EP-4379042-B1 - METHODS FOR THE PREPARATION OF CULTURE MEDIA BASED ON MICROGREENS AND RELATED PRODUCTS

Inventors

• EIBL, Regine
• Eibl, Dieter
• HÜHN, Tilo

Dates

Publication Date

20260506

Application Date

20221129

Claims (13)

1. Method for the preparation of a cell culture medium or fermentation medium, comprising: a) growing microgreens from edible seeds of vegetables, cereals or herbs; b) harvesting the microgreens in an immature state; c) grinding the harvested microgreens to an average particle size of 500 µm or less to provide a mash; d) subjecting the ground mash to a phase separation resulting in a solid phase and a liquid phase; e1) optionally adding water to dilute the liquid phase; and f) sterilising the optionally diluted liquid phase to provide the cell culture medium or fermentation medium.
2. Method for the preparation of a supplement for a cell culture medium or fermentation medium, comprising: a) growing microgreens from edible seeds of vegetables, cereals or herbs; b) harvesting the microgreens in an immature state; c) grinding the harvested microgreens to an average particle size of 500 µm or less to provide a mash; d) subjecting the ground mash to a phase separation resulting in a solid phase and a liquid phase; e2) subjecting the liquid phase to a drying and/or concentration step to provide the supplement for a cell culture medium or fermentation medium.
3. Method according to any one of claims 1 or 2, wherein step a) comprises growing microgreens of corn ( Zea mays ) , preferably sweet corn ( Zea mays convar. saccharata var. rugosa ), under dark and humid conditions.
4. Method according to any of claims 1 to 3, wherein the solid phase is processed independently from the liquid phase and recombined with the liquid phase before step e1) or e2).
5. Method according to claim 4, wherein the solid phase is independently processed by subjecting the same to a saccharification step.
6. Method according to claim 5, wherein the saccharification step includes an enzymatic treatment, acidic treatment, solid catalyst-assisted microwave irradiation, thermal treatment under pressure, steam treatment or combinations thereof.
7. Method according to any of claims 5 or 6, wherein the solid phase is subjected to a liquefaction step prior to the saccharification step, preferably by slurrying the solid phase in water, gelatinising the slurry and hydrolyzing the same in the presence of one or more types of starch hydrolyzing enzymes.
8. Method according to any of claims 1 to 7, wherein the harvested microgreens are subjected to wet grinding in step c), preferably in the presence of water.
9. Method according to any of claims 1 to 8, wherein step c) is performed at temperatures below 65°C, preferably below 40°C, more preferably at temperatures of 0 to 35°C.
10. Method according to any of claims 1 to 9, wherein in step c), the harvested microgreens are ground to an average particle size of 100 µm or less, preferably 50 µm or less, more preferably 30 µm or less.
11. Method according to any of claims 1 to 10, wherein during the entire preparation process, the liquid phase is not subjected to temperatures of 55°C or above, preferably 45°C or above, and/or only the solid phase is subjected to a thermal treatment at 65°C or above, such as 85°C or higher.
12. Method according to any of claims 1 to 11, wherein one or more of the following is further added during or after step c): one or more salts including Ca 2+ , SO 4 2- , Mg 2+ , PO 4 3- , Cl - , NO 3 - and/or NH 4 + ions; one or more vitamins, preferably Vitamin B5; one or more growth regulators, preferably one or more selected from sterile coconut water, a cytokinin and an auxin, more preferably one or more selected from thidiazuron, zeatin or kinetin gibberellic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), indoleacetic acid (IAA) or indolebutyric acid (IBA); and/or one or more phytohormones.
13. Use of microgreens from edible seeds of vegetables, cereals or herbs for the preparation of a cell culture medium or fermentation medium, or as a supplement for a cell culture medium or fermentation medium.

