WO2019008225A1

WO2019008225A1 - Method for producing cell cultures of the family betulaceae, compositions comprising said cell cultures and use of said cell cultures

Info

Publication number: WO2019008225A1
Application number: PCT/FI2018/050485
Authority: WO
Inventors: Riitta PUUPPONEN-PIMIÄ; Liisa Nohynek; Tarja Tamminen; Anna Maria NUUTILA
Original assignee: Teknologian Tutkimuskeskus Vtt Oy
Priority date: 2017-07-07
Filing date: 2018-06-21
Publication date: 2019-01-10
Also published as: FI20175668A; FI20175668A1

Abstract

The present invention relates to a method for producing a cell culture of a plant of the family Betulaceae, said method comprising two stages, where in the first stage birch callus line is cultured in a liquid medium comprising a medium used for plant cell and tissue culture, with or without irradiation, to obtain primary suspension cell culture, and in the second stage the primary suspension cell culture is up-scaled in at least two steps in the liquid medium with or without irradiation.

Description

METHOD FOR PRODUCING CELL CULTURES OF THE FAMILY BETULACEAE, COMPOSITIONS COMPRISING SAID CELL CULTURES AND USE OF SAID CELL CULTURES

FIELD OF THE INVENTION

The present invention relates to a method for producing cell cultures of the family Betulaceae. The invention relates further to cell cultures obtained by the method. The invention relates also to compositions comprising said cell cultures, and to the use of the said cell cultures in cosmetic, hygiene and personal care applications, in food, nutrition, nutraceuticals, pharmaceutical products, pharmaceutical devices and for providing aroma, flavor and/or color to the products.

BACKGROUND OF THE INVENTION

Birch is a thin-leaved deciduous hardwood tree of the genus Betula in the family Betulaceae. Said family also includes alders, hazels, and hornbeams. The genus Betula contains up to 60 known taxa of which 11 are on the IUCN 2011 Green List of Threatened Species. They are typically widespread in the Northern Hemisphere, particularly in northern temperate and boreal climates.

The cosmetic industry uses various plant-derived fractions due to their bioactivities in cosmetic products, and it is highly interested in natural ingredients, such as natural preservatives, antioxidants and colorants, to replace existing synthetic ingredients which might have harmful or even toxic effects in humans. Many of the synthetic ingredients, such as para bens, are under evaluation by the FDA, and it is obvious that within the coming years their use will be forbidden or at least restricted. This will open new markets for alternative compounds, especially for natural compounds. Many cosmetic companies are constantly looking for more sophisticated products for the markets, such as products containing plant cell cultures, for providing bioactive effects. Plant metabolites are typically very complex, and therefore their chemical synthesis may be difficult or even impossible. Plant cells and tissue cultures have been recognized as potential options for replacing whole plants as sources of valuable industrial plant bio-chemicals. In many plant species, somatic cells of mature plant organs can be induced to proliferate in liquid synthetic media supplemented with plant growth regulators (PGR). Some of these proliferating cells are capable of regeneration and they are able to develop into fertile flowering plants. This unique property of plant cells is called totipotency. In cell cultures, totipotent plant cells can either produce somatic embryos that can develop into mature plants or they can establish meristematic regions which are able to regenerate through organogenesis pathway. Embryogenic in vitro cultures have been established for a large number of plant species belonging to tens of families. However, somatic embryogenesis can be efficiently achieved in relatively few plant species. This totipotent nature of plant cells implies that they have a complete gene set that retains the potential to establish and maintain all differentiated higher plant stages. This also means that they are able to produce any organ specific compound, making them highly interesting raw-material e.g. for cosmetics.

The strategy most often used to induce somatic embryos, is to expose excised plant tissue to high concentration of auxin, which belongs to plant growth regulators (PGR). After proliferation, two main cell types are formed, non-embryogenic highly vacuolated cells and small embryogenic cells. When transferred to auxin-free medium somatic embryos develop exclusively from the small clusters of cells called pro-embryogenic masses (PEM). Same morphological alterations are observed in somatic embryogenesis as in zygotic embryogenesis: globular, heart, torpedo and cotyledonous, from which the seedling and finally the adult plant emerge. Somatic embryo development is suppressed in suspension cultures maintained in medium containing high auxin concentration. However, PEMs are continually formed under these conditions. When auxin is removed, cell clusters undergo embryogenesis at high frequency.

Cloudberry cell cultures have been studied and methods for their production have been proposed. WO 2013/124540 Al discloses cosmetic compositions containing cloudberry cell culture preparation, where said compositions have antioxidant and anti-aging effect. It protects the skin from UV radiation effects and it has effect on the procollagen I synthesis of aged fibroblasts. In said method callus is first produced from sterile cuts of cloudberry plant. The material is maintained on a medium favoring continuous growth of the non- differentiated cells. Selected callus is then chosen for suspension culture, which is stepwise scaled up to large scale cultivation in a bioreactor. The biomass is harvested, followed by washing with water and freeze-drying. The freeze-dried powder may be used as such in cosmetic compositions or the powder may be extracted with methanol or ethanol and filtered prior to use. Despite the ongoing research and development in the field, there is a need for new sustainable and ecological approaches to provide valuable and unique cell cultures originating from the family Betulaceae.

SUMMARY OF THE INVENTION

Cell culture technology offers a sustainable approach for utilizing the biosynthetic capacity of the plants of the family Betulaceae to produce e.g. valuable secondary metabolites including phenolic compounds. Even new compounds, which cannot be found in the natural plant, can be produced utilizing modified biosynthetic pathways. Biosynthetic pathways can be further modified using biotechnical methods e.g. elicitation and precursor feeding. Cell cultures of the family Betulaceae offer a highly interesting new type of raw material for skin care products.

Unique cell cultures of the family Betulaceae, particularly birch such as Betula pendula can be produced by the method of the invention, and their chemical composition is completely different from those found in the wood or plant of family Betulaceae.

The cell cultures produce e.g. natural color compounds, such as anthocyanins and carotenoids. In addition, some of the cultures produce polymeric tannins, such as proanthocyanidins and ellagitannins with known strong antimicrobial properties. Very interesting amino acids were also detected particularly in birch cell cultures, such as gamma-aminobutyric acid (GABA), which is an important transmitter in the brain tissue. In green cell culture phytol, a diterpene alcohol, was found, which is used in cosmetic and fragrance industry. For example, the birch cell cultures show also antimicrobial activity against human skin pathogen Staphylococcus aureus and important cosmetic spoilage bacteria Pseudomonas aueroginosa and further, antioxidant activity important for skin health.

Biotechnical tools, namely elicitation and precursor feeding may be used to highly increase e.g. antioxidant activity of the cultures.

In the present invention, in an embodiment, cultivation of one embryogenic birch cell line was up-scaled in a larger bioreactor, showing feasibility to produce high amounts of the plant biomass. The obtained cell culture material with unique properties offers excellent raw material for sophisticated cosmetic products. An object of the invention is to provide a method for producing cell cultures of the family Betulaceae.

Another object of the invention is to provide a method for producing birch cell cultures. Another object of the invention is to provide a method for producing cell cultures of the family Betulaceae, said method being suitable particularly for larger industrial scale.

Another object of the invention is to provide a method for producing birch cell cultures, said method being suitable particularly for larger industrial scale.

Another object of the invention is to provide compositions comprising cell cultures of the family Betulaceae.

Another object of the invention is to provide compositions comprising birch cell cultures.

An object of the invention is to provide cell cultures of the family Betulaceae.

An object of the invention is to provide birch cell cultures. Another object of the invention is to use of the obtained cell cultures of the family Betulaceae in cosmetic, hygiene and personal care products, as well as for food, nutrition, nutraceuticals, pharmaceuticals and pharmaceutical devices, as e.g. natural colorants, antioxidants, preservatives and as natural flavors. Another object of the invention is to use of the obtained birch cell cultures in cosmetic, hygiene and personal care products, as well as for food, nutrition, nutraceuticals, pharmaceuticals and pharmaceutical devices, as e.g. natural colorants, antioxidants, preservatives and as natural flavors. The present invention generally concerns producing cell cultures of the family Betulaceae having unique compositions, where cell cultures are illuminated with LED-lights favoring strong production of anthocyanins in the culture.

The method of the invention for producing cell culture of a plant of the family Betulaceae comprises two stages, where - in the first stage callus line of a plant of the family Betulaceae is cultured in a liquid medium comprising a medium used for plant cell and tissue culture, under irradiation conditions selected from day-night irradiation regime under normal light, under irradiation using LED light carried out as intermitting irradiation, under irradiation using LED light carried out as constant irradiation, and in darkness without irradiation, to obtain primary suspension cell culture, and

- in the second stage the primary suspension cell culture is up-scaled by culturing in at least two steps in a liquid medium comprising a medium used for plant cell and tissue culture; under irradiation conditions selected from day-night irradiation regime under normal light, under irradiation using LED light carried out as intermitting irradiation, under irradiation using LED light carried out as constant irradiation, and in darkness without irradiation, used in the first step or in the second step or in both steps.

