CZ31652U1 - Cultivation soil for the production of natural pigments using microscopic fungi of the genus Monascus - Google Patents

Description

Technical field

The invention relates to a composition of culture medium for the production of natural pigments by means of microscopic fungi of the genus Monascus.

BACKGROUND OF THE INVENTION

In the manufacture of dyes as food additives, labeled with registration codes (E) and designed to enhance the visual attractiveness of foods, restore the original appearance of foods whose color has been affected by processing, storage, packaging or distribution and last but not least color otherwise food (Emerton V. , Choi E .: Additives The Essential Guide to Food Additives (3), 101-319, 2008) employs the latest in biotechnology. The European Food Safety Authority (EFSA) examines the health of all additives, with food colors among the first substances considered. In recent years, it has been suspected that some naturally identical dyes produced synthetically or even natural food dyes (eg cochineal) may affect children's health. The trend nowadays is an increased initiative of food producers and food service providers to remove some synthetic dyes from products (eg E102, E104, El 10, E122) and replace them with natural health-friendly products.

Opinions on the health safety of dyes are constantly evolving and are a much debated issue. For example, the possible toxic interactions between substances in the mixture are insufficiently mapped, there is a lack of knowledge about the mechanisms of toxicity and the establishment of predictive mathematical and biological models. One recent controversy regarding genotoxicity was due to sulfonated azo compound dyes: Amaranth (E 123), Ponceau 4R (E 124), Sunset Yellow FCF (E 110), Tartrazine (E 102), and Azorubine / Carmoisine (E122). (EFSA Journal 2010; 8 (10): 1778). Although it has been shown that the consumption of these additives does not present a risk, EFSA nevertheless recommended further new additional testing and foods containing one or more of these food colors (E 110, E 104, E 122, E 129, E 102, E 124) additional information: "may adversely affect children's activity and attention" (http://www.europarl.europa.eu/sides/getDoc.do?language=en&type=IM-

PRESS & reference = 20080707IPR33563; https: //www.efsa.europa.eu/en/press/news/ans091112).

Biotechnological production of food dyes has the potential to replace the discussed naturally identical dyes produced synthetically from highly purified petroleum products, or natural dyes having their limits in the seasonality of the raw material, less color stability, sensitivity to light, heating, oxidation and often problematic water solubility.

The source of natural colored substances, most often in the form of pigments, are plant and animal species, some microorganisms, lichens and fungi (Velíšek J., Hajšlová J .: Food Chemistry Π, Ossis, Tábor, 602, 2009).

The global natural dye market was estimated at US $ 1,144 million in 2014 with a growth forecast of US $ 1,698 million by 2020, an increase of 6.8%, with carotenoids dominating with a market share of 31.8% .

(http://www.futuremarketinsights.com/reports/global-natural-food-colours-market)

The companies that play an important role in the international global market include the company Chr. Hansen A / S, Sensient Technologies Corporation and Kalsec Inc, DDW, Naturex S.A., ADM (Wild Flavors Inc), ROHA Dyechem Pvt. Ltd., GNT International Β. V., DIC Corporation, and LycoRed Ltd.

In the Czech Republic, only one company ASCOLOR BIOTEC s.r.o. has historically been commercially producing microbial pigment. with Arpink RedTM. Currently, its activity was followed by NATURAL RED a.s., in cooperation with VÚPP, v.v.i., with a pilot production of 3000 l. The producer of this red pigment is a strain of the microscopic sponge Penicillium oxalicum var. Armeniaca. Based on a favorable opinion of the Ministry of Health of the Czech Republic and after submitting the required documentation, JECFA stated that there is no objection to the use of Arpink Red in meat products and meat analogues (up to 100 mg / kg), further into non-alcoholic beverages (up to 100 mg / kg), alcoholic beverages (up to 200 mg / kg), dairy products (up to 150 mg / kg), frozen creams (up to 150 mg / kg), and confectionery (in up to 300 mg / kg).

With regard to the anticipated amendment of the legislative regulations, the JECFA opinion was limited to 31 December 2002. New documents for the legislative procedure are currently being prepared.

