CZ35655U1 - Mixed pigment based on red, purple, orange and brown dyes with antioxidant effect - Google Patents

Description

Mixed pigment based on red, purple, orange and brown dyes with antioxidant effect

Field of technology

The solution relates to a mixed pigment based on red, purple, orange and brown dyes with an antioxidant effect, which is used mainly in food, pharmacy and cosmetics.

State of the art

Czech patent No. 285721 describes a microscopic fungus Penicillium oxalicum var. Armeniaca CCM 8242, obtained from the soil of the valley below Mount Ararat and in patent No. 302696, is a microscopic sponge CCM 8374, both producing exogenous red pigment with various uses in food, pharmacy, cosmetics.

The current use of these strains results in lower pigment yields and unstable quality, which is undesirable.

Both of the above strains were deposited at the CCM International Storage Site, Czech Collection of Microorganisms, Masaryk University in Brno, CZ.

The essence of the technical solution

The above-mentioned shortcomings are eliminated by a mixed pigment based on red, purple, orange and brown dyes with an antioxidant effect, prepared from the biomass of Penicillium oxalicum var. Armeniaca CCM 8242 and CCM 8374 with a minimum content of 10% by weight, mixtures of α and β glucans, 3% by weight, niacin, 7% by weight, pantothenic acid and 1.5% by weight, pyridoxine in dry matter, especially for food, pharmacy and cosmetics, according to a technical solution, the essence of which is that it shows characteristic maxima on spectrophotometric graphs from molecular absorption spectrophotometry in the visible region of the spectrum in the wavelength range 400 nm to 800 nm, in an aqueous solution with a pH value of 7 to 7.5 two maxima, when the first maximum on Lambda maxi is A = 494 nm and the second highest maximum on Lambda max2 is A = 420 nm, and two maxima in alcohol solution, when the first maximum on Lambda maxi is A = 502 nm and the second highest maximum on Lambda max2 is Λ = 415 nm.

Biomass for the production of antioxidant mixed pigments according to the technical solution using strains of microorganisms Penicillium oxalicum var. Armeniaca CCM 8242 and CCM 8374 also contain glucans in a minimum amount of 10% by weight.

Said new mixed pigments are produced by fermenting the inoculum and isolating pigments with antioxidant effects from it.

Mixed pigments according to the technical solution are used mainly in the food industry, eg in sausages, as a dietary supplement, as well as in the pharmaceutical and cosmetic industries, where they stabilize and color the produced product. The antioxidant effect of these pigments significantly extends the usability and life of all products to which it is added.

The mixed pigments according to the technical solution and their production make it possible to obtain the final product in a repeatable stable composition, which is not yet obvious. Their production now takes place with increased air saturation of the fermentation medium, treatment of the fermentation medium with yeast extract (eg HY YEST 412 Kerry) and adjustment of the pore size of the microfiltration device. By modifying the fermentation process, we obtain mycelial biomass, which contains health

-1 CZ 35655 UI beneficial substances, as it contains at least 10% by weight of glucans, see Giant. 2.

Clarification of drawings

In FIG. 1 is a graph showing the spectrophotometric measurement of the composition of the final base product (mass spectrophotometry), which is a red pigment, and the subsequent purple, orange, brown pigment is determined chromatographically.

In FIG. 2 are graphs showing the FT-IR spectra of fungal glucan and Penicillium oxalicum and Pleurotus mycelia (oyster mushroom), where the solid line represents glucans, the decorative line represents Penicillium oxalicum. The spectrum of the sample lies in the wavelength range 4000 to 450 cm ¹ with a resolution of 2 cm. This is a comparison of the spectrum of fungal glucan with glucans contained in the biomass of Penicillium oxalicum var. Armeniaca.

In FIG. 3 is a graph showing the spectrum from a mass spectrophotometer from the analysis of a sample obtained from experimental fermentation number 2 (natural red batch 2 (NR)) performed at the Institute of Microbiology of the Academy of Sciences of the Czech Republic, CZ.

In FIG. 4 is a graph showing the spectrum from a mass spectrophotometer from the analysis of a sample obtained from experimental pilot plant production from fermentation number 125 (natural red batch 125 (NR)) determined at the Food Research Institute in Prague, CZ.

Mixed pigments according to the technical solution were verified in practice with good results in the laboratories of the Institute of Microbiology A in the Czech Republic in Prague, CZ.

