FR3031986A1 - PROCESS FOR ENRICHING PROTEIN FROM BIOMASS OF MICROALGUES - Google Patents

Abstract

The invention relates to a protein enrichment process of a microalga grown in heterotrophy, microalgae of the genus Chlorella, more particularly Chlorella protothecoides, characterized in that it comprises: a first step aimed at limiting the contribution ammonium so as to obtain a biomass of microalgae having a protein content, less than 50% expressed in N. 6.25, preferably less than 30%, more preferably between 20 and 25%; a second step in which the ammonium supply is increased in the fermentation medium so as to obtain a protein content of greater than 50%, preferably greater than 60%, and still more preferably greater than 65%.

Description

The present invention relates to a process for the protein enrichment of microalgae biomass, more particularly of the genus Chlorella, and more particularly of the species Chlorelle protothecoides. The algae, macro and micro, have a specific richness largely unexplored. Their exploitation for food, chemical or bioenergetic purposes is still very marginal. However, they contain components of great value, wealth and abundance. Microalgae are indeed sources of vitamins, lipids, proteins, sugars, pigments and antioxidants. Algae and microalgae are thus of interest to the industrial sector that uses them for the manufacture of food supplements, functional foods, cosmetics, medicines or for aquaculture. Microalgae are primarily photosynthetic microorganisms that colonize all biotopes exposed to light. On an industrial scale, their monoclonal culture is carried out in photobioreactors (autotrophic conditions: in the light with CO2) or for some of them, also in fermenters (heterotrophic conditions: in the dark in the presence of a carbon source). Some species of microalgae are indeed able to grow in the absence of light: Chlorella, Nitzschia, Cyclotella, Tetraselmis, Crypthecodinium, Schizochytrium.

It is moreover estimated that culture in heterotrophic conditions costs 10 times less expensive than in phototrophic conditions because, for those skilled in the art, these heterotrophic conditions allow: the use of fermentors identical to those used for bacteria and yeasts and allow control of all culture parameters. - the production of biomasses in a quantity much higher than what is obtained by culture based on the light. The profitable exploitation of the microalgae generally requires the control of the fermentation conditions making it possible to accumulate its components of interest, such as: the pigments (chlorophyll a, b and c, B-carotene, astaxanthin, lutein, phycocyanin, xanthophylls, phycoerythrin ...) whose demand is growing, both for their remarkable antioxidant properties, as for their contribution of natural colors in the diet, 3031986 2 - lipids, this in order to optimize their content in fatty acids (up to 60%, see 80% by weight of their dry matter) in particular for: - biofuel applications, but also - applications in human or animal nutrition, when the 5 selected microalgae produce polyunsaturated fatty acids or PUFAs called "essential" (ie brought by the diet because they are not naturally produced by the man or the animal), or - the proteins, this in order to optimize their nutritional qualities or for example to promote the intake of amino acids of interest.

In the context of the amino acid supply of interest, it may indeed be advantageous to have sources of proteins rich in arginine and glutamate. Arginine is an amino acid which has many functions in the animal kingdom. Arginine can be degraded and thus serve as a source of energy, carbon and nitrogen for the cell that assimilates it. In various animals, including mammals, arginine is decomposed into ornithine and urea. The latter is a nitrogen molecule that can be eliminated (by excretion in the urine) so as to regulate the amount of nitrogen compounds present in the cells of animal organisms.

Arginine allows the synthesis of nitric oxide (NO) by NO synthetase, thereby mediating vasodilatation of the arteries, which reduces the rigidity of the blood vessels, increases blood flow and thus improves the functioning of the blood vessels. Food supplements that contain arginine are recommended to promote heart health, vascular function, to prevent "platelet aggregation" (risk of blood clots) and to lower blood pressure. The involvement of arginine in wound healing is related to its role in the formation of proline, another important amino acid for collagen synthesis. Arginine is finally a frequently used component, especially by sportsmen, in energy drinks. Glutamic acid is not only one of the building blocks used for protein synthesis, but is also the most common excitatory neurotransmitter in the central nervous system (brain + spinal cord) and is a precursor to GABA in GABAergic neurons.

Under the code of "E620", glutamate is used as a flavor enhancer of foods. It is added to food preparations to enhance their taste.

