Patent Description:
Prior attempts have been made to use plant-based material as components for cell culture media.

<CIT> describes cell culture medium formulations for the in vitro cultivation of animal cells. These media formulations comprise at least one plant-derived peptide and/or at least one plant-derived lipid and/or at least one plant-derived fatty acid. Rice, soy, potato, and corn are named as suitable sources of proteins, peptides, lipids, and/or fatty acids. Wheat is specifically excluded as a source of plant-derived proteins. The plant peptides to be used in the formulations of <CIT> may be prepared by digesting plant extracts with enzymes such as trypsin or chymotrypsin. The plant peptides are to be added to the basal medium at concentrations of about <NUM> - <NUM>/liter.

<CIT> describes the cost-effective production of recombinant glycoproteins such as human erythropoietin using a serum-free culture medium. The culture medium comprises a plant-derived peptone. The examples of <CIT> show a serum-free adaptation medium and a serum-free production medium. Each one of these two media contains <NUM>% soya-peptone.

<CIT> describes animal protein-free cell culture media comprising polyamines and a plant- and/or yeast-derived hydrolysate. The total concentration of the plant- and/or yeast-derived protein hydrolysate in the cell culture medium is about <NUM>% to about <NUM>% (w/v). The plant-derived protein hydrolysate may be selected from a cereal hydrolysate and/or a soy hydrolysate. <CIT> defines the term "hydrolysate" as relating to any enzymatic digest of a vegetable or yeast extract.

<CIT> discloses that a rice bran extract has a cell growth promoting action, when used in cell culture medium. The rice bran extract preferably comprises <NUM>% by mass or more of protein. The rice bran extract may be a molecular weight fractionated product, preferably in the range between <NUM> kDa and 10kDa. In the Examples of <CIT>, it was shown that a rice bran extract with a protein content of <NUM>% improved proliferation of CHO cells when used in concentrations of <NUM> or <NUM>/L, while cytotoxicity was observed at a concentration of <NUM>/L.

Jarunrattanasri and co-workers studied the aroma components present in rice bran protein, especially the aroma components present in partially acid-hydrolyzed vegetable protein made by partial hydrolysis of rice bran protein (<NPL>).

Hansawasdi and co-workers examined the potential prebiotic effects of oligosaccharide mixtures from acidic hydrolysis of rice bran and cassava pulp (<NPL>).

Terada and colleagues describe a culture supplement extracted from rice bran for serum-free culture of mammalian cells (<NPL>). Rice bran was extracted in an alkaline solution and then precipitated with acid.

The inventors of <CIT> disclose a titration of rice hydrolysates as cell culture supplements. The rice hydrolysates were prepared by enzymatic hydrolysis.

<CIT> refers to a cell culture medium devoid of animal proteins and comprising at least one non-animal or plant-derived peptide. The application further refers to a cell culture medium devoid of animal proteins and comprising at least one non-animal-derived or plant-derived lipid or fatty acid.

At present, nutrient media for cell culture are a mixture of about <NUM> to <NUM> high grade and pure ingredients. The high number and the high purity of the ingredients renders such nutrient media very expensive.

When using nutrient media in the food industry, especially when producing cultured meat, it is desirable to use less expensive ingredients. For ethical reasons and for safety reasons, it is also desirable to avoid ingredients from animal origin.

In the prior art, plant-based hydrolysates were used as additives for cell culture media. However, in the prior art, only small amounts of plant-based extracts were used to supplement cell culture media; e.g. only up to <NUM>/L (= <NUM>/L) in <CIT>; about <NUM>% soya-peptone (= <NUM>/L) in <CIT>; between <NUM> and <NUM>/L of a protein-rich rice bran extract were used in <CIT>. In addition, the plant-based extracts used in the prior art were obtained from isolated plant proteins or from protein-rich plant material, which can be grouped as primary food stream material.

The present inventors have now shown for the first time that nutrient media components can be prepared using plant-based protein hydrolysates that were produced from secondary food stream material, i.e. material having lower economic value than primary food stream material.

