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    • 专利摘要:
    The invention relates to an isolated strain of Sporobolomyces roseus with high carotenoid production capacity. There are also disclosed processes for obtaining colored compositions comprising carotenoids and in particular ß-carotene. The invention also discloses the use of these compositions as antioxidant agents and as colored ingredients in edible products. It also relates to processes for reutilizing food industry waste materials or by-products, such as citrus fruit molasses with Sporobolomyces roseus strains.
    公开号:ES2667433A2
    申请号:ES201890002
    申请日:2016-07-28
    公开日:2018-05-10
    发明作者:Izaskun MARAÑÓN GARCÍA；Leire SAN VICENTE LAURENT；Jessica SÁENZ GÓMEZ；Laura OLIVER GARCÍA；Raquel VIRTO RESANO；Carolina González Ferrero；María De La O RODRÍGUEZ GONZÁLEZ；María GARRIDO MARTÍNEZ；José Antonio GARCÍA BARRADO
    申请人:Centro Nacional De Tecnologia Y Seguridad Alimentaria (cnta)；Centro Nac De Tecnologia Y Seguridad Alimentaria Cnta；Centro Tecnologico Agroalimentario Extremadura；Fundacion Tecnalia Research and Innovation；
    IPC主号:
    专利说明:

    image 1
    SPANISH OFFICE OF THE PATENTS AND BRAND
    SPAIN
    image2 Publication number: 2 667 433
    Application number: 201890002
    image3 57 Summary: A strain of Sporobolomyces roseus for the production of compositions with coloring and antioxidant properties. The invention relates to an isolated strain of Sporobolomyces roseus with high carotenoid production capacity. Methods of obtaining colored compositions comprising carotenoids and in particular β-carotene are also disclosed. The invention also discloses the use of these compositions as antioxidant agents and as colored ingredients in edible products. It also refers to procedures for the reuse of waste materials or by-products of the food industry, such as citrus molasses, with strains of Sporobolomyces roseus.

    image4 A strain of Sporobolomyces roseus for the production of compositions with coloring and antioxidant properties DESCRIPTION
    The present invention relates to the fields of microbiology, food technology
    5 and the processes for obtaining dyes from industrial food waste materials, in particular a new strain of Sporobolomyces roseus yeast that can be used in industrial processes for the production of colored compositions and antioxidant ingredients. 10 PREVIOUS TECHNIQUE
    The dyes are used extensively in the food industry to provide an appropriate color to the final products, which are recognized by consumers as food in good condition.
    15 The production of dyes and, in particular, of carotenoid pigments by chemical synthesis implies high costs associated with low production yields.
    In order to improve the production of dyes and in particular carotenoids,
    20 some of them are produced industrially using bacteria, yeasts, fungi and plants. These dyes produced by living organisms are also called "biocolorants." In the particular case of yeasts, carotenoid production is associated with the response to stress conditions. Carotenoids are organic pigments produced by all these organisms from fats and other
    25 basic metabolic organic components. Carotenoids are divided into two classes, xanthophylls (which contain oxygen) and carotenes (which are purely carbohydrates and do not contain oxygen). All carotenoids are tetraterpenoids, which means they are produced from 8 isoprene molecules and contain 40 carbon atoms. According to their structure and chemical synthesis, carotenoids are tetraterpenoids (40
    30 carbon atoms) obtained from the condensation of four terpene units (each having 10 carbon atoms). Among carotenoids, β-carotene, α-carotene and βcriptoxanthin have vitamin A activity in humans. They can also act as antioxidants. Β-carotene is a strongly colored red-orange pigment. Β-carotene biosynthesizes from geranylgeranyl pyrophosphate and comprises beta rings at both ends of the molecule. The intestinal absorption of β-carotene is enhanced if consumed together with fats, since carotenes are fat soluble molecules.
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    Taking advantage of the fact that some microorganisms can produce carotenoids,
    5 It is widely used to reuse waste materials that these microorganisms process as a source of carbon, nitrogen and energy. An example of this is the production of dyes from yeasts, as disclosed in Marova et al. "Use of several waste substrates for carotenoid-rich biomass yeast production", Journal of Environmental Management-2012, vol. No. 95, p .: s338-s342. Marova et al. spread the
    10 influence of various types of waste substrates and stress factors on the production of carotenoids by yeast strains Rhodotorula glutinis, Rhodotorula mucilaginosa and Sporobolomyces roseus. Some of the waste materials used include potato and whey waste substrate from the dairy industry.
    15 An overview of the biotechnological production of carotenoids can be obtained using yeasts from Mata-Gómez et al. "Biotechnological production of carotenoids by yeast: an overview", Microbial Cell Factories-2014, vol. No. 13:12. The authors disclose some of the necessary conditions for yeasts to produce
    20 carotenoids, as well as the factors that affect such production (carbon source, solvents, culture medium, etc.). In addition, Mata-Gómez et al. They list some of the industrial by-products that are used as low-cost substrates, including grape juice, grape must, cane sugar juice, isolated from hydrolyzed mustard waste, corn syrup and whey. Table 1 of the document lists some of the
    25 species of yeasts that have been used to produce carotenoids from agroindustrial wastes, as well as what wastes are the substrates for yeasts. Among these yeasts are the aforementioned yeasts Rhodotorula glutinis and Rhodotorula mucilaginosa, disclosed by Marova et al. (above).
    30 Other carotenogenic yeasts are also used as food ingredients, such as that disclosed in WO2011130576, in which oilseed oil biomass from Rhodotorula mucilaginosa DSM 70398 and other yeast strains, in the form of yeast oil, is used as food ingredient or of whole oleaginous yeast cells. They are mainly used as satiety inducing agents, antioxidants and / or preservatives.
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    Genetically modified and mutated yeast strains are also disclosed in the
    5 patent literature for the production of compositions comprising β-carotene. Examples include patent application US20120142082, in which it is disclosed that some genetically modified Yarrowia lipolytica yeast strains have the ability to produce carotenoids. In addition, the patent summary JPH0622748 refers to the production of stable β-carotene in quality and productivity using strains
    10 selected from Rhodotorula glutinis created by mutagenic treatment.
    Although there are some microorganisms, together with yeasts, to produce carotenoids, there is still a need for new strains and new industrial conditions that allow high yields of carotenoid production to produce biocolourants and antioxidant ingredients, while saving costs and weather.
    SUMMARY OF THE INVENTION
    The inventors have isolated a new strain of Sporobolomyces roseus producing
    20 pigment deposited in the Spanish Type Culture Collection (CECT) on March 25, 2015 with identification reference LGO1107.
    The strain of Sporobolomyces roseus was isolated from an industrial surface in a food factory in Spain. It was deposited, in accordance with the Budapest Treaty, on March 25, 2015 in the Spanish Type Crops Collection (CECT) at the University of Valencia
    C.P. 46980, Professor Agustín Escardino No. 9 Paterna, Valencia (Spain), by depositors: 1) NATIONAL CENTER OF FOOD TECHNOLOGY AND SAFETY (CNTA) (Ctra. Na - 134, Km 50, 31570 SAN ADRIÁN (Navarra), Spain ); 2) TECNALIA RESEARCH & INNOVATION FOUNDATION (Miramón Technology Park, Mikeletegi
    30 Pasealekua, 2, 20009 DONOSTIA -SAN SEBASTIÁN, Spain); 3) EXTREMADURA AGROALIMENTARY TECHNOLOGICAL CENTER (Ctra. Villafranco a Balboa Km 1,2, 06195, VILLAFRANCO DEL GUADIANA (Badajoz), Spain). The Sporobolomyces roseus strain was identified by the depositor with the identification reference LGO1107 and received the CECT reference number, CECT 13123 after the International Deposit Authority declared the strain viable. It is mentioned interchangeably throughout the present description as Sporobolomyces roseus CECT 13123, S. roseus CECT 13123 or simply CECT 13123, S. roseus LGO1107 or simply LGO1107.