Among the inexhaustible number of possible microbial pigment producers that may find application in the food industry, for example, Xanthophyllomyces denh orhous - Astaxanthin, Monascus sp. - Ankaflavin Color supplement, Monascorubramine, Rubropunctatin, Penicillium oxalicum - Anthraquinone, Fusarium sporotrichioides - Lycopene, Saccharomyces neoformans - Melanin, Rhodotorula sp. - Torularhodin, Flavobacterium Zeaxanthin, Agrobacterium auranticum - Astaxanthin, Mycobacterium lacticola - Astaxanthin, Flavobacterium sp. - Zeaxanthin, Blakeslea trispora and Dunaliella sage - ι-Carotene, Blakeslea trispora - Lycopene, Spongiococcum excentricum - Lutein.

(https://www.hindawi.com/joumals/ab/2014/837891/)

By cultivating microscopic fungi, a number of color-important components of the secondary metabolites may be present. of pigments can reach up to several grams per liter in the fermentation medium. The strong properties of the pigment include strong antioxidant activity, which has a positive effect especially in the prevention of cardiovascular and tumor diseases. The application of fungal mycelial biomass as an intermediate in biotechnological production in animal feed or food production is given by the basic strict requirement for health safety in terms of toxin production. This claim can be met to a certain extent by selecting suitable strains and adjusting the composition of the culture medium. The biotechnological preparation of red pigments could be an alternative substitute or supplement to the hitherto used red pigments in pharmacy or food industry (eg cochineal).

There is currently very little information on the commercial or potential utility of pigment-producing fungal cultures in the feed, pharmaceutical or food industries.

In the controlled cultivation of microscopic fungi, a fungal mycelial biomass, which is also a rich source of a number of biologically active substances, is produced in parallel with the preferred dye. The isolated mycelial biomass contains, in addition to a significant amount of fiber, a significant amount of proteins, vitamins, minerals and some immunostimulatory factors. The practical use of fungal mycelial biomass itself has so far been described only very rarely. The use of this mycoprotein biomass in the feed industry as a protein and vitamin supplement under the name “Terlikin” (a protein-protein preparation derived from biomass produced on waste products in potato processing) is reported in the literature. In the food industry, mycoprotein biomass forms the basis of a range of products offered by the Sainsbury supermarket chains in the UK under the name "Sainsbury's Quom" (eg Sainsbury's Quom pie, Quom Swedish style balls). The use of fungal mycelial biomass for the isolation of glucans (Kyanko, V., Canel, RS, Ludemann V., Pose G., Wagner JR: β-Glucan content and hydration properties of filamentous fungi. 49), 1, 2013, 41-45; Park HS, Yu YM, Lee K. K., Maeng PJ, Kim SC, Yu JH: Velvet-mediated repression of β-glucan synthesis in Aspergillus nidulans spores. (5), Article number: 10199 2015). Glucans are the basis of the internal structure of cell walls. Pure glucans can be used as immunostimulators in medical and veterinary applications because they promote the production of monocytes, neurophylls, collagen, elastin and stimulate macrophages to enhance the interleukin response. By immunostimulatory action, glucans are particularly important for ruminants. Glucans also play an important role in lowering cholesterol.

Much attention in the biotechnology of fungal cultures is devoted to the production and isolation of the produced pigments, which could be a substitute for the synthetic naturally identical dyes used. The amount of pigment produced in liquid soils can be influenced by the selection of suitable culture media, by the ratio of the components contained or by the addition of some trace elements.

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The pigment produced can be obtained from the supernatant by microfiltration and nanofiltration techniques in the form of a highly concentrated, stable preparation useful in the food or pharmaceutical industry.

The composition of the broth essentially influences the recovery of the two basic fermentation constituents described above. pigment and mycelial biomass. There is no absolute direct correlation between the production of fungal mycelial biomass and the amount of pigment product formed. An inappropriate ratio of the carbon to nitrogen components of the medium leads to excessive biomass formation at the expense of the preferred pigment component production.

The aim of the technical solution was to find rational conditions for conducting the cultivation process of the fungal culture of the genus Monascus using ethanol and other biologically active substances present in dairy products. The solution also aims to make available the composition of the culture medium in order to accelerate and achieve optimum pigment production in the culture medium.

