히알루론산에 대해 알아야 할 것은 무엇인가요?

루 론 산, 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.

히알루론산의 성질과 분포 1

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. [화학공학] 히알루론산의 응용

2.1 화장품 속의 히알루론산

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.

의약품의 히알루론산 2.2

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.

식품산업에서의 히알루론산 활용 2.3

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 히알루론산 성분 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's의 HA 부족 및 체내의 히알루론산 부족으로 인한 조직과 세포의 노화를 방지합니다.특히 독거노인의 건강유지에 큰 영향을 미치고 있는 것으로 나타났다.히알루론산은 소화와 흡수를 통해 노화를 지연시켜 피부를 촉촉하고, 매끄럽고, 부드럽고, 탄력 있게 해준다 [11];동시에, 구강 히알루론산은 또한 사람들로 하여금 에너지와 젊은 활력으로 가득 차게 할 수 있습니다.현재 경구 히알루론산 미용 및 건강식품은 점점 더 많은 관심을 받고 있으며 점점 더 많은 사람들이 받아들이고 있다.

히알루론산 3 제조

크게 두 가지가 있다methods for preparing hyaluronic acid:하나는 동물 조직에서 추출하는 것이고, 다른 하나는 미생물 발효 [12]를 통해 얻는 것이다.

3.1조직 추출법

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].

3.2발효 생산법

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 참조하십시오.A 군에는 주로 인간 병원균이므로 생산 균주로 사용해서는 안 되는 표원성 연쇄상구균 등이 포함된다;그룹 C 연쇄상구균은 비인체병원균으로서 공업생산에 더욱 적합하다.현재 Streptococcus pyogenes를 이용한 히알루론산의 공업생산은 근년에 이미 공업화단계에 이르렀다.일반적으로 미생물의 방법으로 생산되는 히알루론산의 수율과 품질은 주로 생산 균주의 성능, 배양액 및 발효 공정의 최적화, 발효 공정의 제어, 발효물의 하류 분리 및 정제 등에 따라 달라진다.

3.2.1 고수익 균주의 선발 및 육종

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's 히알루론산생산균주의 육종은 여전히 주로 전통적인 인공돌연변이에 기초하고있다.돌연변이 발생의 일반적인 방법은 다음과 같다:자연 또는 동물로부터 독창적인 균주를 얻고, Streptococcus zooepidemicus로 확인하고 히알루론산을 생성한 후,이 균주를 NTG, 자외선 또는 두 가지의 조합을 위한 출발균주로 사용한다.유전적으로 안정하고 높은 수준의 히알루론산을 생산하는 균주를 지속적인 돌연변이 발생을 위한 시작 균주로 선정한다.이 과정은 히알루론산의 생성 수준을 높이기 위해 여러 번 반복됩니다.그러나 히알루론산을 생산하는 연쇄상구균 (Streptococcus zooepidemicus)의 메커니즘에 대한 심도 있는 연구가 지속적으로 진행되면서, HA의 생합성을 위한 주요 효소의 클로닝과 HA 생산을 증가시키고 분자량을 조절하기 위한 유전자 조작 박테리아의 제작이 현재 연구 및 개발의 초점이 되고 있다.

문화매체의 일치 3.2.2

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.

발효 기술의 최적화 및 발효 공정의 제어 3.2.3

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.

발효 국물중의 히알루론산의 분리 및 정제3.2.4

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 전망

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 $/kg이다.1985년 국제시장에서 HA의 총 판매액은 1억 달러였으며, 1990년에는 2억 달러 이상으로 상승하였고, 1995년에는 6억 달러에 이르렀다.통계에 따르면 히알루론산 관련 제품의 세계 시장은 2004년 약 30억 달러 규모였으며, 의약품과 화장품 스킨케어 제품 첨가제, 의료용 제품이 각각 시장의 절반을 차지하고 있다.유럽, 미국, 일본은 히알루론산 제품의 생산 및 사용이 가장 큰 시장이며, 향후 매년 15%의 고성장을 지속할 것으로 예상된다.현재 연간 국내 히알루론산 수요량은 약 5톤이다.

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) 세균 균주의 낮은 수율:중국 문헌에 따르면 실험실 수준에서의 수율은 약 6~8 g/L에 도달 할 수 있지만 산업에 응용 한 후에는 원료 및 발효 조건 등의 요인으로 인해 수율이 훨씬 낮을 수 있습니다.국제 공장 발효 수준인 8~10 g/L 이상과는 아직 큰 차이가 있다.따라서 전통적인 돌연변이 육종 또는 유전공학적 방법을 이용하여 높은 수율의 박테리아 균주를 얻는 것은 응용을 위한 중요한 연구 방향이다.

(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) 미숙한 발효과정:Kinetics는 현대발효기술을 응용한 기초이다.이를 연구하면 발효 공정의 법칙을 이해하여 생산 공정을 최적화하고 제어하여 높은 기질 전환율과 높은 생산 강도를 얻을 수 있습니다.

(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.

참조:

[1] 링 페이슈 (Ling Peixue).히알루론산 [M.북경:중국경공업출판사, 2000.

[2] 히알루로닉의 로랑 T C. 생화학.Acta Otolaryngol (Stockh) [J], suppl, 1987(442):7-24.

[3] Kendell F E, Heidellberger M, Dawson M H. group A 용혈성 streptococcus [J]의 점액 균주에 의해 정제된 혈청학적으로 비활성 다당류이다.J Biol Chem, 1997 (118):61-69.

[4] 우동루.탄수화물의 생화학 [M.북경:고등교육출판사, 2001.

[5] Pogrel M A, Lowe M A, Stern R. Hyaluronic (히알루론산) in human 침 [J].Aich 구강 Biol, 1996, 41:667-671.

[6] 왕동.일본 [J]의 합성 히알루론산.한국화학공업공학회지 2002(1):16

[7] 장후종, 원연린.「 동물활성성분 [M] 화학 」, 천진:천진과학기술출판사, 1995.

[8] 수홍, 루지화.화장품에서의 히알루론산나트륨 적용 [J.중국생화학약물학회지, 1998:(5):222-223.

[9] 팬밍.히알루론산이 무릎 골관절염 활액내 사이토카인 함량에 미치는 영향.칭다오:Qingdao University, 2005.

[10] 비잉.수술 후 유착방지를 위한 히알루론산 나트륨 필름 연구 [D.칭다오:Qingdao University, 2002.

[11] 양수전, 천귀루이.히알루론산의 특성과 식품에의 응용.외식산업과학기술, 2008, 29(6):317-320.

[12] 이시카와 오사무.닛폰간카이시 [J.간카, 1987 (38):927.

[13] 루, 니앙시, 탄톈웨이.히알루론산 [J]의 제조와 응용.한국기능성고분자학회지, 2001, 14(3):370-376.

[14] Kui Miaomiao, Cui Bo, Sun Hongbo 등.대파기름의 추출 및 결정방법에 관한 연구 진행 (정미숙, 정미숙, 정미숙)산동경공업대학 논문집 2010, 24(3):1-3.