PHA extraction method for adjusting chromaticity of PHA material, product therefrom, and use and preparation methods therefor

Abstract

The present invention relates to the technical field of new materials, and in particular to a PHA extraction method for adjusting the chromaticity of a PHA material, a product therefrom, and a use thereof. The extraction method comprises: carrying out solid-liquid separation on a halophilic bacteria fermentation liquid to obtain a concentrated bacterial liquid; and adjusting the pH of the concentrated bacterial liquid to be acidic, adding a complex enzyme formulation I to the concentrated bacterial liquid, enabling the average particle size of the resulting emulsion to be 1-10 μm under a mechanical action, then adjusting the pH to be alkaline, adding a complex enzyme formulation II to the emulsion, and continuing the mechanical action to obtain a wall-disrupted liquid. The aqueous phase extraction method of the present invention has mild conditions and is suitable for large-scale continuous production. By means of the cooperation of the special enzyme formulations and special equipment, various impurities causing yellowing are reduced, so that the chromaticity of PHA pellets obtained by further processing is low. Moreover, during thermoplastic processing, the decrease of the molecular weight can be effectively inhibited, the increase of MFR is inhibited, the toughness of various formed bodies is improved, and the risk of dissolution in a solvent environment in subsequent applications is small.

Description

A PHA extraction method for adjusting the color of PHA material and its product and application preparation method

[0001] Cross-references

[0002] This application claims priority to Chinese Patent Application No. 2023117410854, filed on December 18, 2023, entitled “A PHA Extraction Method for Adjusting the Colorimetry of PHA Materials, Its Products and Applications,” all disclosures of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to the field of new material technology, and in particular to a PHA extraction method for adjusting the colorimetry of a PHA material, and a product and application thereof. Background Art

[0004] Polyhydroxyalkanoates (PHAs) are intracellular polyesters synthesized through microbial fermentation. They primarily serve as carbon and energy storage substances within organisms. They possess physicochemical properties similar to those of synthetic plastics, but possess many unique properties not found in synthetic plastics, including biodegradability, biocompatibility, optical activity, piezoelectricity, and gas barrier properties. PHAs have broad application prospects in biodegradable packaging, tissue engineering materials, sustained-release materials, electrical materials, and medical materials.

[0005] However, PHA obtained through microbial fermentation has a small particle size, is in powder form, and has a low bulk density, making it difficult to use directly in downstream processing. It must undergo granulation and molding to obtain a relatively regular granular molded body before it can be suitable for downstream product applications. However, during the heat processing melt granulation process, the PHA powder obtained by traditional aqueous extraction has the problem of increased granular color value (from white powder to yellow or brown granules). This is because PHA itself is a high-molecular ester substance with good hydrophobicity. During aqueous extraction, residual organic impurities such as polysaccharides and residual esters of cell membranes are difficult to remove by water washing. These residual organic impurities will produce Maillard reactions and high-temperature oxidative browning reactions during melt processing, resulting in the melt-processed product being yellowish or yellow-brown, thereby limiting its wide application, especially for molded products with relatively high color requirements.

[0006] However, there are currently few technical solutions to improve the color value of polyhydroxyalkanoates after thermal processing. Furthermore, the high degree of thermal degradation during processing leads to a significant decrease in the polyhydroxyalkanoate's melt flow rate (MFR), an increase in melt flow rate (MFR), and a decrease in toughness. This results in poor overall performance of various molded products, significantly impacting processing and use.

[0007] In the prior art, in order to solve the problem of increased color value and deterioration of toughness of polyhydroxyalkanoates after thermoplastic processing, copolymerization, addition of toughening agents, chain extenders, cross-linking agents and other methods are usually used to improve the deterioration of toughness of polymers after thermal processing, as well as the problem of increased melt mass flow rate (MFR). Since the technical solutions for changing toughness or increasing polymers require the use of organic solvents, reaction catalysts, chain extenders, etc., there are often problems such as complex processing equipment and difficult to control processing methods. Another patent CN202310046774.7 shields the color from appearing during thermal processing by adding yellowness regulator silicone oil compounds or modified silicone oil compounds, while increasing the thermal stability and toughness of the product. The disadvantage of this solution is that the introduction of new reagents increases material and process costs, while also increasing the risk of dissolution of the product when introducing solvents in specific applications. For example, silicone oil compounds can be dissolved in alcohol solvents.

