael Mosley, Executive Producer of Living Proof, agreed to fund a film following the collection of the blood from wild Australian crocodiles and the search for the peptide. An amazing new anti-microbial peptide was discovered in the blood, and the BBC and the university are lodging patent rights. Greg Dyke personally announced the discovery to the world's press.

This quote from BBC Director-General Greg Dyke describes the manner in which a unique anti-microbial peptide was discovered: "Tonight I can reveal that Living Proof, our science documentary on BBC ONE, has done something very unusual: they've actually helped find and isolate a protein which kills resistant bacteria and which could form the basis of a new antibiotic. On a trip to film salt-water crocodiles in Australia, our producer noticed something that surprised her; despite the horrendous injuries the crocs inflict on each other, their wounds rarely get infected. She discussed this with a young croc expert who agreed that it would be interesting to try and find out why. So they set off together to collect blood samples from wild crocodiles. After many adventures they got their blood samples and last week a leading research institute isolated, from these samples, what I'm told is a novel anti microbial peptide. In tests this substance kills strains of virulent bacteria that are resistant to all standard antibiotics."

This quote from Dr. James Perran Ross, a croc researcher at the Florida Museum of Natural History, describes the commonplace occurrence of crocodiles surviving traumatic injuries stemming from a unique anti-microbial peptide:"They can sustain the most frightful injuries. In territorial fights they commonly tear each other's legs off. They go away and sulk for a while and seem to heal up. You often find animals in the wild with missing limbs, missing tails -- what must have been very serious injuries. I found one in the wild with the whole of its lower jaw torn off, all healed up and swimming around. It was a bit skinny but had obviously survived that very traumatic event. So I think their inherent toughness is one aspect. They are also long-lived. They routinely live for decades."

This statement from Animal Planet.com explains in real simple detail exactly why crocodiles don't suffer from infections: Surprisingly, very few crocodiles seem to suffer from infections. We recently discovered the secret behind their remarkable ability - an anti-microbial peptide in their blood. Crocodiles have one of the most efficient immune systems of any animal we know, which is a real advantage for them living in bacteria-filled water and mud. Wounds are common from fights or injuries from prey, and being able to fight off potential infection is clearly very important. The only time crocodiles suffer from infections is when they become stressed as their health declines. This affects their immune system and they can suddenly become susceptible to common bacteria they would normally shrug off. This can be seen in captive crocodiles kept in poor conditions, or wild subordinate or injured crocodiles unable to secure a territory and enough food to survive...



PATENTS

Preparation method and application of crocodile-skin collagen peptide
CN103421872

A preparation method of crocodile-skin collagen peptide comprises the following steps: boiling fat-free crocodile skin in a solvent; treating the boiled crocodile skin with a protease to obtain an enzymatic hydrolysate; conducting centrifugal separation on the enzymatic hydrolysate; taking supernate and drying, so as to obtain the crocodile-skin collagen peptide. The crocodile-skin collagen peptide obtained according to the prepartion method can be used as an antioxidant, a humectant, an anti-DNA damage agent, or a whitening additive in cosmetics. The preparation method is simple and convenient, is suitable for mass production, not only can improve the development added value of a crocodile product, but also can reduce environmental pollution, brings high economic value and social value.

BACKGROUND

Collagen is the body of a human or other animal most abundant class of proteins, 25-33% of the total protein content.

Extracellular collagen matrix (ECM) of the main component of the vascular wall to maintain skin elasticity and to keep the hair glossy and soft nails improve lubricity cartilage has an important role.

Because of its unique physical and chemical properties and excellent biocompatibility in many fields has been widely used.

Collagen peptide is a novel collagen products, collagen is collagen-rich material or as raw material for the production.

It has a unique amino acid composition of collagen, but also has a small molecular weight characteristics.

Therefore, it is more easily absorbed through the skin dermis, it is easy to be digested by the digestive tract, in the chemical, food, health care field have a good purpose.

Preparation of collagen peptides on the currently common in pigs, cattle skin and bone, or fish skin, scales, bone access to research as a source of collagen peptide enzymatic treatment.

The crocodile as raw collagen peptide also rarely reported.

Of alligator research also at the level of primary production of leather goods and crafts, its development is not yet complete and some of the waste is also a lack of opportunities for secondary use.

Currently on alligator leather focused on the development of production, processing and waste generated as well as some specifically for eating crocodile skin crocodile meat can not be used in handicrafts processing, if not effectively use these scraps will become waste, not only caused great waste, but also pollute the environment.

Therefore, the development and application of crocodile skin will become the focus and hot, and it will have a huge economic and social benefits.

SUMMARY OF THE INVENTION

The present invention discloses a method for preparing alligator collagen peptides, including: cooking fat has been removed in a solvent alligator; cooked alligator treated with protease to obtain enzymatic hydrolyzate; enzyme was centrifuged the separate the supernatant and dried to obtain alligator collagen peptide.

In the preparation method of the crocodile skin collagen peptide, in a solvent wherein the cooking time may be 30 ± 5 minutes, and wherein said solvent may be water, preferably distilled water, wherein the amount of solvent and the alligator The weight ratio may 1L / (100 ± 2) g.

In the preparation method of the crocodile skin collagen peptide, wherein the protease may be any of trypsin, neutral protease, alkaline protease, protease complexes, acid protease, preferably an alkaline protease, the alkaline protease Preferably the source of Bacillus licheniformis alkaline protease, and wherein the weight ratio of the alkaline protease can be alligator 0.5% to 4%, preferably 4%.

In the preparation method of the crocodile skin collagen peptide, wherein said condition can be treated in the protease activity of the protease, the pH is maintained at 40 ? ± 2 ? temperature hydrolysis of 3 hours ± 5 minutes.

In the preparation method of the crocodile skin collagen peptide, wherein the centrifugation can be centrifuged at 4000rpm at room temperature 20 ± 5 minutes.

In the preparation method of the crocodile skin collagen peptide, wherein the drying may include freeze-drying and spray drying, preferably freeze-dried.

