Patent Description:
It is known the method of the obtainment of the biomass of Galleria mellonella larvae, which includes the cultivation of larvae of the first-second age on the nutrient medium that contains the wheat flour (<NUM>), bakery yeast (<NUM>), milk powder (<NUM>), wax (<NUM>), glycerine (<NUM>), wheat bran (<NUM>), bee honey (<NUM>) and water (<NUM>); and larvae of the third-fourth age - on the medium that contains the wheat bran (<NUM>), wheat flour (<NUM>), bakery yeast (<NUM>), milk powder (<NUM>), wax (<NUM>), glycerine (<NUM>), water (<NUM>) [<NUM>]. Larvae are intended for use as an organism - the host for various pathogens in the scientific researches [<NUM>].

The disadvantage of this method is the use of poor nutrient medium for the cultivation of the larvae of the third-fourth age, which cannot provide a high biological value of the biomass.

It is also known the method of the obtainment of the biomass of Galleria mellonella larvae, including the incubation of the larvae of the last age or the pupae in the containers, in the darkness for <NUM>-<NUM> weeks for the obtainment of the maximum number of eggs, transfer of eggs (twice a week) to the food containers for <NUM> days for the obtainment of larvae. The nutrient medium used for the feeding of larvae has the following composition: corn flour <NUM>, yeast extract <NUM>, soy flour <NUM>, milk powder <NUM>, bee honey <NUM>, glycerine <NUM>, blocks of beeswax. Larvae are characterized by high resistance to the infections of Staphylococcus aureus, Escherichia coli and Candida albicans and are used as the hosts in the study of entomopathogens [<NUM>].

The disadvantage of the method is that the obtained larvae vary significantly in dimensions (from <NUM> to <NUM>) and quality of biomass because of different ages that is unacceptable, when the biomass is used as the raw material for the production of the cosmetic or pharmaceutical substances.

It is known also the method of the obtainment of Galleria mellonella larvae that consists in the fact that the eggs of the insects are placed in the plastic boxes with the nutrient medium, where they are developed in the course of <NUM> days. The used nutrient medium has the following composition: <NUM> of wheat flour, <NUM> of wheat bran, <NUM> of milk powder, <NUM> of beer yeast, <NUM> of wax powder, <NUM> of honey and <NUM> of glycerin. The obtained larvae are used as the host organism for the cultivation of the entomophagous nematodes [<NUM>].

The disadvantage of this method consists in the <NUM>-day duration of the process of growth that leads to a significant increase in the cost of the technology of cultivation of insects.

