NUTRIENT MEDIA FOR CELL CULTURE CONTAINING PLANT PROTEIN HYDROLYSATES

Abstract

The present invention relates to nutritional components suitable for cell culture of eukaryotic cells. These nutritional components can be prepared from plant-based protein hydrolysates. The invention further relates to nutrient media comprising these nutritional components. The nutrient media of the invention can be prepared by replacing serum components, which are common in such media, at least partiallyby the nutritional components of the invention. The nutritional components and the nutrient media of the invention are particularly suitable for use in the food industry, such as in the production of cultured meat.

Claims

1.-15. (canceled)

16. A method for the production of a plant-based protein hydrolysate obtained from secondary food stream, comprising the steps: (a) providing a plant-based material obtained from secondary food stream, wherein said plant-based material obtained from secondary food stream has a protein content ranging from about 4% to about 35% (w/w dry matter) and has a fibre content ranging from about 1% to about 50% (w/w dry matter); (b) adding acid to the plant-based material, thereby preparing a mixture of acid and material; (c) incubating the mixture of step (b) at a temperature in the range between 60 C. and 100 C. for a time period of at least 12 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.

17. The method according to claim 16, wherein the plant-based material obtained from secondary food stream is obtained from rice bran, wheat bran, rye bran, maize bran, spelt bran, or brewer's spent grain.

18. The method according to claim 16, wherein the acid added in step (b) is a concentrated solution of HCl, wherein preferably the concentration of HCl is in the range from 0.1 M to 2 M.

19. The method according to claim 16, wherein step (c) is carried out at a temperature between 75 C. and 85 C., preferably at about 80 C.; and/or for a time period between 12 hours and 48 hours, preferably between 15 hours and 36 hours, more preferably between 18 hours and 30 hours, most preferably for about 24 hours.

20. The method according to claim 16, wherein step (e) is carried out for about 5 to about 20 minutes at about 4000 to 6000g.

21. The method according to claim 16, wherein the sterile filter is a filter with a pore size of 50 m or less.

22. A plant-based protein hydrolysate (obtained from using secondary food streams), obtainable by the method of claim 16.

23. A composition suitable for use as medium component supporting the growth of eukaryotic cells, the composition comprising: (i) a first plant-based protein hydrolysate obtainable by the method of claim 16; and (ii) a food grade premix comprising one or more minerals, one or more vitamins, one or more amino acids, and/or one or more salts; wherein said composition is devoid of animal proteins, wherein preferably the secondary food stream material is or is prepared from rice bran, wheat bran, rye bran, maize bran, spelt bran, or brewer's spent grain.

24. The composition according to claim 23, wherein the first plant-based protein hydrolysate is present in the composition in a concentration in the range from 0.1% to 50% (w/w).

25. The composition according to claim 23, wherein the one or more minerals in the food grade pre-mix are selected from the group consisting of Mg.sup.2+, Ca.sup.2+, Fe.sup.2+, Fe.sup.3+, Cu.sup.3+, Zn.sup.2+, and Mn.sup.2+; the one or more vitamins in the food grade pre-mix are selected from the group consisting of ascorbic acid, biotin, choline, D-calcium pantothenate, folic acid, niacinamide, pyridoxine, pyridoxal, riboflavin, thiamine, vitamin B12, and i-inositol; the one or more amino acids in the food grade pre-mix are selected from the group consisting of glycine, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine; and/or the one or more salts in the food grade pre-mix are selected from the group consisting of NaCl, KCl, Na.sub.2HPO.sub.4, NaH.sub.2PO.sub.4, and NaHCO.sub.3.

26. The composition according to claim 23, wherein the food grade premix is present in the composition in a concentration in the range from 10% to 99.9% (w/w).

27. The composition according to claim 23, further comprising: (iii) a second plant-based protein hydrolysate prepared from primary food stream material by acid hydrolysis, wherein said primary food stream material has a protein content of at least 30% (w/w dry matter) protein and a fibre content of 5% (w/w dry matter) or less, wherein preferably the primary food stream material has been prepared from soy bean, fava bean, wheat, rice, pea, potato, or cotton seed.

28. The composition according to claim 27, wherein the second plant-based protein hydrolysate obtained from primary food stream material is present in a concentration in the range from 0.1% to 50% (w/w).

29. Use of the composition according to claim 23 in the production of cultured meat.

30. A method for the production of a composition which includes (i) a first plant-based protein hydrolysate obtainable by the method of claim 16; and (ii) a food grade premix comprising one or more minerals, one or more vitamins, one or more amino acids, and/or one or more salts; wherein said composition is devoid of animal proteins, wherein preferably the secondary food stream material is or is prepared from rice bran, wheat bran, rye bran, maize bran, spelt bran, or brewer's spent grain, the method comprising: mixing a plant-based protein hydrolysate obtainable by the method of claim 16 with a food grade premix and optionally with a plant-based protein hydrolysate prepared from primary food stream material by acid hydrolysis.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0107] FIG. 1 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) (20% protein content). Two different lots of RBD hydrolysate were tested. At each day of cultivation, viable cell density (in 10.sup.6 cells/mL) and viability of cells were recorded for each experiment.