The present invention also discloses a collagen peptide alligator skin collagen peptides prepared by the process of the above-described collagen peptides alligator the crocodile.

As described above alligator collagen peptide, wherein the average molecular weight of the crocodile skin collagen peptide may be about 1.2KD.

The present invention also discloses an antioxidant for cosmetics, moisturizers, anti-whitening agents or DNA damaging agent, the anti-oxidants, humectants, anti-DNA-damaging agents and whitening agents containing the above-described crocodile skin collagen peptide .

The present invention also discloses the above-described collagen peptides as crocodile skin cosmetics antioxidants, moisturizers, anti-whitening agent or the application of DNA damage additives.

Crocodile skin contains a lot of collagen, processed through modern biotechnology appropriate enzymatic method can obtain high quality collagen peptide.

Preparation methods disclosed herein alligator collagen peptide, unlike the complex processes in the past handling of raw materials, but skipped the collagen extracted from the raw material of the process, the raw material for processing directly to obtain the effectiveness of good quality collagen peptides, to the purpose of simplifying the preparation process.

Alligator collagen peptides prepared this application can not only increase the added value production and processing crocodiles, while reducing environmental pollution, access to good social and economic benefits.

Experiments show that alligator collagen peptides of the present application in anti-oxidant, moisturizing vitro, in vitro anti-DNA damage aspects have a significant effect; in B16 melanoma cells in mice to test strain experiments, crocodile skin collagen of the application peptide on intracellular reactive oxygen scavenging, inhibition of tyrosinase activity and melanin content reduces have a good effect, with a good description of the whitening effect.

Brief Description

Figure 1 shows the different protease 1,1 - dinitrophenylhydrazine impact of three (DPPH) clearance rate and total reducing power - diphenyl-2.

In Figure 1, the abscissa for different proteases (trypsin, neutral protease, alkaline protease, protease complexes, acid protease), respectively around the vertical axis and the total clearance DPPH reducing power.

Figure 2 shows the effect of different hydrolysis time on DPPH clearance rate and total reducing power.

Among them, the abscissa is different hydrolysis time (1h, 2h, 3h, 4h, 5h, 6h), about the vertical axis, respectively DPPH clearance rate and total reducing power.

Figure 3 shows the effect of different amounts of DPPH radical scavenging enzymes and total reducing power.

Wherein the abscissa is the type of enzyme (0.5%, 1%, 2%, 3%, 4%), respectively, around the vertical axis and the total clearance DPPH reducing power.

Figure 4 shows the MALDI-TOF MS spectra alligator collagen peptide of the present application.

Figure 5 is a DNA agarose gel electrophoresis showing the protective effect of crocodile skin collagen peptide in vitro for the application by the hydroxyl radical-induced DNA damage.

Among them, the lane 1:5 mg / mL of crocodile skin collagen peptide group, lane 2:1 mg / mL of crocodile skin collagen peptide group, lane 3:0.1 mg / mL of crocodile skin collagen peptide group, lane 4: Damage control group, lane 5: control group without damage.

Figure 6 shows the alligator collagen peptides of the present application on mouse B16 melanoma cell proliferation.

Abscissa alligator different concentrations of collagen peptide treated group (crocodile skin collagen peptide concentration were 0,12.5,25,50,100,200,500,1000 µg / mL), the vertical axis is the relative cell proliferation.

Figure 7 shows the effect of crocodile skin collagen peptide of the present application in murine B16 melanoma cells in melanin content.

Abscissa alligator different concentrations of collagen peptide treated group (crocodile skin collagen peptide concentration were 0,12.5,25,50,100,200 µg / mL), the vertical axis is the relative cell melanin content.

Figure 8 shows the alligator collagen peptides of the present application activity of tyrosinase in mouse B16 melanoma cells.

Abscissa alligator different concentrations of collagen peptide treated group (crocodile skin collagen peptide concentration were 0,12.5,25,50,100,200 µg / mL), the activity of tyrosinase activity relative to the vertical axis is within the cell .

Figure 9 shows the effect of crocodile skin collagen peptide of the present application in murine B16 melanoma cells ROS content.

FIG sequence diagram showing the small experimental groups with different concentrations of collagen peptide alligator effect with respect to the control group 0µg/mL FIG. Concentration alligator collagen peptides were 25,50,100,200 µg / mL. The shaded part of the control group.

 


Embodiment

The following will be the way of the application of the embodiment described in further detail as to enable those skilled in the art to practice the application.

It should be understood that other embodiments may be used, and may be appropriately changed without departing from the spirit or scope of the present application.

In order to avoid the person skilled in the art that the present application is not necessary to practice the details of the specification may be omitted for certain information known to the person skilled in the art.

Therefore, the following detailed description should not be understood in a limiting sense, and the scope of the present invention is defined only by the appended claims.

The following examples facilitate a better understanding of the present application, it is not intended to limit the scope of the application.

The following embodiment examples of the raw materials used, except where specifically noted, may be obtained from commercially available.

Example

Effect of different protease DPPH clearance and total reducing power

Fresh crocodile skin, scrape the fat, cleaned, cut into small pieces, add the appropriate amount of distilled water (weight alligator with added water ratio of 100g / L) and cook for 30 minutes to cool to room temperature in the cooker, mashed .

After the mashed alligator divided into five equal parts, were placed in five beaker.

In each beaker were added 5% trypsin (Wuxi Jie Ren Biotechnology Co., Ltd.), a neutral protease (derived from Bacillus subtilis neutral protease enzymes, Ltd. Zaozhuang Jarrow), alkaline protease (Bacillus licheniformis Bacillus alkaline protease sources, Ltd. Zaozhuang Jienuo enzyme), protease complex (CN protamex, Ren Jie Biotechnology Co., Ltd. Wuxi), acid protease (derived from Aspergillus niger acid protease enzyme limited Zaozhuang Jarrow company), the specific hydrolysis conditions shown in Table 1.

After 6 hours, the enzyme was heated to boiling for 10 minutes to inactivate the enzyme.

Then centrifuged at 4000rpm at room temperature for 20 minutes, the supernatant solution was for the test.