Examples of technical solutions

Example 1

The production of mixed pigments according to the technical solution takes place from biomass containing at least 10% by weight of glucans after fermentation of microbial strains of Penicillium oxalicum var. Armeniaca CCM 8242 and CCM 8374 according to the basic production scheme:

• RO preparation of DEMI water for production • Steam generator and preparation of hot steam for sterilization • Air compressor for production • Sanitation equipment • Cooling system • Microbiological laboratory, strain work and inoculum preparation • Biotechnological fermenter - obtaining the active substance • Product processing line - centrifuge, MF, NF - Isolation of the active substance • Spray dryer - drying the product on a suitable carrier

The whole fermentation process is discussed under sterile conditions.

Production begins in a microbiological laboratory, where a strain of the microscopic fungus Penicillium oxalicum var. Armeniaca CCM 8242 and CCM 8374 from lyophilized state. It is inoculated into petri dishes and cultured in a thermostat at 28 to 29 ° C for 5 days.

- 2 CZ 35655 UI

The criteria for mycelial assessment are:

• uniform velvety surface of the mycelium • mycelium in green color • rich sporulation • color of agar under mycelium deep red

From the surface of the petri dish, scrape a homogeneous mass of mycelial spores with a laboratory loop and transfer sterile to a 500 ml Erlenmeyer flask. Inoculate the inoculum in flasks on a shaker at 220 to 240 rpm for 36 hours to 48 hours

The fermentation broth for inoculation consists of yeast extract (eg HY YEST 412 Kerry) in the amount of 6 g / l of soil, beet sugar - sucrose in the amount of 18 g / l of soil and the mass of Agar-agar in the amount of 20 g / l of soil.

The selected inoculum obtained is sterile transferred to the inoculation fermentor. The batch volume of the inoculation fermentor is about 0.1% to 2% of the production fermentor volume.

The process conditions in the inoculation fermentor are as follows:

The temperature inside the unit is 28 to 29 ° C, the pressure in the unit is 0.02 to 0.03 MPa, the air consumption is 15 to 30 m ³ of air / m ³ of fermentation medium / h, stirring with a paddle stirrer at a speed of 250 rpm, soil pH 5.8 to 6.2. The fermentation broth for inoculation consists of yeast extract HY YEST 412 Kerry in the amount of 6 g / l of soil, beet sugar - sucrose in the amount of 18 g / l of soil.

It is necessary to maintain the activity of the fungus (activity) in a regular vaccination cycle in test tubes every 3 months. The sponge must be stored at a temperature of 3 to 5 ° C.

We cultivate the inoculum for 36 to 48 hours, the mycelium must be a fluffy structure with long fibers and rich sporulation, evenly suspended in the fermentation medium. During fermentation, we monitor the pH value, the amount of dye, the amount of mycelial biomass and the microbial purity of the sample - monoculture, fungi. After the above time, we sterile transfer the inoculum to the sterilized fermentation broth in the production fermentor.

The process conditions in the production fermenter are as follows:

The inoculum volume for the production fermentor is about 0.1% to 3% of the production fermentor volume.

The temperature inside the unit is 28 to 29 ° C, the pressure in the unit is 0.07 to 0.08 MPa, the air consumption is 30 to 50 m ³ of air / m ³ of fermentation medium / h, stirring with a paddle stirrer at a speed of 250 to 400 rpm / min, soil pH 5.8 to 6.2, oxygen solubility 80 to 100%.

The fermentation broth for inoculation consists of yeast extract in the amount of 6 g / liter of soil HY YEST 412 Kerry, beet sugar - sucrose in the amount of 18 g / l of soil, PPG defoamer as required.

We cultivate the mycelium in this way for 68 to 72 hours, until the sucrose is depleted from the nutrient medium and during fermentation we monitor the pH value, the amount of residual sucrose, the amount of dye, the amount of mycelial biomass and the microbial purity of the sample - monoculture, fungi.

After the fermentation, we increase the temperature in the fermenter stepwise to 45 ° C to 50 ° C and adjust the pH value with ammonia water to pH 8.5 to 9.5 and mix at a speed of 35 to 40 rpm.

The finalization steps follow, ie the removal of mycelia by means of a centrifuge at a speed of 15,000 rpm, or on a filter press, from the fermentation broth with the desired product - red pigments. The mycelial content is about 3 to 5% of the batch volume. This is followed by purification of the medium from mycelial fragments

-3GB 35655 U1 and insolubles on a microfiltration device (membrane pore size is 0.45 to 0.60 pm) and in the final phase concentrating the product to a volume of 1/10 of the batch volume on the nanofiltration unit (pore size 300 to 350 Daltons).

