Culture media based on protein hydrolysate and a process for preparing thereof
The present invention provides culture media and method for the cultivation of non-human metazoan cells for preparing a food product.
1 . A culture medium for the cultivation of cells comprising,
a protein hydrolysate as a source of amino acids,
wherein the protein hydrolysate is prepared by enzymatic hydrolysis of a protein substrate,
wherein the cells are non-human metazoan cells, and
wherein a total input of amino acids from the protein hydrolysate, including amino acids in the form of peptides or suitable bioavailable derivatives, is at least 75% by weight of a total input of all amino acids in the culture medium;
wherein a total input of the protein hydrolysate expressed as dry protein weight introduced into the culture medium is in the range of 8 g/L to 50 g/L.
2 . The culture medium according to claim 1 , wherein a total input of amino acids added to the culture medium separately from the protein hydrolysate is in a range of 0.1 g/L to 10 g/L.
3 . The culture medium according to claim 1 , wherein the protein substrate for the protein hydrolysate comprises at least one of soy, pea, rice, wheat, corn, fava beans, alfalfa, hemp, chickpea, potato, pumpkin, rapeseed, red lentil, Spirulina, Chlorella , sunflower, water lentil, mung bean, or baker's yeast.
4 . The culture medium according to claim 1 , further comprising one or more of vitamins, sugars, minerals, organic amines, micronutrients, iron supplementation compounds, or shear protectants.
5 . The culture medium according to claim 1 , further comprising at least one exogenous signaling protein, wherein the exogenous signaling protein has a concentration of up to 50 mg/L in the culture medium.
6 . The culture medium according to claim 1 , further comprising a source of bioavailable inorganic nitrogen, wherein a total input of the source of bioavailable inorganic nitrogen is in the range of 1 g/L to 10 g/L.
7 . The culture medium according to claim 1 , wherein the protein hydrolysate is thermally treated at the end of hydrolysis to deactivate enzymes and kill microorganisms.
8 . The culture medium according to claim 1 , wherein a total input of the protein hydrolysate expressed as a percentage of the total input of protein hydrolysate into the culture medium is in a range of 0.2% to 25%.
9 . A culture medium for the cultivation of cells comprising:
a protein hydrolysate as a source of amino acids,
wherein the protein hydrolysate is prepared by an enzymatic hydrolysis of a source protein substrate,
wherein the cells are non-human metazoan cells,
wherein a total input of supplemented amino acids, added to the culture medium separately from the protein hydrolysate, is in a range of 0.1 g/L to 10 g/L.
10 . The culture medium according to claim 9 ,
wherein the culture medium comprises at least one essential amino acid, wherein a highest possible conversion efficiency for essential total amino acids is in a range of 30% to 100%, as calculated by equation (1):
H
EAA
=
A
EAAM
∑
A
EAAM
A
EAAC
∑
A
EAAC
*
100
,
(
1
)
where
H EAA is the highest possible conversion efficiency for a particular amino acid,
A EAAM is the content of the particular essential amino acid in 100 g of protein in the culture medium,
ΣA EAAM is a total content of all essential amino acids in 100 g of protein in the culture medium,
A EAAC is the content of the particular essential amino acid in 100 g of cellular protein, and
wherein the concentrations of amino acids in grams per 100 grams of total essential amino acids introduced into the cultivation process is 0.3 g to 6.04 g for His, 0.57 g to 11.41 g for Ile, 1.00 g to 19.91 g for Leu, 0.92 g to 18.34 g for Lys, 0.32 g to 6.49 g for Met, 0.53 g to 10.51 g for Phe, 0.54 g to 10.74 g for Thr, 0.18 g to 3.58 g for Trp, 0.65 g to 12.98 g for Val; and
wherein the highest possible conversion efficiency is determined by the most limiting essential amino acid.
11 . The culture medium according to claim 9 , further comprising a supplemented amino acid comprising at least one of: L-methionine, L-cysteine, L-cystine, L-ornithine, L-tryptophan, L-histidine, or L-threonine, added separately from the protein enzymatic hydrolysate.
12 . The culture medium according to claim 11 , wherein the total input of the protein hydrolysate expressed as dry protein weight introduced into the culture medium is in a range of 8 g/L to 50 g/L.
13 . The culture medium according to claim 9 , further comprising at least one of: vitamins, sugars, minerals, organic amines, micronutrients, iron supplementation compounds, or shear protectants.
14 . The culture medium according to claim 13 , wherein the culture medium comprises an organic amine, wherein the organic amine is at least one of putrescine or ethanolamine.
