Peptides Production method

Peptides Production method

1)Development process

With the development of technology, the method of producing peptides is also constantly developing. In the 1950s and 1960s, people get peptides from animal organs. For example, thymosin is produced by slaughtering the newly born calf, cutting its thymus. It uses the biotechnology of shock separation to separate the peptides in the calf thymus and make a thymosin injection. Thus,human usually use thymosin peptide in human immunity. Nowadays, this peptide is in a phase of elimination. There was a time spreading “mad cow disease” in the world.

This virus mainly phagocytizes proteins in the animals’ brain and destroys brain tissue, cells and nerves.

Once infected with the virus, it is more terrible than cancer, and it may results in becomes a “plant man” or dies soon. There are some  human blood extracted peptides , which side effects are very huge. It is not only easy to have the human body to be infected with hepatitis A, hepatitis B/ hepatitis C/hepatitis D/ HIV/sexually diseases virus, but also has an allergic reaction. Once this allergic reaction appears, life is at stake.

2)Several Peptides synthesis methods

  • Solid phase synthesis, liquid phase synthesis

There’s one company in Silicon Valley that produce peptides in this method. They mainly purchase amino acids produced by some fine chemical plants in the world.Then start to synthesize a single peptide. They are intermediates for pharmaceutical raw materials. we use western medicine formulas to enhance the efficacy and enhance the rate of absorption .the human body can absord.

  • Acidolysis or alkaline hydrolysis

This peptide is mainly found in Japan. The “soybean peptide” produced by the acid hydrolysis method is a “food additive” mainly used for foods for the elderly and children, and its purpose is to enhance the absorption of food by these two groups of people. The only countries in the world that have “soybean peptides” are Japan and China. However, the raw materials, hydrolysis methods and product properties used are quite different. The raw material used in Japan is soybean meal, and the hydrolysis method is acid method. The peptide acid chemical substance produced is difficult to be removed, and it has a bitter taste. It needs to be adsorbed and debittered by activated carbon, and activated carbon inevitably invades the peptibosome.

  • Enzymatically produced bioactive peptide

Enzymatically produced bioactive peptides. They produced the peptide in China.Also they represents the level and trend of peptide research, development, production and innovation in the world today. The enzymatically produced bioactive peptide is a food-grade plant protease. The human body into a small-molecule active polypeptide normally eat this kind of protein . It has strong biological activity and diversity, has attracted attention around the world, and has become a hot spot in the world today. For example, the Wuhan Jiushengtang produced  “soybean peptide” ,  uses the soy protein isolate with a content of more than 90%.

The hydrolysis method is enzymatic method, and the enzyme used is a food-grade plant protease. The “soybean peptide” produced has no bitter taste, and has a pleasant flavor and has no toxic side effects on the human body. The product is a terminal product and has been listed as a free-funded project by the China Innovation Fund. It has achieved industrial production. Its terminal product “Soy Peptide” Oral Liquid is the world’s first soy peptide end product produced by enzymatic method, in the 89th Canton Fair. It is favored by the world’s merchants.

  • Synthesis Process

The specific synthesis consists of the following cycles:

1)Remove protection

The Fmoc protected column and monomer must be protected with an amino protecting group by a piperidine.

2)Activation and cross-linking

The carboxyl group of the next amino acid is activated by an activator.Chemical process commonly used HBTU/HCTU/HITU/HATU+NMM/DIPEA or HOBT+DIC as activator. The activated monomer reacts with the free amino group to form a peptide bond. In this step, a large amount of super-concentration reagent is used to drive the reaction to completion. Cycle: The two steps of the reaction are repeated until the synthesis is complete.

After the end of the amino acid condensation, the appropriate amount of TFA can be used for elution. According to the acidity and alkalinity, a 10% TFA/DCM solution can be selected to 100% TFA, and so on. (Note: This method is personal synthesis experience, there is no literature available for reference; can be referred to as needed. Knowledge is limited, hope to be cautious)

3)HPLC analytical purification

Analytical HPLC uses a column and pump system that can withstand high pressures, allowing the use of very fine particles (3-10 μm). Thus we analyze  the polypeptide within a few minutes. we divide HPLC into two categories: ion exchange and reversed phase. Ion exchange HPLC relies on direct charge interactions between the polypeptide and the solid phase.

A column carries a specific charge in a certain pH range and becomes a ionic body, while a polypeptide or a mixture of polypeptides exhibits opposite charges from its amino acid composition. Separation is a charge interaction that elutes a polypeptide by variable pH, ionic strength, or both, usually with a low ionic strength solution, which is then stepped up or stepped up until the peptide is eluted. An example of ion exchange separation uses a strong cation exchange column. For example, sulfoethylaspartimide is separated by positive charge in acidic pH.

Reversed phase HPLC conditions were the opposite of normal chromatography. The polypeptide is attached to the column by hydrophobic interaction and eluted with reduced ionic strength, such as increasing the hydrophobicity of the eluent. Typically the column consists of a hydrocarbon chain covalently adsorbed onto the silicon, this chain having a length of G4-G8 carbon atoms. Because elution is a hydrophobic effect. Long-chain columns are smaller than short-chain pairs, and high-charged peptides are good.

On the other hand, large hydrophobic peptides are eluted with short-chain columns. However, in general practice, there is not much difference between the two types of column transitions. The other types of carriers are composed of carbohydrates, such as phenyl.