The closest to the claimed invention is the method of the obtainment of the biomass of Galleria mellonella larvae [<NUM>] for the use as the raw materials for the production of cosmetic and pharmaceutical preparations having the keratolytic effect. This process includes three consecutive stages: <NUM>. Preparation of conditions for the passage of the full life cycle under the conditions of artificial cultivation. For the cultivation of the insects are used the premises with the area of <NUM><NUM>. The walls are painted with a special resin that makes it possible the disinfection by the disinfectant solutions. In the premises where the eggs are incubated, the temperature is maintained at the level of <NUM> ± <NUM>, and humidity - <NUM>%. The premises in which the larvae are cultivated are equipped with the dark boxes and provided with the automatic temperature and humidity control systems. In these premises it is maintained the temperature of <NUM>-<NUM> and humidity of <NUM>%; <NUM>. Cultivation of insects. Immediately after the hatching, the larvae are transferred from the incubation rooms to the premises intended for the cultivation of larvae. Larvae are placed in the dark containers, where the necessary conditions are maintained, and the insects receive the necessary nutrition - the nutrient medium of the following composition: yellow corn extract <NUM>,<NUM>-<NUM>,<NUM>, red corn extract <NUM>,<NUM>-<NUM>,<NUM>, glucose <NUM>,<NUM>-<NUM>,<NUM>, fructose <NUM>,<NUM>- <NUM>,<NUM>, sucrose <NUM>,<NUM>-<NUM>,<NUM>, maltose <NUM>,<NUM>-<NUM>,<NUM>, casein <NUM>,<NUM>-<NUM>,<NUM>, retinol (vitamin A) <NUM>-<NUM> un. , tocopherol acetate (vitamin E) <NUM>-<NUM> un. , vitamin C <NUM>,<NUM>-<NUM>,<NUM>, riboflavin (vitamin B<NUM>) <NUM>,<NUM>-<NUM>,<NUM>, calcium pantothenate (vitamin B<NUM>) <NUM>,<NUM>-<NUM>,<NUM>, thiamine (Vitamin B<NUM>) <NUM>,<NUM>-<NUM>,<NUM>, pyridoxine <NUM>,<NUM>-<NUM>,<NUM>, biotin <NUM>,<NUM>-<NUM>,<NUM>, niacin (vitamin B<NUM>) <NUM>,<NUM>-<NUM>,<NUM>, pinobanksin <NUM>,<NUM>-<NUM>,<NUM>, pinocembrin <NUM>,<NUM>-<NUM>,<NUM>, glycerin <NUM>,<NUM>-<NUM>,<NUM>, NaNO<NUM> <NUM>,<NUM>-<NUM>,<NUM>, KH<NUM>PO<NUM> <NUM>,<NUM>-<NUM>,<NUM>, NaCl <NUM>,<NUM>-<NUM>,<NUM>, CaCl<NUM> <NUM>,<NUM>-<NUM>,<NUM>, MgSO<NUM> - <NUM><NUM>O <NUM>,<NUM>-<NUM>,<NUM>, ZnSO<NUM> * <NUM><NUM>O <NUM>,<NUM>-<NUM>,<NUM>, MnSO<NUM> * <NUM><NUM>O <NUM>,<NUM>-<NUM>,<NUM>, CuSO<NUM> * <NUM><NUM>O <NUM>,<NUM><NUM>,<NUM>, FeCl<NUM> * <NUM><NUM>O <NUM>,<NUM>-<NUM>,<NUM>, EDTA <NUM>,<NUM>-<NUM>,<NUM>, yellow wax <NUM>-<NUM>, purified water - up to <NUM>. Collection of larvae. Larvae are collected at the entrance in the <NUM>th age, immediately after the moulting. This biomass is used for the obtainment of the active complex having the keratolytic effect.

The disadvantage of this method is the use of a very complex nutrient medium, consisting of more than <NUM> components, as well as the collection of larvae immediately after moulting, when the amount of the ferment having the keratolytic effect decreases as a result of their use in the process of the removal of the old exoskeleton.

Also, the disadvantage of all methods described above is that the development of the larval stage of the insect Galleria mellonella that includes from <NUM> to <NUM> moults (depending on the given example), occurs in the same containers in which were placed the eggs or the larvae of the first-second age. This leads to the accumulation of the waste products of insects and the decrease of the yield of biomass.

The problem solved by the given invention consists in the fact of the elaboration of a new effective method for the obtainment of the biomass of Galleria mellonella larvae with high biological activity.

The essence of the invention consists in the fact that it is proposed the method of the obtainment of the biomass of Galleria mellonella larvae with high biological activity that consists in the preparation of the conditions for the cultivation of larvae under the artificial conditions, cultivation of larvae on a nutrient medium and collection of larvae, and the nutrient medium for Galleria mellonella larvae has the following composition, g/kg:.

and during the period of the cultivation of larvae, at least <NUM> transfers to fresh nutrient medium are carried out, the larvae are collected at different ages depending on the type of the required biological activity.

The technical result of the invention, in comparison with the closest solution, consists in the fact that the method of the obtainment of the biomass of Galleria mellonella larvae includes a simpler and less expensive medium that provides a larger amount of biomass (<NUM>,<NUM> - <NUM>,<NUM> of the biomass on average for <NUM> compared with <NUM>,<NUM> - <NUM>,<NUM> for the prototype) with a higher biological activity.

The technical result of the invention is due to the fact that during the development of Galleria mellonella larvae, at least two transfers of larvae to a fresh medium are carried out that prevents the effect of inhibition caused by the accumulation of the waste products in the containers for the cultivation of insects.