The novel composition containing plant-based hydrolysates prepared from secondary food stream are much cheaper than the media ingredients of the prior art. These novel compositions can be used as media components and support the growth of eukaryotic cells, and can even be used in the production of cultured meat. In addition, the components and compositions of the present invention can be used for cell cultivation in an industrial scale.

This summary does not necessarily describe all advantages achieved and all problems solved by the present invention.

In a first aspect the present invention relates to a method for the production of a plant-based hydrolysate suitable for cell culture of eukaryotic cells, said plant-based protein hydrolysate being obtained from secondary food stream material, comprising the steps:.

In a second aspect the present invention relates to a plant-based hydrolysate obtainable by the method of the first aspect.

In a third aspect the present invention relates to a composition suitable for use as medium component supporting the growth of eukaryotic cells, the composition comprising:.

wherein said composition is devoid of animal proteins.

In a fourth aspect the present invention relates to a method for cultivating cells, comprising the steps.

In a fifth aspect the present invention relates to a method for the production of the composition of the third aspect, comprising the step(s):.

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

As used herein, the expression material from "primary food stream" refers to plant material that is suitable for human consumption. This material is characterized by high protein content (at least <NUM>% (w/w dry matter) but typically at least <NUM>% (w/w dry matter)) and low fibre content (<NUM>% or less (w/w dry matter), but typically <NUM>% or less (w/w dry matter)). Examples of primary food stream material include, without limitation, protein-rich material derived from soy, fava bean, wheat (e.g. wheat gluten), rice, pea, potato, or cotton seed.

As used herein, the expression material from "secondary food stream" refers to plant material that is generally not used for human consumption. Material from secondary food stream is mostly used as animal feed, as fertilizer or is simply burnt. As compared to material from primary food stream, secondary food stream material has a lower protein content (<NUM>% to <NUM>%, and typically <NUM>% to <NUM>% (w/w dry matter)) and a higher fibre content (<NUM>% to <NUM>%, and typically <NUM>% to <NUM>% (w/w dry matter)). Examples of secondary food stream material include, without limitation, rice bran, defatted rice bran, wheat bran, rye bran, maize bran, spelt bran, or brewer's spent grain.

In the context of the present invention, a "growth factor" refers to a protein or a peptide that promotes proliferation of eukaryotic cells in cell culture. Examples of growth factors usable in the present invention include, without limitation insulin and insulin-like growth factors (IGF).

As used herein, the term "about" refers to numerical values ranging from <NUM>% below the indicated numerical value to <NUM>% above the indicated numerical value. For example, a protein content of "about <NUM>% (w/w dry matter) " encompasses a protein content ranging from <NUM>% (w/w dry matter) to about <NUM>% (w/w dry matter). As a further example, a temperature of "about <NUM>" refers to a temperature range between <NUM> and <NUM>.

The present invention will now be further described. In the following passages different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

In a third aspect the present invention is directed to a composition comprising:.

In a preferred embodiment of the third aspect, the secondary food stream material has a protein content ranging from <NUM>% to <NUM>%, preferably from <NUM>% to <NUM>%, preferably from <NUM>% to <NUM>%, preferably from <NUM>% to <NUM>%, preferably from <NUM>% to <NUM>%, preferably from <NUM>% to <NUM>%, preferably from <NUM>% to <NUM>%, preferably from <NUM>% to <NUM>%, preferably from <NUM>% to <NUM>%, and most preferably from <NUM>% to <NUM>% (w/w dry matter).

In a preferred embodiment of the third aspect, the secondary food stream material has a fibre content ranging from <NUM>% to <NUM>%, preferably from <NUM>% to <NUM>%, preferably from <NUM>% to <NUM>%, preferably from <NUM>% to <NUM>%, and most preferably from <NUM>% to <NUM>% (w/w dry matter).

In all embodiments of the third aspect, the secondary food stream material is or is prepared from rice bran.

Further disclosed herein but not claimed are compositions, in which the secondary food stream material is or is prepared from wheat bran, rye bran, maize bran, spelt bran, or brewer's spent grain.

In a preferred embodiment of the third aspect, the secondary food stream material is or is prepared from.

Further disclosed herein but not claimed are compositions, in which the secondary food stream material is or is prepared from.

