発酵法によるヒアルロン酸粉末の製造方法?

Hyaluronic acid (HA) is a macromolecular polysaccharide that was first isolated and purified from the vitreous humor of cattle by Meyer and others in 1934, hence its other name, hyaluronan [1]. Hyaluronic acid is a homogeneously repeating linear glucosamine polysaccharide composed of 2,000 to 25,000 disaccharides of glucuronic acid and N-acetylglucosamine alternately bound by β-1,3 glycosidic bonds and β-1,4 glycosidic bonds [2].

Hyaluronic acid is an important component of the extracellular matrix (ECM) [1]. Recent studies have shown that hyaluronic acid is not only widely present in the extracellular matrix between cells, but also exists inside the cell, mainly concentrated in the cytoplasm and nucleus of newborn cells [2]. In addition to being found in the vitreous body, hyaluronic acid is also abundant in the synovial fluid of joints and in the spaces between epidermal cells. In terms of quantity, more than 50% of hyaluronic acid is found in the dermis and epidermis of the skin, and about 35% is found in muscles and bones. It is currently believed that hyaluronic acid is mainly found in the inert space filler of soft connective tissue, and plays an important role in the formation of proteoglycan complexes [2].

1ヒアルロン酸の性質

Under the electron microscope, hyaluronic acid molecules are observed to have a linear single-chain structure, and they expand into a random coil structure in an aqueous solution, with a coil diameter of about 500 nm. Each disaccharide unit in the hyaluronic acid molecule contains a carboxyl group, which can dissociate under physiological conditions to form an anion. The mutual repulsion between the anions at equal spatial distances causes the molecule to be in a loose extended state in an aqueous solution, occupying a large amount of space, so it can bind more than 1,000 times its own weight in water [3].

ソースに応じてとヒアルロン酸抽出法分子量は8×105 ~ 5×106[4]。ヒアルロン酸の構造と生物学的活性は、その相対分子量に依存します。低分子ヒアルロン酸は、低濃度では断片的なネットワークを形成しますが、高分子ヒアルロン酸は完全なネットワークを形成します[3]。

Due to the hydrogen bonds within the molecule, hyaluronic acid molecules adopt a single-helix structure in aqueous solution [5]. When the hyaluronic acid concentration in the solution reaches a certain level, the hyaluronic acid molecules interact with each other to form a double-helix structure, and a network structure is formed at higher concentrations [3]. The currently accepted theory of hyaluronic acid structure is the tertiary structure theory, which states that each trisaccharide unit in a hyaluronic acid molecule has a hydrophobic region. When the solution concentration is high, the hydrophobic regions of the hyaluronic acid molecules interact to form a double-helix structure, which is the basis for the aggregation of hyaluronic acid molecules [6].

Hyaluronic acid is characterized by its very high viscosity[2]. At low concentrations or low relative molecular masses, the viscosity of the solution changes little with increasing concentration or Mr. When the viscosity reaches 10 mPa·s after the Mr and concentration increase, the hyaluronic acid molecules begin to intertwine, at which time the viscosity increases rapidly with increasing Mr and concentration[3].

2ヒアルロン酸粉末の製造技術

ヒアルロン酸粉末の製造技術は、抽出、微生物発酵、合成の3つが報告されている[1]。

The extraction method involves extracting hyaluronic acid from human or animal tissues [1]. The extraction method was the first method used to produce hyaluronic acid. Currently, the main raw materials used in production are chicken combs, human umbilical cords and animal eyes. The main process steps include extraction, impurity removal, enzymatic hydrolysis, precipitation and separation. The extraction and purification processes for hyaluronic acid from different tissues differ to some extent [3]. However, due to the limited source of raw materials for the extraction method, the product extraction rate is extremely low (only about 1%), and the process is complex, so it is difficult to reduce production costs. また、because hyaluronic acid is combined with other high molecular substances in animal tissue, it is more difficult to separate and purify, and ヒアルロン酸製品 extracted from animal tissue may cause infection. These factors limit the wide application of the extraction method in industries such as medicine and cosmetics [2, 3].

The synthetic method involves first synthesizing a “hyaluronic acid oxaziridine derivative” using a biological macromolecule, then adding water and hyaluronidase from the testes of sheep or cattle to prepare a complex of the derivative and the enzyme, and finally removing the enzyme to purify the hyaluronic acid [1]. The synthetic method is still in the laboratory research stage and has not yet been applied to industrial production [1].

The microbial fermentation method refers to the use of screened bacteria to carry out fermentation and culture, and the hyaluronic acid product is obtained by isolating and purifying it from the fermentation broth [1]. Due to the above disadvantages of the extraction method and the fact that the synthetic method is not yet mature, the microbial fermentation method has become the most important method for producing hyaluronic acid. The following is a more systematic overview of the microbial fermentation method for producing hyaluronic acid powder.