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    5 This isolated strain had the ability to produce β-carotene (and other carotenes and carotenoids) in high yields from citrus waste materials, in particular from citrus molasses, as will be represented in the examples below. Citrus molasses is a byproduct of citrus juice extraction. As a general rule, the fresh pulp obtained after pressing the fruits is
    10 mix with lime and press to remove moisture. The resulting liquid (pressing juice) is filtered to remove large particles, stylized by heating and concentrated. The resulting product contains approximately 72% dry matter and approximately 64% sugars. Citrus molasses is a thick viscous liquid, from dark brown to almost black, with a very bitter taste. It is often sold to distilleries
    15 or reincorporates in dried citrus pulp, but it can also be provided directly to animals for food or added to the herb silage.
    Therefore, the yeast strain of the invention provides the advantage of adding value to by-products or industrial by-products, such as citrus molasses.
    The invention also relates to a pure culture of yeast comprising the strain of Sporobolomyces roseus as defined above. Pure culture means that there is no other strain of yeast or other microorganism in the culture, but it may optionally comprise compounds from the culture medium in which the cells were grown. Crops
    25 cigars are generally provided as lyophilized cultures.
    Taking into account the ability of the strain as a high-performance producer of β-carotene, as well as a producer of other carotenoids, another aspect of the invention is a method of obtaining a colored composition comprising carotenoids,
    The process comprising fermenting a culture medium composition comprising a carbon source and a nitrogen source with a yeast strain, as defined above, under aerobic conditions; isolate the yeast strain and break the yeast cell wall to release the composition comprising carotenoids.
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    Therefore, the invention also relates to the use of the strain Sporobolomyces roseus CECT 13123 for the production of carotenoids.
    Colored compositions obtained by the action of the Sporobolomyces strain
    5 roseus CECT 13123 imply the advantage of having high concentrations of carotenes, mainly β-carotene, among synthesized carotenoids. This is because the strain has the capacity to produce approximately 2 µg of carotenes per ml of culture medium composition, quantified by UV-Vis as equivalents of β-carotene at 450 nm. In addition, the extraction or separation of carotenoids can be performed to obtain
    10 colored compositions with higher concentrations of β-carotene and / or an isolated fraction of torotrodin carotenoid compound, as will be illustrated below. The other major carotenes comprised in the colored compositions obtained by the action of the Sporobolomyces roseus CECT 13123 strain include γ-carotene and torulene.
    The invention also relates to a process for obtaining β-carotene which comprises carrying out a process as defined above and extracting the β-carotene from the colored composition comprising carotenoids.
    In addition, the invention also encompasses as an aspect a colored composition comprising carotenoids and which can be obtained by a method as defined above, said composition comprising β-carotene, γ-carotene, torulene and torularrodin.
    25 These colored compositions can thus act as coloring agents, which means that they can be used to color any substrate on which they can be comprised, forming part of a more complex composition (such as food, paint, pharmaceutical composition, etc.). ).
    The colored compositions comprising carotenoids of the invention are particular because they are the result of the release of the cellular cytoplasmic contents of the yeast. Therefore, they also comprise cellular compounds of the S. roseus cell, such as fatty acids. Even in the particular embodiments of the process of obtaining colored compositions, in which steps are performed
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    of extraction or separation of carotenoids, the resulting colored compositions comprise some cellular compounds of the S. roseus cell, which are also extracted or separated together with the carotenoids.
    Considering the well known properties of the carotenoid compositions and in particular of β-carotene, another aspect of the invention is the use of the yeast strain as defined above or of the colored composition as defined above, which comprise carotenoids and in particular high amounts of β-carotene, as antioxidant agents. This antioxidant activity can be carried out, for example, within an oral or nutraceutical pharmaceutical composition, the colored composition or the yeast itself being either the active ingredient or the excipient or the appropriate vehicle to preserve the oxidation of other active substances. The antioxidant activity is provided from the yeast itself, for example, when after ingestion by the consumer, the contents of the cytoplasm are supplied after digestion.
    In fact, the production of carotenoids is achieved by subjecting the strain Sporobolomyces roseus CECT 13123 (LGO1107) isolated to culture conditions that stimulate the synthesis of carotenoids. Therefore, another aspect of the invention is the use of a strain as defined above or of a colored composition obtainable as set forth above, as a coloring producing agent. A coloring producing agent should be understood as any compound, composition or cell that provides color to a substrate or matrix where this compound / composition / cell is. Therefore, it is not only a coloring producing agent that the supplied colored composition included in the yeast cells but also the yeast itself provides such coloring effect, due to its inherent color and the composition comprising carotenoids included in its cytoplasm.
    The colored compositions of the invention, as well as the strain of the invention, can be used in food production as colored additives, with the aim of readjusting the coloring of the final food product (edible product). It is generally known that the color of food actively affects the choice of consumers. In the same way, these colored compositions can be used in pharmaceutical or cosmetic compositions, for example for coloring tablets or pills, and even suspensions or liquid solutions.
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    The colored compositions, as well as the strain of the invention, can also be used as food ingredients, in order to be consumed by consumers and to provide them with the associated healthy effects due to their consumption.
    Therefore, it is another aspect of the invention to use a strain as defined above or a colored composition obtainable as set forth above, as an ingredient of an edible product.
    Yet another aspect of the invention is an edible product comprising an effective amount of S. roseus strain LGO1107 as defined above, together with appropriate amounts of other edible ingredients.
    The invention also relates to a method of carotenoid production comprising
    Fermenting a culture medium composition comprising citrus fruit molasses with a strain of Sporobolomyces roseus yeast under aerobic conditions.
    BRIEF DESCRIPTION OF THE DRAWINGS
    20 FIG. 1 shows the growth kinetics (log of CFU / ml) of S. roseus CECT 13123 (LGO1107) in malt yeast medium (YM) at different glucose concentrations; 10 g / l (A), 20 g / l (B) and 40 g / l (C). T (h) means time in hours.
    FIG. 2, in relation to Example 2, shows the performance of Sporobolomyces roseus
    25 CECT 13123 in biomass (g / l), determined by gravimetric measurement after centrifugation and drying, and the production of carotenes (µg / ml) at 240 hours in malt yeast medium (YM) at different glucose concentrations; 10 g / l (YM10), 20 g / l (YM20) and 40 g / l (YM40).
    FIG. 3, in relation to Example 3, is a bar chart with the production of
    30 carotenes compared (µg / ml of molasses) between S. roseus CECT 13123 (depositor reference: LGO1107) and the reference strain CECT 13019 in tangerine molasses at pH
    6. T (h) means time in hours. Data from another S. roseus isolated from vinegar are also represented. As before, T (h) means time in hours.
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    FIG. 4 and 5 show respectively the growth kinetics (log of CFU / ml) of S. roseus CECT 13123 in tangerine molasses in a volume of 3 liters or 30 liters. T (hour) means time in hours. The pH of the medium was also recorded at each time tested. Carotene production was also recorded (mg / l of medium). How
    5 above, T (h) means time in hours.
    FIG. 6 and 7, in relation to Example 4, respectively show the production of carotene (mg / l) of S. roseus CECT 13123 in YM and YUCCA media supplemented differently to observe the influence of glucose on fermentation parameters.
    10 As before, T (h) means time in hours.
    FIG. 8, also in relation to Example 4, shows the effect of the pH of the culture medium on carotene production. As before, T (h) means time in hours.
    15 FIG. 9, in relation to Example 5, is a bar chart showing glucose levels (g / l) and the amounts of carotenes produced (mg / l) over time (T in hours; T (h)) when S. roseus strain CECT 13123 (LGO1107) is grown in orange molasses.
    20 FIG. 10, in relation to Example 6, is another bar chart showing the effect of the pH of the medium on the amount of β-carotene (µg / ml) produced by S. roseus LGO1107 and by the reference S. roseus CECT 13019. Culture medium composition comprises tangerine molasses and the pH tested are pH 3 and pH 6.