In a preferred embodiment of the third aspect, the first plant-based hydrolysate is present in the composition in a concentration in the range from <NUM>% to <NUM>% (w/w).

In a preferred embodiment of the third aspect,.

In a preferred embodiment of the third aspect, the food grade premix is present in the composition in a concentration in the range from <NUM>% to <NUM>% (w/w).

In a preferred embodiment of the third aspect, the composition further comprises:
(iii) a second plant-based hydrolysate prepared from primary food stream material by acid hydrolysis, wherein said primary food stream material has a protein content of at least <NUM>% (w/w dry matter) protein and a fibre content of <NUM>% (w/w dry matter) or less.

In a preferred embodiment of the third aspect, the primary food stream material has a protein content of at least <NUM>%, preferably at least <NUM>%, preferably at least <NUM>%, preferably at least <NUM>%, preferably at least <NUM>%, preferably at least <NUM>%, preferably at least <NUM>%, preferably at least <NUM>%, preferably at least <NUM>, and most preferably at least <NUM>% (w/w dry matter).

In a preferred embodiment of the third aspect, the primary food stream material has a fibre content of <NUM>% (w/w dry matter) or less, preferably <NUM>% (w/w dry matter) or less, preferably <NUM>% (w/w dry matter) or less, and most preferably <NUM>% (w/w dry matter) or less.

In a preferred embodiment of the third aspect, the second plant-based hydrolysate prepared from primary food stream material has been prepared from soy bean, fava bean, wheat, rice, pea, potato, or cotton seed.

In a preferred embodiment of the third aspect, the second plant-based hydrolysate obtained from primary food stream is present in a concentration in the range from <NUM>% to <NUM>% (w/w).

In a preferred embodiment of the third aspect, the second plant-based hydrolysate obtained from primary food stream was prepared by applying the method of the first aspect. This means that a plant-based material obtained from primary food stream, as defined above, is provided and that then steps (b) to (g) of the method of the first aspect are carried out.

In some embodiments of the invention, the composition according to the third aspect is used as a replacement of cell culture medium. An aqueous solution of the composition according to the third aspect can be prepared, thereby obtaining a plant-based medium. This plant-based medium can be used to partially replace a standard cell culture medium. For example, from about <NUM> to about <NUM> (preferably from about <NUM> to about <NUM>, preferably from about <NUM> to about <NUM>, more preferably from about <NUM> to about <NUM>, even more preferably about <NUM>) are removed from <NUM> of a standard cell culture medium and the same volume of the plant-based medium as the volume that was removed is added to the remaining volume of standard cell culture medium as a replacement. For example, <NUM> are removed from <NUM> of a standard cell culture medium and <NUM> of the plant-based medium are added to the remaining <NUM> as a replacement.

In other embodiments of the invention, the composition according to the third aspect is used as the starting material for the preparation of a cell culture medium. Further components (salts, buffer, energy sources (e.g. glucose), trace minerals, essential amino acids, growth factors) can then be added. Osmolality and pH can be adjusted. A person skilled in the art will be well-aware which further components, which pH and which osmolality will be suitable to promote growth of eukaryotic cells.

In a fourth aspect the present invention is directed to a method for cultivating cells, comprising the steps:.

In a preferred embodiment of the fourth aspect, the sterile aqueous solvent is selected from the group consisting of water, phosphate buffer, and a solution of sodium chloride.

In a preferred embodiment of the fourth aspect, the appropriate cells are bovine cells, porcine cells or chicken cells.

In embodiments, in which the stem cells are human embryonic stem cells, it is obligatory that these human embryonic stem cells were prepared by a process that does not require the destruction of the human embryo.

A person skilled in the art of producing cultured meat will be well aware which temperature ranges and which time-periods are required for obtaining cultured meat, taking into consideration the type of cells and the amount of cells used for inoculation.

In a first aspect the present invention is directed to a method for the production of a plant-based hydrolysate suitable for cell culture of eukaryotic cells, said plant-based protein hydrolysate being obtained from secondary food stream, comprising the steps:.