Detection DPPH clearance rate and total reducing power.

DPPH clearance rate and total antioxidant capacity reflects the reducing power.

Table 1 hydrolysis conditions

The results shown in Figure 1.

The results showed that the alkaline protease enzymatic hydrolysis was obtained at the same time had the highest DPPH scavenging rate and the highest total reducing power.

Effect of different hydrolysis time on DPPH clearance and total reducing power

Fresh crocodile skin, scrape the fat, cleaned, cut into small pieces, add the appropriate amount of distilled water (weight alligator with added water ratio of 100g / L) and cook for 30 minutes to cool to room temperature in the cooker, mashed .

After the mashed average alligator into six parts were placed in six beakers.

5% of the added amount of the enzyme alkaline protease (derived from Bacillus licheniformis alkaline protease enzyme Jienuo Zaozhuang Ltd.), at pH11, 40 ? hydrolysis.

Set hydrolysis time was 1h, 2h, 3h, 4h, 5h, 6h, then the enzyme was heated to boiling for 10 minutes to inactivate the enzyme.

Then centrifuged at 4000rpm at room temperature for 20 minutes, the supernatant solution was for the test.

Detection DPPH clearance rate and total reducing power.

The results shown in Figure 2.

The results showed that when the hydrolysis time of 3 hours, hydrolysis was obtained at the same time had the highest DPPH scavenging rate and the highest total reducing power.

Effect of different enzyme and total clearance rate of DPPH restore power

Fresh crocodile skin, scrape the fat, cleaned, cut into small pieces, add the appropriate amount of distilled water (weight alligator with added water ratio of 100g / L) and cook for 30 minutes to cool to room temperature in the cooker, mashed .

After the mashed alligator divided into five equal parts, were placed in five beaker.

Based on the amount of enzyme were added to 0.5% of the fresh weight of the alligator, 1%, 2%, 3%, 4% alkaline protease (derived from Bacillus licheniformis alkaline protease enzyme Zaozhuang Jienuo Limited) at pH11, 40 ? hydrolyzed for 3 hours.
The enzyme was heated to boiling for 10 minutes to inactivate the enzyme.

Then centrifuged at 4000rpm at room temperature for 20 minutes, the supernatant solution was for the test.

Detection DPPH clearance rate and total reducing power.

The results shown in Figure 3.

The results showed that: the enzyme is 4%, taking into account the cost of production, while enzyme solution was to have the highest DPPH scavenging rates and higher total reducing power.

Determination of average molecular weight collagen peptide alligator

The average molecular weight of the following methods alligator collagen peptide were measured: the sample was dissolved powder 0.05g alligator collagen peptide with 1mL0.5% TFA (trifluoroacetic acid), the matrix a-HCCA (a-cyano-4 - hydroxycinnamic acid) was dissolved in saturated TA (0.1% TFA: ACN (acetonitrile) = 2:1), centrifuged for 5 minutes to 12000rpm.

Take 0.3µL alligator collagen peptide powder sample solution plus 0.3µL TFA matrix solution directly after mixing the sample points, each point 0.6µL.
MALDI-TOF MS with the molecular weight determination.
Example 1

After taking fresh alligator 100g, scrape fat, cleaning, cut side length of about 2 cm square pieces, adding 1L of distilled water in a rice cooker and cook 30 minutes after cooling to room temperature, mashed.

Based on the weight of added alligator 4% alkaline protease (derived from Bacillus licheniformis alkaline protease enzyme, Ltd. Zaozhuang Jarrow), at pH11, 40 ? temperature hydrolysis of 3 hours, heated to boiling for 10 minutes so that the enzyme inactivated.

At room temperature and then centrifuged at 4000rpm for 20 minutes, the supernatant was lyophilized to obtain powder crocodile skin collagen peptide.
Figure 4 shows the MALDI-TOF MS spectra obtained in Example 1 alligator collagen peptides.

Molecular weight obtained in Example 1 alligator collagen peptide powder cases of 1.2KD.

Smaller molecular weight, indicating that when the alkaline protease enzyme fully processed.

Hydroxyproline in collagen-specific amino acid hydroxyproline determination of collagen content reflects.

With hydroxyproline test kit (Nanjing Jiancheng Bioengineering Institute) measuring hydroxyproline content by alkaline solution.

Measured hydroxyproline content 6.2µg/mg, described the resulting high alligator collagen peptide powder collagen content.

Crocodile skin collagen peptide powder in water by UV full wavelength around 220nm after scanning absorption peak, which is characteristic of collagen absorption.

Example 2

Fresh alligator 500g, scrape fat, cleaned, cut into small pieces, 5L of distilled water was added 30 minutes after boiling cooling to room temperature, crushed in a rice cooker.

Based on the weight of added alligator 4% alkaline protease (derived from Bacillus licheniformis alkaline protease enzyme, Ltd. Zaozhuang Jarrow), at pH11, 40 ? temperature hydrolysis of 3 hours, heated to boiling for 10 minutes so that the enzyme inactivated.

Then centrifuged at 4000rpm at room temperature for 20 minutes, and the supernatant, spray-dried alligator collagen peptide powder.

Alligator molecular weight collagen peptide powder obtained was 1.2KD.

Smaller molecular weight, indicating that when the alkaline protease enzyme fully processed.

Measured by hydroxyproline content 6.2µg/mg.
Crocodile skin collagen peptide powder in water by UV full wavelength around 220nm after scanning absorption peak.

Example 3: Application of the collagen peptides alligator moisturizing effect in vitro

Humectant moisturizing properties according to the difference of the force of different humectants different water molecules, the ability to absorb moisture and retain water is also different.

The samples were placed in water containing a constant temperature and humidity of the desiccator, weighed regularly to reduce the sample mass through comparative analysis of the different samples to compare the size of the moisture.

