발효법으로 히알루론산 분말을 생산하는 방법은?

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].

히알루론산의 성질 1

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].

출처에 따라히알루론산 (hyaluronic acid) 추출법, 그것의 상대 분자 질량 (Mr)은 8×105에서 5×106[4]이다.히알루론산의 구조와 생물학적 활성은 상대적인 분자 질량에 의존한다.저분자 히알루론산은 저농도에서 파편화된 네트워크를 형성하고, 고분자 히알루론산은 완전한 네트워크를 형성한다 [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].

히알루론산 파우더의 생산 기술 2

히알루론산 분말의 생산 기술은 추출, 미생물 발효, 합성 등 3가지가 보고되어 있다 [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. 게다가,because hyaluronic acid is combined with other high molecular substances in animal tissue, it is more difficult to separate and purify, and 히알루론산 제품 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.

히알루론산 생성 박테리아의 번식 2.1

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)와 Streptococcus zooepidemicus (S. zooepidemicus)도 히알루론산을 생성할 수 있음이 밝혀졌다 [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 돌연변이 생성 번식

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].

원형질 배양 2.1.2

protoplasts 세포벽이 없기 때문에 일반 세포보다 환경 조건의 변화에 민감하고 돌연변이 치료 [7]에 더 강하게 반응한다.NTG와 같은 화학적 돌연변이나 레이저와 같은 물리적 돌연변이를 이용하여 원균주의 원형을 처리하여 고수익의 균주를 얻는 실험이 성공적으로 수행되었다 [7].

유전공학육종 2.1.3

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 A의 점액가스 균주 S43/192/4의 HA 합성 유전자를 1993년에 처음으로 클로네이트하여 대장균 플라스미드로 제작하였고, 대장균에서 성공적으로 발현하여 HA를 합성하였다 [8].이어서 1997년 Streptococcus agalactiae group C의 HA 합성 유전자가 복제되어 대장균에 발현되었다 [8].

Ling Min 등 9)은 Streptococcus equi subsp의 전체 DNA 로부터 sqhas 유전자를 증폭시켰다.으로 건설 된 zooepidemicus plasmid 및 변환 식 대장균 DH5 α, sqHAS 단백질을 성공적으로 표명 했고, 합성 되어 HA 기질의 존재에 있다.장진유 등은 Streptococcus zooepidemicus의 hasB 유전자를 복제하고 이를 대장균에 발현시켜 해당 단백질을 얻었다.

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].

현주유 [12]는 NICE (nisin-controlled gene expression system) inducible expression system을 통해 Streptococcus zooepidemicus hyaluronan synthase 유전자를 Lactococcus lactis에 도입하여 성공적으로 발현시켜 HA를 합성하였다.

발효 조건의 최적화 2.2

연쇄구균은 영양소가 풍부한 매체로 성장해야 하는 까다로운 영양요구량을 가진 세균이다.연쇄구균은 보통 효모 또는 동물 추출물, 펩톤 및 혈청의 혼합물을 포함하는 복합 매체에서 자란다.이러한 매체의 제형에는 항상 포도당 (10~60 g/L), 아미노산, 뉴클레오티드, 다량의 소금, 미량 미네랄과 비타민 [2]이 포함되어 있다.

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.

히알루론산을 응용한 3가지

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 화장품에 들어있는 히알루론산, health products and medical and pharmaceutical fields.

3.1 히알루론산을 화장품에 적용

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].

보건품에 히알루론산 적용 3.2

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 히알루론산의 의학적 치료 적용

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

눈 질환의 경우, 선호되는 치료 경로는 국소 안과 투여입니다.안과 약물의 경우 약물의 생체 이용성은 일정 범위 내에서 액체의 점도와 양의 상관관계가 있습니다.점도를 높이면 약물의 눈 속 거주 시간을 연장시켜 효능을 향상시킬 수 있다.하지만 일부 점도강화제는 눈이 불편해지는 등의 부작용을 일으킬 수 있다.히알루론산은 뉴턴이 아닌 유체 특성과 좋은 생체 적합성으로 인해이 단점을 극복합니다.따라서 개발 및 적용할 가치가 있는 좋은 안과용 의약품 점안제이다 [18].히알루론산은 안약에 사용되는 것 외에도 안구건조증 치료에도 사용할 수 있다.현재 히알루론산은 안구건조증 [19]을 개선하기 위해 다른 다양한 고분자 화합물과 함께 사용되고 있다.

