阅读说明：本技术 用于提取藻蓝蛋白的方法 (Method for extracting phycocyanin ) 是由 O·科尼亚克 A·阿萨姆 J·德摩尔 于 2020-01-10 设计创作，主要内容包括：本发明涉及一种用于借助选择性沉淀提取和纯化通过发酵微藻产生的、特别是由嗜硫原始红藻(Galdieria sulphuraria)产生的藻蓝蛋白的新方法。(The present invention relates to a novel process for the extraction and purification by means of selective precipitation of phycocyanins produced by fermenting microalgae, in particular produced by the sulfurophilous prototheca (galdiiria subpluraria).)

1. A method for extracting phycocyanin from an initial phycocyanin solution, the method comprising the steps of:

a) selective precipitation comprising adjusting the pH of the initial solution to a selected value within a pH range in which the phycocyanin is less soluble (also referred to as unstable range) and concentrating the phycocyanin in the solution to facilitate its precipitation, and

b) recovering the precipitated phycocyanin.

2. The method according to claim 1, characterized in that both actions of pH adjustment and concentration are performed simultaneously or sequentially, by adjusting the pH of the initial solution before concentration or by concentrating the initial solution before adjusting the pH.

3. The method according to any one of claims 1 or 2, characterized in that the initial solution is a crude solution from the lysis of microbial biomass cultivated for the production of phycocyanin.

4. A method according to any one of claims 1 to 3, characterized in that the phycocyanin is stable at acidic pH.

5. Process according to any one of claims 1 to 4, characterized in that the phycocyanin is a phycocyanin of microbial origin produced by a microorganism selected from the species Cyanidioschyzon, Cyanidium or Rhodophyta (Galdieria).

6. The method according to any one of claims 1 to 4, characterized in that the pH of the labile range is from 4.5 to 5.5.

7. Process according to any one of claims 1 to 6, characterized in that said concentration comprises the removal of water in order to obtain a phycocyanin content of at least 15g/L, preferably at least 20g/L, more preferentially at least 30g/L or even at least 40 g/L.

8. Method according to any one of claims 1 to 7, characterized in that the concentration is carried out by tangential filtration with a cut-off threshold that allows retention of the phycocyanin.

9. A process according to any one of claims 1 to 8, characterized in that the recovered phycocyanin is dried and optionally ground.

10. The process according to any one of claims 1 to 8, characterized in that the recovered phycocyanin has a purity index of at least 2, preferably at least 3.

11. The process according to claim 10, characterized in that the recovered phycocyanin has a purity index higher than 4.

12. A purified phycocyanin obtained by the method of any one of claims 1 to 11.

13. Use of the purified phycocyanin according to claim 12 as a food coloring.

14. A food product, characterized in that it comprises a purified phycocyanin according to claim 12.

Technical Field

The present invention relates to a novel process for the extraction and purification by selective precipitation of phycocyanin produced by fermenting microalgae, in particular produced by the sulfurophilous prototheca (galdiiria subpluraria).

Background

Purification of phycobiliproteins extracted from prototheca sulfidophila and Spirulina (Spirulina) by ammonium sulfate precipitation has been described in the literature (Moon et al 2015; Cruz de Jes et al 2006, CN106190853) but is very difficult to apply on an industrial scale because it requires large amounts of ammonium sulfate, which presents significant problems for reprocessing of ammonium sulfate and supernatant.

Other purification methods described to obtain high purity levels, such as chromatography or ion exchange resin purification (JP 2004359638), are very expensive to implement.

The purification of spirulina phycocyanin by adding an acid to a crude phycocyanin solution has been described (TN 2009000406, JP 2004359638, JP 06271783, CN 106190853). However, this method does not allow selective precipitation and therefore purification of phycocyanin, considering that the isoelectric point of phycocyanin of the genus spirulina is about 4.5 and close to the isoelectric point of a large number of other proteins. Similarly, some have described the use of salicylic acid to precipitate phycocyanin from spirulina (WO 2016/030643). The use of such acids results in non-selective precipitation of PC and the resulting precipitate is particularly difficult to redissolve. The same results were obtained with Rhodophyta PC (Galdieria PC).

