Как производить порошок гиалуроновой кислоты методом ферментации?

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 свойства гиалуроновой кислоты

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].

В зависимости от источника иМетод экстракции гиалуроновой кислоты, его относительная молекулярная масса (Mr) 8-5-106[4]. Структура и биологическая активность гиалуроновой кислоты зависят от ее относительной молекулярной массы. Гиалуроновая кислота с низким молекулярным весом образует раздробленную сеть при низких концентрациях, в то время как гиалуроновая кислота с высоким молекулярным весом образует полную сеть [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 технология производства гиалуроновой кислоты порошка

Имеются три метода производства гиалуроновой кислоты, а именно экстракция, микробная ферментация и синтез [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. Кроме того,because hyaluronic acid is combined with other high molecular substances in animal tissue, it is more difficult to separate and purify, and Продукты из гиалуроновой кислоты 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 размножение бактерий, производящих гиалуроническую кислоту

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,   Установлено также, что сегнодимус (S. zooepidemicus) и стрептококк способны производить гиалуроновую кислоту [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 разведение мутагенеза

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 культура протопласта

Поскольку протопласты не имеют клеточных стен, они более чувствительны к изменениям окружающей среды, чем обычные клетки, и более решительно реагируют на мутагенные методы лечения [7]. Были проведены успешные эксперименты с использованием химических мутагенов, таких как NTG или физических мутагенов, таких как лазеры, для обработки протопластов первоначальных штаммов с целью получения высокоурожайных штаммов [7].

2.1.3 генная инженерия

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].

Ген HA синтеза мукоидного газа S43/192/4 Streptococcus agalactiae group A был впервые клонирован и построен в пласмиде Escherichia coli в 1993 году и успешно выраженный в E. coli для синтеза HA [8]. Впоследствии ген HA синтеза Streptococcus agalactiae group C был клонирован и выражен в E. coli в 1997 году [8].

Линг мин и др. [9] усилили ген sqhas из общей ДНК Streptococcus equi subsp. Zooepidemicus, создал выражение plasmid и превратил его в E. coli DH5α, успешно выразил sqHAS белка, и синтезировал га в присутствии субстрата. Чжан цзиню и др. [10] клонировали ген hasB Streptococcus zooepidemicus и выразили его в E. coli, чтобы получить соответствующий белок.

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].

Шэн юй [12] ввел ген синтазы zooepidemicus hyaluronan Streptococcus в Lactococcus lactis через систему индуцируемого выражения NICE (контролируемую низином) и успешно выразил его для синтеза HA.

2.2 оптимизация условий ферментации

Стрептококчи бактерии с высокими требованиями к питанию, которые должны расти на богатых питательными веществами средств массовой информации. Стрептококчи обычно растут на сложных средах, содержащих смесь дрожжей или экстрактов животных, пептона и сыворотки. Состав этих сред всегда включает глюкозу (10-60 г/л), аминокислоты, нуклеотиды, большое количество соли, микроминералов и витаминов [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 применения гиалуроновой кислоты

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 Гиалуроновая кислота в косметике, health products and medical and pharmaceutical fields.

3.1 применение гиалуроновой кислоты в косметике

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 применение гиалуроновой кислоты в медицинских препаратах

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 применение гиалуроновой кислоты при лечении

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

При глазных болезнях предпочтительным направлением лечения является актуальное офтальмологическое администрирование. Что касается офтальмологических препаратов, то биодоступность препарата положительно коррелируется с вязкостью жидкости в определенном диапазоне. Повышение вязкости может продлить срок пребывания препарата в глазу и, таким образом, повысить эффективность. Однако некоторые усилители вязкости могут вызывать побочные эффекты, такие как дискомфорт глаз. Гиалуроновая кислота преодолевает этот недостаток из-за своих не ньютоновских свойств жидкости и хорошей биосовместимости. Поэтому это хороший офтальмологический препарат вязкости, который стоит разрабатывать и применять [18]. В дополнение к применению в глазных капель, гиалуроновая кислота также может быть использована для лечения сухих симптомов глаза. В настоящее время гиалуроновая кислота используется вместе с другими высокомолекулярными полимерами для улучшения симптомов сухих глаз [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].

Перспективы на будущее

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.

Справочные материалы:

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