Wound-healing agent based on strain trichoderma harzianum rifai

FIELD: biotechnology.

SUBSTANCE: wound-healing agent is a concentrate of the culture liquid of strain Trichoderma harzianum Rifai deposited in the Russian National Collection of Industrial Microorganisms under the number of RNCIM: F-180, as a producer of L-lysine of alpha-oxidase, and can be applied as a wound-healing agent for skin lesions.

EFFECT: invention enables to expand the range of means that provide wound healing of skin lesions.

1 tbl, 2 ex

 

The invention relates to biotechnology, microbiological industry, veterinary medicine, cosmetology and is intended for wound healing of skin damage.

Known wound healing probiotic preparation, including the culture of the bacteria Bacillus subtilis and Lactobacterium plantarum (RF Patent No. 2401116). Lack of preparation is the complexity of cultures used and the complexity of making money.

A well-known inhibitor of the herpes virus simple 1-St type (Patent RF №2022012, 1994), a known inhibitor of human immunodeficiency virus (Patent RF №2022011,1994). As inhibitors used metabolite strain Trichoderma harzianum Rifai - VKPM F-180-homogeneous enzyme L-lysine-and-oxidase (RF Patent No. 2022011, 1994). Information about the wound healing property of this drug are missing.

The technical result of the invention is the expansion of the means to heal the wounds that occur when damage to the skin.

The technical result is achieved by the fact that, as a wound healing remedy in case of damage of the skin, apply a concentrate of the culture fluid of strain Trichoderma harzianum Rifai, deposited in Russian national collection of industrial microorganisms under the number F-180.

This strain is known (Copyright certificate №1044043, 1991), however, discovered an unknown level of the I equipment property, ensure the application of cultural liquid concentrate of this strain as a means to heal the wound of skin damage.

For example, you might create a wax composition as a wound-healing agent intended for the treatment of wound of skin damage.

The cultivation conditions of a strain of Trichoderma harzianum Rifai VKPM F-180 and obtaining a concentrate of the culture fluid.

Fermentation of the strain was carried out on the equipment Experienced process plant IBPM RAS. G.K. Skryabin (Pushchino). Used the fermenter type BIOR-01 produced by BLS Bureau, the city of Kirishi, volume 100 l with a fill factor of 0.6. The fermenter equipped with a magnetic stirrer, filters air, temperature and pH. Nutrient medium was prepared directly in the apparatus. For this purpose, the apparatus was filled with 60 l of tap water, contributed 1% wheat bran, stimulant - 0,1%, 1.3% of ammonium sulfate, pH 5.8 to 6.0 installed a 10% HCl solution. Wheat bran and stimulant pre-soaked in 10 litres of water for 4 hours, were sterilized in an autoclave for 1 hour at a temperature of +125°C.

Prepared fermenter was seeded with seed material from the flasks. Sowing dose of at least 5%. Cultivation was carried out at a temperature of +26°C, air flow throughout the fermentation 30 litres per minute, the speed is Esaki 200 rpm. The pH in the growth process was adjusted to 6.5. The duration of the growing 94-98 hours, the final pH from 5.3 to 7.5.

At the end of the fermentation culture liquid was sent to the site pre-treatment, where the fungal mycelium was separated by filtration under vacuum on the suction filter. The weight of the obtained biomass was 3.5 kg Received native solution was subjected to additional separation in the separator of the type RSD at 9000 rpm./min, for hours at a temperature of 2-4°C. Then native solution in a volume of 55 l, obtained after separation of the biomass was concentrated to 1.5 l (concentrate).

The resulting concentrate of the culture fluid with the activity of L-lysine α-oxidase 5.4 U/ml represents an opaque light yellow liquid with a faint mushroom scent, stable during storage. L-lysine α-oxidase, protein, unsaturated fatty acids, mono - and gisagara, starch, cellulose, organic acids, vitamin B1, vitamin B2 (colors), vitamin PP, iron, potassium, calcium, magnesium, sodium, phosphorus, sodium nitrate.

Conditions create a wound healing agent

The choice of the optimal amount of concentrate the culture fluid of strain, providing sufficient anti-inflammatory and wound healing effects were based on earlier studies, the objective of which was defined by the e activity of the enzyme L-lysine α-oxidase in animals (laboratory Guinea pigs). For the development of composition and technology of gel made of model samples with 1%concentration of the enzyme.