The invention also concerns producing the cell cultures in total darkness i.e. without irradiation provided by an irradiation source. In these conditions the anthocyanin production is inhibited.

The invention also relates to compositions comprising cell cultures obtained by the method, said cell cultures comprising one or more of anthocyanins, proanthocyanidins, phenolic acids, ellagitannins, carotenoids, amino acids, vitamins and fatty acids. Said compounds are for example beneficial for human health.

The invention further relates to the use of said cell cultures in cosmetic compositions, hygiene and personal care products, as well as in foods, feeds, pet foods, nutritional compositions, nutraceutical compositions, pharmaceutical compositions, pharmaceutical devices, for example for providing effects beneficial to the health, and as e.g. natural colorants, antioxidants, preservatives and as natural flavors for providing flavor, aroma and/or color to the products.

Characteristic features of the invention are presented in the appended claims.

DEFINITIONS

The term "cell culture" refers here to callus or cell suspension culture, embryogenic or non- embryogenic cell cultures or hairy root cultures originating from a plant of the family Betulaceae, where said plant includes seeds, flowers, samara, roots, leaves and pieces of plant stem, one or more of hypocotyl, cotyledon, leaf section, stem section, root section of a seedling ; stem section including a node or a n internode, and a leaf section of a mature plant.

The term "callus culture" refers here to culture of non-differentiated plant cells, and it may contain embryogenic and/or non-embryogenic cells. "Embryogenic culture/callus" refers to culture containing PEMs and which can go through differentiation via somatic embryogenesis in proper conditions (low auxin content) . With high auxin contents it can be maintained as undifferentiated state. During long-term maintenance as callus or suspension, it may lose embryogenic capacity. The term "non-embryogenic culture/callus" refers here to culture containing often highly vacuolated cells, which are not capable of going through somatic embryogenesis.

The term "a medium used for plant cell and tissue culture" refers here to any common media used in plant cell and tissue culture, such as "N7 medium", "MS medium" (Murashige & Skoog, 1963), "B5 medium" (Gamborg et al ., 1968) and "Woody Plant Medium, WPM" (Smith and McCrown ( 1982/1983) .

The term "N7 medium" refers here to cell culture medium originally developed for birch cell cultures (Simola 1985) . pH of the medium is 5.8. The basal medium is supplemented with various concentrations of plant growth regulators, also called plant growth hormones, mainly 2,4-D (2,4-dichlorophenoxyacetic acid) and KIN (kinetin) . The N7 medium may be also modified, where the N7 medium is supplemented with additions of sucrose, plant growth regulators, the pH may be adjusted, and for obtaining solid medium Agar or Gelrite may be added .

LED light carried out as intermitting irradiation refers here to illumination/irradiation carried out in intermittent periods with LED light irradiation (light period) and without LED light irradiation (dark period), where the dark period may be from 1 to 24 hours and the light period may be from 1 to 24 hours. An example of intermitting irradiation comprises irradiation carried as day-night irradiation, 10- 18/14-6 hours respectively, such as ( 16/8hours) .

Normal light refers here to incandescent light havi ng illumination/irradiation intensity of 50-300 Ix, typical irradiation about 50 \Avr o\/vr ²sec. BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows photos of cell suspension cultures with different colours and phenotypes established using cell and tissue culture methods from the originally heterogeneous, non- embryogenic BpN l cell line (A and B). Embryogenic callus (C) and somatic embryos (D and E) are also presented in Figure 1.

Figure 2 shows UPLC profiles of birch culture BpN l with methyl jasmonate and etephon elicitation compared to non- elicited culture.

Figure 3 shows that elicitation did not have any adverse effects on growth of the birch cell culture BpN l.

Figure 4 shows UPLC profiles of birch BpN l cell culture fed with precursors or supplemented with various PGRs.

Figure 5 presents effects of precursor feeding on the growth of birch cell culture.

Figure 6 shows that acidic birch cell culture extracts prepared from the non-embryogenic cell suspension BpN l obtained as described in Example 1 show strong antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa in concentration 1 mg/ml. Figure 7 shows that hydrothermal extracts from birch embryogenic cell line Bp6/3 (2,5 mg/ml and 5 mg/ml) obtained as described in Example 1, cultivated in normal light also had moderate antimicrobial activity against Staphylococcus aureus.

Figure 8 shows growth of birch cell line BpN l in 10 L bio-reactor.

DETAILED DESCRIPTION OF THE INVENTION

Specific cell cultures may be established from wild or cultured plant materials of the family Betulaceae. Examples of such materials are wild silver birch (Betula pendula) plants. Said plants may be collected for example from almost any region in Finland.

Callus may be produced from sterile pieces of in vitro grown plants of the family Betulaceae, generated from sterilized seeds, or alternatively from surface sterilized pieces of the plant materials. For example, leaf material is suitable for induction of embryogenic cell culture and seed material for non-embryogenic cell culture.

In the present invention, the calli were grown in vitro at sterile conditions, and they were maintained on a medium favoring continuous growth of the non-differentiated cells. During maintenance, different callus lines (cell cultures) may be separated based on callus color. Particularly suitably bright colored callus of uniform quality is suitably selected by sub-culturing.

Typically, at least five different intensely colored callus lines may be selected from the same primary culture, stabilized and maintained.

At least one selected callus line is suitably chosen for suspension culture.

The callus line may be used as such or it may be cryo-preserved. It can be stored in liquid nitrogen for tens of years.

In the present invention, at least one selected callus line of the family Betulaceae (such as intensely colored embryogenic callus line) may be suspension cultured. The suspension culture is up-scaled to produce the cell cultures in amounts suitable for large industrial scale.

It was surprisingly found that using the suspension culture method of the invention, cell cultures of the family Betulaceae having several advantageous effects could be obtained effectively and consistently on a large industrial scale. The obtained cell cultures of the family Betulaceae exhibit valuable bioactivities and other properties. The method is sustainable, and continuously large amounts of the cell culture of the family Betulaceae may be produced .

The phenolic profiles of the obtained cell cultures of the family Betulaceae are unique and different compared to the plant itself. High amounts of natural colorants, i.e. anthocyanins and other valuable phenolic compounds may be produced with the method, in significant amounts. Examples of such valuable compounds are galloyl derivatives, quercetin derivatives, proanthocyanidins including cathecin derivatives, ellagitannins and prodelphidins. The cell cultures also contain natural aromas, flavors, amino acids and lipids with beneficial fatty acid composition, which are desired ingredients for example in cosmetic, nutraceutical, nutritional and hygiene preparations. The cell cultures can be generated around the year, on an industrial scale, with consistent quality and composition, which can be constantly monitored. Plant material of the family Betulaceae

The plant material of the family Betulaceae refers here to seeds, flowers, samara, roots, leaves and pieces of plant stem of the plant, including one or more of hypocotyl, cotyledon, leaf section, stem section, root section of a seedling; stem section including a node or an internode, and a leaf section of a mature plant. Said plant material may originate from wild plants or cultivated plants. Said plant of the family Betulaceae is suitably selected from birches, alders, hazels, hornbeams, hazel-hornbeams and hop-hornbeams. Preferably the plant is birch. Examples of birches are Betula pendula, Betula nana, Betula pubescens and Betula humilis.

Formation of calli

Callus lines may be induced from sterile pieces of in vitro grown plants, generated from sterilized seeds of the plant, or alternatively from surface sterilized pieces of the plant material, such as birch plant. Calli formed on explants (such as callus of birch cells derived from birch plant) are obtained.

In vitro seedlings are obtained by germinating surface sterilized seeds on agar plates (0.5- 1 % agar in water). Sterile seedlings are cut into pieces and callus is induced on growth medium with PGRs. High auxin content, typically 0.5-10 mg/L, is preferable for induction of embryogenic callus.

Alternatively, leaves of young plants or seeds are surface sterilized, typically the leaves are cut into pieces and seeds are cut in half. In both cases callus is induced on growth medium with PGRs. High auxin content, typically 0.5-10 mg/L, is preferable for induction of embryogenic callus.

In the induction of callus lines, the plant material is suitably cut into pieces. The pieces may have size of about 0.5 - 10 cm.

The pieces of the plant material are treated with at least one surface sterilizing solution, preferably with an ethanol solution followed by hypochlorite solution, to obtain surface sterilized pieces. The ethanol solution comprises suitably at least 50 % v/v, preferably at least 70 % v/v of ethanol in water. The hypochlorite solution comprises from 0.5 to 8 % by weight of hypochlorite in water. Suitably the surface sterilized pieces are rinsed with sterile water and dried. The surface sterilized (sterile) pieces may be used for the induction of the calli, however also sterile pieces of in vitro grown plants may be used for the formation of the calli.