The concentrated product is then spray dried on a suitable support. The temperature of the injected material is 35 to 45 ° C and its pH is 9.0 to 9.5. The air temperature at the inlet to the drying chamber is 200 ° C and at the outlet from the drying chamber 98 ° C

The result is a red powder with a bitter taste, together with a pigment of purple, orange and brown, which is determined chromatographically.

Analysis of the final product - red pigment after fermentation of the strain Penicillium oxalicum var. Armeniaca CCM 8242 and CCM 8374 yielded 6 to 10 g red pigment / 1 medium while maintaining optimal fermentation conditions. The absorption value according to the calculation method proposed by the FAO corresponds to 1.05. In this case, the formula 100xAx 100 is used to determine the red pigment concentration

1.05 xW where denotes

And the Lambda value of the maximum absorption at 494 nm,

W weight of crystalline pigment sample 100 mg, 1.05 constant absorption value.

The obtained red pigment was determined spectrometrically using molecular absorption spectrophotometry in the visible region of the electromagnetic spectrum with a frequency range of 400 nm to 800 nm, when 2 characteristic maxima were obtained in an aqueous solution with a pH of 7 to 7.5, the first maximum on Lambda maxi is A = 494, the second highest maximum on Lambda max2 is A = 420 nm.

Further spectrometry was performed with an alcoholic solution of red pigment, where the first maximum on Lambda maxi was A = 502 nm, the second highest maximum on Lambda max2 was A = 415 nm.

The quality (purity) of the pigment is indicated by the value A1 / A2 = 1.3 to 1.5, at which the value of antioxidant activity AK = 2.65.

The quality of the produced red powder of bitter taste, together with the pigment purple, orange and brown, is evidenced by the graph in Fig. 1.

Example 2

The production was carried out according to Example 1, but with the following modifications:

HY-YEST 412 Kerry yeast extract served as a source of nitrogenous substances in the fermentation broth, 30 to 50 m ³ of air / m ³ of fermentation medium / h were used for aeration during the fermentation process, at a temperature of 28 to 29 ° C and agitation during fermentation at 250 up to 400 rpm, the fermented medium already turns red after 22 hours. The fermentation ends at 68 to 72 hours. Microfiltration on membranes with a pore size of 0.45 to 0.60 μm and nanofiltration on membranes with a pore size of 300 to 350 Daltons were used to finalize and process the product.

-4CZ 35655 UI

Example 3

The biomass for the production of mixed pigments according to the technical solution must contain at least 10% by weight of glucans in order to have the expected good use in nutrition due to the increased content of glucans.

The composition of mycelia = biomass (can be used for complex drying) is shown in Table 1.

Table 1

Qbsah / faktor mycelium g / 100 g dry matter dry matter ash 6.5 fat 3.9 proteins 32.1 total dietary fiber 37.0 soluble food fiber 26.8 insoluble food fiber 10.3 Content / factor mycelium vitamins in biomass g / 100 g dry matter niacin 3.7 pantothenic acid 7.10 pyridoxine 1.81

Note:

Fat - the origin of the defoamer

Protein - probably slightly affected by the addition of ammonia

Antioxidant activity (mg AK / 1 g dry matter) (DPPH) = 2.65

-5CZ 35655 UI

Comparison of glucan content in biomass - see Fig. 2 - shows Table 2.

Table 2

Content / factor mycelium g / 100 g dry matter dry matter total glucans 13.25 and - glucans 3.40 β - glucans 9.85 fungi of the genus Pleurotus (mycelial lyophilisate) total glucans 17% and - glucans 3% β - glucans 14%

In FIG. 2 shows the FT-IR spectra of fungal glucan and Penicillium oxalicum mycelia. The FT-IR spectrum of the sample in the wavelength range 4000 to 450 cm ¹ with a resolution of 2 cm ¹ was measured and the spectra were compared with the spectrum of the fungal glucan, where the presence of glucan containing 1,3 and 1,6 bonds was confirmed, the sample further containing fats ( area 3000 cm ¹ ) and proteins (area 1700 cm ¹ ). The obtained mixed pigments according to the technical solution in the form of a red pigment were subjected to analysis as shown in FIG. 2.

Mixed pigment samples (Natural Red) designated as crude extract and original sample were tested by cell-biological techniques (fluorescence microscopy, flow cytometry) on primary human skin fibroblasts. The experiment was performed twice for both samples, the obtained data did not show any significant variance, it is a biological duplicate.

Methodology:

Cells were thawed from stock cultures stored in liquid nitrogen one week before the experiment to achieve optimal physiological status. Subsequently, Ix were passaged to the optimal cell density (confluence approx. 50%) in 24-well plates with an inserted coverslip (Nunc). For bioactivity / toxicity testing, reagents stored at 50 mg / ml (4 ° C) were diluted under sterile conditions in tissue culture medium (D-MEM, 10% FCS) and added at the required concentrations to the fibroblasts tested. The test was run for 24 h / 72 h at 37 ° C, in 5% CO 2 in the atmosphere.