The phycocyanin extraction method generally includes precipitating organic substances other than phycocyanin present in an aqueous crude extract of microalgae fermentation to retain phycocyanin in a supernatant, which is to be filtered, and then precipitating phycocyanin (JP 2004359638). However, some organic compounds, particularly complex polysaccharides such as glycogen, remain soluble under the same conditions as phycocyanin.

In industrial phycocyanin purification processes, a filtration (ultrafiltration) step can be used to remove water, thereby concentrating the phycocyanin and removing small molecules (proteins, ions, organic acids, etc.) that are smaller than the cut-off threshold of the filter used in order to obtain the purest phycocyanin possible. However, the cut-off threshold of the filter is below the size of glycogen, it is not removed and increases the viscosity of the retentate, thereby reducing the filtration rate. The concentration-dependent viscosity effect of glycogen has been demonstrated using purified glycogen from the sulforhodophyta origin (Martinez-Garcia et al, 2017).

Furthermore, the purified phycocyanins obtained retain high levels of these sugars, which may alter the quality of the phycocyanins, in particular their tinctorial power, thus requiring the production and/or use of larger quantities of phycocyanin to achieve the same effect. These residual polysaccharides act as fillers, which add to the manufacturing costs of phycocyanin and may limit the commercial use of the resulting phycocyanin, for example in the preparation of food products with a low sugar content.

The aim is to improve the process for the extraction and purification of phycocyanin extracted from biomass both from a quality point of view and from an industrial and economic point of view, notably by reducing the residual sugar content, in particular the residual glycogen content, in the final product.

Disclosure of Invention

The method according to the invention comprises the selective precipitation of phycocyanin directly from crude extracts containing phycocyanin under conditions that preserve the integrity of phycobiliproteins and allow the major impurities (in particular polysaccharides, including glycogen) to remain in solution.

This selective precipitation results from the combined action of two factors, simultaneously or sequentially in any order, on the one hand the pH of the solution and on the other hand the concentration of phycocyanin.

The method according to the invention is particularly suitable for the purification of acidic pH-resistant phycocyanins produced by prototheca sulfidophila.

Drawings

Figure 1 shows the mass of the precipitate obtained at different phycocyanin concentrations and different pH in the initial solution.

FIG.2 shows the variation of phycocyanin concentration in the supernatant with pH after recovery of the precipitate for different phycocyanin concentrations.

Examples

Materials and methods

The strain is as follows: prototheca sulphurophila (also known as thermorhodophyta) UTEX # 2919.

The culture conditions are as follows:

biomass was obtained by fed-batch fermentation using the conditions described in patent WO 2017050918 a 1.

Extraction conditions are as follows:

use ofThe cells were mechanically ground by KD ball mill (Willy a. bachofen AG Maschinenfabrik). Since Phycocyanin (PC) is a hydrophilic molecule, it is prepared by using alkali (NaOH, KOH, NH)₄OH, etc.) or acids (H)₂SO₄Citric acid, etc.) to a desired value and extracting it with water. After separating cell debris by centrifugation at 10000g for 10min at room temperature, the crude PC extract was recovered. The crude extract is concentrated by tangential filtration with a ceramic or organic membrane having a cut-off threshold that allows retention of phycocyanin. The sample is then centrifuged to separate the pellet from the supernatant. The mass of the precipitate was measured with a precision balance. The pellet was resuspended in an aqueous solution of pH 7 which was allowed to re-dissolve in order to quantify the precipitated phycocyanin.

PC determines:

estimation of phycocyanin content and purity index was performed by making absorbance measurements using the method described by Moon et al (Moon et al, Korean j. chem. eng.,2014, 1-6).