In order to better simulate the actual conditions of the skin, 3M tape affixed to the slides, each test substance (distilled water, respectively (negative control), 0.3% hyaluronic acid (positive control), prepared as in Example 1 crocodile embodiment skin collagen peptide and shark skin collagen peptide) in aqueous solution (solid-liquid ratio of 1:4) coating on it, put it filled with saturated ammonium sulfate solution (relative humidity 85%) of the dryer in every one ~ 2 hours were weighed to calculate the rate of water loss in the period, and the results are shown in Table 2.
Water loss rate formula is:

Water loss rate = (m0-mt) / m0 × 100%

In which the sample is placed t mt water quality of the water quality of the initial hours after, m0 is the sample.

Table 2 shows that, compared with other test substances, the rate of water loss per unit time of the present application prepared in Example 1 alligator collagen peptide and dehydration interval smaller sum.

Crocodile skin collagen at the first 6-7 hours due to the lost moisture in the negative control, the rate of water loss per unit of time than the present application peptide prepared in Example 1 is small, this also shows that prepared in Example 1 of the present application alligator collagen peptide can be more durable and stably retain moisture.

Table 2 compares the test object table outside the moisturizing effect

Example 4: The application alligator collagen peptides in vitro ability to protect against DNA damage

To study prepared in Example 1 alligator collagen peptides in vitro by the hydroxyl radical induced DNA damage protective effect, we constructed a hydroxyl radical DNA damage in vitro model.

Prepare the experimental group and the control group without injury damage control samples:
Experimental sample preparation: 1µL were added to pET-32aDNA, 2µL phosphate buffer solution (PBS) (50mM, pH7.4), 5µL alligator different concentrations of collagen peptide (respectively 5mg/mL, 1mg/mL, 0.1 mg / mL), 1µL8mM FeSO4 and 1µL8.8mM H2O2;

Sample preparation without injury control group: the turn to join 1µL of pET-32aDNA and 9µLPBS (50mM, pH7.4);

Prepare damage control samples: in order to join 1µL of pET-32aDNA, 7µLPBS (50mM, pH7.4), 1µL8mM FeSO4, 1µL8.8mM H2O2.

After mixing each of the samples were incubated for 30 minutes 37 ?, and then electrophoresed on 0.8% agarose gel electrophoresis conditions are provided: control the voltage is not higher than 5V/cm (voltage value V / bipolar plate of the electrophoresis distance between), when the front end of the lane bromophenol blue ran from 2/3 to stop electrophoresis.

Lane 1 collagen peptide containing alligator 5mg/mL, lane 2 contains 1mg/mL alligator collagen peptide, lane 3 with alligator collagen peptide 0.1mg/mL, lane 4 is damage control, lane 5 No injury control group.

The results are shown in Figure 5.

Figure 5 shows: the plasmid DNA was only added without damage control (lane 5) the results of electrophoresis of DNA and the presence of ring-opening supercoiled DNA.

Damage control group (lane 4) in FeSO4 and H2O2 produces hydroxyl radicals, on supercoiled DNA damage, so that it becomes an open-loop DNA, thus electrophoresis only one band open loop DNA.
Alligator different concentrations of collagen peptide (lanes 1-3) of the electrophoresis results can be seen, a high concentration of collagen peptide alligator scavenge free radicals and thus can well protect DNA from damage.

Experiments show that alligator collagen peptides in vitro ability to protect DNA from damage.
Example 5: alligator collagen peptide of this application on mouse B16 melanoma cells whitening effect

1, crocodile skin collagen peptide on mouse B16 melanoma cell proliferation

With RPMI1640 medium (containing 10% newborn calf serum, penicillin 100U/mL, streptomycin 100µg/mL), CO2 incubator at 37 ?, CO2 = 5%, and the cells were cultured in a humidified condition.

When the cells were grown to near confluency, after 0.25% trypsin, the cells were collected and adjusted to a concentration of 104/mL in 96-well cell culture plate.

Each hole by adding 200µL of mouse B16 melanoma cells single cell suspension overnight.

When the cells adherent discard the original medium, were added with 0,12.5,25,50,100,200,500,1000 µg / mL of the application obtained in Example 2 alligator collagen peptide RPMI1640 medium 200µL.
After cultured for 48h discard broth.

At the end of 4 hours, with pH7.4 PBS buffer (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 2mM KH2PO4) and washed two times, each well was added 20µL of 0.5mg/mL MTT and 180µL of fresh RPMI1640 culture medium, After the CO2 incubator at 37 ?, CO2 = 5% and saturated humidity for 4 hours the cells, the supernatant was discarded.

Was added to each well 200µL DMSO, at 37 ? for shock 10 minutes, so that blue-purple formazan crystals dissolved completely.

Measured immediately 570nm absorbance with a microplate reader.

Each concentrations three replicates averaged.
Each experiment were taken in the same passage cells.
The results shown in Figure 6.

Figure 6 shows: on mouse B16 melanoma cell proliferation 0 ~ 200µg/mL alligator collagen peptide had no effect, indicating no cytotoxicity.

2, the impact of crocodile skin collagen peptide in mouse B16 melanoma cells melanin content

Cell culture methods above.

When the cells were grown to near confluency, and 0.25% trypsin, and cell concentration was adjusted to collect, 2 × 104 were seeded in 6-well plates overnight, the medium was changed to be adherent, were added with 0,12.5,25,50,100 , 200µg/mL alligator collagen peptide medium 2mL.

After cultured for 48 hours discard broth.

Washed with PBS buffer, pH7.4 twice 1.0mL10% DMSO was added a solution of 1.0M NaOH, 80 ? for 2 hours, 1000g centrifugation for 10 minutes, the supernatant was measured at 405nm absorbance.
Each concentrations three replicates averaged.
Each experiment were taken in the same passage cells.
The results are shown in Figure 7.

Figure 7 shows that: in the concentration range of 0 to effect 200µg/mL alligator collagen peptide under the mouse B16 melanoma cells was inhibited melanin content, and showing a concentration-dependent inhibition.

3, crocodile skin collagen peptide tyrosine activity of B16 melanoma cells in mice

Cell culture methods above.

When the cells were grown to near confluency, and 0.25% trypsin, and cell concentration was adjusted to collect, 2 × 104 were seeded in 6-well plates overnight, the medium was changed to be adherent, were added with 0,12.5,25,50,100 , 200µg/mL alligator collagen peptide medium 2mL.