[0008] Therefore, in order to prepare polyhydroxyalkanoate resin products with better quality, it is urgent to reduce the chromaticity of PHA pellets, improve the toughness and thermal degradation degree of the polymer after thermal processing, and reduce the risk of dissolution in solvent environment. Summary of the Invention

[0009] In further research, the inventors discovered that by combining specialized enzyme preparations with specialized equipment, they could remove the various impurities that cause the yellow color of granulation at the source. This method is more economical, environmentally friendly, and more amenable to industrialization. Based on this principle, they developed a method for adjusting the color of PHA materials, as well as a product and application.

[0010] Specifically, the present invention provides a PHA extraction method for adjusting the color of a PHA material, comprising:

[0011] The halophilic bacteria fermentation liquid is subjected to solid-liquid separation to obtain a concentrated bacterial liquid;

[0012] The pH of the concentrated bacterial liquid is adjusted to acidic, the first complex enzyme preparation is added thereto, and the mixture is mixed under mechanical action so that the average particle size of the obtained emulsion is 1 to 10 μm. The pH is then adjusted to alkaline, the second complex enzyme preparation is added thereto, and the cell walls are further broken by mechanical action to obtain a broken cell liquid.

[0013] The PHAs referred to in this invention refer to polyhydroxyalkanoates, which can be divided into homopolymers and copolymers based on the monomer composition. Based on the number of carbon atoms in the monomers, they can be short-chain PHAs (i.e., monomers containing C3-C5 hydroxy fatty acids) or medium-chain PHAs (i.e., monomers containing C6-C18 hydroxy fatty acids), but are not limited thereto.

[0014] PHA can be a homopolymer, including but not limited to polyhydroxypropionate (PHP), polyhydroxybutyrate (PHB), polyhydroxyoctanoate (PHO) polyhydroxyvalerate (PHV), etc., for example, poly-3-hydroxybutyrate (P3HB), poly-4-hydroxybutyrate (P4HB), poly-3-hydroxypropionate (P3HP) or poly-3-hydroxyvalerate (P3HV), etc.

[0015] PHA can be a copolymer, for example, a copolymer of 3-hydroxybutyric acid and 4-hydroxybutyric acid (P34HB), a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid (PHBHHx), a copolymer of 3-hydroxybutyric acid and 3-hydroxyvaleric acid (PHBV), a copolymer of 3-hydroxyoctanoic acid and 3-hydroxyhexanoic acid (P3HO3HHx), a terpolymer of 3-hydroxybutyric acid, 3-hydroxyvaleric acid and 3-hydroxyhexanoic acid (PHBVHHx), a terpolymer of 3-hydroxybutyric acid, 4-hydroxybutyric acid and 3-hydroxyvaleric acid (P3HB-co-4HB-co-3HV), etc.

[0016] The halophilic bacteria fermentation broth of the present invention is used as a raw material for producing polyhydroxyalkanoates, wherein the halophilic bacteria include halophilic bacteria and / or the halophilic archaea. Preferably, the halophilic bacteria are halophilic bacteria, more preferably bacteria of the genus Halomonas and their derivative strains or combinations thereof, and more preferably any species under the genus Halomonas, such as any species of Halomonas bluephagenesis, Halomonas campaniensis, Halomonas aydingkolgenesis, Halomonas aerodenitrificans, and Halomonas halocynthiae. More preferably, Halomonas bluephagenesis TD01, with a deposit registration number of CGMCC No. 4353, was obtained from Tsinghua University.

[0017] According to the PHA extraction method for adjusting the color of the PHA material provided by the present invention, the mechanical action is carried out in a high-speed mechanical mixing device, which includes a colloid mill, an ultrafine grinder, a homogenizing emulsification pump, or a high-pressure homogenizer. The mechanical mixing device can maximize the effect of the enzyme preparation and avoid insufficient enzyme action caused by uneven mass transfer.