    25 FIG. 11, in relation to Example 9, is a chromatogram of carotenoids extracted from Sporobolomyces roseus CECT 13123. Method of detection and quantification by HPLC-DAD. mUA are units of milliabsorbance (Y axis); The elution volume is in ml (X axis). The peaks corresponding to tolurene, β-carotene, γ-carotene and torularrodin are represented for a sample extracted from the fermentation of PDA medium (signaled form of
    30 Potato Dextrose Agar, dextrose agar and potato) by CECT 13123.
    FIG. 12, in relation to example 11, is a comparison of the carotenoid content between the colored composition of the invention and that disclosed in Davoli et. al., which describes
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    a strain of Sporobolomyces roseus that is apparently similar to that of the present invention.
    FIG. 13, in relation to example 11, is a comparison of the fatty acid content between the colored composition of the invention and that disclosed in Davoli et. al., which describes a strain of Sporobolomyces roseus that is apparently similar to that of the present invention.
    DETAILED DESCRIPTION OF THE INVENTION
    All terms as used herein in the present application, unless otherwise indicated, will be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms that are used in the present application are those set forth below and are intended to be applied uniformly throughout the specification and the claims, unless a definition expressly stated otherwise. Provide a broader definition. The following definitions are included for the purpose of compression.
    By "food ingredient" means any substance that becomes part of a food product, either directly or indirectly, during some phase of its processing, storage or packaging, or that can be consumed alone as food. Included in this definition, "food additive" means any substance added to a food for a specific purpose in that food: the intended use of which results, directly or indirectly, that becomes a component of the food or that otherwise affect the characteristics of any food. The strain of the invention
    or the colored composition that can be obtained from it can be used or conceived independently as a food ingredient as a whole or even specifically as a food additive. By way of example, if the composition comprising carotenoids (i.e., β-carotene) is in a final food to be consumed and with the aim of producing a particular effect on organisms (for example, stimulating the synthesis of vitamin A) , then it is conceived as an ingredient. If, on the other hand, the composition is added to the food to preserve it until its expiration date, it is conceived as an additive. The concentration in the final food will be adjusted accordingly depending on the desired function.
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    By "color additive" should be understood any substance or composition that can be added to something else to cause a color change. A color additive (also referred to herein as a coloring agent or agent that produces coloration) is any colorant, pigment, substance or composition that, when added or applied to a food, drug or cosmetic, or to the human body, has the capacity (only through reactions with other substances) to impart color. A "colored composition comprising carotenoids" should therefore be understood as a composition with the ability to change the color of any substrate (eg, another composition) where it is added.
    By "antioxidant agent" is meant a molecule or composition that inhibits the oxidation of other molecules. Oxidation is a chemical reaction that involves the loss of electrons or an increase in the oxidation state. Oxidation reactions can produce free radicals. In turn, these radicals can initiate chain reactions. When the chain reaction occurs in a cell, this can cause damage or death of the cell. Antioxidants, and in particular carotenoids, interrupt these chain reactions by eliminating intermediate free radicals and inhibiting other oxidation reactions. They do this by oxidizing themselves, so antioxidants are often reducing agents that have the ability to inactivate reactive oxygen species (ROS, acronym for reactive oxygen species).
    By "edible product" should be understood any food that is ingested orally by consumers. Examples of foods include fruit juices, soft drinks, milkshakes, dairy products (milk, yogurt and butter), vegetable creams and pastry products, among others. The "effective amount of the colored composition or of the yeast strain" refers to a non-toxic amount of this composition that produces, in the composition in which it is added, a color change in addition to acting as an oxidizing agent or simply As an edible ingredient. An "edible ingredient" is any compound of an edible product that can be consumed orally.
    "Oral pharmaceutical compositions" or "nutraceutical compositions" comprising the effective amounts of any of the colored composition or yeast strain include tablets, pills, capsules and suspensions or liquid solutions. The strain or colored compositions are added in this type of compositions in an amount
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    effective non-toxic, capable of producing the desired effect without being dangerous for the consumer. These pharmaceutical compositions are accompanied (or comprise) excipients and / or pharmaceutical vehicles. The nutraceutical compositions are nutrients and / or vitamins and / or concentrated mineral sources ingested by animals or humans to complete their diet. Cosmetic compositions in which effective amounts of any of the colored composition or yeast strain are included, comprise gels, creams, ointments, serums, powders, foams, lotions, bars, ointments, pastes, shampoos, bath gels, liquid body products or liquid facial products, in which any cosmetically acceptable excipient and / or vehicle is accompanying
    The colored composition of the invention or strain CECT 13123. These cosmetic compositions benefit from the effective amounts of any of the colored composition or yeast strain, in particular due to its antioxidant activity.
    By "β-carotene equivalents" the total amount of carotenes of
    Any type in a solution that provides the same absorbance units (U.A.) that will correspond to a solution with pure β-carotene (without another carotene-like compound).
    The invention provides an isolated strain of Sporobolomyces roseus with valuable advantages.
    20 over other strains of Sporobolomyces roseus, as it is a producer of high-performance carotenoids in aerobic conditions and in particular a high producer of β-carotene, which is a pigment widely used in many industries, including the food industry. In addition, this strain has the ability to grow on substrates containing agroindustrial waste material, such as by-products or by-products of
    25 citrus fruits, including citrus molasses. In that sense, the strain also has the ability to manufacture carotenoids from these secondary products, in particular carotenes (mainly β-carotene), adding value to them while reducing the impact of waste material.
    30 The strain of Sporobolomyces roseus CECT 13123 (LGO1107) is used to produce carotenoids. The strain CECT 13123 can also be used in a process for obtaining colored compositions comprising said carotenoids (among which β-carotene is present), said method comprising fermenting a culture medium composition that provides the elementary nutrients for the cultivation of the yeast strain in aerobic conditions. These elementary nutritional ingredients are a source of carbon, such as carbohydrates (for energy and basic components for cell walls) and a source of nitrogen, such as amino acids, used primarily for protein synthesis. Since the
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    Carotenoids are non-secreted compounds that remain within the yeast cell, the rupture of the yeast cell wall or extraction in any way from inside the cell, it is mandatory to release the carotenoids (colored composition comprising carotenoids).
    In a particular embodiment, the process for obtaining colored compositions is characterized by comprising the steps of:
    (a) preparation of a preculture of the strain of Sporobolomyces roseus CECT 13123 as defined above;
    (b) inoculation of a culture medium composition comprising a source of 15 carbon and one of nitrogen with the preculture of step (a);
    (C) aerobic fermentation of the carbon source of the culture medium composition;
    (d)  yeast strain isolation; Y
    (and) rupture of the yeast cell wall to release the composition comprising 20 carotenoids.
    It is easy to observe that the above mentioned method can be stopped at (d) if desired. Then, a method of isolating the strain of the invention is performed with the colored composition that constitutes the cytoplasm of the cell. As will be indicated to
    Then, these whole isolated cells are, as such, coloring producing agents, due to their inherent coloration.
    In still a more particular embodiment, the preculture is a small-scale preliminary culture of the strain used to inoculate the culture medium composition in which the fermentation will take place. In another particular embodiment, the preculture of the strain of the invention can be carried out in a yeast culture medium with a modified pH of between 3 and 6. With this preculture it is intended to prepare the strain for effective cultivation in the fermentation medium, in which the production of carotenoids will take place. An example of this yeast culture medium to prepare the preculture
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    it is the YUCCA medium, which comprises glucose 10 g / l, yeast extract 3 g / l, KH2PO4 2 g / l, MgSO4 0.5 g / l (pH 6), where the strain is grown in the culture conditions aerobics explained below.
    In addition, in another more particular embodiment, optionally in combination with any previous or subsequent embodiment, the culture medium composition of step (b), which comprises the source of carbon and nitrogen, additionally comprises or consists of molasses citric fruits. Examples of molasses include those of tangerine, orange, lemon, grapefruit, lime and mixtures thereof. More particularly, fruit molasses
    10 citrus is tangerine molasses or orange molasses.