In addition to glutamate, the Codex Alimentarius has also recognized as taste enhancers its salts of sodium (E621), potassium (E622), calcium (E623), ammonium (E624) and magnesium (E625). Glutamate (or its salts) is often present in ready-made dishes (soups, 5 sauces, chips, ready meals). It is also commonly used in Asian cooking. It is currently frequently used in combination with aromas in appetizers (bacon taste, cheese taste). This enhances the taste of bacon, cheese, etc. It is rare to find an aperitif that does not contain any. It is also found in some drug capsules but not for its taste functions. Finally, it is the majority component of kitchen auxiliaries (cubic broths, sauces, sauces, etc.). In order to exploit the metabolic richness of microalgae, the first fermentation processes that make it possible to obtain high cell densities (HCD for High-Cell-Density) have thus been extensively worked out in order to obtain yields and productivities. maximum in protein or lipids. The objective of these HCD cultures was to obtain the highest possible concentration of the desired product in the shortest time.

This precept is true, for example, for the biosynthesis of astaxanthin by Chlorella zofingiensis, where the growth of the microalgae has been directly correlated with the production of this compound (Wang and Peng, 2008, World J. Microbiol., Biotechnol., 24 (9), 1915-1922). However, maintaining growth at its maximum rate (ie, in h -1) is not always correlated with the high production of the desired product. It quickly became apparent to specialists in the field that, for example, microalgae must be subjected to a nutritional stress which limits their growth when they wish to produce large lipid reserves. It is therefore now decoupling growth / production in fermentative processes and control of the cell growth rate. In general, a person skilled in the art chooses to control the growth of microalgae by controlling the fermentation conditions (Tp, pH, etc.), or by the regulated supply of nutritional components of the fermentation medium (semi-continuous conditions called " fedbatch ").

If he chooses to control the growth of heterotrophic microalgae by the supply of carbon sources, the person skilled in the art generally chooses to adapt the carbon source (pure glucose, acetate, ethanol, etc.) to the microalgae. (C. cohnii, Euglena gracilis ...) depending on the metabolite produced (for example a polyunsaturated fatty acid type DHA). Temperature may also be a key parameter: it has for example been reported that the synthesis of polyunsaturated fatty acids in certain microalgae species, such as EPA by Chlorella minutissima, is favored at a lower temperature than that required for optimal growth of said microalgae; - In contrast, the yield of lutein is higher in Chlorella protothecoides grown in heterotrophy, when the production temperature is increased from 24 to 35 ° C.

10 Chlorella protothecoides is justly recognized as one of the best oil-producing microalgae. In heterotrophic conditions, it rapidly converts carbohydrates into triglycerides (more than 50% of its dry matter).

To optimize this production of triglycerides, those skilled in the art are led to optimize the carbon flow towards the production of oil, by acting on the nutritional environment of the fermentation medium. It is thus known that the accumulation of oil occurs during a sufficient carbon supply, but under conditions of nitrogen deficiency.

The C / N ratio is therefore decisive here, and it is accepted that the best results are obtained by acting directly on the nitrogen content, the glucose content not being limiting. Not surprisingly, this nitrogen deficiency affects cell growth, resulting in a 30% lower growth rate than the normal growth rate of the microalgae (Xiong et al., Plant Physiology, 2010, 154, pp1001-1011). To explain this result, Xiong et al, in the article cited above, in fact demonstrate that if we divide Chlorella biomass into its main components, ie carbohydrates, lipids, proteins, DNA and RNA (representing 85 % of its dry matter), if the C / N ratio has no impact on the DNA, RNA and carbohydrate content, it becomes preeminent for the protein and lipid content. Chlorella cells cultured with a low C / N ratio contain 25.8% protein and 25.23% lipid, while a high C / N ratio allows the synthesis of 53.8% of the protein. lipids and 10.5% protein.

In order to optimize its production of oil, it is therefore essential for those skilled in the art to control the carbon flow by deviating it towards the production of oil, to the detriment of the production of proteins; the carbon flux is redistributed and accumulates in lipid reserve substances when the microalgae are placed in nitrogen-deficient medium. But Chlorella protothecoides can also be advantageously chosen to produce proteins. Given the analysis made by those skilled in the art as to the management of the C / N ratio for the production of oil (aiming at a high C / N ratio), the skilled person is therefore led to favor a C ratio. / N low, and thus: make a significant contribution of nitrogen source to the fermentation medium, while maintaining constant the carbon source feed that will be converted into proteins and stimulate the growth of the microalgae. It is thus chosen to modify the carbon flow towards the production of proteins (and therefore of biomasses), to the detriment of the production of reserve lipids.