[0018] And / or, the complex enzyme preparation one comprises two or more of lysozyme, nuclease, snailase and glucanase; preferably, the complex enzyme preparation one is mainly composed of snailase and lysozyme; further preferably, the complex enzyme preparation one comprises 4 to 6 parts of snailase and 2 to 4 parts of lysozyme by weight;

[0019] And / or, the second complex enzyme preparation includes two or more of alkaline protease, amylase, peptidase and nuclease; preferably, by weight, the second complex enzyme preparation is mainly composed of alkaline protease and peptidase; further preferably, the second complex enzyme preparation includes 3 to 6 parts of alkaline protease and 3 to 4 parts of peptidase.

[0020] The high-pressure homogenizer is also called a "high-pressure fluid nano-homogenizer". The present invention uses a high-pressure homogenizer to make the lysis mixture in a suspension state flow at high speed through a cavity with a special internal structure (high-pressure homogenization cavity) under ultra-high pressure (up to 60,000 psi), causing the lysis mixture to undergo a series of changes in physical, chemical, and structural properties, thereby achieving a homogenization effect.

[0021] The colloid mill of the present invention is made of stainless steel and relies on the relative movement of the toothed inclined surfaces of the grinding discs, one of which rotates at high speed and the other is stationary, so that the lysis mixture passing through the toothed inclined surfaces is subjected to very large shear force and friction force. At the same time, under the action of complex forces such as high-frequency vibration and high vortex, the lysis mixture is effectively ground, emulsified, crushed, dispersed and homogenized, thereby achieving a fine ultrafine particle crushing effect.

[0022] The ultrafine pulverizer of the present invention has the same working principle as the above-mentioned colloid mill.

[0023] The homogenizing emulsifying pump in the present invention can efficiently, quickly and evenly transfer one or more phases (liquid, solid, gas) into another immiscible continuous phase (usually liquid), while under normal circumstances the phases are immiscible. When external energy is input, the two materials are reorganized into a homogeneous phase. Due to the high tangential speed generated by the high-speed rotation of the rotor and the strong kinetic energy brought by the high-frequency mechanical effect, the material is subjected to strong mechanical and hydraulic shearing, centrifugal extrusion, liquid layer friction, impact tearing and turbulence in the narrow gap between the stator and rotor, forming a suspension (solid / liquid), emulsion (liquid / liquid) and foam (gas / liquid). As a result, the immiscible solid phase, liquid phase and gas phase can be instantly and evenly dispersed and emulsified, and a stable emulsified product is obtained through high-frequency circulation.

[0024] The mechanical action referred to in the present invention is achieved by the above-mentioned specific high-speed mechanical mixing equipment, while the mixing and stirring in other situations in the extraction method can adopt the above-mentioned specific equipment or conventional mixing devices, such as electric stirrers, etc., and conventional mixing can be achieved by controlling a certain stirring speed.

[0025] According to the PHA extraction method for adjusting the color of the PHA material provided by the present invention, the amount of the compound enzyme preparation 1 added is 0.01% to 0.1% (w / v) of the volume of the material solution during enzymatic hydrolysis; the pH of the concentrated bacterial solution is adjusted to acidic by adjusting the pH value to 5.5 to 6.5;

[0026] And / or, the addition amount of the second complex enzyme preparation is 0.01% to 0.1% (w / v) of the volume of the feed solution during enzymatic hydrolysis; preferably, the pH is adjusted to alkaline by adjusting the pH value to 8.0 to 12.0, preferably 9.0 to 11.0.

[0027] The PHA extraction method for adjusting the color of the PHA material provided by the present invention comprises: performing solid-liquid separation on the cell wall-breaking liquid to obtain a crude PHA product, and performing enzymatic hydrolysis and purification of the crude PHA product using a composite enzyme preparation three under alkaline conditions;

[0028] Preferably, the complex enzyme preparation three comprises two or more of phospholipase, lipohydrolase, phosphatase, glycosidase, nuclease and polysaccharide hydrolase.