    This culture medium composition comprising a carbon source and a nitrogen source (whether or not molasses) refers to a culture medium that contains digestible energy, carbon and nitrogen used by the yeast strain. For example, suitable sources of carbon and energy include glucose, xylose, fructose, sucrose, lactose, galactose and mixtures thereof. Suitable sources of nitrogen may include, but are not limited to, yeast extract, meat extract, corn macerate water (AMM), meat, soy or casein peptones, protein hydrolysates from protein-rich agri-food by-products, such as flour
    20 soy, whey, dried distillery grains with soluble (GSDS) and mixtures thereof.
    As stated above, these molasses are obtained as byproducts of citrus juice extraction. When citrus molasses are used as a culture medium, they are previously treated to reduce the solids content. This
    Treatment may include steps such as precipitation and / or filtration, and / or decantation of solid citrus fruit particles in order to separate solids that will not be used in fermentation. In a particular embodiment, the citrus molasses is clarified to a solid residue that is in the range of 1% to 10% by weight, in particular 2.5% to 5% by weight.
    The invention also provides a method for the production of carotenoids comprising fermenting a culture medium composition comprising citrus molasses with a strain of Sporobolomyces roseus yeast under aerobic conditions and breaking the yeast cell wall to recover the carotenoids. . As stated


    previously, a particular embodiment of this process is carried out with the strain LGO1107 which uses as a culture medium a composition that comprises or consists of citrus molasses.
    5 In order to release the carotenoids inside yeast cells, the cell wall has to be broken, understood as breaking both the formation of pores in the cell wall or the cell membrane of yeasts and the formation of any other deterioration in the wall or the cell membrane of yeasts that allows the release of cytoplasmic contents. In a particular embodiment, the rupture is performed by centrifugation, by
    10 grinding procedures or with high voltage electrical impulses, combined with the use of organic solvents, which means in an organic solvent.
    In a particular embodiment of the process, the rupture of the yeast cell wall is carried out in a liquid medium comprising the aprotic polar solvent.
    Dimethyl sulfoxide (abbreviated DMSO), which can optionally be preheated from 40 ° C to 50 ° C. Ultrasound treatment and vortexing are also additional techniques suitable for breaking the cell wall of yeasts. The liquid media may be the solvent itself or a known yeast culture medium, such as the YUCCA medium or the YM medium, said apolar organic solvent comprising.
    In another particular embodiment, optionally in combination with any of the previous or subsequent embodiments, the rupture of the yeast cell wall is performed by milling procedures in an organic solvent selected from diethyl ether, ethyl acetate, acetone, hexane, ethanol, petroleum ether and combinations of
    25 same. In yet another particular embodiment, the break is performed with high voltage electrical impulses in any of these organic solvents.
    By linking with the additional techniques or steps to recover the compositions comprising carotenoids, in another more particular embodiment, the process comprises 30 additional steps to achieve the isolation of specific carotene compounds, such as purified β-carotene. "Purified β-carotene" should be understood within the meaning of the present invention, as a composition that is recovered by any of the technologies mentioned above or below, and in which the weight percentage


    (in g / g) of β-carotene in relation to the total amount of carotenes isolated is at least 90%.
    In a particular embodiment of the method of obtaining β-carotene, or obtaining
    5 compositions colored with carotenoids (mainly carotenes), the method comprises additional liquid extraction stages or extraction stages in combination with distillation, or other techniques that the person skilled in the art will know and that can be used for the isolation of the different carotenes.
    10 Bearing in mind the particular chemical and physical properties of carotenoids, fractions enriched with particular carotenoids can be obtained by applying various distillation series or as an alternative by several solvent extraction stages (solid or liquid-liquid phase extraction), or as alternative using chromatographic techniques that the person skilled in the art will know. The examples are provided to
    15 continuation.
    Therefore, in another particular embodiment of the procedure, after the rupture of the yeast cell wall, an additional step is performed to recover the carotenoids. In particular, the recovery is carried out by means of an extraction step in an apolar organic solvent (non-polar organic solvent). In a particular embodiment, the organic solvent is a nonpolar organic solvent selected from diethyl ether, ethyl acetate, acetone, hexane, ethanol, petroleum ether and combinations thereof. The use of the organic solvent allows the recovery of the carotenoids or the colored composition produced by the yeast strain from other compounds of the cell cytoplasm
    25 yeast Depending on the solvent used, it could also be used for the purpose of separating (fractionating) individual carotenoid compounds produced by the strain.
    In a particular embodiment, the solution comprising carotenoids is dried to obtain carotenoids in dry form or, alternatively, the solution comprising carotenoids
    30 is subjected to precipitation of carotenoids, which are later recovered from the precipitate.
    Any of the ways in which the colored composition comprising carotenoids is provided can be used as a coloring producing agent or as an antioxidant within more complex compositions (foods, oral or nutraceutical pharmaceutical compositions).
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    In a more particular embodiment, obtaining or releasing the colored composition
    5 comprising carotenoids is performed by: -isolating yeast cells from the fermented culture medium of step (c) by centrifugation means; -the resuspension and rupture of yeast cells in an organic solvent, in particular dimethylsulfoxide (DMSO), and in which the solvent, in particular, is preheated
    10 at a temperature ranging from 40 to 50 ° C; -extracting the resuspended and broken yeast cells with an organic solvent selected from diethyl ether, ethyl acetate, acetone, hexane, ethanol, petroleum ether and combinations thereof, in particular the diethyl ether / ethyl acetate combination (1: 1) ; and - centrifugation of the mixture to obtain two phases, and
    15 -the recovery of the upper organic phase consisting of the colored composition comprising carotenoids.
    In any of the methods of obtaining colored compositions comprising carotenoids or obtaining β-carotene, step (b) is carried out with
    A strain inoculum concentration of 103 to 108 colony forming units per ml of culture (CFU / ml), in particular 104 to 107 CFU / ml. In a more particular embodiment, it is 105 to 106 CFU / ml of culture medium. In a more particular embodiment, the total volume of the culture medium composition is 15 to 60 liters (l). 103 to 108 includes 103, 104, 105, 106, 107 and 108 CFU / ml.
    In another particular embodiment of the process of the invention, optionally in combination with any previous or subsequent embodiment, the fermentation step of the culture medium is carried out in a temperature range of 20 ° C to 35 ° C, more particularly 20 ° C to 30 ° C and even more particularly from 20 ° C to 25 ° C. From 20 ° C to
    30 35 ° C means any selected temperature of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35 ° C.
    In another particular embodiment, optionally in combination with any previous or subsequent embodiment, the fermentation step of the culture medium is carried out in a
    5
    10
    fifteen
    twenty
    25
    30


    pH range from 2.0 to 8.0, more particularly from 3.0 to 6.0. Therefore, the pH is selected from 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0.
    When aerobic conditions are mentioned in the present invention, these comprise in particular embodiments of oxygenation (proportion of an oxygen source) at a flow rate of 2 to 10 l / min, in particular 5 l / min, of 20% v / v -30% v / v, in which v / v means a volume of air containing up to 21% O2 per volume of culture medium, and constant agitation of the culture medium composition, thus helping homogeneous oxygenation of the entire volume.
    In another particular embodiment of the procedures, the fermentation stage is carried out during a period that varies between 7 and 12 days, more particularly for 10 days.
    Through all these embodiments of the processes, colored compositions comprising carotenoids, in particular comprising β-carotene, can be obtained. Therefore, in a particular embodiment the colored compositions can be obtained by a process comprising fermenting a culture medium composition comprising a carbon source and a nitrogen source with the strain of Sporobolomyces roseus CECT 13123, or with a pure culture from it under aerobic conditions and breaking the yeast cell wall to release the composition comprising carotenoids.
    More particularly, colored compositions can be obtained by:
    (to)  the preparation of a preculture of the strain of Sporobolomyces roseus CECT 13123, or a pure culture thereof;
    (b) the inoculation of a culture medium composition comprising a carbon and a nitrogen source with the preculture of step (a), said composition of citrus fruit molasses or even consisting only of these citrus molasses ;
    (C) the aerobic fermentation of the carbon source of the culture medium composition;
    (d)  the isolation of the yeast strain, and
    (and) the rupture of the yeast cell wall to release the colored composition comprising carotenoids from inside the cells.