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 전망

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.

참조:

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[2] CHONG B F, BLANK L M, MCLAUGHLIN R, 그 외.미생물의 히알루론산 생산 [J.Appl Microbiol Biotechnol, 2005, 66(4):341-351.

[3] 류 롱.Streptococcus zooepidemicus 발효에 의한 히알루론산 생산 공정 제어 및 최적화 [D].장쑤 우시:장난대학, 2009.

[4] 장규안, 링페이슈, 린홍 외.히알루론산 (J.중국제약산업학회지, 2006, 37(1):15-16.

[5] 스턴 R, 아사리 A A, 스가하라 K N. 히알루로난의 조각:정보가 풍부한 시스템 [J].Eur J Cell Biol, 2006, 85(8):699-715.

[6] 바르부치 R, 람포니 S, 보르자키엘로 A 외.골관절염 치료에 쓰이는 히알루론산 하이드로겔.Biomaterials, 2002, 23(23):4503-4513.

[7] 쉬옌리, 궈쑤핑, 루안일홍.히알루론산 생성 세균 번식 개요 [J.식품의약품안전처, 2006, 8(10):22-24.

[8] 정슈링, 왕펑산, 링페이슈.hyaluronic acid synthase의 연구 진행 [J.제약생명공학, 2004, 11(6):413-416.

[9] 링민, 황리보, 황근 외.Streptococcus equi subsp의 히알루론산 합성효소 유전자의 분자의 cloning과 발현.zooepidemicus다 [J다]산업미생물학, 2003, 33(2):4-8.

장진위, 우샤오밍, 하오닝 등 (10)Streptococcus zooepidemicus의 히알루론산 합성 관련 유전자 hasB의 Cloning 및 특성 (J.중국생명공학회지, 2005, 25(7):86-91.

[11] 재조합 Lactococcus lactis [J]에 의한 CHIEN L J, LEE C K. 히알루론산 생산.Appl Microbiol Biotechnol, 2007, 77(2):339-346.

[12] Lactococcus 락티스 (lactis) 에서의 히알루론산 합성 경로 확립 및 미생물에 합성한 히알루론산의 상대 분자량 조절 메커니즘에 대한 기초 연구 [D] 현 J.제남:산동대학, 2009.

[13] 시이 L, 왕 F S, 구 X P 등.발효에 의한 hyaluronan 생산에 대한 연구 조사 (J.중국과학기술학회, 2006, 2(4):268-271.

[14] LIU L, DU G C, CHEN J 등.Streptococcus zooepi-demicus의 회분식 배양에서 과산화수소와 아스코르베이트를 첨가하여 저분자량 히알루론산을 미생물의 생산 [J.바이오자원기술, 2009, 100(1):362-367.

[15] 구풍산, 링페이슈, 구수핑 외.히알루론산의 생리학적 기능과 화장품 및 미용 및 건강식품에의 응용.중국 비즈니스 산업, 2002 (9):45-46.

[16] 송용민, 궈쑤핑, 루안일홍 외.신자원 식품-히알루론산 [J.식품의약품안전처, 2009, 11(5):56-59.

[17] 완서유, 링 페이슈, 장천민.히알루론산 [J]의 연구 및 응용의 새로운 개발.식품의약품안전처, 2006, 8(12):1-3.

[18] Ling Peixue, Guan Huashi, Rong Xiaohua 등이 있다.안과 약물 전달 시스템의 연구 진행 [J.중국약학학회지 2006, 41(1):7-9.

[19] 링 페이슈, 장천민, 리키 외.안과약물에서의 히알루론산의 응용 및 연구 진행 [J.한의학과 임상, 2004, 4(9):697-699.

[20] 링 페이슈, 허연리, 장칭.골관절염에 대한 히알루론산의 치료효과.식품의약품안전처, 2005, 7(1):1-3.