[0029] According to the PHA extraction method for adjusting the color of the PHA material provided by the present invention, the addition amount of the composite enzyme preparation three is 0.01% to 0.1% (w / v) of the feed liquid volume during enzymatic hydrolysis; before the enzymatic hydrolysis and purification of the composite enzyme preparation three, an appropriate amount of water is added to the crude PHA product so that the feed liquid volume during enzymatic hydrolysis and purification is the same as the feed liquid volume during enzymatic hydrolysis of the composite enzyme preparation one or the composite enzyme preparation two;

[0030] The temperature during enzymatic hydrolysis and purification is 30°C to 80°C, preferably 40°C to 60°C;

[0031] The pH value during enzymatic purification is 8.5 to 13.0, preferably 10.0 to 12.0.

[0032] The PHA extraction method for adjusting the color of the PHA material provided by the present invention comprises: adding a surfactant to the material after the enzymatic hydrolysis and purification, mixing and stirring, and performing solid-liquid separation, washing and drying the obtained liquid to obtain PHA;

[0033] Preferably, the surfactant is mainly composed of anionic surfactants; the anionic surfactant is preferably one or more of sodium dodecylbenzenesulfonate, sodium lauryl sulfate, sodium octadecyl sulfate, etc.;

[0034] More preferably, the amount of surfactant added is 0-5% (w / v), preferably 0.02%-0.5% (w / v), of the volume of the enzymatic hydrolysis and purification.

[0035] The PHA extraction method for adjusting the color of a PHA material provided by the present invention comprises:

[0036] (1) taking a halophilic bacteria fermentation liquid and performing solid-liquid separation to obtain a concentrated bacterial liquid with an impurity removal rate of ≥90%;

[0037] (2) After heating the concentrated bacterial solution obtained in step (1), the first complex enzyme preparation is added, and the mixture is mixed using a high-speed mechanical mixing device for 3 to 30 minutes, stirred at the temperature, and the pH is adjusted. Then, the second complex enzyme preparation is added, and the mixture is mixed using a high-speed mechanical mixing device for 3 to 30 minutes, stirred at the temperature, and the cell wall-broken solution is obtained;

[0038] When using high-speed mechanical mixing equipment, the method adopted by the present invention can make the average particle size of the emulsion 1-10 μm. However, due to the agglomeration phenomenon of the product itself after crushing, the actual particle size detected during testing after sampling will be larger.

[0039] (3) performing solid-liquid separation on the broken cell liquid obtained in step (2) to obtain a crude PHA product with an impurity removal rate of ≥70%;

[0040] (4) The crude PHA obtained in step (3) is subjected to enzymatic hydrolysis and purification using the composite enzyme preparation 3 for a certain period of time, and then a surfactant is added thereto and mixed and stirred for a certain period of time;

[0041] (5) The liquid obtained in step (4) is subjected to solid-liquid separation to remove more than 90% of impurities, washed, and dried to obtain pure PHA.

[0042] In step (5), the solid-liquid separation can be carried out by centrifugation or filtration.

[0043] Among them, the filtration method can use equipment such as plate and frame filter press, flat plate centrifuge, etc. RO water is used to clean the filter cake online or offline, and the moisture content of the filter cake is <50%.

[0044] Among them, the filtration method can use equipment such as cup centrifuge, stack centrifuge, etc., RO water is used to clean the heavy phase online or offline, and the solid content of the concentrate is 15-40%, preferably 20-30%.

[0045] There is no particular limitation on the drying method of the filter cake or concentrated liquid, and methods such as room temperature ventilation drying, heating oven drying, spray drying, and flash evaporation may be selected.

[0046] The PHA extraction method for adjusting the color of a PHA material provided by the present invention comprises:

[0047] In step (1), the solid-liquid separation can be carried out by centrifugation or filtration, preferably centrifugation;

[0048] and / or, in step (1), the solid content of the concentrated bacterial solution is 13-30%, preferably 15-25%;

[0049] and / or, in step (2), heating to a temperature of 30°C to 95°C, preferably 40°C to 80°C, more preferably 45°C to 70°C;

[0050] And / or, in step (2), stirring is continued during the heat preservation in each step, the stirring speed is 100 to 1000 rpm / min, and the stirring time is 30 to 90 min;

[0051] And / or, in step (3), the solid-liquid separation is performed by centrifugation or filtration;

[0052] And / or, in step (4), the stirring speed required for enzymatic purification is 100 to 1000 rpm / min, and the stirring time is 10 to 60 min.