In a preferred embodiment of the first aspect, the plant-based material obtained from secondary food stream has a protein content ranging from <NUM>% to <NUM>%, preferably from <NUM>% to <NUM>%, preferably from <NUM>% to <NUM>%, preferably from <NUM>% to <NUM>%, preferably from <NUM>% to <NUM>%, preferably from <NUM>% to <NUM>%, preferably from <NUM>% to <NUM>%, preferably from <NUM>% to <NUM>%, preferably from <NUM>% to <NUM>%, and most preferably from <NUM>% to <NUM>% (w/w dry matter).

In a preferred embodiment of the first aspect, the plant-based material obtained from secondary food stream has a fibre content ranging from <NUM>% to <NUM>%, preferably from <NUM>% to <NUM>%, preferably from <NUM>% to <NUM>%, preferably from <NUM>% to <NUM>%, and most preferably from <NUM>% to <NUM>% (w/w dry matter).

In all embodiments of the first aspect, the plant-based material obtained from secondary food stream is or is obtained from rice bran.

Further disclosed herein but not claimed are methods, in which the secondary food stream material is or is prepared from wheat bran, rye bran, maize bran, spelt bran, or brewer's spent grain.

In a preferred embodiment of the first aspect, the plant-based material obtained from secondary food stream is or is prepared from.

Further disclosed herein but not claimed are methods, in which the secondary food stream material is or is prepared from.

In a preferred embodiment of the first aspect, the concentration of HCl is in the range from <NUM> to <NUM>, more preferably in the range from <NUM> to about <NUM>, and most preferably at about <NUM>.

In a preferred embodiment of the first aspect, the incubation of step (c) is carried out at a temperature in the range between <NUM> and <NUM>, preferably at a temperature in the range between <NUM> and <NUM>, preferably at a temperature in the range between <NUM> and <NUM>, preferably at a temperature in the range between <NUM> and <NUM>, preferably at a temperature in the range between <NUM> and <NUM>, preferably at a temperature in the range between <NUM> and <NUM>, preferably at a temperature in the range between <NUM> and <NUM>, preferably at a temperature in the range between <NUM> and <NUM>, preferably at a temperature in the range between <NUM> and <NUM>, and most preferably at about <NUM>.

In a preferred embodiment of the first aspect, step (c) is carried out for a time period between <NUM> hours and <NUM> hours, and most preferably for about <NUM> hours.

In a preferred embodiment of the first aspect, step (e) is carried out for about <NUM> to about <NUM> at about <NUM> to <NUM> x g, preferably for about <NUM> to <NUM> at about <NUM> to about <NUM> x g; most preferably for about <NUM> at about <NUM> x g.

In a preferred embodiment of the first aspect, the sterile filter is a filter with a pore size of <NUM> or less, preferably <NUM> or less, more preferably <NUM> or less, and even more preferably, <NUM> or less.

In preferred embodiments of the first aspect, several filtration steps with increasingly smaller pore sizes are used; e.g. a first filtration step with a filter having a pore size of <NUM> or less, a second filtration step with a filter having a pore size of <NUM> or less, a third filtration step with a filter having a pore size of <NUM> or less, and a fourth filtration step with a filter having a pore size of <NUM> or less.

As stated above, the method of the first aspect and its preferred embodiments are also suitable for producing a plant-based hydrolysate obtained from primary food stream when using a plant-based material obtained from primary food stream as starting material.

In a second aspect the present invention is directed to a plant-based hydrolysate obtainable by the method of the first aspect.

In a preferred embodiment of the second aspect, the plant-based hydrolysate has been obtained by the method of the first aspect.

In a fifth aspect the present invention is directed to a method for the production of the composition of the third aspect, comprising the step(s):.

In a preferred embodiment of the fifth aspect, the plant-based hydrolysate prepared from secondary food stream has been obtained by the method of the first aspect.

In preferred embodiments of the fifth aspect, the secondary food stream material is as defined in the embodiments of the third aspect.

In a preferred embodiment of the fifth aspect, the food grade premix is defined as in the embodiments of the third aspect of the invention.

In preferred embodiments of the fifth aspect, the primary food stream material is as defined in the embodiments of the third aspect.

The following figures and examples are merely illustrative of the present invention and should not be construed to limit the scope of the invention as indicated by the appended claims in any way.

