ヒアルロン酸について知っておくべきことは?

ヒアルロン酸, also known as hyaluronan, is a viscous polysaccharide substance formed by the repeated alternation of (1–3) –2–acetamido–2–deoxy–β–D–glucose linked via β–1,4 and β–1,3 glycosidic bonds with (1–4) –O–β–D–glucuronic acid [1]. The molecular weight ranges from tens of thousands to millions. The molar ratio of N-acetylglucosamine to glucuronic acid in the molecule is 1:1, and its structure is shown in Figure 1 [2].

In 1937, Kendell et al. [3] extracted HA from the fermentation broth of a strain of bacteria. Since then, there has been extensive and in-depth research on the distribution, physical and chemical properties, chemical structure, production process, and applications in medicine, cosmetics, and health food.

1ヒアルロン酸の性質と分布

Hyaluronic acid has many properties in common with other viscous polysaccharides: it is white, an amorphous solid at room temperature, odourless and tasteless, highly hygroscopic, slightly soluble in water and insoluble in organic solvents. However, hyaluronic acid has unique properties compared to other sugars. This is because the glycosidic bonds between glucuronic acid and N-acetylglucosamine on the straight chain axis of hyaluronic acid, hydrogen bonds and interactions with the aqueous solution cause hyaluronic acid to form a rigid helical column with a radius of 200 nm in space. On the one hand, the inner surface of the column is strongly hydrophilic due to the large number of hydroxyl groups; on the other hand, the continuous and directional arrangement of the hydroxyl groups also creates highly hydrophobic areas on the hyaluronic acid molecule chain. It is precisely the hydrophilic and hydrophobic properties of the HA molecule that allow a continuous three-dimensional honeycomb network structure to form even at HA concentrations below 10-3. Water molecules are fixed in the spiral column within this honeycomb network by polar bonds and hydrogen bonds with hyaluronic acid molecules, and are not easily lost. Therefore, hyaluronic acid can absorb about 1000 times its own weight in water like a molecular sponge, and is internationally recognized as the best moisturizer [4].

In nature, HA is widely distributed in various tissues of higher animals. HA has now been isolated from umbilical cords, skin, human serum, chicken combs, synovial fluid, brain cartilage, eye vitreous humor, chicken embryos, human urine, and arterial and venous walls. In mammals, the highest amounts of hyaluronic acid are found in the vitreous body, synovial fluid and umbilical cord [5], while the amount of HA in the comb of a rooster is similar to that in synovial fluid [6]. HA is bound to proteins and coexists with other mucopolysaccharides. It exists in a dissolved form in the vitreous body and synovial fluid, and in a gel form in the comb of a rooster and umbilical cord. Hyaluronic acid is not only found in the tissues of higher animals, but also in some bacteria. Bacteria with high HA content have been found, mainly Pseudomonas aeruginosa, Clostridium perfringens and Streptococcus hemolyticus types A, B and C [7].

2. ヒアルロン酸の応用

2.1化粧品におけるヒアルロン酸

Hyaluronic acid has a good moisturizing effect and is a natural moisturizing ingredient additive in high-end cosmetics. Because it is a natural factor widely present in human skin tissue and has good compatibility, it can be added to almost any beauty cosmetic, from the original creams, lotions, and lotions to the current bath salts, powders, lipsticks, etc. [8]. Its superior performance has attracted widespread attention from the international cosmetics industry. When cosmetics containing HA are applied to the skin surface, hyaluronic acid can form a viscoelastic hydrated film on the skin surface, which can smooth and moisturize the stratum corneum of the skin, and block the invasion of foreign bacteria, dust and ultraviolet rays, protecting the skin from damage. At the same time, hyaluronic acid can penetrate the epidermis of the skin, slightly expand the capillaries, increase blood circulation, improve intermediary metabolism, and promote the absorption of skin nutrients and the discharge of waste. In addition, hyaluronic acid has a strong wrinkle-reducing effect, increases skin elasticity, and delays skin aging. This is the dual effect of hyaluronic acid on skin moisturization and nutrition.

2.2医学におけるヒアルロン酸

Hyaluronic acid and its salts are the main components of connective tissue such as the human body's intercellular matrix, the vitreous body of the eye, articular cartilage, and synovial fluid of the joints [9]. Due to its unique physical and chemical properties and physiological functions, hyaluronic acid has been widely used in medicine. In the treatment of joint diseases, supplementing the body with exogenous hyaluronic acid can restore the lubricating function of synovial fluid, creating time for the diseased joint to repair itself, promote joint repair and functional improvement; in ophthalmic surgery, it can effectively support the anterior chamber after being injected into the anterior chamber, and can form a protective film on the surface of other tissues in the eye and the surface of surgical equipment to prevent mechanical damage to the equipment; in surgical operations, hyaluronic acid can be used to prevent postoperative adhesion and promote skin wound healing; as a medium, HA is also widely used in eye drops [10]. In addition, hyaluronic acid can be combined with other drugs to achieve targeted and timed release by using the targeting effect of HA receptors. Therefore, with the gradual development of medical technology, the application of hyaluronic acid in the medical field will become more and more extensive.