The present invention relates to a process for enriching biomass proteins of microalgae, more particularly of the genus Chlorella, more particularly of the species Chlorella protothecoides. The present invention relates to a process for enriching the biomass proteins of certain microalgae, more particularly Chlorella protothecoides, proteins whose arginine and glutamine content is remarkably high. The present invention more particularly covers a method for producing biomass of microalgae enriched in proteins, characterized in that it consists in increasing the ammonium input in a biomass previously deficient in nitrogen.

Indeed, in the context of the invention, the Applicant Company has instead chosen to explore an original way by proposing an alternative solution to that conventionally envisaged by those skilled in the art, or even completely the opposite of that that he would have chosen. The invention thus relates to a protein enrichment process of a microalga grown in heterotrophy, a microalgae of the Chlorella genus, more particularly to Chlorella protothecoides, a heterotrophic culture method which comprises: a first step aimed at limiting the contribution ammonium in order to obtain a biomass of microalgae having a protein content of less than 50% (3/0) expressed in N. 6.25, preferably less than 30%, preferably between 20 and 25 (3/0; a second step in which the ammonium feed in the fermentation medium is increased so as to obtain a protein content greater than 50%, preferably greater than 60%, more preferably still greater than 65%. will be exemplified below, a preferred embodiment of the method 5 according to the invention may consist in carrying out the pH regulation in the first step with an NH 3 / KOH mixture thus limiting the ammonium intake and thus favoring the production of a low protein content, then using pH regulation with NH3 alone in the second stage, in order to replenish the medium of ammonium fermentation. The NH3 / KOH mixture will be such as to limit the intake of ammonium. For example, for the same concentration of NH 3 between the first and second stages, the mixture may comprise NH 3 / KOH ratios which are in the order of about 1: 1, such as about 70-45% NH 3 and 55% KOH, preferably about 65-55% v / v NH3 and 35-45% KOH, the amounts being expressed in moles. By "about" is meant a range of value comprising + or - 10% of the indicated value, preferably + or - 5% thereof. For example, about 10 is between 9 and 11, preferably between 9.5 and 10.5. In this preferred embodiment, the supply of NH3 is then multiplied by about 1.5 to 2, and the resulting rate of nitrogen consumption is multiplied by 5. This second step is a step during which the increase in the supply of ammonium to a biomass previously deficient in nitrogen causes a specific overconsumption of this salt by this biomass and leads remarkably to boost the protein content, to a content greater than 50%, preferably greater than 50%; 60%, preferably greater than 65% (°) / 0 expressed in N, 6, 25).

It is thus found that the specific rate of nitrogen consumption, which falls to a value of less than 0.005 g / g / hr during the nitrogen deficiency phase, increases to a value of greater than 0.01 g / g / g. h after the lifting of the nitrogen deficiency. In a preferred embodiment, the growth rate is kept substantially constant. For example, during these two phases, the growth rate is maintained at 0.07 hr-1 to 0.09 hr-1, preferably about 0.08 hr-1. To exemplify this concept, the invention more specifically covers a heterotrophic Chlorella protothecoides culture method comprising: a batch phase after inoculation of the fermenter supplying 20 g / l of glucose. An exponential fed-batch phase with a growth rate set at 0.08 h -1, triggered when the glucose supplied in batch is totally consumed, during which the ammonium intake is limited by using a regulation of pH using a mixture of NH3 and KOH as mentioned above in order to obtain a biomass having less than 25% of proteins (expressed in N. 6.25), then a fed phase -batch exponential with the same growth rate set at 0.08 h-1 during which the ammonium deficiency is lifted by regulating the pH using a 100% ammonia solution. For example, the second fed-batch phase where ammonium deficiency is lifted is initiated when about 2 kg of dry glucose has been introduced. Within the meaning of the invention, the essential criterion is that the cellular stress caused by the nitrogen deficiency of the fermentation medium, and then a lifting of this stress, under specific conditions, trigger a consumption of nitrogen introduced to boost the protein content of the biomass produced.