2.1ヒアルロン酸産生細菌の繁殖

The earliest discovered microorganism to produce hyaluronic acid was Streptococcus pyogenes, which was discovered in 1937 to be able to produce hyaluronic acid [7]. Subsequently, in 1939, it was discovered that Streptococcus equisimilis and S. zooepidemicus were also able to produce hyaluronic acid [7]. Since wild-type Streptococcus can produce hyaluronic acid,   equisimilisやstreptococcus zooepidemicus (s . zooepidemicus)もヒアルロン酸を産生することがわかった[7]。

Since wild-type Streptococcus has disadvantages such as the ability to produce hyaluronidase, express other extracellular proteins, and low hyaluronic acid production [2], wild-type strains must be modified by various means in actual production to meet the needs of industrial production.

2.1.1変異原繁殖

Mutagens mainly include physical mutagens, chemical mutagens and biological mutagens. At present, the mutagens used in the breeding of hyaluronic acid-producing strains mainly include ultraviolet light, 60Co γ rays and nitroguanidine (NTG) [7]. Many research reports have shown that by treating some original strains that can produce hyaluronic acid, such as Streptococcus zooepidemicus and Streptococcus equi, with various mutagenic treatments, excellent strains with high hyaluronic acid production, or relatively high molecular weight hyaluronic acid, or negative reactions after pre-treatment with hyaluronidase, or non-hemolysis, or a combination of the above characteristics can be obtained [7].

2.1.2原形質体文化

プロトプラストは細胞壁を持たないため、通常の細胞よりも環境条件の変化に敏感であり、変異原性治療に対してより強く反応する[7]。ntgなどの化学的変異原やレーザーなどの物理的変異原を用いて、原株の原細胞を治療し、高収量株を得る実験が成功している[7]。

2.1.3遺伝子工学育種

The gene encoding the enzyme involved in the hyaluronic acid synthesis pathway in Streptococcus is located on a single reverse transcriptase and is called the has operon. In Streptococcus pyogenes, the has operon consists of three genes: hasA (1248 bp), encoding hyaluronic acid synthase (42.0 U), hasB (1204 bp), encoding UDP-glucose dehydrogenase (47.0 U), and hasC (915 bp), encoding UDP-glucose pyrophosphorylase (33.7 U) [2]. Although it is not yet clear how hyaluronic acid chains are transported across the cell membrane, the expression of hyaluronic acid synthase and UDP-glucose dehydrogenase in Enterococcus faecalis, Escherichia coli and Bacillus subtilis is sufficient to direct hyaluronic acid production and transport [2]. Therefore, hyaluronic acid can be produced simply by transferring the hasA and hasB genes into the host cell and having them expressed in the host cell [7].

streptococcus agalactiae group aのmucoid gas株s 43/192/4のha合成遺伝子は、1993年に最初にクローン・構築され、大腸菌のプラスミドになり、大腸菌でha合成に成功した[8]。その後、streptococcus agalactiae group cのha合成遺伝子がクローンされ、1997年に大腸菌で発現した[8]。

ling minら[9]は、streptococcus equi subspの全dnaからsqhas遺伝子を増幅した。zooepidemicus、表現plasmid大腸菌な形にDH5α、sqHASタンパク質を表明しており、合成されたHAれ基板上の存在に関する。zhang jinyuら[10]は、streptococcus zooepidemicusのhasb遺伝子をクローニングし、それを大腸菌で発現させて対応するタンパク質を得た。

The Chien research group in Taiwan Province of China introduced the hasA and hasB genes of Streptococcus zooepidemicus into Lactococcus lactis through the NICE inducible expression system, and successfully obtained an engineered strain that produces hyaluronic acid [11].

sheng juyu[12]は、streptococcus zooepidemicusのヒアルロン酸合成酵素遺伝子を、nice (nisin-controlled gene expression system)誘導性発現系を介してlactococcus lactisに導入し、ha合成に成功した。

2.2発酵条件の最適化

レンサ球菌は、栄養価の高い培地で生育する必要のある、栄養要件の厳しい細菌である。レンサ球菌は通常、酵母または動物抽出物、ペプトンおよび血清の混合物を含む複雑な培地上で生育する。これらの媒体の組成は、常にグルコース(10 - 60 g/ l)、アミノ酸、ヌクレオチド、大量の塩、微量ミネラルおよびビタミンを含みます[2]。

The pH and temperature are very important for the growth of Streptococcus zooepidemicus and the production of hyaluronic acid. Some studies have shown that the conditions of pH 6.7 ± 0.2 and temperature 37 °C are most suitable for the growth of Streptococcus zooepidemicus and the production of hyaluronic acid [13]. The stirring rate also affects the production of hyaluronic acid. Studies have shown that under conditions of low agitation rates, lactic acid production is high and hyaluronic acid production is low [13]. High-speed agitation can reduce the effect of lactic acid synthesis and increase hyaluronic acid production, but it can also destroy hyaluronic acid polymers and reduce their relative molecular mass [13]. The initial glucose concentration has a significant effect on the relative molecular mass of hyaluronic acid. Research shows that when the initial glucose concentration is increased from 20 g/L to 40 g/L, the relative molecular mass of hyaluronic acid also increases from (2.1±0.1)×106 to (3.1±0.1)×106 [13].