[0053] And / or, in step (4), after adding the surfactant, the required stirring speed is 100 to 1000 rpm / min, and the stirring time is 10 to 60 min.

[0054] The present invention also provides a PHA product obtained by the PHA extraction method for adjusting the color of the PHA material as described above;

[0055] Preferably, the total nitrogen content of the PHA obtained by the extraction method is within 0.02%.

[0056] The present invention also provides a PHA pellet or PHA material obtained by melt processing the PHA obtained by the PHA extraction method for adjusting the color of the PHA material as described above or the PHA product as described above;

[0057] Preferably, the B value of the PHA pellets is within 22;

[0058] The present invention provides a PHA extraction method for adjusting the color of a PHA material, as well as its products and applications. By selecting an economical, environmentally friendly, and more industrially amenable aqueous extraction method, the method employs mild conditions and is suitable for large-scale continuous production. The method utilizes a special enzyme preparation and specific equipment to remove, at the source, various impurities that cause yellowing during melt processing, such as residual organic impurities such as polysaccharides and residual esters of cell membranes. The resulting PHA product has a higher purity, and the color of the PHA pellets obtained by further processing is reduced. Furthermore, the PHA product obtained by this extraction method can effectively suppress the reduction of the PHA molecular weight and the increase in the melt mass flow rate (MFR) during thermoplastic processing, thereby improving the toughness of various molded bodies. Furthermore, the PHA product is more stable during processing of various molded bodies, and has a low risk of dissolution in solvent environments in subsequent applications. DETAILED DESCRIPTION

[0059] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0060] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0061] The complex enzyme preparations of the present invention, including complex enzyme preparation 1, complex enzyme preparation 2 and complex enzyme preparation 3, are all prepared by weighing various enzyme preparation powders according to corresponding proportions and pre-mixing them in a mixer before use.

[0062] The comparison of the English abbreviations and the Chinese full names of the present invention is shown in the following table:

[0063] Example 1

[0064] (1) Take 4 L of fermentation broth of PHB-producing halophilic bacteria and centrifuge it at 8000 rpm for 10 min. Discard the supernatant and collect the bacterial precipitate. Resuspend it in water to 4 L and centrifuge it again at 8000 rpm for 10 min. Discard the supernatant and collect the bacterial precipitate.

[0065] (2) The bacterial precipitate obtained in step (1) was resuspended to 3.2 L, heated to 52° C., the pH value was adjusted to 6.0, 0.04% of a composite enzyme preparation 1 (consisting of 4 parts of snail enzyme, 4 parts of lysozyme and 2 parts of nuclease by mass) was added, and the mixture was treated with a colloid mill for 5 min to make the average particle size of the emulsion 1 to 10 μm, and then stirred at 400 rpm / min and 52° C. for 45 min; then the pH value was adjusted to 10.5, 0.02% (volume percentage) of a composite enzyme preparation 2 (consisting of 6 parts of alkaline protease, 3 parts of peptidase and 1 part of amylase by mass) was added, the mixture was treated with a colloid mill for 5 min, and then stirred at 400 rpm / min and 52° C. for 45 min.

[0066] (3) The obtained solution in step (2) was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the crude PHA product was collected. An appropriate amount of water was added to the crude PHA product to prepare a 3.2 L mixed solution, and the mixture was stirred and mixed.

[0067] (4) The crude PHA mixture of step (3) was heated to 45° C., the pH value was adjusted to 8.5, 0.02% of complex enzyme preparation III (consisting of 5 parts of phospholipase, 3 parts of polysaccharide hydrolase and 2 parts of nuclease by mass) was added and stirred at 400 rpm / min for 30 min; 0.05% of sodium lauryl sulfate was added and stirred at 400 rpm / min for 30 min to proceed to the next step.

[0068] (5) The PHA obtained in step (4) was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, the precipitate was collected and resuspended in water to 4 L, and the mixture was centrifuged again at 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected and dried in an oven at 60°C to obtain a PHA powder product.