This strategy therefore goes against the technical prejudice that to increase the protein content of the biomass, it is inevitable to increase nitrogen input from the beginning of the crop. Moreover, these operating conditions are reflected here not only by an increase in the protein richness, but also lead to a remarkable increase in the content of arginine and glutamate. More particularly, as will be exemplified hereinafter, the heterotrophic culture of microalgae of the Chlorelle protothecoides species comprising a culture step with a nitrogen deficiency and then an ammonium pulse results in the production of more than 45% of glutamic acid and arginine on the total amino acids. Thus, the present invention also relates to a process for enriching the glutamic acid and / or arginine content of a microalga grown in heterotrophy, preferably a microalgae of the Chlorella protothecoides species, the process comprising the heterotrophic culture of said microalgae which comprises a step to limit the intake of ammonium so as to obtain a low protein biomass of microalgae, followed by a step in which the growth rate is maintained and the feed intake is increased. ammonium. These cultivation conditions thus result in the preparation of a biomass of microalgae comprising more than 45% of glutamic acid and arginine on the total amino acids.

The invention will be better understood with the aid of the following examples, which are intended to be illustrative and non-limiting. DESCRIPTION OF THE FIGURES FIG. 1 illustrates the evolution of N.6, 25 depending on the glucose consumed. Figure 2 illustrates the evolution of the specific rate of consumption of nitrogen (qN) as a function of the glucose consumed. Figure 3 illustrates the evolution of N.6,25 as a function of time and the amount of each amino acid in% of weight of dry biomass as a function of time.

Figure 4 illustrates the evolution of N.6,25 as a function of time and the amount of total or particular fatty acids in% dry biomass weight as a function of time. Figure 5 illustrates the evolution of N.6,25 as a function of time and the amount of total or particular sugars in% of weight of dry biomass as a function of time.

EXAMPLES Example 1: Preparation of C. high protein protein-rich biomass with high content of glutamic acid and arginine The strain used is Chlorella protothecoides (strain CCAP211 / 8D - The Culture Collection of Algae and Protozoa, Scotland, UK). ). Preculture: - 150 ml of medium in a 500 ml Erlenmeyer flask; - Composition of the medium: 40 g / l of glucose + 10 g / l of yeast extract.

The incubation takes place under the following conditions: time: 72 h; temperature: 28 ° C; agitation: 110 rpm (Infors Multitron Incubator).

Culture in batch mode and then fed batch Preparation and initial batch medium prepare and filter a KOH mixture at 400 g / I (41%) / NH3 at 20% v / v (59%); sterilize 20 L fermenter at 121 ° C / 20 min; inoculate with 2 500 ml preculture Erlenmeyer flasks (OD600 nm 15); PH control at 5.2 with KOH / NH3; agitation of 300 rpm start; aeration: 15 L / min of air; 30% pO regulation by varying the stirring; temperature: 28 ° C Glucose feed: 500 g / I 5 ammonium sulfate: 25 g / I sodium phosphate monobasic: 17 g / I potassium phosphate monobasic: 23 g / I magnesium sulfate heptahydrate: 20 g / I iron sulfate : 120 mg / I 10 calcium nitrate: 610 mg / I trace element solution: 45 ml / 1 vitamin solution: 3.6 ml / 1 trace element solution (for 2 liters) Ingredients (g) CuSO4, 5 H2O 0 , 22 ZnSO4, 7 H2O 28 MnSO4, 1 H2O 16 FeSO4, 7 H2O 2.2 Citric acid 60 H2O qs 2 15 Vitamin solution Ingredients (g / 1) Thiamine HCI 13.5 Biotin 0.7 Pyridoxine 6.75 Conducts the fermentation bring the equivalent of 20 g / l of glucose before inoculation when the glucose concentration is = 0 g / l, start feeding the glucose in fed-batch; use a rate to set the growth rate at 0.08 h-1. pH control at 5.2 with the mixture 41% KOH / 59% NH3 when 2 kg of glucose are consumed by the microalgae, the pH is changed by NH3 alone, when the biomass reaches 100 g / l by weight of dry matter, and that about 3.5 kg of glucose were sent, the glucose supply is stopped.

Results Two tests were carried out under these same conditions and the results are shown in Table I and the following graphs: Table I. Test 1 Test 2 F2 140519 F5 140623 Final title (%) Final title (%) (for 3.6 kg of glucose (for 3.4 kg of glucose consumed) consumed) N.6.25 66.0 65.7 Total amino acids 44.7 43.2 Arg and Glu content 46 46 total amino acids total fat 10.2 10.1 Total sugars 20.3 21.7 Biomass staining Yellow Yellow Figure 1 illustrates the evolution of N.6,25 as a function of the glucose consumed.