15 . The culture medium according to claim 13 , wherein the culture medium comprises a micronutrient, wherein the micronutrient comprises at least one of: spermine, spermidine, putrescine, thymidine, L-ornithine, ethanolamine, myo-inositol, or choline.
16 . The culture medium according to claim 13 , wherein the culture medium comprises at least one iron supplementation compound, wherein the at least one iron supplementation compound comprises iron in oxidation state iron (III) or iron (II).
17 . The culture medium according to claim 13 , wherein the culture medium comprises a shear protectant, wherein the shear protectant comprises at least one of: polyethylene glycol (PEG), methyl cellulose (MC), (hydroxypropyl)methyl cellulose (HPMC), hydroxypropyl cellulose (HPC), carboxymethyl cellulose (CMC), or dextran sulfate.
18 . A culture medium for the cultivation of cells,
comprising a protein hydrolysate as a source of amino acids,
wherein the protein hydrolysate is prepared by an enzymatic hydrolysis of a protein substrate,
wherein the cells are non-human metazoan cells,
wherein a total input of protein hydrolysate, expressed as dry protein weight introduced into the culture medium is in a range of 8 g/L to 50 g/L.
19 . The culture medium according to claim 18 ,
wherein the enzymatic hydrolysis occurs in a hydrolysis reaction mixture comprising the enzymes and the protein substrate,
wherein a total enzyme concentration in the hydrolysis reaction mixture is in a range of 0.05 to 5% expressed as a ratio of the concentration of the enzymes in the hydrolysis reaction mixture to the concentration of the protein substrate in the hydrolysis reaction mixture.
20 . The culture medium according to claim 18 , wherein a degree of the enzymatic hydrolysis of the protein substrate, defined as a percentage of hydrolyzed peptide bonds out of a total amount of peptide bonds present in the substrate at the start of the hydrolysis reaction, is in a range of 10% to 60%.
21 . The culture medium according to claim 18 , wherein the cells are anchorage independent, defined as being able to survive and grow in suspension conditions without attachment to any surface, and/or the cells are able to survive and grow as a suspension of cell clumps, cell aggregates, spheroids, or organoids.
22 . The culture medium according to claim 18 , wherein the concentration of protein in the reaction mixture of hydrolysis is in a range of 30 g/L to 130 g/L.
23 . The culture medium according to claim 18 , wherein an amount of the amino acid source in a range of 20% to 100% is converted during hydrolysis into free amino acids and short peptides smaller than 500 Da.
24 . A method for preparing a culture medium for the cultivation of cells comprising:
comprising providing a protein hydrolysate as a source of amino acids, wherein the protein hydrolysate is prepared by enzymatic hydrolysis of a protein substrate, adding the protein hydrolysate to the culture medium in an amount, expressed as dry protein weight, in a range of 8 g/L to 50 g/L; and
wherein a total input of amino acids from the protein hydrolysate, including amino acids in the form of peptides, bioavailable derivatives, or a combination thereof, is at least 75% by weight of a total input of all amino acids in the culture medium;
wherein the cells are non-human metazoan cells.
25 . The method for preparing a culture medium according to claim 24 ,
wherein at least two types of enzymes are used for the enzymatic hydrolysis, wherein the at least two types of enzymes comprise:
i) at least one endoprotease and at least one exoprotease, or
ii) at least one of the following types of enzymes: serine protease, cysteine protease, metalloprotease, glutamic protease, or aspartic protease.
26 . The method for preparing the culture medium according to claim 24 ,
wherein the culture medium has an osmolality in a range of 200 mOsm/kg to 400 mOsm/kg.
27 . The method for preparation of the culture medium according to claim 24 , wherein the enzymatic hydrolysis of the protein substrate occurs in a hydrolysis reaction mixture comprising the protein substrate and an enzyme, wherein the protein substrate is at a concentration in a range of 30 g/L to 130 g/L.
28 . The method for preparation of the culture medium according to claim 24 , wherein the culture medium is further filtered using a filter having a pore size in a range of 0.001 μm to 10 μm.
29 . The method for preparation of the culture medium according to claim 24 , wherein the protein substrate is subjected to an initial thermal pretreatment to improve solubility and susceptibility to hydrolysis; wherein a temperature of the initial thermal pretreatment is in a range of 75° C. to 95° C. for a duration in a range of 5 minutes to 120 minutes.
30 . The method for preparation of the culture medium according to claim 24 , wherein the protein substrate is further enzymatically treated by a phytase enzyme.