[0069] Example 2

[0070] (1) Take 4 L of fermentation broth of P(3HB-co-3HV) halophilic bacteria and centrifuge it at 8000 rpm for 10 min. Discard the supernatant and collect the bacterial precipitate. Resuspend it in water to 4 L and centrifuge it again at 8000 rpm for 10 min. Discard the supernatant and collect the bacterial precipitate.

[0071] (2) The bacterial precipitate obtained in step (1) was resuspended to 3.2 L, heated to 52° C., the pH value was adjusted to 6.0, 0.03% of a composite enzyme preparation 1 (consisting of 6 parts of snail enzyme, 3 parts of lysozyme and 1 part of glucanase by mass) was added, and the mixture was treated with a colloid mill for 5 min to make the average particle size of the emulsion 1 to 10 μm, and then stirred at 400 rpm / min and 52° C. for 45 min; then the pH value was adjusted to 10.5, 0.03% (volume percentage) of a composite enzyme preparation 2 (consisting of 3 parts of alkaline protease, 3 parts of peptidase, 3 parts of nuclease and 1 part of amylase by mass) was added, the mixture was treated with a colloid mill for 5 min, and then stirred at 400 rpm / min and 52° C. for 45 min.

[0072] (3) The obtained solution in step (2) was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the crude PHA product was collected. An appropriate amount of water was added to the crude PHA product to prepare a 3.2 L mixed solution, and the mixture was stirred and mixed.

[0073] (4) The crude PHA mixture of step (3) was heated to 45° C., the pH value was adjusted to 8.5, 0.02% of complex enzyme preparation III (consisting of 5 parts of phospholipase, 3 parts of lipase, and 2 parts of polysaccharide hydrolase by mass) was added and stirred at 400 rpm / min for 30 min; 0.04% of sodium dodecylbenzene sulfonate was added and stirred at 400 rpm / min for 30 min to proceed to the next step.

[0074] (5) The PHA obtained in step (4) was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, the precipitate was collected and resuspended in water to 4 L, and the mixture was centrifuged again at 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected and dried in an oven at 60°C to obtain a PHA powder product.

[0075] Example 3

[0076] (1) Take 4 L of fermentation broth of P34HB-producing halophilic bacteria and centrifuge it at 8000 rpm for 10 min. Discard the supernatant and collect the bacterial precipitate. Resuspend it in water to 4 L and centrifuge it again at 8000 rpm for 10 min. Discard the supernatant and collect the bacterial precipitate.

[0077] (2) The bacterial precipitate obtained in step (1) was resuspended to 3.2 L, heated to 52° C., the pH value was adjusted to 6.0, 0.04% of a composite enzyme preparation 1 (consisting of 6 parts of snail enzyme and 4 parts of lysozyme by mass) was added, and the mixture was treated with a colloid mill for 5 min to reduce the average particle size of the emulsion to 1 to 10 μm, followed by stirring at 400 rpm / min and 52° C. for 45 min; the pH value was then adjusted to 10.5, 0.04% (volume percentage) of a composite enzyme preparation 2 (consisting of 6 parts of alkaline protease and 4 parts of peptidase by mass) was added, the mixture was treated with a colloid mill for 5 min, and then stirred at 400 rpm / min and 52° C. for 45 min.

[0078] (3) The obtained solution in step (2) was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the crude PHA product was collected. An appropriate amount of water was added to the crude PHA product to prepare a 3.2 L mixed solution, and the mixture was stirred and mixed.

[0079] (4) The crude PHA mixture of step (3) was heated to 45° C., the pH value was adjusted to 8.5, 0.03% of complex enzyme preparation III (4 parts of polysaccharide hydrolase, 2 parts of phospholipase and 4 parts of lipolyase by mass) was added and stirred at 400 rpm / min for 30 min; 0.04% of sodium dodecylbenzene sulfonate was added and stirred at 400 rpm / min for 30 min to proceed to the next step.

[0080] (5) The PHA obtained in step (4) was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, the precipitate was collected and resuspended in water to 4 L, and the mixture was centrifuged again at 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected and dried in an oven at 60°C to obtain a PHA powder product.

[0081] Example 4

[0082] (1) Take 3.2 tons of fermentation liquid of P34HB halophilic bacteria and put it into the tank. Use a disc centrifuge to wash it 4 times with 1 minute and 1 wash, and concentrate the heavy phase to 0.8 tons.