    The colored compositions thus obtained comprise β-carotene, among other carotenoids (including xanthophylls and other carotenes). The colored compositions thus obtained comprise in particular β-carotene, γ-carotene, torulene and torularrodin. In a particular embodiment of the colored compositions of the invention the weight percentage of β-carotene is at least 40% in relation to the total carotenes in the composition (w / w in grams). The weight percentage indicated here is calculated by recovering the carotenes of the colored composition by breaking the yeast cells with preheated DMSO at 50 ° C and extracting the broken yeast cells with diethyl ether / ethyl
    10 acetate (1: 1). This is the analytical reference method proposed by the inventors to compare the results obtained with other yeast strains.
    In another particular embodiment, the colored composition comprising carotenoids comprises β-carotene in a weight percentage ranging from 48 to 50%; γ-carotene in a 15 percent by weight ranging from 10 to 12%; torulene in a percentage by weight that varies from 26 to 28% and torularrodin in a percentage by weight that varies from 12 to 14%, all percentages by weight in relation to the total amount of carotenes that amounts to 100%. As indicated above, the colored composition comprising carotenoids also comprises other carotenoids, in particular xanthophylls. These percentages by weight
    20 of carotenes in the colored composition are determined, as indicated above, recovering carotenes with the process comprising breaking with DMSO and extraction with diethyl ether / ethyl acetate.
    Considering the known antioxidant properties of β-carotene, the compositions
    Colored of the invention are proposed for use as antioxidant agents, as well as coloring producing agents within complex compositions. Examples of complex compositions include edible compositions (food), nutraceutical and pharmaceutical compositions, or cosmetic compositions.
    Non-limiting examples of products in which the colored compositions of the invention can be added include pharmaceutical compositions (in particular oral pharmaceutical compositions), nutraceutical compositions, cosmetic compositions and foods, in particular fruit juices, soft drinks, milkshakes, dairy products (milk, yogurt, butter), vegetable creams and pastry products among others.
    image15
    Throughout the description and the claims it is not intended that the word "comprises" and the variations of the word, exclude other technical characteristics, additives, components
    or stages In addition, the word "understand" encompasses the case of "consists of". The objectives, advantages and additional features of the invention will be obvious to those skilled in the art after examination of the description or can be learned through the practice of the invention. The following examples are provided by way of illustration and are not intended
    10 limiting the present invention. In addition, the present invention includes all possible combinations of particular and preferred embodiments described herein. 15 EXAMPLES
    Example 1. Isolation of the strain Sporobolomyces roseus CECT 13123 (LGO1107)
    The Sporobolomyces roseus strain of the invention was isolated from a surface in a food industry, according to the following procedure:
    Five bacterial colonies were selected from Rodac contact plates with chloramphenicol sabouraud agar incubated for five days at 25 ° C and isolated by their red color. Several incubations were made in Dextrose and Potato Agar (ADP) before
    25 analysis
    Identification was performed using the restriction fragment length polymorphism (RFLP) technique of the internal transcribed spacer (ITS) and internal transcribed spacer) and
    30 sequencing the appropriate rRNA region to know the specific strain with the Basic Local Alignment Search Tool (BLAST).
    image16
    Table 1. Molecular weight using RFLP-ITS
    Code Gender and species STI Hhal Haell Hinfl Internal sample
    LGO1107 Sporobolomyces 610 300 + 200 + 80 600 280 + 120 + 95 + 95 sp
    Table 2. Sequencing results
    Internal Profile Code by RFLP Homology of BLAST% sample
    LGO1107 Sporobolomyces sp 99.9% roseus
    All these data confirmed that the isolated strain was a strain of S. roseus.
    As indicated above, the isolated strain LGO1107 was deposited in the Spanish Collection 10 of Type Crops (CECT) and received the reference number CECT 13123.
    Example 2. Cultivation and production of carotenoids of Sporobolomyces roseus CECT 13123 (LGO1107) in YM broth.
    15 To better characterize the new strain isolated, its growth was analyzed in a Malta Yeast Broth (YM) formulated with the addition of different amounts of glucose (main sugar in the molasses by-product): glucose 10.00, 20.00 and 40.00 g / l. Under these conditions, carotene production was also evaluated.
    20 The composition of YM broth was: Peptic digestion of animal tissue (5.00 g / l), yeast extract (3.00 g / l), Malt extract (3.00 g / l), Dextrose (10, 00 g / l).
    In FIG. 1 (A to C) growth kinetics and carotene production are represented. Carotene production was determined as set forth in Example 8, 25, the procedure comprising breaking with DMSO and extraction with diethyl ether / ethyl acetate (1: 1) and quantification with a UV-Vis spectrophotometry test at 450 nm .


    FIG. 2 shows the production of Sporobolomyces roseus CECT13123 in biomass (g / l), determined by gravimetric measurement after centrifugation and drying, and carotene production at 240 hours.
    5 The conclusion from these data is that Sporobolomyces roseus CECT13123 produces high amounts of carotenes in this culture medium.
    Example 3. Colored compositions from the fermentation of tangerine molasses with yeast strains. Comparison of Sporobolomyces roseus CECT 13123 with others
    10 strains of Sporobolomyces roseus
    Sporobolomyces roseus CECT 13123 (LGO1107), the reference Sporobolomyces roseus CECT 13019 strain (commercially available) and another strain of Sporobolomyces roseus isolated from vinegar were grown and tested to obtain colored compositions
    15 comprising carotenoids.
    The strains (previously maintained at 4 ° C in dextrose and potato agar) were inoculated in a YUCCA synthetic broth (culture medium composition) comprising yeast extract (2 g / l) glucose 10 g / l, KH2PO4 10 g / l, MgSO4 2 g / l (pH 6). These were grown
    20 for 24 h at 23 ° C with constant oxygenation and stirring.
    On a laboratory scale, 105-6 CFU / ml of each strain were inoculated from the precultures, in 3 μl of tangerine molasses. The fermentation was carried out for 10 days (240 h) under light conditions and at a temperature of 23 ° C. The stirring was at 200 rpm
    25 (Biostat ® B Plus) and oxygenation was kept constant with a flow of 25% v / v using Biostat ® B Plus.
    Mandarin molasses was obtained from a supplier of citrus fruit derivatives and its composition was as follows: moisture (95 g / 100 g), nitrogen (0.32 / 100 g), ashes (0.17
    30 g / 100 g), carbohydrates (4.5 g / 100 g), fructose (0.10 g / 100 g), glucose (3.9 g / 100 g), sucrose (0.32 g / 100 g) and pH 3.4. The molasses was clarified by filtration and the pH was adjusted to 6 before inoculation of the strains.
    image17
    In FIG. 3, the total carotene production between the three different strains of S. roseus in tangerine molasses at pH 6 in a 3 l fermenter is represented with bars. The amounts of carotene were determined as in Example 8, with the procedure comprising breaking with DMSO and extraction with Diethyl ether / Ethyl Acetate (1: 1), and the
    5 quantification by a UV-Vis spectrophotometry test at 450 nm, as equivalents of β-carotene.
    Sporobolomyces roseus CECT 13123 (LGO1107) appears as the best carotene producer in mandarin molasses with modified pH, even better than the CECT reference
    10 13019. The results of FIG. 3 show that S. roseus LGO1107 produced a greater amount of colored composition comprising carotenoids, in particular carotenes, from citrus molasses than the CECT reference 13019. In addition, the production of LGO1107 was faster than the production of CECT 13019 , given that in each of the times tested a higher production was observed.
    15 In order to further characterize the growth kinetics and behavior of Sporobolomyces roseus CECT 13123, various pH adjustments were made in the tangerine molasses where CECT 13123 was inoculated as indicated above. The log of the CFU / ml and the production of carotene (mg / l) were recorded. The data is
    20 depicted in FIG. 4, which shows the growth kinetics (log of CFU / ml) versus time in hours of S. roseus CECT 13123 in tangerine molasses in a volume of 3 liters. The pH of the medium at each time is also represented.