After cultured for 48 hours discard broth.

Washed with PBS buffer, pH7.4 2, the volume fraction of each well of PBS buffer 900µL 1% TritonX-100, and then added 100µL1.0mg/mL L-DOPA, sonicate for 2 minutes, followed by culture at 30 ? 30 minutes, and the absorbance was measured at 475nm.

Each concentrations three replicates averaged.

Each experiment were taken in the same passage cells.

The results are shown in Figure 8.

Figure 8 shows that: in the concentration range of 0 to effect 200µg/mL alligator collagen peptide, the tyrosine kinase activity of mouse B16 melanoma cells was inhibited and exhibits concentration-dependent inhibition.

4, the impact of crocodile skin collagen peptide in mouse B16 melanoma cells reactive oxygen species (ROS) content

Cell culture methods above.

When the cells were grown to near confluency, and 0.25% trypsin, and cell concentration was adjusted to collect, 2 × 104 were seeded in 6-well plates overnight, the medium was changed to be adherent, were added with 0,25,50,100,200 µg / mL collagen peptide alligator medium 2mL.

After cultured for 48 hours discard broth.

With PBS buffer pH7.4 were washed two times, cells were trypsinized and collected in a 1.5mL tube Ep.

Then wash the remaining cells in 1mL PBS once, Ep collected into the same tube, 800g centrifugal six minutes, the supernatant after the cells were resuspended with 1mL PBS, centrifuged again to the supernatant.

After the cells were resuspended in 1mL PBS, adding 10mM probe DCFH-DA (dichloro-fluorescein diacetate) 1µL, incubated for 30 min at 37 ?.

The supernatant was then centrifuged, resuspended cells with 1mL PBS.

Flow cytometry to FS (forward scattering intensity) and SS (side scattered intensity) as a parameter to select live cells, which was defined in the Gate (gate), the cells Gate (gate) for ROS in the analysis.

The results are shown in Figure 9.

Figure 9 were different concentrations of crocodile skin collagen peptide in the experimental group (25,50,100,200 µg / mL of crocodile skin collagen peptide) compared to the control group (0µg/mL alligator collagen peptide) effects chart .

The shaded portion represents the control group.

As can be seen from Figure 9 relative to the control group, the experimental group significantly the curve to the left.

And the greater the concentration, the more obvious the left, reflecting lower ROS levels, indicating crocodile skin collagen peptide in mouse B16 melanoma cells reactive oxygen species (ROS) has the ability to clear, and in a concentration-dependent manner.

Crocodile skin collagen peptide concentrations higher ROS scavenging ability is stronger.

These results suggest that alligator collagen peptides of the present application on mouse B16 melanoma cells without cytotoxic, and can suppress levels in mouse melanoma B16 melanoma cells, inhibition may be mainly through scavenging reactive oxygen species and inhibition of tyrosine activity to be achieved.

Therefore, crocodile skin collagen peptide of the present application has a good whitening effect, can be used as a whitening cosmetic additives.

In summary, the above is only preferred embodiments of the present application only, not intended to limit the scope of the present application, therefore, where any modifications within the spirit and principle of the application made by the equivalent replacement, or improvement should be included within the scope of protection of the present application.



Composition for crocodile collagen antibacterial peptide and preparation method thereof
CN103239705

The invention discloses a composition for crocodile collagen antibacterial peptide and a preparation method thereof and aims to provide a composition which has a good curative effect on treatment of arthritis and other osteoarticular diseases. According to the technical key points, the composition is prepared from the following raw materials in percentage by mass: 1-90 percent of crocodile collagen antibacterial peptide, 1-50 percent of D-glucosamine and 1-50 percent of chondroitin sulfate, wherein the sum of the content of the components is 100 percent. The preparation method comprises the following steps: 1) weighing the crocodile collagen antibacterial peptide, D-glucosamine, chondroitin sulfate, maltodextrin and an adhesive; and 2) preparing the weighed crocodile collagen antibacterial peptide and D-glucosamine into fine particles through a traditional spraying method, mixing the fine particles with chondroitin sulfate particles, fully stirring, and adding the maltodextrin and the adhesive. The invention belongs to the technical field of biological medicine.

Technical field

The present invention relates to an antimicrobial peptide crocodile collagen composition of the present invention also relates to a method for preparing the composition, the field of biotechnology medicine.

BACKGROUND

Collagen mainly in bone, cartilage, skin and tendon tissue in animals, accounting for 25% of mammal itself contained to 35% of the total protein plays a role in the bracket and protection.

Collagen polypeptide is a collagen hydrolyzate, the molecular weight is much lower than that of collagen, which peptide has been disconnected, can be directly absorbed by the body to use.

Currently, a large number of studies show that the hydrolysis products of bone collagen peptides obtained by proteolysis with a variety of animal biological activity, e.g., lowering blood pressure, anti-oxidation, the prevention and treatment of arthritis, and anti-aging.

Antimicrobial peptides are involved in innate immunity, with a broad spectrum antimicrobial active substances, abundant in plants and animals, has antibacterial and strong, safe, non-toxic, water-soluble, good thermal stability, a wide range of effects, etc., but also health effects.

Crocodiles and dinosaurs, is one of the oldest animals on the planet, there is a "living fossil".

Crocodile is also one of the longest life expectancy of animals on Earth, life of up to two hundred years old, is longer than the tortoise and turtle.

In addition, the crocodile is the largest animal on Earth shape, currently found in Africa's largest crocodile body length of 12 meters and weight up to 8 tons.

Necessarily need such a large body of super bone to support.

The study found that the crocodile bone to bone hardness than hard times, often require the use of the processing characteristics of alloy steel imported from Germany.

The study found that crocodile bone meal contains a lot of calcium and phosphorus as well as the activity of collagen, etc., can also be used for the prevention and treatment of osteoporosis, calcium deficiency, such as infants and young children.

Meanwhile, the crocodile tail collagen rich in pectin, which can effectively prevent osteoporosis, and soothing beauty effect.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a method for improving human bone joints and preparation of biological products of the composition.