The choice of structural components for the manufacture of a gel made with regard to therapeutic, technological, and consumer demands for drugs in this group (Army O.A., gavagan M.A., Levchuk T.A., Okhotnikov SCI excipients used in the technology of soft medicinal forms (ointments, gels, liniments, creams) // Chemical and pharmaceutical journal, T, No. 9, 2005. - P.45-48; the Army O.A., Suslin S.N. Krasnyk I.I. Substantiation of the gel Evkalimina based on a comparative study of rheological parameters redkostej acrylic polymers. // Bulletin of the Russian peoples friendship University, series "Medicine", specialty "pharmacy", №4 (28), 2004. S-222). As bases for gel selected hydrophilic base redkostej acrylic polymers (Mars and Carbopol) and derivatives of methyl cellulose (MC).

Thus, as a result of complex physical, chemical, biological and rheological studies developed the following composition of the gel with L-lysine α-oxidase. The composition of wound healing products, wt.%:

Example 1

The carbopol (974 P NF)

European Pharmacopoeia 2001 Art."Carbomers"0,8
The concentrate of the culture fluid with
L-lysine α-oxidase activity 5.4 U/ml0,8
A solution of sodium hydroxide (30%) (GOST 4328-77)to pH 6,0-6,5
Purified water (FS 42-2619-97)to 100.0

Example # 2

The carbopol (974 P NF)

European Pharmacopoeia 2001 Art."Carbomers"1,2
The concentrate of the culture fluid with
L-lysine α-oxidase activity 5.4 U/ml1,2
A solution of sodium hydroxide (30%) (GOST 4328-77)to pH 6,0-6,5
Purified water (FS 42-2619-97)to 100.0

Manufacturer of gel begin with preparation of wound healing agents and bases. In operation, the substrate preparation is the process of dissolution and swelling, as well as the regulation of the pH value with sodium hydroxide.

Manufacturing fundamentals “RAP: water”. In production capacity measure calculated amount of water ooisensors Brine (carbopol) layer on the surface of the water and leave to swell for 1 hour, then the system is stirred using a mechanical stirrer (ECROS-8100) with a speed of about 100-120./min to obtain a homogeneous gel. After that, with constant stirring, add the calculated amount of sodium hydroxide, to obtain the given pH of the medium (5.5 to 6.5). The introduction of a neutralizing agent leads to a thickening system and increases the level of viscosity. The ingredients are thoroughly mixed using a high speed stirrer (ECROS-8100). The obtained gel leave until completion of structure formation within 4-6 hours

Manufacturer of gel concentrate culture liquid of Trichoderma harzianum Rifai-F-180 and the study of wound healing agent.

The culture fluid (concentrate k.zh.) make the base gel and homogenized on a paddle stirrer (ECROS-8100, 120.min) for 10 minutes. Conducted an experiment on 20 Guinea pigs, males, weighing 320±15 g (table.). It is established that the gel concentrate culture liquid of Trichoderma harzianum Rifai-F-180 (1%) has a wound healing effect, reducing the average time of healing of wounds in animals by 5.4 days compared with the control group (without treatment) and 4.2 days compared with placebo gel, respectively.

From the table it is seen that the difference in terms of treatment of animals belonging to different experimental groups, reliable.

Table
The healing time of wounds (in days)
The form of the medicationAnimals in groupTime to wound healing (days)t1t2tptto
The control without treatment (K)512,0±0,4 (11,6÷12,4)of 5.43,41,2-
Gel with L-lysine α-oxidase 1% carbopol(1)56,60±0,48 (6,12÷7,08)--2-4,2-5,4
Gel with L-lysine α-oxidase 1% phospholipid basis (2)58,60±0,48 (8,12÷9,02)2--2,2-3,4
The gel of carbopol (without the addition of L-lysine α-oxidase)(p)5 10,80±0,32 (10,48÷11, 12)4,22,2--1,2

Note: t1the time of wound healing with the use of the gel with 1% L-lysine α-oxidase on the carbopol;

t2the time of wound healing with the use of the gel with 1% L-lysine α-oxidase on phospholipid basis;

tp- time healing on the carbopol without L-lysine α-oxidase;

ttothe time of wound healing (control) without treatment.

The obtained experimental results showed a high healing activity of the gel with 1% of a concentrate of the culture fluid of strain Trichoderma harzianum Rifai-F-180.

We offer a wound healing agent has a pronounced healing effect, encourages earlier wound healing in animals and can be used in the presence of purulent-necrotic masses in the wound in veterinary medicine.

The concentration of the culture fluid of strain Trichoderma harzianum Rifai, deposited in Russian national collection of industrial microorganisms under the number F-180, as a wound healing agent when damage to the skin.



 

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