Liu et al. [14] reported that during batch fermentation of Streptococcus zooepidemicus, hydrogen peroxide (1.0 mmol/g HA) and ascorbic acid (0.5 mmol/g HA) were added at 8 h and 12 h, respectively, to cause the redox depolymerization of hyaluronic acid, resulting in a decrease in relative molecular mass and The yield increased from 5.0 g/L to 6.5 g/L.

3ヒアルロン酸の応用

Due to the many properties of hyaluronic acid mentioned above, it has been widely used in many fields. The following mainly summarizes the application of 化粧品におけるヒアルロン酸, health products and medical and pharmaceutical fields.

3.1化粧品におけるヒアルロン酸の応用

Hyaluronic acid is mainly found in the extracellular matrix between cells, where it has the function of maintaining the extracellular space of tissue cells, accelerating the flow of nutrients, and maintaining the tissue. First, compared with traditional moisturizers, hyaluronic acid has a better moisturizing effect and has the advantages of being non-greasy and not clogging pores. Second, an aqueous solution of hyaluronic acid has strong viscoelasticity and lubricity, which helps to form a breathable moisturizing film on the skin surface to keep the skin moisturized. Third, small molecules of hyaluronic acid can enter the dermis, promote blood microcirculation, and help the skin absorb nutrients, which can have a cosmetic and health-promoting effect. Finally, hyaluronic acid can remove active oxygen free radicals in the skin caused by ultraviolet radiation, providing sun protection and repair[15].

Due to the many advantages of hyaluronic acid, it is widely used in cosmetics as the ideal natural moisturizing factor to moisturize, emollient, anti-wrinkle and sunscreen. The usual addition amount is 0.05% to 0.50% [15].

3.2健康製品におけるヒアルロン酸の応用

Since hyaluronic acid has various properties such as water retention, lubrication, promoting wound healing and protecting cells, a decrease in hyaluronic acid in the body can lead to many problems such as arthritis, skin aging, and increased wrinkles. Therefore, oral supplementation of hyaluronic acid to supplement endogenous. Hyaluronic acid is currently considered to be one of the effective ways to maintain beauty and health and prolong life [16].

The theoretical basis for oral hyaluronic acid is that after oral digestion, hyaluronic acid can increase the precursors for the synthesis of hyaluronic acid in the body, thereby increasing the amount of hyaluronic acid synthesized in the body and targeting it to tissues such as the skin to exert its effect. At present, a variety of oral hyaluronic acid products have been launched, such as tablets, capsules and oral liquids [16].

3.3医療におけるヒアルロン酸の応用

Hyaluronic acid is widely used in ophthalmology, orthopedics and many other medical fields due to its unique viscoelasticity, biocompatibility and non-immunogenicity [17].

眼疾患の場合、優先的な治療経路は眼の局所投与である。眼科薬の場合、薬剤の生物学的利用能は一定範囲内の液体の粘度と正の相関がある。粘度を上げると、目の中の薬剤の滞留時間が長くなり、効果が向上します。しかし、いくつかの粘度増強剤は、目の不快感などの副作用を引き起こす可能性があります。ヒアルロン酸は、非ニュートン流体の特性と優れた生体適合性により、この欠点を克服します。したがって、開発および適用する価値のある優れた眼科薬粘度剤である[18]。ヒアルロン酸は目薬に使用されるだけでなく、ドライアイの症状の治療にも使用できます。現在、ヒアルロン酸は、ドライアイの症状を改善するために、他の様々な高分子ポリマーと一緒に使用されています[19]。

In addition to its presence in the vitreous body, hyaluronic acid is also the main component of articular cartilage and synovial fluid. When the body develops osteoarthritis, rheumatoid arthritis and other joint diseases, the production and metabolism of hyaluronic acid in the joint is abnormal, and the concentration and relative molecular weight of hyaluronic acid in the synovial fluid are significantly reduced, which disrupts cartilage degradation. This has led to the development of viscoelastic complementary therapy, which treats joint diseases by supplementing exogenous hyaluronic acid. This therapy is becoming increasingly popular with doctors and patients alike because of its long-lasting efficacy and few side effects[20].