2.3食品産業におけるヒアルロン酸の応用

The amount of hyaluronic acid in the human body is about 18g, and it plays a vital role in the physiological activities of the human body. If the ヒアルロン酸コンテンツ in the joint cavity, blood vessels, heart, eyes, brain and other tissues and organs decreases, it may lead to diseases such as arthritis, atherosclerosis, pulse disorders and brain atrophy. Oral intake of hyaluronic acid can supplement the body&#haの39の欠如と体内のヒアルロン酸の欠如によって引き起こされる組織や細胞の老化を防ぎます。特に、高齢者の健康維持に大きな効果があります。ヒアルロン酸は、消化と吸収によって老化を遅らせることができ、肌に潤いを与え、滑らかで、柔らかく、弾力があります[11];同時に、経口ヒアルロン酸は、人々に活力と若々しい活力を与えます。現在、経口ヒアルロン酸美容健康食品はますます注目され、より多くの人々に受け入れられています。

3ヒアルロン酸の準備

主に2つありますmethods for preparing hyaluronic acid1つは動物組織から抽出する方法であり、もう1つは微生物発酵によって得る方法である[12]。

3.1組織抽出法

HA is widely found in the interstitial matrix of various animal tissues, such as skin, joint synovial fluid, cartilage, eye vitreous body, rooster comb, chicken embryo, etc. Among them, the human umbilical cord, rooster comb, joint synovial fluid and eye vitreous body have the highest hyaluronic acid content. The general method for extracting HA from tissues is as follows: animal tissue is first homogenized, then dissolved in water and a dilute salt solution. The solution is precipitated with hexadecylpyridinium chloride, and the resulting precipitate is dissolved in sodium chloride solution. The crude product is then precipitated with a threefold ethanol solution, and purified repeatedly using hexadecylpyridinium chloride and ethanol, or by gel and ion exchange chromatography. The hyaluronic acid obtained from humans and animals has a higher viscosity and better moisturizing properties, and its relative molecular weight is generally greater than 600,000. At present, most of the hyaluronic acid used in high-end cosmetics and medical-grade ophthalmology is still obtained by this method. However, the traditional extraction method is costly, the source is limited, and the purification is complicated, making it difficult to meet the ever-increasing demand [13].

3.2発酵製法

Since the 1970s, some people have been using microbial fermentation to produce hyaluronic acid. In 1985, Shiseido of Japan first reported the use of Streptococcus zooepidemicus to produce hyaluronic acid. The commonly reported hyaluronic acid-producing bacteria are mainly Group A and Group C streptococci in the Berger' sマニュアル。a群は主に人の病原体で、生産株として使用されるべきではない発疹性連鎖球菌などを含みます;グループcレンサ球菌は非ヒト病原体であり、工業生産に適している。現在、化膿連鎖球菌を用いたヒアルロン酸の工業生産は、すでに近年工業化の段階に達しています。一般的に、微生物によるヒアルロン酸の生産量と品質は、生産菌株の性能、培地と発酵プロセスの最適化、発酵プロセスの制御、発酵の下流での分離と精製などの側面に依存します。

3.2.1高収量系統の選択と育種

The breeding of high-yielding hyaluronic acid strains is a very important task that must be carried out over the long term. At present, China&#ヒアルロン酸生産株の39の繁殖は、依然として伝統的な人工突然変異に主に基づいています。一般的な突然変異誘発法は、自然または動物からオリジナルの株を採取し、それをズエピデミック連鎖球菌として同定し、ヒアルロン酸を生成し、この株をntg、紫外線、またはその組み合わせの出発株として用いる。遺伝的に安定で高レベルのヒアルロン酸を産生する株を、突然変異を継続するための開始株として選択する。このプロセスは、ヒアルロン酸の生産レベルを上げるために何度も繰り返されます。しかし、遊菌streptococcus zoepidemicusがヒアルロン酸を産生するメカニズムの継続的な詳細な研究に伴い、ha生合成のための重要な酵素のクローニングと、haの産生を増加させ、その分子量を制御するための遺伝子操作細菌の構築が、現在の研究開発の焦点となっている。

3.2.2培地のマッチング

The requirements of Streptococcus zooepidemicus for the production of hyaluronic acid are extremely demanding. Currently, the main carbon source for industrial production of hyaluronic acid is glucose, and the nitrogen source is yeast powder, peptone, beef extract or compound organic nitrogen source. However, when the medium is not properly matched, resulting in an imbalance in the ratio of nitrogen and carbon sources or a lack of growth factors, the metabolic pathway of Streptococcus zooepidemicus will change, and more of the substrate glucose will be converted into by-products such as lactic acid or acetic acid, affecting the fermentation production level of hyaluronic acid. Therefore, the choice of culture medium is extremely important.