Following procedure may suitably be used to produce sterile in vitro plantlets. In an embodiment, the surface sterilized seeds are transferred to a solid medium used for plant cell and tissue culture without plant growth regulators, followed by incubating with the medium under sterile conditions, at the temperature of 20 - 28°C, under day-night illumination period under normal light (light-dark irradiation regime), for the time of 1 - 4 weeks, whereby roots and new leaves (in vitro grown plants) formed on the explants are obtained. Typically, the day-night photoperiod under normal light refers to 10-18/14-6 hours, respectively, such as 16 : 8 photoperiod, with irradiation 30-60 μιηοΙ/ιη²5. Basic plant cell culture media are suitable, such as N7, MS, WPM or B5. Preferably the solid medium used for plant cell and tissue culture is e.g. N7 medium, with added sucrose and agar. Typically, sucrose (such as 2% w/vol) is added and the medium is solidified with agar. Suitably the incubation is carried out in sealed sterile boxes. Preferably the temperature is in the range of 20 - 25°C. Suitably the incubation is carried out for 1 -3 weeks.

Sterile pieces of the in vitro grown plants, such as birch plants, generated from sterilized pieces of the plant material, or pieces of surface sterilized plant material are incubated on a solid medium, suitably N7 medium, with plant growth regulators (PGR) favoring development of undifferentiated plant material called callus. Callus formation is induced by combination of auxins and cytokines. Suitable auxins are 2,4-D (2,4-dichlorophenoxyacetic acid), NAA (a-naphthaleneacetic acid) and IAA (indole-3-acetic acid), and cytokine is suitably KIN (Kinetin). The callus is grown in vitro at sterile conditions, and it is maintained on a medium favoring continuous growth of the non-differentiated cells, suitably N7 medium with proper hormone content, typically auxin 0.1-10 mg/L and cytokine 0-2 mg/L. Different cell lines (cell cultures) may be separated based on callus color. Said cell lines (or cell cultures) may be used as such or they may be cryo-preserved (in liquid nitrogen) for later use.

The day-night (light-dark) irradiation regime with normal light refers typically to a photoperiod of 10-18/14-6 hours, respectively, such as 16 : 8, with irradiation 30-60 μιηοΙ/ιη²5. The LED light or lights as defined on page 19 of this specification are preferably used. Preferably the temperature is 20 - 25°C.

Preferably the incubation time is 1 - 4 weeks.

Preferably the pH is in the range of 3-7, preferably 4 - 6, particularly preferably - 5-6.

Preferably the medium is a modified N7 medium, where sucrose, gelrite and plant growth regulators are added and where the pH is adjusted into the range of 3-7, preferably of 5 - 6. Typically, said modified N7 medium comprisesl-3 % w/v (2 % w/v) of sucrose, 0.1- 5 g/l (lg/L) casein hydrolysate and 6-9% w/v (8 g^L"l) of gerlite and plant growth regulators.

The plant growth regulators (PGR, plant growth hormones) are selected from cytokines, auxins and combinations thereof. Suitably the cytokine is kinetin, and a suitable auxin is 2,4-dichlorophenoxyacetic acid (2,4-D) or a-naphthaleneacetic acid (NAA). Suitably, 0- 1.0 ppm of kinetin and 0.01 - 5 ppm of auxin is used. Preferably the PGRs are used in amounts of 1-3 mg/L (4.5-13.5 μΜ) 2,4-D and 0-0.7 mg/L (0-3.2 μΜ) kinetin for inducing and maintaining the embryogenic calli.

The calli formed on the explants are separated from the explants. They may be used as such or they may be cryo-preserved (in liquid nitrogen) for later use. The cell lines can be cry-preserved and stored in liquid nitrogen for tens of years without losing viability or embryogenic capacity.

Subculturing The separated calli (optionally taken out from cryo-storage) are transferred to a fresh solid medium. Suitably the same medium (such as N7) as used in the inducing of callus is used in the subculturing. Also, other commonly used plant cell culture media may be used, such as MS, B5 or WPM media. The calli are sub-cultured regularly favoring soft productive biomass. Cultured plant cells are totipotent, and when treated with PGRs the cells multiply continuously producing biomass consisting of identical cells.

Different callus lines may be separated based on the color of the callus, for sub-culturing.

The sub-culturing is carried out suitably as follows. The calli formed on the explants are separated from the explant, or taken out from cryo-storage, and transferred to fresh solid medium. They are maintained under day-night irradiation regime under normal light or under irradiation using LED light carried out as intermitting irradiation or as constant irradiation, or at constant dark. Preferably LED light is used, particularly preferably intermitting irradiation with LED light.

During each sub-culturing the medium is replaced with a fresh medium with pH adjustment to 3-7, preferably to 4-6, particularly preferably to 5-6.

The separated callus is sub-cultured every 2-6 weeks, preferably 3-4 weeks, suitably by gentle division, for 2 to 8 months to obtain sub-cultured callus line. Any dark brown material is removed. Preferably the solid medium is a modified N7 medium, where sucrose (1-3 %), gelrite (6-9%), casein hydrolysate and plant growth regulator(s) are added and where the pH is adjusted into the range of 3-7, preferably of 5-6. Combinations of auxin and cytokine are included in said medium. Preferably, 2,4-dichlorophenoxyacetic acid and kinetin are used. Suitably the amounts are 0-0.5 ppm of kinetin and 0.01 - 5 ppm of auxin. Casein hydrolysate is omitted gradually, in 2-4 sub-culturing passages. The temperature of 20 - 28°C, preferably 20-25°C is used. Sub-cultured callus lines are obtained.

Embryogenic and non-embryogenic callus lines are cultivated in different PGR combinations. Suitably 1-3 mg/L 2,4-dichlorophenoxyacetic acid and 0-0.7 mg/L kinetin are used for maintaining the embryogenic callus line.

For maintaining the non-embryogenic callus line, the medium is supplemented with 0.1- 0.5 mg/L of 2,4- dichlorophenoxyacetic acid and 0-0.03 mg/L of kinetin.

The day-night (light-dark) irradiation regime with normal light refers typically to a photoperiod of 10-18/14-6 hours, respectively, such as 16 : 8, with irradiation 30-60 μιηοΙ/ιη²5.

The LED light or lights as defined on page 19 of this specification are preferably used. The callus line is an embryogenic or non-embryogenic callus line produced from material of the plant.

The cell lines obtained may be used as such or they may be cry-preserved (in liquid nitrogen) for later use. Method for producing cell cultures of the family Betulaceae

The method of the invention for producing cell cultures of a plant of the family Betulaceae is particularly suitable for larger industrial scale, providing plant cell culture of consistent quality in high amounts, such as birch cell culture. Up-scaling of the primary suspension cell culture is realized by suspension culturing carried out in at least two steps.

The method of the invention for producing cell cultures of a plant of the family Betulaceae comprises two stages, where

- in the first stage callus line of a plant of the family Betulaceae is cultured in a liquid medium comprising a medium used for plant cell and tissue culture, under irradiation conditions selected from day-night irradiation regime under normal light, under irradiation using LED light carried out as intermitting irradiation, under irradiation using LED light carried out as constant irradiation, and in darkness without irradiation, to obtain primary suspension cell culture, and

Darkness or constant darkness refers to irradiation conditions where no irradiation source is used. Constant irradiation refers to irradiation carried out without interruptions.

In one preferable embodiment, the second up-scaling stage comprises at least three steps.

In another preferable embodiment, the second up-scaling stage comprises at least four steps.

Preferably LED light is used, particularly preferably intermitting irradiation with LED light at least in the first stage, or in the first step or the second step of the second stage. In a preferable embodiment, LED light irradiation carried out as day-night irradiation is used. Day-night irradiation refers typically to a photoperiod of 10-18/14-6 hours, respectively, such as 16 : 8 respectively.

First stage: Generation of primary suspension cell culture of the family Betulaceae

In the first stage of the method generation of a primary suspension cell culture of a plant of the family Betulaceae is carried out. The first stage of the method comprises generation of primary suspension cell culture, where callus line of the family Betulaceae, such as birch, is cultured in a liquid medium comprising a medium used for plant cell and tissue culture, to generate inoculum for larger scale. For carrying out the suspension culture at least one callus line of the family Betulaceae is introduced in a liquid medium comprising a medium used for plant cell and tissue culture, and water.

Said liquid medium has a pH of 3-7 and at temperature of 20 - 28°C.