In order to solve the above problems, the first aspect of the present invention, a provision is this: the crocodile collagen compositions of antimicrobial peptides, the mass percentage of material comprising: an antimicrobial peptide crocodile collagen?

90%, D-glucosamine 1?
50%, chondroitin sulfate, a?
50%, of the components is 100%.

Preferably, the collagen compositions of antimicrobial peptides crocodile, the composition comprising a raw material mass percentage: 10 crocodile antimicrobial peptides of collagen?
80%, D-glucosamine 5?
40%, chondroitin sulfate, 5?
40%, of the components is 100%.

Better, the crocodile antimicrobial peptides of collagen composition, the composition comprising the mass percentage of raw material: crocodile collagen antimicrobial peptides 25?
65%, D-glucosamine 10?
30%, chondroitin sulfate, 10?
30, of the components is 100%.

One aspect of the present invention is to provide such a: preparation of the compositions of antimicrobial peptides crocodile collagen, comprising the following steps in sequence: 1) Weigh crocodile bone, grinding, water was added at a mass ratio of 1:1, After boiling placed to maintain 15min 4 ? after cooling, the solidified fat on top of the cooled removal, preparation of bone cement; 2) bone cement with distilled water mass ratio of 1:1 into the flask, placed in a constant temperature water bath enzymatic 1-5h; 3) after enzymatic hydrolysis, in 80 ~ C water bath for 20min to inactivate the enzyme; 4) will destroy the enzyme digestion solution was cooled to room temperature after removal of non-hydrolyzed bone particles, and then 4 ?, 10000r/min centrifuged 15min, to remove solids, the supernatant was collected, freeze-dried using a vacuum freeze drier to a powder for use.

Crocodile above for preparing antimicrobial peptides of collagen composition, wherein the step 2) of the enzymatic complex used in the animal enzyme is a protease or protease or alkaline protease or flavor papain or pepsin, or neutral or one of the proteases trypsin; Step 2) when the reaction temperature is 37-60 / ?; Step 2) When the enzyme was added to keep the pH value of the solution to maintain the pH at 3-8 between; Step 2) the quality of the enzymatic hydrolysis with bone cement ratio :1:0.005 -0 .01

Compared with the prior art, the invention has the following advantages: The present invention crocodile bone collagen is extracted, having a good effect in improving the treatment of arthritis and other diseases of bones and joints, for the treatment of bone and joint diseases, provides a new way .

Brief Description

Figure 1 is a degree of hydrolysis of seven kinds of the enzyme treatment time trend;

Figure 2 is an enzyme addition on the effects of different degree of hydrolysis.

Where: Animal protamex 1; Flavourzyme 2; trypsin 3; alkaline protease 4; neutral protease 5; pepsin 6; papain 7.

Specific embodiments

Hereinafter with reference to specific embodiments, further detailed description of the invention according to the requirements for the work, but does not constitute any limitation of the present invention, any person to do a limited number of modifications are within the claims of the invention scope is still present invention. protection within the scope of the claims.

Example 1

An alligator antimicrobial peptides of the present invention, the collagen composition comprising the ingredients: antimicrobial peptides of collagen crocodile 1g, D-glucosamine 49g, chondroitin sulfate 50g.

Example 2

An alligator collagen antimicrobial peptides of the present invention is a composition comprising the following ingredients: antimicrobial peptides of collagen crocodile 90g, D-glucosamine 5g, chondroitin sulfate 5g.

Example 3

An alligator collagen antimicrobial peptides of the present invention is a composition comprising the following ingredients: antimicrobial peptides of collagen crocodile 50g, D-glucosamine 20g, chondroitin sulfate 30g.

Example 4

An alligator antimicrobial peptides of the present invention, the collagen composition comprising the ingredients: antimicrobial peptides of collagen crocodile 1g, D-glucosamine 49g, chondroitin sulfate 50g.

Example 5

1 Materials and methods

1.1 Materials and Reagents

Crocodile bone provided by Guangxi Qinzhou Histon Aquaculture Development Co. Wuchuan special branch.
Pepsin (8.6 × 10 U / g), trypsin 05.2 × 10 U / g), papain (2.5 × 104U / g), agar Sinopharm Chemical Reagent Co., Ltd.; alkaline protease (9.2 × 104U / g), neutral protease (6.2 × 10 U / g), flavor protease (4.2 × 10 U / g), animal protamex (8.9 × 10 U / g) Novozymes (China) Investment Co., Ltd.; Staphylococcus aureus and enteritis Salmonella China Food Fermentation Industry Research Institute; nutrition and brain heart infusion broth medium Beijing Land Bridge Technology Co., Ltd..

1.2 Instruments and Equipment

Incubator (Sanyo, Japan); Bone Crusher (Friends of Langfang, Hebei Machinery Corporation); LGJ-30 vacuum freeze dryer (Beijing Fourth Ring Scientific Instrument Co., Ltd.); Oxford Cup (Xinxiang City, Henan Xinhua detection Instrument) ; electric heated water bath (Beijing Medical Equipment Factory); two biological safety cabinet (Singapore Esco Technologies Ltd.).

1.3 Methods

1.3.1 crocodile collagen hydrolyzate

Weigh 200g crocodile bone, trimmed, cleaned and crushed into small particles, water is approximately the size of 2mm, an equal quality, maintaining the boiling 15min, then set at 4 ? ambient cooling, the oil solidified on cooling after the upper removal, repeated twice, to obtain experimental bone cement.

200g bone cement is then added to distilled water, placed in a constant temperature water bath digestion.

7-protease (protease complex animal flavor protease, alkaline protease, papain, pepsin, trypsin and neutral protease) digestion.
Enzymatic hydrolysis of each bone crocodile employed pH and temperature conditions are shown in Table 1, the bone quality of the water used in hydrolysis ratio is 1:1.

Reaction process to continue adding acid to maintain the pH of the solution.

After hydrolysis, at 80 ? water bath for 20min to inactivate the enzyme.