Example 1: effect of concentration and pH on Phycocyanin (PC) precipitation and purification

PC of initial concentration 1g/L and a crude phycocyanin solution of initial purity 1.6 were concentrated by tangential filtration at pH 4 to obtain a retentate of concentration 20g/L, then 30g/L and then 45 g/L. An increase in the purity of the product can be observed during the filtration, which does not exceed a value of 2, however, despite the degree of concentration of the product. As can be seen in FIG.1, for a concentration of 20g/L, the precipitation of phycocyanin was low at pH 4 and increased slightly as the pH increased towards higher values (FIG. 1). At the same time, measurements of the concentration of soluble phycocyanin in the supernatant during this pH increase showed a relatively small decrease. This phenomenon of precipitation due to pH changes is much more pronounced for PC concentrations of 30g/L and maxima appear at values of 4.5 and 5.5 (FIGS. 1 and 2). After further filtration and concentration of phycocyanin to soluble values of up to 40g/L, formation of a significant precipitate was observed during filtration even before the pH change (fig. 1). As before, the pH change increases the precipitation of phycocyanin.

By tangential filtration, the purity of the phycocyanin decreases and the purity of the precipitate collected and redissolved at pH 7 is reversed. This indicates that phycocyanin precipitates preferentially and that phycocyanin can be redissolved under more favorable pH conditions.

Table 1 reports the measurement of phycocyanin purity after redissolving the precipitated phycocyanin precipitate at pH 7.5.

TABLE 1

Example 2: concentration and pH effects on precipitation and purification of PC from enzymatically digested samples.

In this example, the crude solution was subjected to enzymatic digestion to degrade glycogen present. Enzymatic degradation was performed with α 1-4 glucuronidase ("Pectinex Ultra SP-L") and α 1-6 glucosidase ("Novozyme 26062") at room temperature and pH 4.

Enrichment was performed by tangential filtration to reach a phycocyanin concentration of several tens g/L, and then pH adjustment was performed, thereby causing precipitation. Samples of pH 4.5, 5 and 5.5 were taken, and the residual soluble phycocyanin was measured and the precipitated phycocyanin was measured after collecting the precipitate and re-dissolving with a buffer solution of pH 7.5.

Similarly to the previous examples, the precipitation was remarkable for a pH range comprised between 4.5 and 5.5, and the purity level in this case reached values higher than 3.8 after re-dissolution of the precipitated phycocyanin. Enzymatic digestion of glycogen does not affect purification by precipitation. The purity index values of phycocyanin after precipitation by adjustment to an unstable pH and also after re-dissolution of the phycocyanin precipitate are given in table 2 below.

TABLE 2

Reference to the literature

-Cruz de Jesùs et al.,Int J Food Nutr Sci(2016)3(3):1-0

-Martinez-Garcia et al.,Int J Biol Macromol.(2016)89:12-8

-Martinez-Garcia et al.,Carbohydrate Polymers(2017)169:75-82

-Moon et al.,2014Korean J.Chem.Eng.,2014,1-6

-TN 2009000406,US 6,074,854,US 5,817,498,US 2017/159090,WO 2009/075682,WO 2016/030643,WO 2017/050917,WO 2017/050918,WO 2017/093345,WO 2018/178334

Detailed Description

The present invention relates to a process for extracting phycocyanin from a solution containing one or more phycocyanin (also called initial phycocyanin solution), comprising a selective precipitation step which comprises, on the one hand, adjusting the pH of the initial solution to a selected value within the pH range in which said phycocyanin is less soluble (also called unstable range), and, on the other hand, concentrating the phycocyanin in the solution to facilitate its precipitation, and then a step of recovering the precipitated phycocyanin.

Both the acts of pH adjustment and concentration may be performed simultaneously or sequentially, by adjusting the pH of the initial solution prior to concentration or by concentrating the initial solution prior to adjusting the pH.