The suspension culture is carried out under irradiation conditions selected from day-night irradiation regime under normal light, under irradiation using LED light carried out as intermitting irradiation, under irradiation using LED light carried out as constant irradiation, and in darkness without irradiation. Preferably LED light is used, particularly preferably intermitting irradiation with LED light. The suspension culture is initiated suitably with 0.1- 20 g of a selected callus line, such as birch callus line, in 10-200 ml of the liquid medium. The suspension culture is sub-cultured every 7 - 15 days intervals and stepwise up-scaled to 50-500 ml, preferably to 200-500 ml culture. A suitable vessel, such as an Erlenmeyer flask may be used. Suitably more than one replicate suspensions are cultured in the flasks, preferably at least two replicate suspensions. The sub-culturing is carried out in the liquid medium as defined below. Any visually detectable clumps are removed, preferably after 2 - 4 sub-culturing, to provide a homogeneous suspension by visual inspection. Sieving etc. may be used. After 2-10, preferably after 4-8 sub-culturing, primary suspension cell culture is obtained as a finely divided and homogeneous suspension, when inspected visually. In an embodiment, the cells may be harvested using methods known in the art, and they may be suspended in the fresh medium to obtain a homogeneous suspension. The primary suspension cell culture of a plant of the family Betulaceae (inoculum) is used in the second stage.

The water used in the method is selected from sterilized MQ-water, RO-water, and tap water, preferably high purity sterilized MQ-water or RO-water.

Any medium used for plant cell and tissue culture may be used in the method, such as N7,

MS, WPM or B5 medium, preferably the medium used for plant cell and tissue culture is a modified N7 medium. Said modified N7 medium comprises sucrose, suitably 1-3 % w/v.

Said modified N7 medium comprises plant growth regulators selected from cytokines, auxins and combinations thereof. Suitably the cytokine is kinetin, and a suitable auxin is 2,4-dichlorophenoxyacetic (2,4-D), a-naphthaleneacetic acid (NAA) or indole-3-acetic acid

(IAA). Suitably 0- 0.5 ppm of kinetin and 0.01 - 5 ppm of auxin is used. The pH of modified

N7 medium is adjusted into the range of 3-7, preferably of 5-6.

The day-night (light-dark) irradiation regime under normal light refers typically to a photoperiod of 10-18/14-6 hours, respectively, such as 16 : 8; with irradiation 30-60 μιηοΙ/ιη²5.

Preferably the temperature is 22-25°C. The first stage is suitably carried out in a vessel comprising glass or plastic, allowing the irradiation pass through the walls of the vessel. Suitably glass flasks used in the art are selected, such as Erlenmeyer flasks etc., or alternatively plastic flasks or bags may be used, which allow passing of the irradiation to the mixture/suspension. The LED light or lights as defined on page 19 of this specification are preferably used.

Second stage: Up-scaling of the primary suspension cell culture

The second stage of the method comprises up-scaling of the primary suspension cell culture (inoculum from the first stage), where the up-scaling is carried out in at least two steps.

In the first step, the inoculum obtained from the first stage is sub-cultured, suitably every 10±5 days, and up-scaled to a volume of not more than 1000 ml. Suitably a vessel of 200- 1000 ml is used, preferably of 200-500 ml containing about 30 to 80 ml of the primary suspension culture, using the same medium and temperature as in the first stage. During this first step the culture is exposed to irradiation conditions selected from day-night irradiation regime under normal light, under irradiation using LED light carried out as intermitting irradiation, under irradiation using LED light carried out as constant irradiation, and in darkness without irradiation, to yield cell culture from the first step. Preferably the cells are harvested using methods known in the art, and they may be suspended in the fresh medium to provide an inoculum.

In the second step, the inoculum from the first step is up-scaled to a volume of not more than 20 I and made up to with fresh medium to obtain initial cell density of 10-50 g L ^_1, preferably 25-40 g L^_1. Suitably at least one bio-reactor having the volume of 1-20 L, preferably 1-10 L is used. The suspension is grown in the bio-reactor or bioreactors for 7-15 days, with optionally from 1 to 3 additions of the fresh medium.

Suitably cultivation conditions, where the temperature is 20-28°C, preferably 22-26°C are used.

Suitably the dissolved oxygen (DO) is at least 10%, preferably at least 20 %. Agitation speed, depending on cultivation vessel, is in the range of 20 to 300 rpm.

The pH is suitably uncontrolled. The aeration of 0.25 - 3.0 I min-1 may be used. A suspension cell culture from the second step is obtained. Preferably the same medium as in the first stage is used .

During this second step the culture may be irradiated using irradiation conditions selected from day-night irradiation regime under normal light, under irradiation using LED light carried out as intermitting irradiation, under irradiation using LED light carried out as constant irradiation, and in darkness without irradiation, preferably LED light is used. In an embodiment where the second stage comprises more than two steps, the second step may alternatively be carried out exposed to no irradiation. The suspension cell culture from the second step may be obtained as the final product or alternatively it may be used as inoculum in a third step. The cells may also be harvested using methods known in the art and the harvested cells may be suspended in the fresh medium to provide the inoculum. The second stage may comprise a third step. In the third step the inoculum from the second step is up-scaled to volume of not more than 70 I, made up to with the fresh medium to obtain initial cell density of 10-50 g L ^_1, preferably 25-40 g L ^_1, suitably using a bio-reactor having the volume of 10-70 L, preferably 10-50 L. The suspension is grown in the bio- reactor for 7-15 days, with optionally one addition of fresh medium. Suitably cultivation conditions, where the temperature is 20-28°C, preferably from 22 to 26°C, and dissolved oxygen (DO) at least 10%, preferably at least 20 % are used. The agitation speed, depending on cultivation vessel, is suitably in the range of 20 - 150 rpm. The pH is suitably uncontrolled. The aeration is preferably 0.3 - 2 I min ^_1 The pressure is suitably in the range 0.008 to 0.2 bar, depending on cultivation vessel. Preferably the same medium as in the first stage is used. During this third step the culture may be irradiated using irradiation conditions selected from day-night irradiation regime under normal light, under irradiation using LED light carried out as intermitting irradiation, under irradiation using LED light carried out as constant irradiation, and in darkness without irradiation, preferably LED light is used, particularly preferably intermitting irradiation with LED light. In an embodiment where the second stage comprises more than three steps, the third step may be carried out exposed to no irradiation.

A suspension cell culture from the third step is obtained as the final product or alternatively it may be used as inoculum in a fourth step. The cells may also be harvested using methods known in the art. The harvested cells may be suspended in the fresh medium to provide the inoculum.

The second stage may comprise a fourth step. In the fourth steps the inoculum from the third step is up-scaled to volume of not more than 500 I made up to with fresh medium to obtain initial cell density of 10-50 g L ^_1, preferably 25-40 g L ^_1, suitably using a bio-reactor having the volume of 300-500 L. The suspension is grown in the bio-reactor for 7-15 days, with optionally one addition of fresh medium, which may comprise 2- to 4-fold by weight of sucrose compared to the medium used in the first stage. Suitably cultivation conditions, where the temperature is from 20-28°C, preferably 22 to 26°C, and dissolved oxygen (DO) at least 10 %, preferably at least 20 %, are used. The agitation speed is suitably in the range of 50 - 100 rpm. The pH is suitably uncontrolled. The aeration is 10 - 20 I min^-1. The pressure is preferably 0.2 bars. Preferably the same medium as in the first stage is used. During this fourth step the culture is irradiated using irradiation conditions selected from day-night irradiation regime under normal light, under irradiation using LED light carried out as intermitting irradiation, under irradiation using LED light carried out as constant irradiation, and in darkness without irradiation, preferably LED light is used, particularly preferably intermitting irradiation with LED light. A suspension cell culture comprising biomass is obtained. The cells may also be harvested using methods known in the art. The harvested cells may be suspended in the fresh medium.

The LED light or lights as defined on page 19 of this specification are preferably used.

The second stage may also comprise further up-scaling steps.

In the method of the invention the final product, the suspension cell culture of the family Betulaceae, comprising the biomass, obtained in the second stage, may be used as such, or alternatively it may be dried, suitably using freeze-drying or spray-drying methods known in the art, to provide a finely divided powder. Alternatively, the cells may also be harvested using methods known in the art, and they may be used as such or dried.

Dried powder may be used as such as cosmetic ingredient. Alternatively, solvent extraction methods, such as hydrothermal extraction process may be utilized to concentrate biomolecules.

The pH of the N7 medium used in the method may preferably be adjusted to 3.5-6, in all steps before autoclaving the medium, using 0.1 M NaOH.