3.2.3発酵技術の最適化と発酵プロセスの制御

Streptococcus zooepidemicus, which produces hyaluronic acid, is a facultative anaerobe, and hyaluronic acid is the main component of the capsule of Streptococcus zooepidemicus. When there is too much dissolved oxygen in the fermentation broth, it will cause damage to the bacteria. Insufficient dissolved oxygen will result in a lack of stimulation of Streptococcus zooepidemicus, which will lead to the bacteria not producing capsules and reduce the production of hyaluronic acid. The main process factors affecting the dissolved oxygen level in the fermentation broth are the rotational speed and the amount of aeration. In addition, different temperatures, rotational speeds and pH values have a significant effect on the molecular weight of hyaluronic acid. Studying the fermentation kinetics of hyaluronic acid can help us understand the laws of the fermentation production process, so that we can optimize and control the fermentation process to improve the production level and efficiency of hyaluronic acid.

3.2.4発酵スープ中のヒアルロン酸の分離と精製

The downstream separation and purification process is a key link in obtaining high molecular weight and high purity hyaluronic acid, and it is the bottleneck restricting the production of high-standard HA in many companies. The hyaluronic acid fermentation broth is very viscous and is a complex multi-phase system. The fluid properties are non-Newtonian. The main suspended solids in the fermentation broth are: fermentation product hyaluronic acid, bacterial cells, various impurity proteins and residual components in the culture medium and other metabolites. It is difficult to quickly and effectively separate HA from other components in the fermentation broth. At the same time, autolysis of the bacteria in the fermentation broth can produce hyaluronidase to degrade HA, which greatly reduces the molecular weight of HA [14]. Therefore, finding a suitable separation and purification process is of great significance for the preparation of high-standard hyaluronic acid.

4展望

Fermentation is a cost-effective method for producing hyaluronic acid, and the extraction process is simple. It has been scaled up for production and has broad market prospects. The international market price for cosmetic-grade HA is 1000 to 2000 $/kg, while the price for pharmaceutical-grade HA is 6000 to 20000 $/ kg。1985年には国際市場におけるhaの総売上高は1億ドルであったが、1990年には2億ドルを超え、1995年には6億ドルに達した。統計によると、ヒアルロン酸関連製品の世界市場は、2004年には約30億ドルであり、医薬品、化粧品のスキンケア用添加剤と医療製品がそれぞれ市場の半分を占めています。欧州、米国、日本はヒアルロン酸製品の最大の生産・使用市場であり、今後も年率15%の成長が見込まれています。現在、ヒアルロン酸の年間国内需要は約5トンです。

Since the 1980s, the production of hyaluronic acid by fermentation has been achieved abroad, and the number of manufacturers has increased from more than ten to hundreds, and the scale is also getting larger and larger. In contrast, in China, the production level and production efficiency of hyaluronic acid are relatively low, and there are few manufacturers that produce HA by microbial fermentation, which seriously restricts the development and application of hyaluronic acid in China. At present, the main bottlenecks in the development of hyaluronic acid production in China and the problems that need to be solved are mainly the following:

(1) 細菌株の低収率:中国の文献によると、実験室レベルの収率は約6 ~ 8 g/ lに達するが、産業に適用した後、原料や発酵条件などの要因により、収率が大幅に低下する可能性がある。国際工場の発酵レベルは8 ~ 10 g/ l以上とまだ大きな差があります。そのため、従来の突然変異育種法や遺伝子工学的手法を用いて高収量の細菌株を得ることは、応用研究の重要な方向性である。

(2) Small and uneven molecules: There are many factors that affect the molecular size of hyaluronic acid. According to literature reports, during the fermentation process, dissolved oxygen, pH, and temperature can all affect the molecular size.

(3)未熟な発酵プロセス:速度論は現代の発酵技術の応用の基礎である。これを研究することで、発酵プロセスの法則を理解することができ、生産プロセスを最適化して制御し、高い基質変換速度と高い生産強度を得ることができます。

(4) The fermentation mechanism is not clear: According to existing literature reports, only a very small number of substrates can be converted into hyaluronic acid. Although the research on the synthesis pathway of hyaluronic acid has been carried out at the molecular level, there is no feasible and effective theory that can greatly increase the yield of hyaluronic acid.

参照:

一石二鳥です凌ペッシェ。[1]ヒアルロン酸间学[M] .北京:中国軽工業出版、2000年。

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