Typical operations are often composed of two buffers, 0.1% TFA-H2o and 80% acetonitrile 0.1% TFA–H2o dilute acetonitrile. Mix with a linear ladder at a rate varying from 0.5% to 1.0% per minute. The columns for common analysis and purification are 4.6×250mm (3-10μm) and 22×250mm (10μm). If radial column is used, the size is 8×100 (3-10μm) and 25×250mm (10μm)

A large variety of buffers contain many different reagents, such as heptafluorobutyric acid, 0.1% phosphoric acid, dilute He formic acid (5-6%, pH 2-4), 10-100 mM NH4HCO3, sodium acetate/ammonia, TFA/TEA, sodium phosphate or Potassium, isovalerol. Many different combinations can form buffers, but one thing to note: Silicon reversed phase columns cannot be exposed to high pH or even slightly alkaline pH for extended periods of time because they can damage the column.

Do not confuse the peptide content and purity. The purity of the peptide may be 100%, and  the anti-ion amount of the charged group (such as Arg, Lys) determine the peptide content and the hydrophilicity of the peptide. This is the nature of the synthetic peptide itself.

  • Enzymatic peptide

An enzymatic polypeptide is a polypeptide got from protease degradation of a protein.

The polypeptide bean obtained by proteinase degradation of egg protein, milk protein, casein, fish protein, kiln protein and the like has the physiological function of promoting, enhancing and regulating immunity. When we introduce such enzymatic polypeptides into the circulatory system and human tissues, they can stimulate the body’s immune system to undergo a specific immune response.

After the polypeptide enters the human body, the body use it as an antigen, and can directly stimulate B cells to produce antibodies without the aid of T cells. However, when the polypeptide enters the human body, it can induce and promote T cell differentiation and maturation; stimulate B cells to produce and secrete immunoglobulin (antibody) to participate in the human immune response;

Improve the activity of natural killer cells (NK), kill tumor cells without antibody participation, exert broad-spectrum anti-tumor, anti-infective effects, participate in immune regulation; it is not easy to phagocytize pathogens  ,such as parasites, malignant Tumor cells, etc.;Also, stimulate the killing ability of N cells;

Stimulate the phagocytic ability of phagocytic cells, mainly improve their phagocytic function, secretory function, participate in immune response, immune regulation process, and anti-infection, anti-tumor;

Enhance red blood cell immune function; increase interleukin-2 Production level and receptor expression level of (1L-2);Enhance the production of peripheral monocyte Y-interferon; Enhance SOD activity in serum;Can significantly increase lymphocyte function;

It can effectively prevent radiation nuclear radiotherapy and chemotherapy and air, water, food, environmental pollution poisoning bean white blood cell reduction;Also can effectively inhibit the growth of tumor cells;What’s more it prevent the decrease of CD4+ caused by radiotherapy and chemotherapy of malignant tumors.In short, the enzymatic peptide is an edible immune agent, which can comprehensively enhance the immune function and immune regulation of the human body. Therefore ,it is a modern new immunizing agent and a treasure of modern immunity.

1)Origin of Enzymatic peptide 

We call the method of catalyzing a protein with a biological enzyme  is enzymatic method, and we call  polypeptide obtained by catalyzing a protein by a biological enzyme catalysis is an “enzymatic polypeptide”.

The term enzymatic peptide originally originated in 1996, and the peptide scientist Zou Yuandong has achieved great success with bioenzyme-catalyzed protein-derived peptides.At that time, the news of the People’s Daily’s overseas edition of the external report attracted worldwide attention.In the following 14 years, the mainstream media in China successively reported Zou Yuandong as a major example of China’s independent innovation.

In 2006, Guangming Daily reported on the topic of “Zou Yuandong and his enzymatic peptides” for three consecutive days. ” Science and Technology Daily” reported Zou Yuandong with a full-page version of 8000 words on the topic of “Zou Yuandong’s Independent Innovation Enzyme Peptide”. Xinhua News Agency Xinhuanet, People’s Daily People’s Daily Online, CCTV, China central television, CNR China radio network, People’s Liberation Army Daily, China Academy of Sciences, China Center for Disease Control, China Medicine News,The China Intellectual Property News ,reported on the deeds of Zou Yuandong’s independent innovation of enzymatic peptides in a prominent section. We  call  it ” the enzymatic peptide “for this purpose and is famous for this.

2)The Enzymatic peptide Characteristics

In tradition, too many ways to obtain peptides. The traditional methods mainly include: acid method, alkali method, electric method, artificial grafting method, gene expression method and the like. However, in terms of process technology, the limitations of these synthetic processes are responsible for the lack of products in these synthetic processes.

The enzymatic method has made breakthrough and innovation on the basis of traditional methods. And has adapted to the requirements of low-carbon economy and green environmental protection. Enzymatic method is to use a biological enzyme to catalyze a protein to obtain polypeptide, is a protein degradation and artificially synthesized polypeptide.

The enzymatic method is milder and more environmentally friendly than the acid method,  alkali method and electric method. The production process simple, investment low and effect fast. Suitable for industrial production. The representative products like Sanjiu protein peptide, Jiusheng brand bitter gourd polypeptide……

Use the enzymatic method obtains the polypeptide, the molecular weight is easy to control.  Itself has rich green properties, peptide has no bitter taste. and the peptide small molecular weight (the molecular weight is mostly below 1000). These small molecular peptides directly absorbed, needn’t to  digested. It has the power, carrier, transport, transmitter and nutritional functions, especially its strong activity and diversity, which is an important biological function.


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