The first step is suitably carried out in a vessel comprising glass or plastic allowing the irradiation pass through the walls of the vessel. Suitably glass flasks used in the art are selected. The second and further up-scaling steps may be carried out in vessels, such as bioreactors allowing the cultivation to final culture volume and passing of the irradiation to the reaction mixture. Suitably vessels comprising glass or plastic material may be used. Chemical elicitators

Chemical elicitators may be used to boost secondary metabolite production. Suitable chemical elicitors are e.g. methyl jasmonate, abscisic acid, salicylic acid, etephon and chitosan. the chemical elicitators, such as methyl jasmonate or ethephon may be used in amounts 5-50 ppm and 50-500 ppm respectively. Preferably methyl jasmonate is used. Elicitors stimulate formation of phenolic compounds and increase the amount of total phenolics typically three times in the cell material and at least two times in the medium. Elicitors can be used in all cultivation stages, but when up-scaling culture system their use is preferable in the final up-scaling stage (second stage). Elicitors are added to the cell cultures during exponential growth phase, in days 4-9, preferably on day 6.

Precursor feeding

Precursor feeding may also be used to boost secondary metabolite production. Suitably a precursor selected from mannitol (1-10 wt%), betuligenol (0.1 -5 mM), phenylalanine (0.1 -5 mM) and combinations thereof is used. Elicitors can be used in all cultivation stages, but when up-scaling culture system their use is preferable in the final up-scaling stage (second stage). Precursors are added to the cultures either at the beginning (day 0) or at the exponential growth phase in days 4-9, preferably on day 6.

PGRs

Content of phenolic compounds can be affected by changing the PRGs and their concentration of the growth medium. Especially by replacing the auxin 2,4-D by NAA or IAA (0,5 - 10 mg/L) increases the content of phenolic several times.

LED light

LED (light emitting diode) light is used in the method. Preferably the LED light is used in all stages and all steps.

The LED light has color temperature of 2700-3000K.

In an embodiment, the LED light comprises an effectual spectrum comprising at least 25 % of an integral in the wave length range of 630 nm-760 nm (red light integral), calculated from the total effectual spectrum. Red light is generally classified as "warm light shade". In an embodiment, the LED light comprises a total effectual spectrum in the wave length range of 400-800nm.

In an embodiment, the LED light comprises less than 75 % of an integral in the wave length range of 400-450 nm (violet light integral) or of an integral in the wave length range of 450-490 nm (blue light integral) or of an integral in the wave length range of 490-560 nm (green light integral) or of an integral in the wave length range of 560-590 nm (orange light integral) or a combination thereof.

In the method irradiation source is preferably LED light with illumination intensity over 500 Ix, preferable over 800 Ix.

One LED light (source) or combination of LED lights (light sources, several light sources) may be used .

Said LED lights may be arranged in a panel surrounding or enclosing the reaction vessel, or in any other way to provide the reaction mixture the required irradiation.

The final product suspension cell culture, comprising the biomass, obtained in the second stage, may be used as such in the manufacture of products, or alternatively it may be dried, suitably using spray-drying or freeze-drying methods known in the art to provide a finely divided powder.

Alternatively, solvent extraction methods, including hydrothermal extraction technology, may be used to concentrate biomolecules from the wet or dry biomass. Alternatively, the cells may also be harvested, and used as such or dried. The biomass may be separated using technique known in the art. Suitably a filter press or vacuum filtration is used, where the biomass is filtered and washed with sterile water. The biomass filter cake is suitably dried, for example using freeze-drying. The dry biomass may be ground to finely divided powder and stored in sealed packages, preferably at temperatures of about -20°C.

Cell cultures of the family Betulaceae, such as birch cell cultures are obtained. Cell cultures of the family Betulaceae

The cell cultures of a plant of the family Betulaceae obtained with the method of the present invention exhibit high bioactivities and other beneficial and interesting properties. The chemical composition of the cell cultures of plants of the family Betulaceae, obtained by the method, particularly of birch cell cultures, is clearly different from the one natural birch plant.

Using irradiation with the specific LED light in the manufacturing method, high amounts of cell lines and suspension cultures with different colors and phenotypes can be established from the originally heterogeneous, non-embryogenic cell lines and embryogenic cell lines. Suspension cultures with intense red, reddish, yellow, yellowish, bright green or pale green color are obtained. Unique chemical composition can be obtained in a consistent manner. Natural birch plants contain typically hydroxycinnamic acids and their derivatives, anthocyanins, flavonols, ellagic acid derivatives and ellagitannins, gallic acid derivatives and cathechins. Procyanidins are not found in the natural plants. The birch cell cultures obtained with the method of the invention may comprise 0.1-10 wt% (dry weight) of anthocyanins and 0.1-5 wt% (dry weight) of proanthocyanidins.

The birch cell cultures obtained by the method of the invention comprise significant amounts of various organic acids, oligomeric aromatics, aromatics, natural acidic pectins, carotenoids, plant sterols, phytol, and polyphenols, such as ellagitannins and proanthocyanidins (procyanidins and prodelphinidins). Further, said birch cell cultures comprise typically proteins, fatty acids such as a-linoleic acid and carbohydrates, such as sucrose. Also, amino acids were also detected particularly in birch cell cultures, such as gamma-aminobutyric acid (GABA), which is an important transmitter in the brain tissue.

Anthocyanins exhibit antioxidant and anti-inflammatory effect, in addition to intensive color, aroma and flavor, and thus they are beneficial for use in cosmetic, hygiene and personal care applications, in food, feeds, pet foods, nutritional products, nutraceuticals, pharmaceutical products and pharmaceutical devices, and for providing aroma, flavor and/or color to the products. Antioxidant activity is also essential for example in delaying skin aging processes. With the method of the invention the anthocyanin content can be increased over seven-fold, particularly with the red-colored birch cell lines, compared to the manufacture under normal light conditions, and further, the content of other phenolic compounds is increased. The cells obtained with the method are more intact and contain less water when compared with the ones manufactured under normal light conditions. They provide an advantage as being more effective and economic in downstream processing.

The phenolic compounds, such as flavonoids exhibit antimicrobial activity. Antimicrobial activity is beneficial in cosmetic applications, where for example in cosmetic products it helps to balance skin microbiota and prevents product contaminations. The invention provides compositions comprising birch cell cultures obtained by the method, said birch cell cultures comprising anthocyanins, proanthocyanidins, phenolic acids, vitamins, amino acids and fatty acids beneficial for human health.

The invention relates also to compositions comprising cell cultures of the family Betulaceae, particularly birch cell cultures, obtained by the method of the invention, where the composition is selected from cosmetic compositions, hygiene and personal care products, as well as in foods, feeds, pet foods nutritional compositions, nutraceutical compositions, and pharmaceutical compositions and pharmaceutical devices. The invention relates also to the use of said cell cultures of the family Betulaceae, particularly birch cell cultures in compositions selected from cosmetic compositions, hygiene and personal care products, as well as in foods, feeds, pet foods nutritional compositions, nutraceutical compositions, and pharmaceutical compositions and pharmaceutical devices.

The cosmetic compositions may contain cosmetically acceptable substances in addition to said cell culture(s). Said compositions may be used as day creams, foundation creams, peeling creams, lipsticks, color cosmetics, skin serums, mascaras, products for hair and/or scalp care, washing products for skin or hair, and as products for skin hygiene.

The cosmetic composition can be, for example, an emulsion cream, such as a day cream or a foundation cream; peeling cream; a lipstick; a skin serum or a hair cosmetics product, such as a composition for hair conditioning or a composition for scalp treatment. The cosmetic composition may contain the cell culture in powder form in an amount of 0.001 to 25 % by weight, more preferably the amount of the powder is 0.01 to 5 % by weight, and most preferably 0.01-1 % by weight.

The emulsion cream containing the cell culture can be of the type oil-in-water emulsion, water-in-oil emulsion, water-oil-water emulsion or a micro-emulsion. The emulsion cream composition may contain the cell culture in powder form, preferably 0.001 to 25 % by weight, more preferably 0.01 to 5 % by weight and most preferably 0.01 to 1 % by weight.