Pupae of Galleria mellonella are taken out the refrigerator, where were stored at the temperature of <NUM>-<NUM> and are left at the indoor temperature for <NUM> hours. With the passage of this time, the pupae are transferred to the closed containers containing a small amount of nutrient medium (<NUM>/<NUM> of the volume of the container) for their subsequent development and transition to the phase of imago and oviposition. For the initiation of the hatching of larvae from eggs in the dark containers, it is maintained the temperature of <NUM> and the relative humidity of <NUM>%. The first instar larvae appear <NUM> hours after the pupae are placed in the containers. The process is monitored, over a period of time during which <NUM>% yield is provided, but not more than <NUM> hours.

The used nutrient medium has the following composition, g/kg:.

New containers with nutrient medium are prepared. <NUM> hours after the start of the hatching, the larvae of the first generation are selected by the tweezers from the containers in which they appeared and are placed in the prepared containers.

Growth of larvae lasts <NUM> days since the date of the first transfer. The temperature of the container is maintained at the level of <NUM> and the relative humidity constitutes <NUM>% in the dark conditions.

During the given period, on the <NUM>th day and the <NUM>th day, two transfers of larvae to a fresh nutrient medium are carried out.

For the obtainment of the biomass of larvae with a high keratolytic activity, the collection of larvae occurs on the <NUM>th day of cultivation, before the beginning of the <NUM>th moulting of larvae. Larvae are washed twice by four times volumes of physiological solution and twice more by four times volumes of purified water.

In the obtained biomass are determined the quantitative and qualitative indicators. The obtained results are demonstrated in the table <NUM>.

The data in the Table <NUM> prove that the use of a simplified medium leads to a significant reduction of the cost of the production of larvae (the cost of food in the proposed solution constitutes <NUM>% of the cost of the prototype medium), and the amount of the biomass produced using one gram of medium is <NUM>,<NUM>% more compared to the prototype. At the same time the keratolytic activity of the biomass is doubled.

Pupae of Galleria mellonella are taken out the refrigerator, where were stored at the temperature of <NUM>-<NUM> and are left at the indoor temperature for <NUM> hours. With the passage of this time, the pupae are transferred to the closed containers containing a small amount of the nutrient medium (<NUM>/<NUM> of the volume of the container) for their subsequent development and transition to the phase of imago and oviposition. For the initiation of the hatching of larvae from eggs in the dark containers, it is maintained the temperature of <NUM> and a relative humidity of <NUM>%. The first instar larvae appear <NUM> hours after the pupae are placed in the containers. The process is monitored, over a period of time during which <NUM>% yield is provided, but not more than <NUM> hours.

For the obtainment of the biomass of larvae with a high antioxidant activity, the collection of larvae occurs on the <NUM>th day of cultivation. Larvae are washed twice by four times volumes of physiological solution and twice more by four times volumes of purified water.

The data in the Table <NUM> demonstrate that the amount of the biomass obtained using one gram of medium is increased by <NUM>,<NUM>% compared with the prototype. Also, it is increased the antioxidant activity of the biomass by <NUM>,<NUM> times.

For the obtainment of the biomass of larvae with a high anti-inflammatory activity, the collection of larvae occurs on the <NUM>st day of cultivation. Larvae are washed twice by four times volumes of physiological solution and twice more by four times volumes of purified water.

Claim 1:
Method of obtainment of the biomass of Galleria Mellonella larvae with high biological activity which consists in placing the pupae in containers with nutrient medium, maintaining them at a temperature of <NUM>
and a humidity of <NUM>% until the hatching of the first instar larvae, transferring the larvae after <NUM> hours after hatching in other containers with nutrient medium, their cultivation at a temperature of <NUM>
and humidity of <NUM>%, the nutrient cultivation medium having the following composition, g/kg:

<TAB>

characterized in that the cultivation of larvae takes place optimally for <NUM>-<NUM> days, respectively: <NUM> days - for biomass with high keratolytic activity; <NUM> days - for biomass with high antioxidant activity and <NUM> days - for biomass with high anti-inflammatory activity, with the transfer of larvae at least <NUM> times to fresh nutrient medium and the collection of larvae.