Description

FIELD OF INVENTION This invention relates to methods and/or techniques for a cost-effective and sustainable preparation of cell culture media or fermentation media on the basis of microgreens from edible seeds of vegetables, cereals or herbs. In certain embodiments, the invention relates to the use of microgreens for the preparation of a cell culture medium or fermentation medium, or as a supplement for a cell culture medium or fermentation medium. BACKGROUND OF THE INVENTION Culture media are a source of nutrients and growth factors required for the growth of microorganisms and cells ex-vivo under laboratory conditions. Many types of culture media, which may be classified based on their nutrient composition, consistency or their use in life science laboratories, have been developed to grow selective or desired microorganisms, cells or small plants. Typically, a general distinction is being made between defined and undefined media. A defined medium (also known as chemically defined medium or synthetic medium) is a chemically characterised medium produced from pure chemical substances, wherein no yeast, animal, or plant tissue is present. As long as their composition is defined, such media may also be prepared by using purified plant extracts, such as plant proteins (see WO 2021/148 955 A1) or plant peptides and lipids (see WO 98/15614). On the other hand, an undefined medium (i.e. a basal or complex medium) is characterised in that it contains a carbon source, water, various salts a source of amino acids and nitrogen in unknown quantities. As an example, the addition of organic supplements from natural origin (i.e. coconut water, yeast extract, malt extract, potato extract, banana homogenisate, algal compounds) to prepare undefined plant culture media is reviewed in the publication Z. Molnár et al, Acta Biologica Szegediensis 2011, 55(1), 123-127. US 2011/0212489 A1 discloses a culture medium which comprises a hydrolyzate of plant seeds containing protein and oil (including sunflower seeds). US 2004/0185561 A1 discloses a cell culture medium which comprises a non-hydrolyzed plant extract (e.g. from nuts, peas, or potatoes) and is tested for its growth-promoting effect as part of a cultivation of animal cells, especially keratinocytes. Examples of the use of plant material for the manufacture of culture media are disclosed in Cejas et al., Journal of Food Science and Technology 2017, 54 (13), 4464-4472; S. Khalil "Evaluation of Plant-Based Products for their Suitability as Bacteriological Media", PhD Thesis, University of Karachi, Pakistan, 2017; and Mourad et al. Microbes Environ. 2018, 33 (1), 40-49. An overview of advances in microgreen research is provided in "Ongoing Research on Microgreens: Nutritional Properties, Shelf-Life, Sustainable Production, Innovative Growing and Processing Approaches", M. Paradiso and M. Renna (Eds.) Foods 2020, MDPI. In commercial cell culture technology, culture media play a very important role in the efficiency and economic viability of production processes at industrial scale. Recent publications suggest that plant cell cultures or their extracts may be used as foodstuffs (see, e.g., E. Nordlund et al., Food Res Int. 2018, 107, 297-305 and R. Eibl et al., Appl. Microbiol. Biotechnol. 2018, 102, 8661-8675) and be manufactured with less energy and lower possible impact on the environment compared to whole plants, and independently of location and season. However, the use of defined cell culture media requires elaborate purification protocols and/or chemicals with a high degree of purity, which is a major cost driver in the transfer of such processes to a commercially relevant scale. While the addition of organic supplements from natural sources (e.g. to prepare undefined plant culture media) may advantageously influence cell survival, adhesion and proliferation, it still remains desirable to effectively produce culture media which contain macronutrients, micronutrients, vitamins, amino acids or nitrogen supplements, source(s) of carbon and growth regulators at reduced processing costs. Moreover, taking into account that common culture media used in the food science sector are usually derived from pharmaceutical science with a different set of legal requirements for approval, a direct transfer of such technologies to commercial food science for the preparation of cell culture-based food is a major challenge (due to the necessary declaration as novel food, for example). For instance, the addition of hormone supplements for control of cell differentiation may be subject to declaration depending on the application and region. Hence, it would be desirable to provide culture media for food applications which may be produced sustainably and inexpensively, and which do not solely rely upon chemical in-vitro synthesis of such hormone additives. SUMMARY OF THE INVENTION The present invention solves this object with the subject matter of the claims as defined herein. The advantages of