    On a pilot scale, Sporobolomyces roseus CECT 13123 (LGO1107) was also inoculated into
    25 same initial concentration as in the laboratory scale but in a final volume of tangerine molasses of 30 l. The fermentation was carried out for 10 days at 23 ° C, 680 rpm (RB-50 Biological Reactor, Energesa S.A.) and constant oxygen supply. The results are represented in FIG. 5, in which growth kinetics (log of CFU / ml) are observed versus time in hours of S. roseus CECT 13123. The stationary phase is
    30 reached 72 h and growth data between 96 h and 240 h are not represented.
    Both FIGs. 4 and 5 show that the new strain had the ability to grow with high yields in tangerine molasses and also had the ability to produce carotenes in high concentrations.
    image18
    The strain of Sporobolomyces roseus CECT 13123 (LGO1107) was therefore also useful for carrying out fermentation at industrial levels. Without linking to any theory, the inventors believe that this is due in particular to the fact that this strain has a high growth rate in citrus molasses in relation to other strains of
    5 Sporobolomyces roseus.
    Example 4: Influence of fermentation conditions on carotene production by Sporobolomyces roseus CECT 13123
    10 The fermentation conditions for the optimization of carotene production were studied.
    The production of carotenes in YM and YUCCA media with different glucose concentrations was analyzed. The culture conditions and inoculation were the same
    15 than those indicated in Example 3 (only the culture media were changed). The data is represented in FIG. 6, for YM complemented and in FIG. 7, for the YUCCA medium complemented. The higher the glucose content, the higher the carotene production (in mg / l) considering the total cultivation time (240 hours).
    The effect of the pH of the culture medium can be seen in FIG. 8. The growth of CECT 13123 in YUCCA supplemented with glucose 20 g / l was tested at various pH (pH 3, 5 and 7). The production of carotenes at various culture times (mainly of β-carotene among other carotenes) was determined as set forth above (as in Example 8, the procedure comprising breaking with DMSO and extraction with Diethyl ether / Ethyl
    Acetate (1: 1), and quantification with a UV-Vis spectrophotometry test at 450 nm). Surprisingly, carotene production is achieved even at a low pH (those of citrus molasses). The best production in YUCCA was obtained at pH 5-7.
    30 Example 5. Cultivation of Sporobolomyces roseus CECT 13123 (LGO1107) in orange molasses
    In order to determine if the strain of LGO1107 had the ability to grow and produce carotenoids, the culture was carried out in orange molasses at pH 5.5, and the recovery of carotenes and quantification as indicated in Example 8, the procedure comprising breaking with DMSO, extraction with Diethyl Ether / Ethyl Acetate (1: 1) and quantification with a UV-Vis spectrophotometry test at 450 nm as equivalents of β-carotene.
    image19
    5 LGO1107 had the ability to also grow in orange molasses. The production of carotenes (mainly of β-carotene, among other carotenes) is represented in FIG. 9. In this figure, the first bars are the amounts of carotene (mg / l of medium) produced in each sampled time (in hours). The second bars correspond to the
    10 glucose levels (g / l of medium) in the culture medium at the same time sampled.
    This example shows the ability of the strain of the invention to ferment another citrus fruit material (orange molasses), which makes it a versatile strain useful in various waste materials of citrus fruit industries.
    Example 6. Effect of pH of the culture medium composition comprising tangerine molasses
    Sporobolomyces roseus CECT13123 (LGO1107) and CECT 13019 were grown in tangerine molasses as indicated in Example 3.
    The culture was carried out in an Erlenmeyer (0.3 l medium volume) for 240 hours. To control the fermentation procedure, cell growth and pH were monitored daily during the fermentation time.
    The pH of the media was adjusted to 3 or 6. As can be seen in FIG. 10, the strain of the invention (LGO1107) had the ability to produce carotenes even in acidic conditions (pH 3), where it had the ability to grow. This was not the result with the reference strain CECT 13019. As set forth above, at pH 6 the strain of the invention
    30 produces greater amounts of carotene than the reference strain. (Carotene recovery and quantification, as equivalents of β-carotene, as indicated in Example 8, the procedure comprising breaking with DMSO and extraction with Diethyl ether / Ethyl Acetate (1: 1), and a UV spectrophotometry test -Vis at 450 nm).
    image20
    Therefore, Sporobolomyces roseus CECT 13123 (LGO1107) had the ability to grow in tangerine molasses with higher yields than the CECT 13019 reference, although the latter also had the ability to grow in this medium and also produced carotenoids. However, Sporobolomyces roseus LGO1107, with a high yield of
    5 production of carotenoids and a better adaptation to this particular medium, is an advantageous strain with a real industrial contribution.
    These data demonstrate the versatility of the strain of the invention compared to the reference strain in relation to the culture media. Therefore, the strain of the invention 10 is not only capable of growing in various culture media comprising citrus fruit molasses, it also has the ability to produce carotenoids in all these analyzed conditions. The production of carotenoids, even under severe stress conditions (for example pH 3), makes the strain CECT 13123 (LGO1107) a really interesting strain for the food industry, specifically in the revaluation of waste material
    15 from the citrus food industry.
    Example 7. Comparison of Sporobolomyces roseus CECT 13123 (LGO1107) with Rhodotorula mucilaginosa DSM 70398
    The strain LGO1107 was also compared with Rhodotorula mucilaginosa, another yeast known to produce carotenoids (as disclosed in WO2011130576), in a laboratory test in malt yeast medium (YM) supplemented with glucose at different concentrations (10, 20 and 40 g / l). The inoculums were performed at a concentration of 105 cells / ml and the culture for 240 hours in Erlenmeyer flasks, pH 6 in a
    25 fermenter of 3 l. The data is represented in Table 3 below.
    Table 3. Comparison of the production in equivalents of β-Carotene (µg / g of dry biomass)
    Broth R. mucilaginous DSM S. roseus LGO1107 70398
    YM + 10 g / L glucose 6.39 10.00 YM + 20 g / L glucose 11.18 18.40 YM + 40 g / L glucose 13.09 25.28 (quantified by UV-Vis as equivalent of β-carotene at 450 nm) (µg / g of biomass
    30 dry)


    From this comparison, the advantage of using S. roseus CECT 13123 (LGO1107) as a carotenoid producer is directly deduced. Under the same culture conditions, it had the capacity to produce greater amounts of carotenes (mainly β
    5 carotene) than another yeast also known as a high carotene producer.
    All the data presented here allow us to conclude that an industrial fermentation procedure could be established by adding value to the citrus by-products of the food industry. This was carried out with a strain of Sporobolomyces
    10 roseus, which is a producer of high carotenoids in relation to other yeast strains and can produce such carotenoids in a medium comprising molasses from agroindustrial waste materials, even under severe stress conditions for yeasts, such as a pH low.
    Example 8. Effect of the methods of cell rupture and extraction of carotenes for the recovery of carotenes from dry biomass isolated from R. glutinis, R. mucilaginosa or S. roseus.
    The inventors also focused on the provision of a method for a determination
    20 of conventional carotene recovery after fermentation with yeast strains, and also in the provision of an acceptable recovery method in the food industry, where the colored compositions of the invention can be used.
    Different methods are identified in the literature as useful to achieve the rupture of the
    25 yeast cell wall and also a large amount of organic solvents and combinations for the extraction of carotenes from inside the cell. Table 4 below summarizes the main carotene recovery methods (in the columns, the sequential procedural steps).
    30


    Table 4. Carotene recovery methods
    BREAK OF THE CELL WALL
    DMSODMSO GRINDINGHIGH VOLTAGE ELECTRICAL IMPULSES
    Ultrasound treatment Vortex Agitation
    TemperatureTemperature
    Organic solventOrganic solvent
    CAROTENE EXTRACTION
    Centrifugation Filtration
    Organic solvent
    Agitation
    Centrifugation
    Saline solution
    Decanting
    CAROTENE CONCENTRATION
    Solvent evaporation
    5 The inventors want to indicate that the recovery of carotenoids depends on the specific conditions of the methods of rupture and extraction of carotenes (temperature, solvents, time and type of agitation ...) but also on the strain of yeast cells or bacteria, which also has a strong influence due to differences in
    10 wall resistance of each strain.