<FIG> shows a cell culture experiment with CHO cells. CHO cells were grown in reference medium or in media, in which components of the reference medium were replaced by a hydrolysate of defatted rice bran (RBD) (<NUM>% protein content). Two different lots of RBD hydrolysate were tested. At each day of cultivation, viable cell density (in <NUM><NUM> cells/mL) and viability of cells were recorded for each experiment.

The plant material (either from primary food stream or from secondary food stream) was hydrolysed using the following protocol:.

Plant hydrolysates were prepared from the following starting materials (protein content is indicated in brackets): rye bran (<NUM>%), wheat bran (<NUM>%), fava bean (<NUM>%), spelt bran (<NUM>%), legria (<NUM>%), stabilized rice bran (<NUM>%), and defatted rice bran (<NUM>%).

The above-described protocol was also carried out in a larger scale, in which all materials were used in <NUM>-fold amount:.

Cell culture experiments were carried out by Xell AG in their fermenters in Bielefeld as contract work. CHO-K1 cells were grown in reference medium or in a medium containing a replacement with a hydrolysate of defatted rice bran (RBD) (<NUM>% protein content). The hydrolysates of defatted rice bran had been prepared according to the acid hydrolysis protocol described in Example <NUM> above. Two different lots of RBD hydrolysate were tested.

Growth curves for the reference medium <NUM>:<NUM> and for the two experiments with two separate lots of RBD hydrolysate are shown in <FIG>. Reference medium <NUM>:<NUM> means that <NUM> per <NUM> of reference medium were replaced by distilled water. RBD and RBD1 both refer to a medium composition, in which <NUM> of defatted rice bran medium were added to <NUM> reference medium. Glucose concentration in all media variants was adjusted to either <NUM>/L (for RBD and RBD1) or <NUM>/L (for Reference <NUM>:<NUM>), depending of glucose concentration of the food grade materials used. Glutamine was added to a final concentration of <NUM>. Cells were cultivated in <NUM> plain shake flasks with a working volume of <NUM> (in growth curves) in a <NUM> incubator with an atmosphere of <NUM> % CO<NUM>.

As can be seen from the growth curves, media containing a replacement of RBD hydrolysate allow growth of CHO cells. No inhibitory effect and no cytotoxicity were observed.

However, the maximum cell density was lower when using a medium containing a replacement of rice bran hydrolysate (about <NUM> × <NUM><NUM> cells/mL for the first lot; about <NUM> x <NUM><NUM> cells/mL for the second lot) as compared to the reference medium (<NUM> × <NUM><NUM> cells/mL). In addition, maximum cell density was reached only after longer cultivation times (at day <NUM> for the first lot; at day <NUM> for the second lot) as compared to the reference medium (at day <NUM>).

Claim 1:
A method for the production of a plant-based protein hydrolysate suitable for cell culture of eukaryotic cells, said plant-based protein hydrolysate being obtained from secondary food stream, the method comprising the steps:
(a) providing a plant-based material obtained from secondary food stream, wherein the plant-based material obtained from secondary food stream is obtained from rice bran, wherein said plant-based material obtained from secondary food stream has a protein content ranging from <NUM>% to <NUM>% (w/w dry matter) and has a fibre content ranging from <NUM>% to <NUM>% (w/w dry matter);
(b) adding a concentrated solution of HCl to the plant-based material, wherein the concentration of HCl is in the range from <NUM> to <NUM>, thereby preparing a mixture of acid and material;
(c) incubating the mixture of step (b) at a temperature in the range between <NUM> and <NUM> for a time period of between <NUM> hours and <NUM> hours, thereby preparing an acid-hydrolysate;
(d) adding an alkaline solution to the acid-hydrolysate of step (c) in an amount sufficient to neutralize the acid-hydrolysate;
(e) centrifuging the neutralized acid-hydrolysate of step (d) to precipitate insoluble material;
(f) recovering the supernatant of the centrifugation of step (e); and
(g) filtering the supernatant recovered in step (f) through a sterile filter or a combination of sterile filters, thereby obtaining the plant-based protein hydrolysate suitable for cell culture of eukaryotic cells.