Surprisingly, the different concentration conditions mean that only phycocyanin precipitates and other products, in particular polysaccharides, which can be described as impurities, remain in solution.

Therefore, it is possible to recover the precipitated phycocyanins by separating them from the solution and, if necessary, drying them to obtain purified phycocyanin powder.

The process according to the invention not only allows extraction of the phycocyanin from the solution, but also its purification in the same step, the phycocyanin obtained being particularly pure and low in residual sugars.

The method according to the invention is particularly suitable for purifying a phycocyanin solution extracted from a phycocyanin producing microbial culture that also produces glycogen, in particular in the context of an industrial phycocyanin producing process comprising culturing the microorganism and then recovering the biomass produced to extract the phycocyanin and recovering the phycocyanin from this biomass.

The method is particularly suitable for phycocyanin produced by a high-level glycogen producing microorganism, and is particularly suitable for extracting and purifying phycocyanin from biomass containing more than 10% glycogen based on total dry matter.

Phycocyanin-producing microorganisms are well known and include algae (or microalgae) of the order cyaniliales. The order Cyanidales includes the family Cyanididaceae or Galdiiacea, which are themselves subdivided into the genera Cyanididocyzyzon, Cyanidium or Rhodophyta (Galdieria), among which the species Cyanididocyzyzon merolene 10D, Cyanididocyzyzon merolene DBV201, Rhodophyta (Cyanidium caldarium), Cyanidium dayield, Cyanidium maximum, Cyanidium partitum, Cyanidium rumpens, Galdiia dayield, Galdiia partita or Thiophthal origin. The strain thiotropic primary red algae (also known as thermorhodophyta) UTEX 2919 may be mentioned in particular.

There may also be mentioned known phycocyanin producers, such as filamentous cyanobacteria of the genus Arthrospira (Arthrospira), which are industrially cultured under the generic name of the genus Spirulina.

Among the microorganisms mentioned above, microorganisms which produce phycocyanin having a high glycogen content, particularly Cyanidioschyzon, Cyanidium and Rhodophyta species, more particularly Thiophthalomorph.

Industrial methods for culturing phycocyanin-producing microorganisms are well known to those skilled in the art. Patent applications WO 2017/093345, WO 2017/050917 may be mentioned in particular.

The recovery of phycocyanin from biomass is also known to the skilled person. Patent application WO 2018/178334 may be mentioned in particular. It usually requires a step of cellular, mechanical or enzymatic lysis in order to release the produced phycocyanin in the cell compartment of the microorganism. This cleavage is advantageously carried out at a pH which favours the dissolution of phycocyanin. Such cell lysis will generally produce a phycocyanin solution comprising suspended organic material (referred to as a crude suspension) which can be separated by conventional filtration methods. A crude phycocyanin solution is then obtained, which may be further purified by conventional ultrafiltration methods to remove low molecular weight organic residues to obtain a refined solution from which phycocyanin may be obtained by conventional precipitation and drying methods. Tangential filtration on ceramic or organic membranes, such as polyethersulfone hollow fibers, may be mentioned in particular. The threshold of these filters can be selected to separate molecules with molecular weights above or below the target phycobiliprotein.

The method according to the invention is particularly suitable for the purification of acidic pH-resistant phycocyanin solutions, in particular phycocyanin as described in application WO 2017/050918.

In particular, the method according to the invention is used for the purification of acidic pH-resistant phycocyanins produced by prototheca sulfidophila, more particularly in an industrial process for the production of these phycocyanins for the cultivation of prototheca sulfidophila by means of fermenters.

The process is advantageously carried out to extract phycocyanin from a crude juice obtained from the biomass of a phycocyanin producing microorganism.

Advantageously, the initial phycocyanin solution, in particular the crude juice, comprises from 0.1 to 10g/L of phycocyanin.