For FACS flow cytometry (FACS), cells were detached from the culture plate with 0.1% trypsin solution, immediately labeled with fluorescent dye DAPI identifying necrotic and apoptotic cells (HY YEST 412 Kerry) and subsequently analyzed by FACS Aria (Becton Dickinson). Data were analyzed using FlowJo software.

For fluorescence microscopy, cells were fixed with 4% PFA in PBS for 10 min, permeabilized with 0.01% Triton X-100 in PBS, RT, blocked with 5% BSA in PBS, and labeled with a monoclonal antibody to Lamp-3 (MEM 259) and fluorescently labeled phthaloidin-Alexa 594. Mice

-6CZ 35655 UI monoclonal antibody was subsequently identified using a secondary GAM-Alexa 488 antibody. Visualization was performed using an Olympus inverted fluorescence microscope using a DP50 color sensitive camera (40x objective).

Flow cytometric:

Dot-plot analysis shows the result of one of the duplicates for 24 h incubation with concentrations covering the range of 8 to 1000 micrograms / ml.

Conclusion:

No significant cytotoxicity was detected by flow cytometry in the concentration range of 8 to 1000 micrograms / ml. The highest tested concentration is extreme, only a few substances of the secondary metabolite type are thus bio-neutral. Similar data were obtained in 24h and 72h experiments in biological duplicate.

Fluorine scenario microscopy:

Primary skin fibroblasts were tested for a concentration range of Natural Red extracts at a concentration of 1 to 500 micrograms / ml. The integrity and pattern of the actin cytoskeleton (which is extremely sensitive to environmental influences), the late endosomal / lysosomal vesicular system and nuclei were visualized.

Conclusion:

The chosen combination of features is suitable for sensitive identification of cell physiology disorders. This was not observed, except perhaps the highest concentration, which led to a slight change in cell adhesion and elongation, the endosomal / lysosomal system was not affected, necrosis or apoptosis were observed (at both time points - 24 and 72 h), in both fractions in biological duplicate.

The studied Natural Red fractions show extraordinary biocompatibility / absence of cytotoxicity to the primary human skin fibroblast cell line. Significant cell pathology was not recorded in a wide concentration range applied in a multi-day format, tested using sensitive quantitative (flow cytometry) and qualitative techniques (fluorescence microscopy).

Industrial applicability

The solution concerns new mixed pigments red, purple, orange and brown with antioxidant effect, created after cultivation and fermentation of biomass from strains of Penicillium oxalicum var. Armeniaca CCM 8242 and CCM 8374 for use in the food, pharmaceutical and cosmetic industries.

-7 CZ 35655 UI

List of abbreviations that are used in the text:

FT-IR spectra - infrared fluorescence detector

RO - reverse osmosis

DEMI - demineralized

MF- microfiltration

NF - nanofiltration

PPG - polypropylene glycol

DPPH - diphenylpicrylhydrazyl

CO2 - carbon dioxide

FCS - fluorescence correlation spectroscopy

FACS - fluorescence flow cytometry

DAPI - 4,6-diamidine-2-phenylindole - fluorescent dye

PFA - plasma proteins

PBS - buffer (phosphates) for tissue cultures

Triton X-100 - nonionic surfactant

RT - reverse transcriptase

BSA - bovine serum albumin

MEM - monoclonal antibody

GAM-Alexa 488 - antibody name.

Claims (1)

PROTECTION REQUIREMENTS 1. Mixed pigment based on red, purple, orange and brown dye with antioxidant effect prepared from biomass of strains of microorganisms Penicillium oxalicum var. Armeniaca CCM 8242 and CCM 8374 with a minimum content of 10% by weight, mixtures of α and β glucans, 3% by weight, niacin, 7% by weight, pantothenic acid and 1.5% by weight, pyridoxine in dry matter, especially for food, pharmacy and cosmetics, characterized by showing characteristic maxima on spectrophotometric graphs from molecular absorption spectrophotometry in the visible region of the spectrum ίο in the wavelength range 400 nm to 800 nm, in an aqueous solution of pH 7 to 7.5 two maxima, the first maximum on Lambda maxi is A = 494 nm and the second highest maximum on Lambda max2 is A = 420 nm, and two maxima in the alcohol solution, where the first maximum on Lambda maxi is A = 502 nm and the second highest maximum on Lambda max2 is A = 415 nm.