In the emulsion creams the cell culture can be combined also with synthetic or natural vitamins or their combinations. Examples of these vitamins are retinol and A-vitamin palmitate. On the other hand, also different E-vitamins and E-vitamin derivatives, like tocopherol, C-vitamin and its derivatives, like ascorbyl palmitate and magnesium ascorbyl phosphate, panthenol and other B-vitamins and/or biotin may be used. The composition may contain synthetic and/ or natural vitamins preferably 0.01 to 10 % by weight, more preferably 0.02 to 5 % by weight. The emulsion cream compositions may contain, in addition, one or more adjuvants acceptable in the field of cosmetics, such as preservation agents, thickening agents, moisturizing agents, and other suitable additives, such as for example perfumes and/or coloring agents. Suitable preservation agents are for example parabens, phenoxyethanol, imidazolidinyl urea and methyl dibromo glutaronitrile. These preservation agents can be used alone or combined with each other. Any thickening agent suitable in the field of cosmetics can be used provided, that it is compatible with the other components of the composition, for example xanthan gum and hydroxyethylcellulose, hydroxypropylmethylcellulose, Sclerotium gum, Chondrus Crispus, polyacrylates, polyacrylamides, cetearyl dimethicone crosspolymer and magnesium aluminum silicate. Thickening agents can be used alone or in combination with each other. Suitable moisturizing agents are for example heptyl undecylenate, hyaluronic acid or sodium PCA. In the emulsion creams, also different skin conditioning agents, for example tocopheryl acetate and ethyl hexylglycerin may be used. In addition, one or more compounds acting as an emulsifier, i.e. a compound dispersing and stabilizing the oil in water may be needed in the emulsion cream composition. Useful emulsifiers are all non-ionic emulsifiers accepted by the cosmetic legislation, such as, for example, glyceryl stearate, PEG-5 glyceryl stearate, PEG-1 00 stearate, PEG-30 dipolyhydroxystearate, lecithin, hydrogenated lecithin and PEG- 8 bees wax, steareth-21, steareth-2, sorbitan olivate as well as a mixture of a fatty glucoside, such as for example cetearyl, cocoyl, or myristyl glucoside and a fatty alcohol, such as for example cetearyl, cetyl, stearyl, octyldodecanol, caprylic/capric triglyceride or myristyl alcohol. In addition, of anionic emulsifiers, for example stearic acid, sodium hydroxide and triethanolamine are useful. Also, different chelating agents, like disodium EDTA and citric acid may be used.

The peeling cream compositions contain the cell culture preferably 0.001 to 25 % by weight, more preferably 0.01 to 5 % by weight and most preferably 0.01 to 1 % by weight.

The peeling cream compositions may contain in addition to the cell culture also other substances, which are acceptable in the field of cosmetics and which are traditional components of peeling cream compositions. Peeling cream compositions can also contain emulsifiers like PEG-8, behenyl alcohol, arachidyl glucoside and arachidyl alcohol, thickening agents like ethylcellulose and various vitamins and derivatives of those, like tocopherol, ascorbyl palmitate and ascorbic acid.

The peeling cream compositions can in addition contain one or more adjuvants and/or additives acceptable in the field of cosmetics, such as preservation agent. Suitable preservation agents are for example parabens and phenoxyethanol. These preservation agents can be used alone or in combination with each other.

The lipstick compositions contain the cell culture preferably 0.001 to 25 % by weight, more preferably 0.01 to 5 % by weight and most preferably 0.01 to 2 % by weight.

The lipstick compositions may contain in addition to the cell culture also other substances, like different waxes, oils, coloring and pearlescent agents, which are acceptable in the field of cosmetics and which are traditional components of lipstick compositions. Usable waxes are the natural waxes, such as bees wax, candelilla, carnauba, cereal based waxes, jojoba wax and their derivatives, in addition paraffin waxes and synthetic polyethylenes. Lipstick compositions can also contain emulsifiers like PEG-8, behenyl alcohol, arachidyl glucoside and arachidyl alcohol, thickening agents like ethylcellulose and various vitamins and derivatives of those, like tocopherol, ascorbyl palmitate and ascorbic acid.

The lipstick compositions can in addition contain one or more adjuvants and/or additives acceptable in the field of cosmetics, such as preservation agent. Suitable preservation agents are for example parabens and phenoxyethanol. These preservation agents can be used alone or in combination with each other. The compositions intended for hair or scalp care may contain the cell culture, preferably 0.001 to 10 % by weight, more preferably 0.01 to 5 % by weight and most preferably 0.01 to 2 % by weight. Further, the compositions intended for hair or scalp care may also contain different caring agents. These caring agents are typically in the amount of 0.1 to 40 % by weight. Preferably the caring agent content of a product is 0.1 to 20 % by weight. The compositions intended for hair or scalp care may contain in addition to birch cell culture, also other substances acceptable in the field of cosmetics, like cationically active substances, such as cetrimonium chloride, in addition an emulsion forming substance, such as for example cetyl alcohol, cetearyl alcohol, ceteareth-20. In addition, the compositions intended for hair or scalp care can contain one or more adjuvants acceptable in the field of cosmetics, such as a cellulose derivative, ethanol and/or water. Also, oils, waxes and fatty alcohols can be present in the compositions, according to the invention, intended for hair or scalp care.

The serum compositions may contain the cell culture preferably 0.001 to 25 % by weight, more preferably 0.01 to 5 % by weight and most preferably 0.01 to 2 % by weight.

The serum compositions may contain also other substances acceptable in the field of cosmetics such as emulsifying agents, chelating agents, solvents, preservatives, stabilizers together with substances affecting the skin permeability of the composition. These substances may be e.g. methylpropanediol, glycerin, phenoxyethanol, hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer, xanthan gum, propanediol, ammonium acryloyldimethyltaurate/VP copolymer, polyisobutene, disodium EDTA, lecithin, glucose, hydrogenated phosphatidylcholine, inulin lauryl carbamate, PEG-7, trimethylolpropane coconut ether, chondrus crispus, ethyl pyrrolidone, and cellulose gum. Additionally, the serum compositions may contain different moisturizing and skin conditioning agents such as heptyl undecylenate, ethylhexylglycerin, caprylyl glycol, ethylhexyl cocoate, peat extract and glycolipids.

As indicated above, the cell culture is preferably used in the form of freeze-dried powder, and the amounts given above are calculated for said powder. However, in case methanol or ethanol extracts or hydrothermal extracts are used, it is within the expertise of a person skilled in the art to adapt the amounts correspondingly.

The phenolic profile of the cell cultures of the present invention, particularly of the birch cell culture is unique and different compared to the plant, such as birch plant, as said cell cultures may contain proanthocyanidins, gallic acid derivatives, kampherol derivatives, quercetin derivatives. Some of the culture lines produce high amounts of natural colorants, i.e. anthocyanins. The cell cultures produced by the method of the invention, in addition, contain vitamin E (a-tocopherol), unique carbohydrate composition and unique fatty acid composition. The cell cultures also contain natural flavors and lipids with beneficial fatty acid composition, which are desired ingredients in cosmetic and hygiene preparations. The cell cultures can be generated around the year at different scales in consistent quality, which can be constantly monitored. It was surprisingly found that several advantageous effects may be achieved with the present invention.

The method for producing birch cell cultures and the birch cell cultures obtained by the invention provide remarkable advantages. The cell culture material can be produced through the whole year with no seasonal variations, low yield and quality variation, no agrochemical contamination, no impact of pests and diseases, easy product isolation and purification and possibility to produce novel compounds. The cell cultures contain several antioxidants (carotenoids and phenolic compounds) which prevent too early skin aging. Many of the phenolic compounds also act as UV screen and are thus beneficial in sunbathing. Birch cell culture shows multifunctional benefits in cosmetics: natural preservative nature, natural antioxidant and colorant. Birch cell cultures are also rich in gamma-aminobutyric acid, which is an important neurotransmitter in brain tissue, which could be utilized in pharma applications.

EXAMPLES

The following examples are illustrative embodiments of the present invention, as described above, and they are not meant to limit the invention in any way.

Example 1

GENERATING BIRCH SUSPENSION CELL CULTURE Three cell lines of birch (Betula pendula) were established from leaves and seeds. The cell lines were cryopreserved and stored in liquid nitrogen for more than 20 years. The cell lines, BpN l (non-embryogenic), Bp6/3 (embryogenic) and Bp A/K (embryogenic), were taken out of cryo-storage, and several callus cultures were established showing high viability.

The callus cultures were maintained on solidified N7 medium, supplemented with the plant growth regulators 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin in petri dishes, sealed with parafilm. PGR combination with 0.1 ppm 2,4-D + 0.02 ppm kinetin was used for N l cell line and 2 ppm 2.4-D + 0.5 ppm kinetin was used for 6/3 and A/K embryogenic cell lines. Sub-culturing time was 2-4 weeks. The following cultivation parameters were used : T = +25°C, photoperiod = 16 : 8 h (light:dark), irradiation about 50

Some cell cultures of the line N l were also cultivated in total darkness or in continuous light (normal light). Suspension cultures were established from the callus cultures. Suspensions were cultivated in 250 ml Erlenmeyer flasks. Content of the growth medium was 70 ml. Suspensions were sub-cultured to fresh medium every 10 - 12 days. About 3 g (FW) of the cells was transferred to fresh medium (70 ml in 250 ml flasks). Following cultivation parameters were used : shaking 110 rpm, T = +25°C, photoperiod = 16: 8 h (light:dark), irradiation about 50

Some suspensions were also cultivated in total darkness or in continuous light. Highly coloured callus cultures and suspensions, red, green and yellow in colour were formed and maintained stably at these conditions. The suspensions were occasionally sieved (using 1.0 mm metal sieve) to increase the amount of highly coloured small cell aggregates.