[0083] (2) The concentrated solution obtained in step (1) was added with water to a volume of 3.2t, heated to 52°C, adjusted to a pH of 6.0, added with 0.04% of a composite enzyme preparation 1 (consisting of 6 parts of snail enzyme, 2 parts of lysozyme and 2 parts of nuclease by mass), and circulated for 30 min using a homogenizing emulsification pump to adjust the average particle size of the emulsion to 1 to 10 μm, and then stirred at 400 rpm / min and 52°C for 45 min; then the pH was adjusted to 10.5, and 0.04% (volume percentage) of a composite enzyme preparation 2 (consisting of 6 parts of alkaline protease and 4 parts of peptidase by mass) was added, and treated with a colloid mill for 5 min, and then stirred at 400 rpm / min and 52°C for 45 min.

[0084] (3) The liquid obtained in step (2) was washed twice with a disc centrifuge, the volume was fixed to 3.2t, and stirred to mix.

[0085] (4) The crude PHA mixture of step (3) was heated to 45° C., the pH value was adjusted to 8.5, 0.03% of complex enzyme preparation III (4 parts of polysaccharide hydrolase, 2 parts of phospholipase and 4 parts of lipolyase by mass) was added and stirred at 400 rpm / min for 30 min; 0.05% of sodium lauryl sulfate was added and stirred at 400 rpm / min for 30 min to proceed to the next step.

[0086] (5) The PHA obtained in step (4) was washed four times with a disc centrifuge, the heavy phase was concentrated to 0.8t, and the emulsion was spray-dried to obtain a PHA powder product.

[0087] Example 5

[0088] (1) Take 4 L of fermentation broth of P (3HB-co-4HB-co-3HV) halophilic bacteria and centrifuge it at 8000 rpm for 10 min. Discard the supernatant and collect the bacterial precipitate. Resuspend it in water to 4 L and centrifuge it again at 8000 rpm for 10 min. Discard the supernatant and collect the bacterial precipitate.

[0089] (2) The bacterial precipitate obtained in step (1) was resuspended to 3.2 L, heated to 52° C., the pH value was adjusted to 6.0, 0.04% of a composite enzyme preparation 1 (consisting of 4 parts of snail enzyme, 4 parts of lysozyme and 2 parts of nuclease by mass) was added, and the mixture was treated with a colloid mill for 5 min to make the average particle size of the emulsion 1 to 10 μm, and then stirred at 400 rpm / min and 52° C. for 45 min; then the pH value was adjusted to 10.5, 0.02% (volume percentage) of a composite enzyme preparation 2 (consisting of 6 parts of alkaline protease, 3 parts of peptidase and 1 part of amylase by mass) was added, the mixture was treated with a colloid mill for 5 min, and then stirred at 400 rpm / min and 52° C. for 45 min.

[0090] (3) The obtained solution in step (2) was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the crude PHA product was collected. An appropriate amount of water was added to the crude PHA product to prepare a 3.2 L mixed solution, and the mixture was stirred and mixed.

[0091] (4) The crude PHA mixture of step (3) was heated to 45°C, the pH value was adjusted to 8.5, 0.02% of complex enzyme preparation III (consisting of 5 parts of phospholipase, 3 parts of lipase and 2 parts of nuclease by mass) was added and stirred at 400 rpm / min for 30 minutes; 0.04% of sodium dodecyl sulfate was added and stirred at 400 rpm / min for 30 minutes to proceed to the next step.

[0092] (5) The PHA obtained in step (4) was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, the precipitate was collected and resuspended in water to 4 L, and the mixture was centrifuged again at 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected and dried in an oven at 60°C to obtain a PHA powder product.

[0093] Example 6

[0094] The method is basically the same as Example 1, except that the colloid mill used in the enzymatic hydrolysis of the complex enzyme preparation in step (2) is replaced by the ultrafine grinder.

[0095] Example 7

[0096] The method is basically the same as Example 1, except that the colloid mill used in the enzymatic hydrolysis of the complex enzyme preparation in step (2) is replaced by the high-pressure homogenizer.