10 These two tests show remarkable results: the obtaining of a yellow biomass with a N6,25 rate of more than 65 `Vo. Figure 2 illustrates the evolution of the specific rate of consumption of nitrogen (qN) as a function of the glucose consumed. It appears that the specific rate of nitrogen consumption is at its maximum after the lifting of the nitrogen limitation (at 2 kg of glucose consumed), and then gradually decreases. Similar speeds between the two trials also reflect the good repeatability of the protocol. A complete analysis of the amino acids present in the biomass was performed on a sample taken just before the limitation was lifted and on several samples after the pulse. The results are shown in Figure 3. It is noted that just before the lifting of the limitation in nitrogen, the sum of the amino acids is low (16.3%), and there is no predominance among the different amino acids .

One hour after the lifting of the nitrogen limitation, it is noted that the amino acid that knows the strongest rise is glutamic acid, followed by arginine. The level of other amino acids also increases but much less. The increase in N6.25 is therefore mainly correlated with the increase in glutamic acid and arginine. In addition to these analyzes, a complete analysis of the fatty acids present in the biomass was performed on a sample taken just before the lifting of the nitrogen limitation and on several samples after the pulse. The results are shown in Figure 4.

The total fatty acid content in the biomass which is 19.2% before pulse decreases to 10.2%. The major fatty acid that follows this curve is oleic acid. Fatty acids accumulate in the biomass when it is deficient in nitrogen. A complete analysis of the sugars present in the biomass was also carried out on a sample taken just before the lifting of the nitrogen limitation and on several samples after the lifting of the nitrogen limitation. The results are shown in Figure 5. The total sugar content in the biomass, which is 37.5% before the lifting of the nitrogen limitation, decreases to 20% and then stagnates. The major sugar that follows this curve is glucose. The sugars are therefore also stored in the biomass when it is deficient in nitrogen. The level of sugars seems to stabilize afterwards, in contrast to the fatty acid content which is only decreasing.

The salt loading of the biomass was measured by the measurement of the residue at calcination: it is here of 9 'Vo.

Claims (8)

REVENDICATIONS1. Process for the enrichment in proteins of a microalga grown in heterotrophy, microalgae of the Chlorella genus, more particularly Chlorella protothecoides, characterized in that it comprises: a first step aimed at limiting the intake of ammonium so as to obtain a biomass of microalgae having a protein content of less than 50% (3/0, expressed as N. 6.25, preferably less than 30%, more preferably between 20 and 25%; a second stage in which the supply of ammonium in the fermentation medium so as to obtain a protein content greater than 50%, preferably greater than 60%, preferably still greater than 65%. 2. Method according to claim 1, characterized in that the pH regulation is carried out in the first step by an NH3 / KOH mixture, and is carried out in the second by NH3 alone. 3. Method according to either of claims 1 and 2, characterized in that the mixture NH3 / KOH is about 70-45% NH3 and 30-55% KOH, preferably about 65-55% v. NH3 and 35-45% KOH, the amounts being expressed in moles. 4. Method according to any one of claims 1 to 3, characterized in that the ammonium intake is multiplied by about 1.5 to 2. 5. Method according to any one of claims 1 to 4, characterized in that in the first step, the specific rate of consumption of nitrogen by the microalgae is less than 0.005 g / g / h and that in the second step it is greater than 0.01 g / g / h. 6. Method according to any one of claims 1 to 5, characterized in that the growth rate is kept substantially constant during the first and second phase, preferably maintained at 0.07 h -1 to 0.09 h -1 preferably about 0.08 h -1. 3031986 13 7. Method according to any one of claims 1 to 6, characterized in that it comprises: a batch phase, after seeding of the fermenter, providing 20 g / l of glucose, an exponential fed-batch phase with a growth rate set at 0.08 h 1, triggered when the glucose brought in batch is totally consumed, during which the ammonium intake is limited by using a pH regulation using a mixture of NH3 and KOH in order to obtain a biomass having less than 25% of proteins (expressed in N. 6.25), then an exponential fed-batch phase with the same growth rate set at 0.08 h -1 during the ammonium limitation is lifted by regulating the pH with a 100% ammonia solution. 15 8. Method according to any one of claims 1 to 7, characterized in that it leads to produce a biomass of microalgae with more than 45% of glutamic acid and arginine on total amino acids component said biomass.