Table 1 protease enzyme crocodile bone pH and temperature conditions

1.3.2 determine the time each Protease

Water mass ratio of 1:1 in the bone, which (as a percentage of bone mass) with an amount of 0.5% of enzyme under the conditions, to determine the optimal hydrolysis time seven kinds of proteases.
1.3.3 to determine the amount of each of the protease enzyme

The bone and the water mass ratio of 1:1 for 4h protease hydrolysis conditions, comparing the amount of enzyme (% of bone percentage) of 0.50%, 0.75%, 1.00%, 1.25%, 1.50% when the degree of hydrolysis identify seven kinds of enzymatic hydrolysis of collagen protein crocodile optimal enzyme dosage used.

1.3.4 Separation drying

The enzymatic hydrolysis was destroyed after cooling to room temperature, remove unhydrolyzed bone particles, and then 4 ?, 10000r/min centrifuge 15min, remove solids, the supernatant was collected by vacuum freeze-dried into a powder freeze drying machine spare .

1-3.5 protein determination methods

Using GB 5009.5-2010 "Determination of Protein Foods" Kjeldahl method.

1.3.6 Determination of amino acid nitrogen

Pipette 5g two enzymatic supernatant were placed 250mL Erlenmeyer flask, add water, 40mL; one of which add 3 drops of neutral red indicator, 0.100mol / L NaOH solution was titrated to amber as the end point, then consumption NaOH solution is referred to as volume V1.

Another added 3 drops of neutral formaldehyde l0mL and Barry phenolphthalein indicator, shake, standing l min.

Then 0.100mol / L NaOH solution was titrated to a pale blue, the volume of consumption of NaOH solution is denoted by V2.

After titration using equation (1) is calculated.
Amino nitrogen content /% = (V2-V1) × N × 0.014 × 100 / m (1)

Where: N NaOH standard solution was equivalent concentration; m is the mass of the sample solution rather sample, g.

1.3.7 Determination of the degree of hydrolysis

The degree of hydrolysis (DH) as an indicator of screening hydrolyzate, using equation (2) calculation of DH.

DH /% = enzymatic liquid form of free amino acid content × 100 / enzymatic solution, total nitrogen content (2)

Preparation of 1-3.8 bacterial suspension

Saved inoculated into the nutrient broth agar medium, and cultured at 37 ? for 24h, two successive activation, the activated species of 10-5 sequentially diluted with saline to prepare a bacterial suspension.

1.3.9 Determination of antibacterial activity

Oxford cup method was determined by enzymatic liquid antibacterial activity.

The medium was poured into the dish, 0.1mL draw bacterial suspension was added to the plates, coating evenly.

Each tablet put 3 cups of Oxford.

After the dissolution of the enzyme solution with 0.45um membrane filter sterilized and then added 1.50um suitable concentration of enzyme solution in each cup of Oxford, separate blank.

Placed in 37 ? incubator, the size of the zone of inhibition was measured after 24h.

Each experiment was repeated three times.

2 Results and analysis

2.1 The Protease crocodile determine the optimum hydrolysis time of bone

Figure 1 shows that, under the action of seven kinds of enzymes, with time, the degree of hydrolysis basically stabilized trend emerged after a first rise, the reason may be asked to extend the hydrolysis with one hand collagen content of the substrate is reduced enzyme, the other is decreased enzyme activity.

Water mass ratio of 1:1 in the bone, which (as a percentage of bone mass) amount of enzyme used as the conditions under 0.50%, the composite animal protease, alkaline protease pepsin and have a high degree of hydrolysis.

Animal compound protease hydrolysis of pepsin, trypsin and protease flavor 4h substantially no increase after determining the optimal hydrolysis time 4h these four kinds of enzymes; degree of hydrolysis of the alkaline protease, neutral protease and papain in Basically not increase after 5h, 5h therefore determine the optimum hydrolysis time these three enzymes.

2.2 The determination of the amount of protease
Figure 2 shows that, when a substrate concentration, amount of enzyme substrate is not saturated with the increase of the amount of enzyme added, increasing the degree of hydrolysis; saturated amount of enzyme substrate, along with the increasing amount of enzyme , substantially no change in the degree of hydrolysis.

At the same concentration of substrate and hydrolysis time, the composite animal with the highest degree of hydrolysis of the protease, pepsin followed.

Animal compound protease, pepsin, papain, protease and flavor addition level of 1.25%, after the hydrolysis stabilized variation, determining the optimal amount of 1.25% of these four enzymes; alkaline protease, trypsin and neutral protease dosage 1.00% after changing the degree of hydrolysis stabilized, and therefore determine the best dosage of 1.00% for the three kinds of enzymes.
By comparing Figures 1, can be seen in the same hydrolysis conditions, the composite animal with the highest degree of hydrolysis of the protease, and alkaline hydrolysis of the protease pepsin is higher, while the hydrolysis of neutral protease and papain degree low.

2.3 inhibitory effect of different antimicrobial peptides hydrolyzate

In the seven kinds of the enzyme and the optimum amount of hydrolysis time crocodile hydrolyzed collagen, hydrolyzed by centrifugation and freeze dried, and measured inhibitory effect on Staphylococcus aureus and Salmonella enteritidis.
The antibacterial activity of different peptides experimental protease enzyme obtained results in Table 2.

Table 2 various protease enzyme degrees bacteriostatic activity of the two size

From Table 2, the flavor and neutral protease enzymatic solution Staphylococcus aureus has antibacterial effect, the inhibition zone diameter was 6.03mm and 7.97mm; animal protease complex flavor and trypsin for Salmonella enteritidis with antibacterial effect, the inhibition zone diameter were 8.67,9.10,9.03 mm, visible, flavor protease enzyme solution to this bacterium has the highest antibacterial activity.

About inhibitory mechanism of antimicrobial peptides, generally considered to be antimicrobial peptides will destroy the bacterial cell membrane or cell wall, causing the cell contents spill leaving bacterial death m.