The essence of the technical solution

These deficiencies are eliminated by the culture medium for the production of natural pigments by means of microscopic fungi of the genus Monascus, according to the present invention, which consists in that it contains ethanol as the main carbon source in actual concentrations of 0.2 to 1% by volume. % in 3 to 20 wt. Kluyveromyces marxianus yeast, further comprising a major nitrogen source of protein hydrolysates of 0.1 to 0.5 wt. together with a minor proportion of yeast extract at a concentration of 0.05 to 0.1% by weight, for the function of the growth agents. The culture medium, for the content of actual concentrations of ethanol 0.2 to 1% by volume, contains 3 to 20% by weight. fermented milk product solutions containing 1,5 to 8% ethanol by volume. The culture medium, in the case of a loss of nutrients, further comprises the addition of a fermented solution of dairy products to ethanol, changing the adjusted pH range of 4.5 to 6.

The following examples of carrying out the fermentation process on soils with variable sources of carbon and nitrogen, the solution, merely illustrate, without limiting it in any way. The use of these cultivation media according to the solution has been successfully tested by the inventors in practice at the Applicant in the Food Research Institute in Prague, v.v.i., CZ.

Examples of technical solutions

Example 1

Comparison of assimilable carbon sources for the production of natural pigment by the culture of the microscopic fungus Monascus.

Table 1 shows the results of cultivation experiments on nutrient media with different content of carbonaceous substances. A serial culture experiment was carried out in Erlenmeyer flasks with a useful filling of 500 ml of broth containing equal amounts of protein hydrolyzate 0.2 wt. % and yeast extract 0.1 wt. and various additions of assimilable carbon sources and ethanol at two different concentrations. The experiments were performed on a rotary shaker (100 RPM) at 30 ° C without pH adjustment. After 100 hours of culture, the individual variants from this serial experiment were evaluated for pigment production by measuring absorbance A494. In the ethanol culture variant, an ethanol source in the form of 20 wt. deproteinized whey of 8% v / v ethanol fermented with Kluveromyces marxianus yeast culture so that the final concentration of ethanol in the medium in Erlenmayer flasks is 1 or 2% v / v. The best results were obtained in culture medium variants containing 2 wt. sucrose and 1 vol% ethanol. By comparing the values obtained for the individual carbon sources, it is evident that the higher content of carbon source in the case of a 4% wt. and in a variant of the nutrient broth with ethanol of 2% by volume, it slows down pigment production.

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Table 1. Composition of culture media with different contents of assimilable sources

C-source Sucrose Maltose Glucose Ethanol % 2 4 2 4 2 4 1 2 Yeast extract (%) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Vysokoprotein. hydrolyzate (%) 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Absorbance (494 nm) 2,974 0.540 0.337 0.695 0.238 0.490 2,458 2,077

Example 2

Comparative cultivation experiment on deproteinized whey with microscopic sponge Monascus purpureus with different variants of soil composition.

Cultivation experiments with Monascus purpureus strain were conducted in Erlenmayer flasks with a useful loading of 500 ml culture medium. The soil base was deproteinized whey 5 wt. The culture broth in both variants I and II further contained the same amounts of yeast extract 0.1% by weight, but different amounts of protein hydrolyzate 0.2, respectively. 0.4 wt. The main carbon source in both soil variants was ethanol contained in the fermented milk fermentation culture of Kluyveromyces marxianus. Before the start of the cultivation experiment, ethanol was added in the form of a dairy culture to the nutrient medium so that the initial concentration of ethanol was 0.6% by volume in both nutrient media. After ethanol introduction, cultivation was started on a rotary shaker (100 RPM) at 29 . During the 144-hour cultivation periodically the absorbance value was determined to assess the production of natural pigments and the actual concentrations of ethanol were monitored. It can be seen from Table 2 that the highest absorbance was measured in the Nutrient Option II, where the ratio of nitrogen nutrients to the carbon source contained was higher (53 mg of amino nitrogen / ml and 106 mg of amino nitrogen / ml of ethanol respectively).