Several cell suspension cultures with different colours and phenotypes were established using cell and tissue culture methods from the originally heterogeneous, non-embryogenic BpN l cell line: yellowish, reddish, pale and bright green (Figure 1). Cell suspensions of two embryogenic dark red cell lines Bp6/3 and BpA/K were also established. Their embryogenic capacity was tested by transferring them to growth medium with lower concentration of plant growth regulators (PGR) (2 ppm 2.4-D + 0.5 ppm kinetin - 0.1 ppm 2,4-D + 0.02 ppm kinetin). In these conditions the cultures produced somatic embryos in high amounts as shown in Figure 1. The established cell cultures were maintained as callus cultures on plates.

After 4 - 8 sub-culturing the cell lines in suspension were used as inoculum in bioreactor up-scaling.

The compositions of the cell culture materials were screened by several chemical methods. The procedure included a rough separation into solid cell wall and suspended/dissolved fractions. The solid fractions were analyzed for selected samples after extraction by two- stage acid hydrolysis for cellulose and hemicellulose quantitation by HPAEC/PAD, lignin as hydrolysis residue and acid soluble lignin by UV spectroscopy and elemental analysis. The protein content was estimated from the nitrogen content. Lignin content was corrected for protein impurity via the nitrogen analysis, too. The carbohydrate compositions of the solutions were analyzed by slightly modifying the methods for solid material. The solutions were analyzed by slightly modifying the methods for solid material. The soluble fractions were analyzed by UPLC and UPLC-MS. Total phenolics contents were measured by Folin Ciocalteu method as gallic acid equivalents by spectrophotometry (Singleton and Rossi, 1965).

In addition, the soluble fractions were analyzed by alkaline SEC to detect the molar mass distribution of the UV active (aromatic) components.

For screening purposes, a phloroglucinol staining method was set up at VTT for this project. In this method, lignin content of the fresh or dried sample was assessed by staining with saturated phloroglucinol (1,3,5-benzenetriol) in 20% HCI on well plates. The method has been utilised in the detection of lignin in various applications including cell cultures and microscopy (e.g. Ogita et al. 2012). Reaction of phloroglucinol with lignin gives an intensive red colour and this colour development is related to the presence of coniferaldehyde groups (Harkin 1966).

From the selected cell lines, more detailed analyses were performed. Chemical composition of BpN l suspension cultures were analysed in detail in late stationary phase. The samples contained significant amounts of lignin, as determined by standard methods (Klason and acid-soluble), even when corrected for protein contaminants in Klason. However, Py-GC/MS did not detect degradation products typical to lignin (or only minor amount). Alkaline SEC was used to follow the distribution of UV- active components in the various fractions (medium, MeOH-extract, cells). These may be mono/oligomeric phenolics, lignin, tannins and protein (amino acids, peptides). Carbohydrates in the cells were mainly pectins and hemicelluloses, but also cellulose.

GC-MS analysis of methanol extracts showed presence of various organic acids, fatty acids and phytol, which is diterpene alcohol and precursor of vitamin E. Phytol was detected especially in the dark green cultures. Oxalic, benzoic, succinic, glyceric, fumaric, malonic, hydroxymalic and isocitric acids were detected as organic acids and palmitic (16: 0), palmitoleic (16: ln-7), stearic (18 : 0), oleic (18: 1), linoleic (18 : 2n-6) and alpha-linolenic acids (18: 3n-3) were detected as fatty acids in the cultures.

Comparison of the cell cultures showed that one of the embryogenic cultures BpA/K produced phenolic polymers called ellagitannins, and the other embryogenic line was rich in phenolic polymers called proanthocyanidins. All red birch cell cultures produced natural colourants, anthocyanins and also carotenoids identified by UPLC-DAD. Phloroglucinol staining method for screening lignin content in the cell culture samples was successfully set up based on the tests with lignin preparates. However, fresh cell culture samples did not indicate presence of lignin. Formation of pale red colour was observed in the freeze-dried samples containing both medium and cells. This could be a consequence of the release of lignin due to the rupture of cells during lyophilisation. In line with other chemical analyses, it was concluded that no clear evidence of the presence of lignin in the cultured cells or in medium was observed.

Example 2

ELICITATION EXPERIMENTS These experiments were carried out using suspension culture originating from the non- embryogenic BpN l cultures obtained in Example 1 Stable suspensions with small cell aggregates were used in these experiments (typically 4-8 sub-culturing in suspension needed). Chemical elicitors used were methyl jasmonate (11.2 ppm and 22.4 ppm) and etephon (100 ppm and 200 ppm). In these experiments growth curves of the stable cell suspension was first measured in shake flasks in order to find out the right point for elicitation, which is known to be the late exponential growth phase. The elicitors were added at day 6 to the culture medium. The experiments were carried out in shake flask scale (100 ml shake flasks). The culture samples were collected in at different time points. Phenolic profiles were analyzed by UPLC-DAD. Total phenolics were measures by Folin Ciocalteu method as gallic acid equivalent by spectrophotometry. Antioxidant activity was evaluated from methanol extracts by DPPH radical scavenging method described by Malterud et a/. 1993.

The results are presented in Figures 2 and 3 and Table 1. Figure 2 shows UPLC profiles of birch culture BpN l (non-embryogenic) with methyl jasmonate and etephon elicitation compared to non- elicited culture. MeJ 1 = 11,2 ppm; MeJ 2 = 22,4 ppm; Etephon 1 = 100 ppm and Etephon 2 = 200 ppm.

UPLC-DAD chromatograms clearly showed that the amount of phenolic compunds increased compared to control, especially when methyl jasmonate (MeJ) was used as an elicitor. Total phenolic measurement (Table 1) shows that the lower concentration was more efficient compared to the higher concentration. In Table 1 content of total phenolics and DPPH activity of elicitated birch cell cultures compared to non-treated culture is presented. Total phenolic content and DPPH activity were measured at day 12, which was end-point of cultivation. Cloudberry (berry fruit) was used as a reference, which is known to be very good radical scavenger.

The methyl jasmonate (MeJ) treatment increased the amount of total phenolics in the cell material about three times and in the medium (secreted compounds) about two times. In addition, SEC analysis also showed that methyl jasmonate elicitated cultures differed from the others in terms of molar mass distributions, indicating that the composition of the polyphenols had been affected.

Table 1.

The results showed that elicitation with methyl jasmonate had strong effect on

antioxidant activity measured as radical scavenging activity. Almost seven times increase was detected in methyl jasmonate elicited cell culture compared to control culture (Table 1). The results are in good line with the total phenolics. The lower concentration was more efficient compared to the higher value. Figure 3 shows that elicitation did not have any adverse effects on growth of the birch cell culture BpN l.

As a conclusion, elicitation seems to be a powerful method for increasing the amount of phenolic compounds and antioxidant activity in birch cell cultures, important for cosmetic applications. Example 3

PRECURSOR FEEDING AND PGRs

These experiments were carried out with non-embryogenic BpN l cell suspension line obtained as described in Example 1. The compounds fed to the culture were 1) Mannitol (5%); 2) Betuligenol (1 mM) and 6) Phenylalanine (1 mM). In additions, effects of following PGRs were studied 1) NAA + KIN (5 mg/L + KIN 1 mg/L); 2) 2,4-D + KIN (1 mg/L + 1 mg/L); 3) Reference medium 2,4-D + KIN (0,1 mg/L + 0,02 mg/L) and 4) medium without PGRs. UPLC profiles, total phenolic content, radical scavenging activity and growth curves were measured as in example 2.

Experiments were carried out in shake flasks ( 100 ml). Precursors and PGRs were fed to the cell cultures at the beginning of cultivation (day 0). Samples were analysed at the end of cultivation (day 11). Results are shown in Figures 4 and 5 and Table 2.

Figure 4. shows UPLC profiles of birch BpN l cell culture fed with precursors or supplemented with various PGRs. Interesting peaks or totally new peaks are circled.

The chromatogram showed the metabolism of the cells can clearly be changed by precursor feeding or by changing the PGRs. Replacing 2,4-D by NAA seemed to increase intensity of peaks of many phenolics (circled in Figure 4). Totally new peaks were formed by betuligenol and phenylalanine feeding. Figure 5 presents effects of precursor feeding on the growth of birch cell culture.

Table 2 presents contents of total phenolics and DPPH activity of birch cell cultures after precursor feeding and supplementation with various PGRs. Total phenolic content and DPPH activity are measured at day 11, which was end-point of cultivation. Cloudberry (berry fruit) was used as a reference, which is known to be very good radical scavenger.

Table 2.