Concentration comprises removing water in order to obtain a phycocyanin content of at least 15g/L, preferably at least 20g/L, more preferentially at least 30g/L or even at least 40 g/L.

This concentration can be defined as% volume loss based on the phycocyanin content in the initial solution.

In an industrial phycocyanin production process, the crude juice will advantageously contain at least 1g/L of phycocyanin. In this case, concentrating will include removing at least 93% of the initial liquid volume.

Concentration is accomplished by any known method that allows for the removal of water under conditions that preserve the integrity of the phycocyanin. Mention may be made of the water evaporation process, in particular to promote this evaporation under reduced pressure under temperature conditions which guarantee the integrity of the phycocyanin without affecting its coloring power. Mention may also be made of methods that allow the removal of liquids, such as tangential filtration with a pore size that allows water and small molecules in solution to pass through but retains proteins.

These filtration methods and the devices for their implementation are well known to those skilled in the art, in particular the Spectrum Labs TFF system from Repligen. For phycocyanin, it is advantageous to select filters with a pore size of 50kD to 100kD, in particular polyethersulfone or polysulfone filters.

pH adjustment involves adding an acid or base to the initial or concentrated solution in order to achieve a pH value in the unstable range. The range of instability will depend on the phycocyanin to be purified and in particular the microorganism from which it is produced. Typically, this instability ranges from 4.5 to 5.5, especially for acidic pH-resistant phycocyanins as described above.

For these acid pH resistant phycocyanins, cell lysis is performed at an acid pH, preferably below 4.5, usually about 4, even as low as 3.

The pH adjustment then consisted of: base is added to achieve a pH in the unstable range.

According to a first embodiment of the invention, the method first comprises concentrating the initial juice. In this case, the concentration is carried out at a pH which favours the dissolution of phycocyanin (i.e.outside the unstable range). For the above-mentioned acidic pH-resistant phycocyanins, these pH values favoring the solubilization of phycocyanin will advantageously be lower than 4 or higher than 5.

According to another preferred embodiment of the invention, the method comprises first adjusting the pH to an unstable range and then concentrating the solution until the phycocyanin precipitates.

The method may then be described as obtaining a solution of unstable pH from the initial solution, and then concentrating the solution of unstable pH to cause precipitation of phycocyanin. When precipitation of phycocyanin is observed, a percentage of volume reduction will be reached.

This selective precipitation step is advantageously carried out at room temperature. Of course, the person skilled in the art will be able to vary the temperature in a manner that facilitates precipitation, for example by lowering the temperature to carry out the second part of the step (concentration or pH adjustment), during which precipitation takes place.

The polysaccharide, in particular glycogen, in the solution can then be recovered by conventional polysaccharide precipitation methods, for example by addition of ethanol (Martinez-Garcia et al, 2016), which can subsequently also be purified.

According to a particular embodiment of the invention, the polysaccharides contained in the initial solution with phycocyanin are subjected to an enzymatic cleavage, which favours their retention in solution. These traces of polysaccharide, which are already low and which are reduced even further when they are cleaved into even more soluble low molecular weight oligosaccharides, are likely to be carried away by the precipitation of phycocyanin. Furthermore, when the concentration step is performed by tangential filtration, the low molecular weight oligosaccharides are removed together with other small molecules in the solution, which is advantageous for obtaining a solution with an even higher phycocyanin content.

In particular, the enzymatic cleavage of glycogen is carried out at room temperature at a pH of less than or equal to 5, preferably of about 4.5.

These temperature and pH conditions are particularly suitable for retaining phycocyanin during the enzymatic reaction.

The enzyme active at acidic pH and room temperature is selected from known enzymesAn enzyme having α 1-4 glucuronidase, α 1-4 glucosidase (or α -glucosidase) activity. There will be mentioned in particular pectinases known to degrade pectin, and in particular pectinases extracted from filamentous fungi such as Aspergillus (Aspergillus), more in particular from Aspergillus aculeatus (Aspergillus aculeatus), such as the pectinases under the name given by the Novozymes companyEnzymes are sold.