Comparative culture experiment with the production of natural pigments in cultures of microscopic fungi Monascus purpureus on ethanol contained in 5 wt. deproteinized whey (different ratios of nutrients nitrogen and carbon)

Soil components AND II Deprotein whey (5% w / w) 500 500 Yeast extract (% by weight) 0.1 0.1 Vysokoprotein. hydrolyzate (wt.%) 0.2 0.4 Ethanol (% v / v) - initial concentration 0.6 0.6 Time (hours) Actual ethanol content in nutrient medium (% by volume) 0 72 0.324 (faint red) 0.278 (faint red) 96 red red

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Soil components AND II 120 0,866 1,460 144 1,132 2,902 264 4,244 7,600 Time (hours) Actual ethanol content in nutrient medium (% by volume) 0 (initial concentration) 0.6 0.6 72 0.41 0.52 120 0.21 0.22 144 0.05 0.004

Example 3

Production of natural pigments of the microscopic fungus Monascus purpureus on nutrient media containing various sources of ethanol (ethanol contained in the fermented yeast culture of a mixture of milk and whey).

Comparative culture experiments with a Monascus purpureus microscopic culture were performed on a rotary shaker (100 RPM) at 29 ° C using various nutrient media variants containing 3 wt. % whey deproteinized and additions 0.1 wt. % yeast extract, 0.4 wt. % protein hydrolyzate and 1 wt. sucrose. The contained sucrose in all soil variants was used to ferment the microscopic fungus culture. After 96 hours, when the contained sucrose was exhausted, a different carbonaceous ethanol source was added to the culture medium variants II and III. In the variant II of the broth (96 to 120 hours in divided doses) it was ethanol contained in the alkalized fermented milk and whey solution using the yeast Kluyveromyces marxianus and in the comparative variant ΙΠ commercial concentrated ethanol was used as the carbon source. Option I served as a control medium, i.e. the cultivation continued without the addition of ethanol. Ethanol contents and the amount of pigment produced, expressed as absorbance at 494 nm, were monitored in a continuous culture on a rotary shaker. While in variant I, where no ethanol supplement was present, there was a continuous increase in pH and a very slight increase in the measured amount of pigments (possibly due to its release from the inside of cells), in the other variants II and III growth continued and was accompanied by media and increasing the production of pigments. In variant II supplemented with an ethanolized dairy product, increased pigment production in the medium was observed, indicating that ethanol produced by the yeast culture of Kluveromyces marxianus in the dairy product solution may be a very good carbon source for pigment production.

Table 3 Microbial Pigment Production by Microscopic Monascus Sponge on Various Ethanol Sources (Commercial Ethanol and Ethanol Contained in Fermented Dairy Product)

AND Control Π III Time (throw.) Rf % PH A494 EtO H (obj. %) Rf% PH A494 EtOH (obj. %) Rf % pH A494 EtOH (obj. %) 0 4.9 5.08 ON 0 4.9 5.08 ON 0 4.9 5.08 ON 0 96 4.4 7.03 1,289 0 4.4 7.08 1,455 0 4.4 7.06 1,311 0

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After at- dání etha- nolu * 0 4.8 7.49 ** 0.58 4.6 7.08 *** 0.60 120 4.1 7.39 1,391 0.04 4.0 6.15 3,020 0.56 4.4 6.8 1,704 0.59 144 4.4 7.62 1,630 0.04 3.5 5,52 6,370 0.37 4,5 6.54 3,075 0.38

* without the addition of ethanol, ** addition of ethanol contained in the fermented dairy product, *** addition of ethanol of concentrated commercial

Industrial applicability

The cultivation medium for the production of natural pigments with the help of microscopic fungi of the genus Monascus 5 and their possible other derivatives expands the range of suitable soils with limited input of ballast substances while reducing the costs of its preparation.

Claims (3)

PROTECTION REQUIREMENTS Culture medium for the production of natural pigments by means of microscopic fungi of the genus Monascus, characterized in that it contains ethanol as the main carbon source 10 at actual concentrations of 0.2 to 1% by volume, obtained by fermentation of lactose contained in 3 to 20% by weight. Kluyveromyces marxianus yeast, further containing protein hydrolysates at a concentration of 0.1 to 0.5 wt. together with a minor proportion of yeast extract at a concentration of 0.05 to 0.1 wt. The culture medium according to claim 1, characterized in that it contains actual contents 15 concentrations of ethanol 0.2 to 1% by volume contain 3 to 20% by weight. fermented milk product solutions containing 1,5 to 8% ethanol by volume. The culture medium according to claim 1 or 2, characterized in that, in the case of a loss of nutrients, it further comprises the addition of a fermented solution of ethanolized dairy products at a change of the adjusted pH range of 4.5 to 6.