Staphylococcus aureus used in this study belongs to Gram-positive bacteria, Gram-negative bacteria are Salmonella enteritidis, therefore, can be judged, animal compound and trypsin hydrolysis crocodile collagen antimicrobial peptides may get only leather Gram-negative bacteria play a role; neutral protease hydrolyzed collagen obtained crocodile antimicrobial peptides may only against Gram-positive bacteria play a role; while Flavourzyme inhibitory peptide may get Gram-positive bacteria and negative bacteria are works.

3 Conclusion

In this study, antimicrobial peptides biological enzyme way to get the optimum temperature and pH on the enzyme, the bone under water ratio of 1:1, using animal protease enzyme complex, the degree of hydrolysis resulting hydrolyzate the highest, the best hydrolysis time was 4h, the optimum amount of enzyme added (as a percentage of bone mass) of 1.25%.
Neutral protease enzyme liquid collagen solution obtained crocodile Staphylococcus aureus has the highest antimicrobial activity, protease enzymatic hydrolysis liquid flavor crocodile collagen obtained for Salmonella enteritidis has the highest antibacterial activity.

This study developed a natural preservative provides a theoretical reference, and for the future of collagen antimicrobial spectrum antimicrobial peptide experiment, purification and structural analysis provides a reliable basis.

D-Glucosamine:

The latest medical study found that a lack of body Glucosamine will directly lead to the occurrence of various bone and joint diseases, and the process of Glucosamine loss in people 35 years of age had begun.
Glucosamine is not only controls the body's bone and joint health, but also controls the metabolic balance synovial articular cartilage.

Glucosamine can be a lot of birth for the body and replenish synovial fluid, so as to continuously lubricate the articular cartilage surface, reducing wear and tear, so that the joints flexible.
Add enough of synovial fluid, articular cartilage also provides, enough material carriers.

Glucosamine by strong stimulation of chondrocytes, synthesis of human collagen and hyaluronic acid, has been worn continuously repair articular cartilage, and can generate a new articular cartilage and synovial membrane, thereby restoring the normal function of joints and incompetence inability to function.

Meanwhile, the damage to the articular cartilage cells long-term use of anti-inflammatory and analgesic drugs caused, but also has a good repair.
Glucosamine is a joint cavity, "scavenger", not only to suppress non-specific inflammatory factors, relieve pain, and can eliminate intra-articular harmful enzymes, the body's joints and improve immunity.

Bring to enhance immunity by supplementing Glucosamine is an important prerequisite for the elimination of arthritis.

Procurement of raw materials can be extracted from Qingdao Green Biotechnology Co., Ltd., Zhejiang Aoxing Biotechnology Co., Ltd., many domestic companies.

Chondroitin sulfate:

Chondroitin sulfate is a glycosaminoglycan, the D-glucuronic acid and N-acetyl galactosamine in ß-1, 4 - glycosidic bonds repeating disaccharide units composed of polysaccharides connected together, and N - the half-acetamido lactose or C-4 position of the C-6-hydroxyl acid esterification.
Chondroitin sulfate is isolated from animal cartilage mucopolysaccharide substances has an important role in cardiovascular diseases, prevention and treatment of joint diseases; while chondroitin sulfate protective effect on bone, prevent bone hard and brittle, to prevent calcification increased, making the rigid skeleton recovery flexibility, to restore bone hyperplasia have a greater help to narrow a range of hard and brittle bones outline presents an expanded state.
Application as a health food in the U.S. has been popular for many years.

After years of use, chondroitin sulfate has been shown to improve degenerative arthritis, rheumatoid arthritis have some effect, so the market continued to show a rapid increase in momentum.

Consumers in the U.S. alone amount of up to about 600 tons annually.

China is the largest chondroitin sulfate raw material producing countries, domestic Shandong, Hebei is the largest distribution center.
Raw materials can be sourced from Jiaxing Jie biological, Hebei Sanxin Group, Yantai Dongcheng Biochemicals, Qingdao Green-Extract Biology, Qingdao Jiulong biological, Sinochem (Qingdao) Co., and other companies.



Composition of crocodile hemoglobin peptide and preparation method of composition
CN103230587

The invention discloses a composition of crocodile hemoglobin peptide and a preparation method of the composition, and aims at providing a composition which has the advantages of reinforcing the human body immunity and the oxidization resistance, reducing the formation number of radicals in a human body and improving the cardiovascular disease. The composition is technically characterized by comprising the following raw materials by weight percent: 1-90 percent of crocodile hemoglobin peptide, 1-50 percent of protein derived from crocodile meat and 1-50 percent of coenzyme Q10, and the percent sum of all components is 100 percent. The preparation method comprises the steps of: 1. weighing the crocodile hemoglobin peptide, the protein derived from crocodile meat, the coenzyme Q10, maltodextrin and an adhesive; and 2. preparing the weighed crocodile hemoglobin peptide and protein polypeptide derived from crocodile meat through a conventional spraying method into fine particles, then mixing with the coenzyme Q10 together, fully mixing, and adding the maltodextrin and the adhesive. The composition belongs to the technical field of biological medicines.



Crocodile meat protein antioxidative peptide powder and preparation method thereof
CN102793052

The invention discloses crocodile meat protein antioxidative peptide powder which belongs to the field of functional food. The invention further discloses a preparation method of the crocodile meat protein antioxidative peptide powder, comprising the following steps: grinding crocodile meat into a meat paste, then adding water and conducting homogenate; adding compound protease according to a weight ratio of the compound protease to the crocodile meat paste being 1:500-1000 to conduct enzymatic hydrolysis reaction, wherein the compound protease comprises papain, AS1398 neutral protease and flavourzyme; and carrying out centrifugation and filtration on the hydrolysate, and drying the filtrate. According to the invention, the crocodile meat protein antioxidative peptide powder disclosed herein has strong antioxidant property, excellent processing characteristics, high dissolvability, and high emulsification activity; in addition, the crocodile meat protein antioxidative peptide powder disclosed herein is prepared by using the food-grade compound protease, thus the organoleptic properties are excellent, the enzymatic hydrolysis product is creamy white, the whole color is consistent, and there is no foreign odor; and the safety is high, and the cost is low.





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