    To compare different strains and methods, the results in the tables below are related to the dry biomass used in each rupture and extraction procedure.
    The following Tables 5 and 6 are only illustrative examples not related to the S. roseus CECT 13123 strain of the invention. But they are included to point out the importance of considering comparative experimental conditions.
    image21
    Table 5. (Reference example) Comparison of carotene extraction solvents
    from R. glutinis grown in YM broth after the use of DMSO (50 ° C) and vortexing
    (quantified by UV-Vis as equivalents of β-carotene at 450 nm).
    SOLVENT CARCENE CONCENTRATION (µg / g
    of dry sediment) Diethyl ether 97.88 ± 8.82 Diethyl ether / Ethyl Acetate (1: 1) 128.07 ± 6.53 Petroleum Ether 114.50 ± 9.27 Petroleum Ether / Acetone (1: 2) 120.61 ± 6.03
    5
    This table illustrates the variability due to the nature of the solvent used in the
    extraction. Diethyl ether / ethyl acetate (1: 1) allows the highest extraction yields.
    Table 6. (Reference example) Comparison of carotene recovery in the
    10 methods with DMSO from R. mucilaginosa and R. glutinis (quantified by UV-Vis as
    equivalents of β-carotene at 450 nm).
    TIME METHOD
    CAROTEN CONCENTRATION (hours) (µg / mL)
    R. mucilaginous
    R. DMSO swallow (50 ° C) and stirring 24 0.0636 ± 0.0190 vortex 48 0.212 ± 0.00160 0.799 ± 0.016 Hexane / Acetone / Ethanol 73 0.326 ± 0.00620 1.035 ± 0.059
    (2: 1: 1) as solvent 96 0.317 ± 0.00190 1.499 ± 0.122
    Organic 162 0.620 ± 0.0303 1.852 ± 0.0550 168 0.612 ± 0.470 1.403 ± 0.204
    DMSO (50 ° C) and vortex agitation Diethyl ether / Ethyl Acetate
    (1: 1) as organic solvent
    24 0.190 ± 0.054
    48 0.623 ± 0.0052.31 ± 0.046
    73 0.948 ± 0.0182.99 ± 0.169
    96 0.921 ± 0.0054.31 ± 0.347
    162 1.79 ± 0.0865.32 ± 0.156
    168 1.76 ± 0.1345.03 ± 0.582
    The data from this table allow us to conclude that both methods are good for
    15 carotene recovery. The table also shows that despite the different species of yeast and the differences in the resistance of their cell wall, the method comprising vortexing and extraction with Diethyl ether / Ethyl Acetate (1: 1), organic solvent is the best in terms of concentration of carotenes. This last method in particular is the one proposed and used by the inventors as the analytical method of


    reference to compare the production of carotenoids (mainly carotenes) between species and strains of yeast.
    Since this method is the one that has been used for the quantification of carotenes in Example 2-7, it is the one that is disclosed in more detail:
    The culture medium where the yeast strains were grown was centrifuged for 10 min at 10,000 rpm and washed. The resulting sediment was resuspended in DMSO (preheated to 50 ° C) to cause the yeast cell wall to rupture. Carotenoids are
    10 extracted with a solvent mixture of diethyl ether / ethyl acetate (1/1 v / v) and stirred in a genoGrinder® for 5 min at 480 min-1. The extraction was performed twice and finally the supernatants were mixed. NaCl was added to stimulate phase separation during centrifugation (10 min, 10,000 rpm).
    A known volume of this upper phase was evaporated with nitrogen flow. The extracted carotenoids were dissolved in acetone (4 ml), the solution was filtered (0.45 micrometers) and the absorbance was measured at 454 nm (UV-Vis spectrophotometer). The calibration standard was performed with dilutions of β-carotene (0.2-10 μg / ml). With this procedure, the concentration of carotenes is determined / quantified by UV-Vis
    20 as equivalents of β-carotene at 450 nm. As stated above, "β-carotene equivalents" is the total amount of carotenes of any type in a solution that provides the same absorbance units (AU) that will correspond to a solution with pure β-carotene (without another compound of carotene type).
    25 For industrial purposes, in particular due to food industry regulations, cell wall rupture and carotene extraction cannot be performed with DMSO. Therefore, alternative methods have been tested, in particular the combination of cell rupture with grinding or with high voltage pulses with extraction with acetone as organic solvent.
    30 The following Table 7, which refers to Sporobolomyces roseus CECT 13123, details the recovery of carotenes (mg / g of biomass) obtained in two fermentation tests (1 and 2) and using different recovery procedures.


    Table 7. Comparison of carotene recovery from Sporobolomyces roseus CECT 13123 (quantified by UV-Vis as beta-carotene equivalents at 450 nm) (mg / g of biomass)
    EXPERIMENT RECOVERY PROCEDURE
    OF FERMENTATION DMSO (50 ° C) and vortex agitation Diethyl ether / Ethyl AcetateGrinding Acetone as organic solventHigh voltage electrical impulses
    (1: 1) as solvent Acetonehow
    organic organic solvent
    1 0.065 0.075 0.091
    2 0.043 0.050 0.059
    5 This table 7 allows us to conclude that when the scale of laboratory procedures is increased (cell wall rupture with DMSO), recovery is maintained or even improved.
    10 Example 9. Identification of the carotene composition
    The total amount of carotene compounds was measured by UV-Vis spectrometry as equivalents of β-carotene at 454 nm. This method has been used in all the previous examples when reference was made to the quantification of carotenes.
    In addition, the identification and quantification of the individual carotene compounds was performed by High Performance Liquid Chromatography (Agilent HPLC, column C18, gradient of the mobile phase of acetone (A) and water (B) at 1 ml / min with an injection volume of 20 microliters) and diode beam detection at two different wavelengths
    20 (450 nm and 490 nm). The main compounds identified in each of the samples tested from the fermentation of PDA medium by CECT 13123 were: β-carotene, γ-carotene, torulene and torularrodin. No reference standard for the quantification of torulene is available; therefore, this compound was quantified using a β-carotene pattern according to the literature.
    25 The results obtained for Sporobolomyces roseus CECT 13123 in several fermentations in PDA medium at 168 hours are shown in Table 8, in which


    establish the intervals of percentages by weight of each of the carotenes detected. The data is also represented in the chromatogram of FIG. eleven
    Table 8. Composition of carotenes from Sporobolomyces roseus CECT 13123
    Compound % Range (weight / weight, total compounds
    carotene carotene is 100% carotene in the sample)
    (Quantification by HPLC / beta-carotene equivalents
    by UV-Vis measurement)
    β-carotene 48 - 50
    γ-carotene 10-12
    Torulene 26-28
    Torularrodin 12-14
    The total carotene production and relative composition depends on several aspects, such as the composition of the medium (i.e. the type and concentration of sugar and nitrogen), stress factors (light, oxygen, etc.) and the time of fermentation. This is because the production of carotenes by the yeast strain is in response to stress conditions. Therefore, depending on this stress, a quantitative pattern of carotenes is obtained within the ranges of Table 8. β-carotene is always obtained with at least 40% by weight and the rest of the indicated carotenes are also present in the colored composition that can be obtained by the action of
    15 Sporobolomyces roseus CECT 13123.
    Example 10. Isolation of carotenoid fractions
    Solid phase extraction (on silica) was performed to obtain different compositions
    20 dyes One composition was related to the torularrodin fraction and another composition was related to β-carotene, torulene and γ-carotene.
    As the person skilled in the art will observe, if desired, β-carotene can be further purified from the rest of the carotenes (torulene and γ-carotene), by means of several series of
    Distillation or, alternatively, by several stages of solvent extraction (solid or liquid-liquid phase extraction) or, alternatively, by chromatography techniques.