In addition, hyaluronic acid is also widely used in drug delivery systems as various carriers (such as anti-tumor targeted drug carriers, non-viral vectors for gene therapy, and carriers for peptide and protein drugs), as implant materials in surgery, and in the treatment of recurrent oral ulcers [17].

4展望

As hyaluronic acid is gradually being applied in various fields, the microbial fermentation method for producing hyaluronic acid powder will gradually replace the extraction method and become the main method for the industrial production of hyaluronic acid. The beginning of hyaluronic acid production in a foreign host indicates that the production of hyaluronic acid has entered the stage of applying modern biotechnology. In the future, strains that can produce hyaluronic acid with different relative molecular masses will be selected, and through the continuous optimization of fermentation conditions, hyaluronic acid products that can be used in different fields will be provided. Hyaluronic acid will also be used more and more widely in many fields.

参照:

一石二鳥です凌ペッシェ。[1]ヒアルロン酸の研究と応用[m]。北京:People&#^『医術辞典』医術出版社、2010年。1開拓民たちはキリスト教を信奉した。

[2] chong b f, blank l m, mclaughlin r, et al。微生物ヒアルロン酸産生[j]。^ a b c d e f g h i l biotechnol, 2005, 66(4): 341-351。

[3]劉龍。streptococcus zooepidemicus発酵によるヒアルロン酸生産のプロセス制御と最適化[d]。^ a b「無錫、江蘇省:江南大学、2009年。

[4] jiang qiuyan, ling peixue, lin hong, et al。ヒアルロン酸の熱分解に関する研究[j]。中国医薬産業ジャーナル、2006年、37(1):15-16。

[5] stern r, asari a a, sugahara k n .ヒアルロン酸断片:情報豊富な系[j]。^ eur j cell biol, 2006, 85(8): 699-715。

[6] barbucci r, lamponi s, borzacchiello a, et al。変形性関節症の治療におけるヒアルロン酸ハイドロゲル。^ a b c d e f g h i(2002年)、453 - 453頁。

【7】石延麗、郭雪平、欒一紅。ヒアルロン酸産生細菌の繁殖の概要[j]。^ a b c d e f g h i(2006年)、22-24頁。

【8】鄭雪嶺、王鳳山、凌peixue。ヒアルロン酸合成酵素の研究成果[j]。^ a b c d e f g h i(2004)、416 -416頁。

[9] ling min, huang ribo, huang kun, et al。streptococcus equi subsp.のヒアルロン酸合成酵素遺伝子の分子クローニングと発現。zooepidemicus [J]。^産業微生物学、2003年、33(2):4-8。

[10] zhang jinyu, wu xiaoming, hao ning, et al。streptococcus zooepidemicusのヒアルロン酸合成関連遺伝子hasbのクローン・解析[j]。中国生物学会誌,2005,25(7):86-91。

[11] chien l j, lee c k .組換えlactococcus lactisによるヒアルロン酸産生[j]。^ a b c d e f g h i l l biotechnol, 2007, 77(2): 339-346。

[12] sheng j . lactococcus lactisにおけるヒアルロン酸合成経路の確立と微生物合成ヒアルロン酸の相対分子量調節機構の予備的研究[d]。2009年-山東大学教授。

[13] shi y l, wang f s, guo x p, et al。発酵によるヒアルロン酸産生の研究[j]。^『日本近代建築史』第2巻、日本建築学会、2006年(平成18年)、268-271頁。

[14] liu l, du g c, chen j, et al。streptococcus zoepiのバッチ培養で過酸化水素とアスコルビン酸を添加した低分子ヒアルロン酸の微生物生産- demicus [j]。」。bioresource technology, 2009, 100(1): 362-367。

[15] guo fengxian, ling peixue, guo xueping, et al。ヒアルロン酸の生理機能と化粧品、美容・健康食品への応用[j]。^ a b c d e f g h『人事興信録』第9版、45-46頁。

[16] song yongmin, guo xueping, luan yihong, et al。新資源食品-ヒアルロン酸[j]。^ a b c d e f g h i i(2009年)、56-59頁。

【17】ワン・スウ、リン・ベハク、張天民。ヒアルロン酸の研究と応用における新たな展開[j]。2006年(平成18年)12月1日:1-3号線を廃止。

[18] ling peixue, guan huashi, rong xiaohua, et al。眼科薬物送達システムの研究進展[j]。中国医薬ジャーナル2006,41(1):7-9。

[19] ling peixue, zhang tianmin, li qi, et al。眼科薬におけるヒアルロン酸の応用と研究の進展[j]。中国医学と臨床,2004,4(9):697-699。

【20】凌peixue、何yanli、張清。変形性関節症に対するヒアルロン酸の治療効果[j]。^「food and drugs, 2005, 7(1): 1-3」。food and drugs(2005年7月1日). 2011年7月1日閲覧。