Changing the growth regulators from 2,4 -D + KIN to NAA + KIN seemed to have slight positive effects on total phenolics and radical scavenging activity compared to reference culture (Table 2). This was very positive observation, as utilization of auxin NAA is expected to be more suitable in cosmetics than 2,4-D, although their concentrations are traces when the cells are harvested after cultivation. Mannitol had dramatic increasing effects on total phenolics and DPPH activity. However, it also depressed the cell growth strongly (Figure 5) and is thus not suitable for further application, especially in such high concentrations. As a conclusion, precursor feeding and changing PGRs seems to be an interesting approach to change metabolism of the cultured birch cell, and positive effects an phenolics and antioxidant activity, important for cosmetics, can be detected.

Example 4

ANTIMICROBIAL ACTIVITY OF THE BIRCH CELL CULTURE EXTRACTS

Antimicrobial activity of the cell culture extracts was measured using plate count method which measures growth curves in liquid culture (Nohynek et al. 2006). Acetone extracts were prepared as described by Puupponen-Pimia et al. 2016 with slight modifications. Hydrothermal extracts were prepared as described in WO 2016/097488 Al . Acidic birch cell culture extracts prepared from the non-embryogenic cell suspension BpN l obtained as described in Example 1 showed strong antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa in concentration 1 mg/ml (effect was not caused only by low pH), as can be seen in Figure 6.

Antimicrobial activity of aceton extracts of birch cell culture BpN l (KAS N-l) (1 mg/ml) against Staphylococcus aureus is presented. C18 column was used to purify raw acetone extracts. Chloramphenicol (5C^g/ml) was used as a control.

Hydrothermal extracts from birch embryogenic cell line Bp6/3 (2,5 mg/ml and 5 mg/ml) obtained as described in Example 1, cultivated in normal light also had moderate antimicrobial activity against Staphylococcus aureus (Figure 7). The results show that extracts prepared from birch cell cultures have strong to moderate antimicrobial activity against severe human skin pathogen, Staphylococcus aureus, and thus they have potential as natural preservatives or ingredients supporting healthy skin microbiota in cosmetics. Example 5

CULTIVATION OF BIRCH CELL CULTURES IN BIOREACTORS WITH/WITHOUT LED ILLUMINATION

Cultivation systems for birch cell suspension cultures were up-scaled in 1) 10 L steel bioreactor without light and 2) 2 L glass bioreactor with/without LED light. Total phenolics, DPPH activity and antimicrobial activity were evaluated from the glass bioreactor samples with/without LED illumination.

Cultivation system for BpN l cell suspension, obtained in Example 1, which was yellowish- greenish in colour, was successfully scaled-up in 10 L steel bio-reactor. The inoculum was produced in 250 ml shake flasks with 60 ml working volume. Altogether 14 shake flasks were needed to inoculate the bioreactor. Each shake flask produced about 13 g biomass (sieved fresh weight) in one week. Working volume of the bioreactor was 5 L. The amount of inoculum was 30 g/L and the cultivation time was 7 days. Following parameters were used : Agitation speed 100 rpm; Temperature 24°C; Aeration with pressurized air 2 L/min; Dissolved oxygen level 104 (at the beginning, 54 at the end); CC level 0.04 at the beginning and pH 5.59 at the beginning, 6.70 at the end (not adjusted) .

Growth was very good in steel bioreactor, and the cells showed high viability also in bioreactor cultivation. Figure 8 shows growth of birch cell line BpN l in 10 L bio-reactor. The result showed, that birch cell culture without anthocyanin colour compounds, can be easily up-scaled in steel bio-reactor.

Cultivation of embryogenic red birch cell line Bp 6/3, obtained in Example 1, as up-scaled in 2 L glass bio-reactor with and without LED light. Inoculum was produced in shake flasks as described above. The amount of inoculum was 46 g (sieved fresh weight) for LED cultivation and 47 g (sieved fresh weight) for cultivation in normal light of the fermenter hall. Working volume was 1,5 L and cultivation time 10 days. Cultivation parameters were the following : Dissolved oxygen level 20 %; Agitation speed lOOrpm; Aeration 1,5 L/min with pressurized air. After cultivation, total phenolics and DPPH radical scavenging activity were analysed as in Example 2 and antimicrobial activity as in Example 3.

Both cultures grew very well in glass bio-reactors and the viability stayed at over 95% (FDA staining). Interestingly, LED light clearly increased biomass production. Fresh weight after cultivation was 297 g and dry weight 13 g in LED lights. Fresh weight was 202.4 g and dry weight 7.85 g in normal light conditions. Interestingly, the cells grown under LED light contained less water (dry weight 4.4 %) than the cells grown without LED (dry weight 3.8 %).

LED lights clearly increased the amount of total phenolics. GAE (gallic acid equivalent) (mg/g) were the following : Glass bioreactor + LED 14.5; glass bioreactor + fermenter hall normal light 6.7 and. LED illumination strongly increased intensity of red coloured anthocyanins in bioreactor during 10 d cultivation compared to the cultivation in normal pilot hall light. Anthocyanin content of the birch cells measured by UPLC-DAD was ten times higher when cultured under LED light (28.8 mg/g dry weight) compared to culturing under normal fermenter hall light conditions (1.1. mg/g dry weight).

Also, DPPH radical scavenging activity measured as ARP (AntiRadicalPower or AntiRadicalPotential)) (l/ICso), where ICso denotes half maximum inhibitory concentration) was increased by LED illumination in the following way: Glass bioreactor + LED 10.6; glass bioreactor + fermenter hall normal light 3.6.

Acetone extracts of LED illuminated samples had slightly higher antimicrobial activity (0.5- 1 logarithmic unit) against Staphylococcus aureus compared to samples cultivated under normal fermenter hall light. As a conclusion, LED light seemed to be an essential parameter in upscaling red-coloured birch cell cultures. It has major effects on biomass formation and anthocyanin content and stability. It also increases the content of total phenolics, antioxidant and antimicrobial activity.

Claims

1. A method for producing cell culture of a plant of the family Betulaceae, characterized in that the method comprises two stages, where

2. The method according to claim 1, characterized in that in the second stage the primary birch suspension cell culture is up-scaled by culturing in three steps.

3. The method according to claim 1, characterized in that in the second stage the primary birch suspension cell culture is up-scaled by culturing in four steps.

4. The method according to any one of claims 1 - 3, characterized in that the plant of the family Betulaceae is selected from birches, alders, hazels, hornbeams, hazel- hornbeams and hop-hornbeams, preferably the plant is birch.

5. The method according to any one of claims 1 - 4, characterized in that callus line is an embryogenic or non-embryogenic callus line produced material of the plant.

6. The method according to any one of claims 1- 5, characterized in that the medium used for plant cell and tissue culture is selected N7 medium, MS medium, B5 medium and Woody Plant Medium WPM, preferably N7 medium is used.

7. The method according to claim 6, characterized in that the N7 medium is a modified medium comprising at least one component selected from sucrose, agar, gelrite, plant growth regulators and combinations thereof, and the pH of modified MS medium is adjusted into the range of 3-7.

8. The method according to any one of claims 1 - 7, characterized in that the plant growth regulator is selected from cytokines, auxins and combinations thereof.

9. The method according to any one of claims 1 - 8, characterized in that the LED light has color temperature of 2700-3000K.

10. The method according to any one of claims 1 - 9, characterized in that the LED light comprises an effectual spectrum comprising at least 25 % of an integral in the wave length range of 630 nm-760 nm, calculated from the total effectual spectrum.

11. The method according to any one of claims 1- 10, characterized in that the irradiation source is LED light with illumination intensity over 500 Ix, preferable over 800 Ix.

12. Cell cultures of a plant of the family Betulaceae, characterized in that said cell cultures are obtained by the method according to any one of claims 1-11.

13. The cell culture of a plant of the family Betulaceae according to claim 12, characterized in that said plant is birch and cell culture comprises 0.1-10 wt% (dry weight) of anthocyanins and 0.1-10 wt% (dry weight) of proanthocyanidins.

14. Composition comprising the cell culture according to claim 12 or 13, where the composition is selected from cosmetic compositions, hygiene and personal care products, foods, feeds, pet foods, nutritional compositions, nutraceutical compositions, pharmaceutical compositions and pharmaceutical devices.

15. The composition according to claim 14, characterized in that the cosmetic composition is selected from day creams, night creams, foundation creams, peeling creams, lipsticks, color cosmetics, skin serums, mascaras, products for hair and/or scalp care, washing products for skin or hair, and skin hygiene products.

16. The composition according to claim 15, characterized in that the cosmetic composition comprises the cell culture of a plant of the family Betulaceae in powder form in an amount of 0.001 to 25 % by weight, more preferably the amount of the powder is 0.01 to 5 % by weight, and most preferably 0.01-1 % by weight.

17. Use of the cell culture of a plant of the family Betulaceae according to claim 12 or 13 in cosmetic compositions, hygiene and personal care products, in foods, feeds, pet foods, nutritional compositions, nutraceutical compositions, pharmaceutical compositions, and pharmaceutical devices, and for providing aroma, flavor and/or color to the products.