EXAMPLES

1. Acid Hydrolysis of Plant Material

[0108] The plant material (either from primary food stream or from secondary food stream) was hydrolysed using the following protocol: [0109] 5 g of plant material was added to an empty 100 ml flask. [0110] 50 mL of 0.5 M HCl was added to the plant material. [0111] The mixture of plant material and HCl was mixed and incubated at 80 C. for about 24 hours. [0112] Different solutions of NaOH were slowly and consecutively added until a pH of 7.0 was reached: First a 5 M NaOH solution was added until a pH of about 5 was reached; then a 1 M NaOH solution was added until a pH of about 6 was reached; finally, a 0.1 M NaOH solution was added until pH 7.0 was reached. [0113] The neutralized mixture was transferred to a new 100 ml flask. [0114] Distilled water was added up to a total volume of 100 mL. [0115] The flask was closed by a lid and was shaken. The temperature was adjusted to 20 C. [0116] The mixture was transferred to centrifugation tubes (2 tubes with a size of 50 mL each). The tubes were centrifuged at 4000 rpm for 10 min in an Allegra X-15R centrifuge (Beckman Coulter); corresponding to 5250g. [0117] The supernatant was collected and filtered through a 10 m Teflon filter, thereby obtaining a sterile solution of plant hydrolysate.

[0118] Plant hydrolysates were prepared from the following starting materials (protein content is indicated in brackets): rye bran (17.1%), wheat bran (14.3%), fava bean (66%), spelt bran (19.7%), legria (21.2%), stabilized rice bran (15.1%), and defatted rice bran (19.0%).

[0119] The above-described protocol was also carried out in a larger scale, in which all materials were used in 50-fold amount: [0120] 250 g of plant material was added to an empty 5 L flask. [0121] 2.5 L of 0.5 M HCl was added to the plant material. [0122] The mixture of plant material and HCl was mixed and incubated at 80 C. for about 24 hours. [0123] Different solutions of NaOH were slowly and consecutively added until a pH of 7.0 was reached: First a 5 M NaOH solution was added until a pH of about 5 was reached; then a 1 M NaOH solution was added until a pH of about 6 was reached; finally, a 0.1 M NaOH solution was added until pH 7.0 was reached. [0124] The neutralized mixture was transferred to a new 5 L flask. [0125] Distilled water was added up to a total volume of 5 L. [0126] The flask was closed by a lid and was shaken. The temperature was adjusted to 20 C. [0127] The mixture was transferred to centrifugation tubes (8 tubes with a size of 750 mL each). The tubes were centrifuged for 10 min at 5250g.

[0128] The supernatant was collected and filtered through a 10 m Teflon filter, thereby obtaining a sterile solution of plant hydrolysate.

2. Cell Culture Experiments

[0129] Materials: [0130] CHO-K1 cells Chinese hamster ovary cell line, subclone K1 [0131] Reference medium Liquid SBF medium, obtainable from Xell AG (Bielefeld, Germany) upon request under the product number 2052-0001

[0132] 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) (20% protein content). The hydrolysates of defatted rice bran had been prepared according to the acid hydrolysis protocol described in Example 1 above. Two different lots of RBD hydrolysate were tested.

[0133] Growth curves for the reference medium 1:3 and for the two experiments with two separate lots of RBD hydrolysate are shown in FIG. 1. Reference medium 1:3 means that 300 mL per 1 L of reference medium were replaced by distilled water. RBD and RBD1 both refer to a medium composition, in which 300 mL of defatted rice bran medium were added to 700 mL reference medium. Glucose concentration in all media variants was adjusted to either 4.5 g/L (for RBD and RBD1) or 6.2 g/L (for Reference 1:3), depending of glucose concentration of the food grade materials used. Glutamine was added to a final concentration of 6 mM. Cells were cultivated in 125 mL plain shake flasks with a working volume of 50 mL (in growth curves) in a 37 C. incubator with an atmosphere of 5% CO.sub.2.

[0134] 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.

[0135] However, the maximum cell density was lower when using a medium containing a replacement of rice bran hydrolysate (about 410.sup.6 cells/mL for the first lot; about 3.510.sup.6 cells/mL for the second lot) as compared to the reference medium (510.sup.6 cells/mL). In addition, maximum cell density was reached only after longer cultivation times (at day 7 for the first lot; at day 8 for the second lot) as compared to the reference medium (at day 6).

[0136] In summary, the above results provide a proof of principle that a hydrolysate from rice bran (i.e. from a secondary food stream material) can be used as a supplement in cell culture media and supports the growth of eukaryotic cells.