In addition to α 1-4 glucuronidase or α 1-4 glucosidase, enzymatic cleavage of glycogen can also be achieved with α 1-6 glucosidase. Alpha 1-6 glucosidases active under the pH and temperature conditions set forth above are also known to the skilled person. In particular, these are pullulanases which are known to hydrolyze the α 1-6 glycosidic bonds of amylopectin, in particular to remove starch branches.

These are generally enzymes extracted from bacteria, in particular from the genus Bacillus (Bacillus). US 6,074,854, US 5,817,498 and WO 2009/075682 describe such pullulanases extracted from Bacillus deramificans or Bacillus acidophilus (Bacillus acidopulullyticus). Commercially available pullulanases are also known, in particular under the names "Promozyme D2" (Novozymes), "Novozym 26062" (Novozymes) and "Optimax L1000" (DuPont-Genencor).

It is noted that pullulanase/alpha-amylase mixtures are described in the prior art, but in particular for the production of glucose syrup from starch (US 2017/159090).

The skilled person will know how to determine appropriate reaction conditions to optimally reduce the amount of glycogen as a function of the initial glycogen content in the solution to be treated, the amount of enzyme used and the desired purity of the phycocyanin produced.

Recovery of the solid precipitated phycocyanin is carried out by any method known to the skilled person, such as filtration or centrifugation.

The skilled person will be able to envisage any method of recovering solids to reduce the volume to be treated by filtration or centrifugation.

This recovery can be performed discontinuously, batchwise or continuously, with the addition of an initial solution to compensate for the recovery of the solid phycocyanin.

These successive recovery steps will advantageously be carried out in the case of concentration by tangential filtration of the solution of unstable pH, the person skilled in the art being able to adjust the respective flow rates of water removal and of solution supply of unstable pH to promote precipitation of phycocyanin.

Such a continuous process would be particularly suitable for treating the initial solution, wherein the polysaccharides, in particular glycogen, would undergo enzymatic cleavage, which facilitates their removal by tangential filtration together with water and other soluble small molecules.

The present invention also relates to a method for producing phycocyanin by microbial fermentation, comprising the steps of: (i) culturing the microorganism to obtain a biomass enriched in phycocyanins, (ii) recovering the biomass and performing cell lysis to solubilize the released phycocyanins in the cell particle suspension, (iii) clarifying the previously obtained suspension to obtain a crude phycocyanin solution, and (iv) recovering the phycocyanins from the previously obtained crude solution, characterized in that the recovery of the phycocyanins comprises a selective precipitation step as defined above.

The recovered solids may then be dried by any suitable method and, if desired, ground.

The recovered solid comprising phycocyanin may also be subjected to purification by methods known to the skilled person, such as diafiltration.

Methods of fermentation culture, biomass recovery, lysis and clarification are well known to the skilled person, in particular those described in patent applications WO 2017/050917, WO 2017/093345 and WO 2018/178334.

The selective precipitation carried out according to the invention reduces the energy required for producing phycocyanin powder from the starting solution, in particular from the crude juice, both in terms of the amount of material to be treated and in terms of the energy required for drying the solid phycocyanin and grinding it.

The phycocyanin obtained by this process has a purity index of at least 2, preferably at least 3, or even higher than 4.

This purity index was measured by absorbance measurements using the method described by Moon et al (2014).

Advantageously, the phycocyanin obtained is phycocyanin having a glycogen/phycocyanin ratio (by dry weight) lower than 6, advantageously lower than 4, preferably lower than 3, more preferentially lower than 2.5, even more preferentially lower than 1.

The invention also relates to the use of the phycocyanin obtained as a colorant, in particular as a food colorant. The invention also relates to a solid or liquid food, in particular a beverage, comprising a low glycogen phycocyanin according to the invention.