    Example 11. Comparison of the colored composition of the invention with that described in Davoli et al. "Carotenoids and fatty acids in red yeasts Sporobolomyces roseus and Rhodutorula glutinis" Appl. Biochem And Microbiol. 2004, vol. 40, p. 392-397.
    In an attempt to determine whether the colored composition that can be obtained by the process of the invention (based on strain LGO1107) has any difference with the related prior art, a series of experiments were carried out. In particular, the inventors wanted to determine if the colored composition of the invention has any
    Difference in the composition of carotenoids and fatty acids with respect to the colored composition described in Davoli et al. (cited above), which describes the strain Sporobolomyces roseus D99040.
    As can be seen in FIG. 12 and FIG. 13, there are clear differences between the 15 colored compositions obtained by the two strains.
    FIG. 12 represents the clear difference in terms of the proportion of carotenoids between the two compositions. The strain selection has a statistically significant effect on all carotenoid compounds in the 95.0% confidence interval. The composition obtained by the strain described in Davoli et al. It has a higher proportion of β-carotene and torulene, compared to the composition obtained by strain LGO1107. Additionally, the composition obtained by the strain described in Davoli et al. It has a lower proportion of torularrodin compared to the composition obtained by strain LGO1107. It should be noted that the contents in β
    Carotene are also clearly distinct in conventional experiments.
    It should be noted at this point that differences in the proportion of types of carotenoids have an influence on the final color of the composition as well as its antioxidant activity. Torularrodin, which has a higher proportion in the colored composition
    30 obtained from strain LGO1107, has a more potent effect on the uptake of peroxyl radicals than other carotenoids such as β-carotene; In addition, it inhibits substrate degradation by singlet oxygen more efficiently than β-carotene (Sakaki, H., “Effect of active oxygen species on the productivity of torularhodin by Rhodotorula glutinis No. 21” J. Biosci. Bioeng. 2002 , vol. 93, p. 338-340).
    image22
    FIG. 13 represents the clear difference in terms of proportions of fatty acids between the two compositions. The composition obtained by the strain described in Davoli et al. It has a different proportion of C18 fatty acids compared to composition 5 obtained by strain LGO1107. In particular, it is observed that the fraction of C18: 2 (linoleic acid) is much higher in the colored composition obtained by strain D99040, while the fraction of C18: 1 (oleic) is much larger in the colored composition obtained by the strain LGO1107. It has been suggested that the degree of unsaturation is proportionally related to the oxidation rate. Therefore, the composition of
    10 LGO1107, due to its higher proportion of oleic acid, could promote the stability of the colored composition as an antioxidant.
    Finally, it should also be noted that the strain of the present invention is much more effective than the strain disclosed in Davoli et al. (cited above) in terms of
    15 production of carotenoids, since the production in a straight flask is 108.9 μg / g of dry matter for D99040, while in the case of the strain of the invention (LGO1107), the production is 6599.6 ± 1108.5 μg / g.
    Therefore, it is expected that the colored composition that can be obtained by the process of the invention has a different behavior than the composition disclosed in Davoli et al.
    REFERENCES CITED IN THE APPLICATION
    25 -Marova et al. "Use of several waste substrates for carotenoid-rich biomass yeast production", Journal of Environmental Management-2012, vol. No. 95, p .: s338-s342 -Mata-Gómez et al. "Biotechnological production of carotenoids by yeast: an overview", Microbial Cell Factories-2014, vol. No. 13:12. -Document US20120142082
    30 -Document JPH0622748 -Document WO2011130576 -Davoli et al. "Carotenoids and fatty acids in red yeasts Sporobolomyces roseus and Rhodutorula glutinis" Appl. Biochem And Microbiol. 2004, vol. 40, p. 392-397
    权利要求:
    Claims (12)
    [1]
    image 1
    -Sakaki, H., “Effect of active oxygen species on the productivity of torularhodin by Rhodotorula glutinis No. 21” J. Biosci. Bioeng 2002, vol. 93, p. 338-340
    35
    image2
    1. An isolated yeast strain of Sporobolomyces roseus deposited on March 25
    2015 in the Spanish Type Culture Collection (CECT) with identification reference 5 LGO1107 and which received the reference number CECT 13123.
    [2]
    2. A method of obtaining a colored composition comprising carotenoids, the process comprising: -ferring under aerobic conditions a culture medium composition that
    10 comprises a carbon source and a nitrogen source with a yeast strain as defined in claim 1; -isolate the yeast strain; and breaking the yeast cell wall to release the composition comprising carotenoids.
    fifteen
    [3]
    3. The method according to claim 2, comprising the steps of:
    (to)  preparation of a preculture of a strain as defined in claim 1;
    (b) inoculation of a culture medium composition comprising a carbon source and a nitrogen source with the preculture of step (a);
    20 (c) aerobic fermentation of the carbon source of the culture medium composition; Y
    (d)  yeast strain isolation, and
    (and) rupture of the yeast cell wall to release the composition comprising
    carotenoids 25
    [4]
    Four.  The method according to claim 3, wherein step (b) is carried out with a strain inoculum concentration of 103 to 108 CFU / ml of culture medium.
    [5]
    5. The method according to any of claims 2-4, wherein the
    The fermentation of the culture medium is carried out in a temperature range of 20 ° C to 35 ° C.
    36
    image3
    [6]
    6. The process according to any of claims 2-5, wherein the fermentation of the culture medium is carried out in a pH range of 2.0 to 8.0.
    [7]
    7. The method according to any of claims 2-6, wherein the
    5 aerobic conditions include oxygenation at a flow rate of 2 to 10 l / min of 20% v / v30% v / v, in which v / v means volume of air containing up to 21% O2 per volume of medium of culture.
    [8]
    8. The method according to any of claims 2-7, wherein the
    The culture medium composition comprising a carbon source and a nitrogen source is a culture medium comprising citrus fruit molasses.
    [9]
    9. A colored composition comprising carotenoids obtainable by a
    The method as defined in any of claims 2-8, said β-carotene, γ-carotene, torulene and torularrodin composition comprising.
    [10]
    10. The colored composition according to claim 9, wherein the weight percentage of β-carotene is at least 40% in relation to the total amount of carotenes in the composition (w / w), said carotenes are recovered breaking yeast cells with
    20 preheated dimethylsulfoxide at 50 ° C and extracting the broken yeast cell with diethyl ether / ethyl acetate (1: 1).
    [11]
    11. Use of a strain as defined in claim 1, or of a colored composition
    as defined in any of claims 9-10, as antioxidant agents. 25
    [12]
    12. Use of a strain as defined in claim 1, or of a colored composition as defined in any of claims 9-10, as a coloring producing agent.
    13. The use according to claim 12, wherein the coloring effect is applied to a product selected from the group consisting of an edible product, an oral pharmaceutical composition, a nutraceutical composition and a cosmetic composition.
    37
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    同族专利:
    公开号 | 公开日
    ES2667433B1|2019-03-13|
    ES2667433R1|2018-06-06|
    WO2017017188A1|2017-02-02|
    EP3124597A1|2017-02-01|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    JP2810990B2|1992-07-08|1998-10-15|三菱石油株式会社|Yeast producing β-carotene and method for producing β-carotene by yeast|
    US8846374B2|2006-12-12|2014-09-30|E I Du Pont De Nemours And Company|Carotenoid production in a recombinant oleaginous yeast|
    US20110256268A1|2010-04-14|2011-10-20|Solazyme, Inc.|Oleaginous Yeast Food Compositions|
    WO2012078256A1|2010-10-26|2012-06-14|Kansas State University Research Foundation|Fermentation process to produce natural carotenoids and carotenoid-enriched feed products|WO2021163194A1|2020-02-10|2021-08-19|C16 Biosciences, Inc.|Microbially produced palm oil substitutes|
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    PCT/EP2016/068009|WO2017017188A1|2015-07-29|2016-07-28|A sporobolomyces roseus strain for the production of compositions with colorant and antioxidant properties|
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