[0097] Comparative Example 1

[0098] The method is basically the same as Example 1, except that the colloid mill used in the enzymatic hydrolysis of the complex enzyme preparation in step (2) is replaced by an electric stirrer, and the speed of the electric stirrer is maintained at 3000 rpm / min for mechanical action.

[0099] Comparative Example 2

[0100] The method is basically the same as Example 1, except that the colloid mill used in the second enzymatic hydrolysis of the complex enzyme preparation in step (2) is replaced by an electric stirrer, and the speed of the electric stirrer is maintained at 3000 rpm / min for mechanical action.

[0101] Comparative Example 3

[0102] The process is basically the same as Example 1, except that step (1) is as follows:

[0103] (2) The bacterial precipitate obtained in step (1) was resuspended to 3.2 L, heated to 52° C., the pH value was adjusted to 10.5, 0.02% (volume percentage) of complex enzyme preparation II (consisting of 6 parts of alkaline protease, 3 parts of peptidase and 1 part of amylase by mass) was added, and the mixture was treated with a colloid mill for 5 min to make the average particle size of the emulsion 1 to 10 μm, and then stirred at 400 rpm / min and 52° C. for 45 min; then the pH value was adjusted to 6.0, 0.04% of complex enzyme preparation I (consisting of 4 parts of snail enzyme, 4 parts of lysozyme and 2 parts of nuclease by mass) was added, the mixture was treated with a colloid mill for 5 min, and then stirred at 400 rpm / min and 52° C. for 45 min.

[0104] The PHA powder products obtained in the above examples and comparative examples were tested for purity, weight-average molecular weight, and total nitrogen. The powders were melt-granulated according to conventional processes and then tested for melt index, weight-average molecular weight, and color. The testing methods are as follows:

[0105] PHA powder purity (GC) test method: refer to CN115894981A.

[0106] PHA powder / pellet weight average molecular weight measurement method: Weight average molecular weight (Mw): Measured using a gel permeation chromatograph (HPLC GPC system, manufactured by Shimadzu Corporation) using a chloroform solution and in terms of polystyrene. The column used in the gel permeation chromatograph can be any column suitable for measuring weight average molecular weight.

[0107] PHA powder total nitrogen test method: Use Kjeldahl nitrogen analyzer to determine according to the national standard GB / T22427.10-2008 method.

[0108] The melt index (MFR) of PHA powder and pellets, also known as melt mass flow rate (MFR), is measured using a melt flow rate tester according to the national standard GB / T3682.1, using a 2.16kg load and a die diameter of 2.095±0.005mm. The unit of melt flow rate is g / 10min. For materials with a molar ratio of PHB to the second component less than 6.5%, the test temperature is 190°C; for materials with a molar ratio of the second component or the sum of the second and third components of 10±1%, the test temperature is 180°C; and for materials with a molar ratio of the second component or the sum of the second and third components of 15±1%, the test temperature is 175°C.

[0109] PHA granule color test method: CR-910 desktop spectrophotometer (Beijing Kemei Runda Instrument Equipment Co., Ltd.) was used for measurement after calibration with L\A\B standard color plates.

[0110] The test results are shown in the following table:

[0111] Table 1

[0112] Table 2

[0113] The chromaticity values ​​L, A, and B are interpreted as follows:

[0114] L, A, and B on the colorimeter represent the chromaticity values ​​of the object's color, and are also the color space coordinates of the color. Any color has a unique coordinate value. Specifically:

[0115] L: It represents the depth of black and white, ranging from 0 to 100, with no negative values. The larger the L, the whiter (brighter), and the smaller the L, the blacker (darker).

[0116] A: It represents red and green, with positive and negative values. +A represents redder, and -A represents greener (not red enough).

[0117] B: It represents yellow-blue, and can be positive or negative. +B represents yellowish, and -B represents bluish.

[0118] The above data demonstrates that, through the coordinated use of specialized enzyme preparations and specialized equipment, various impurities that cause yellowing during melt processing can be removed at the source, ensuring the purity, yield, and molecular weight of the PHA powder. Furthermore, when the PHA powder is melt-processed into PHA pellets, a decrease in weight-average molecular weight and an increase in melt index are avoided, resulting in a significant decrease in the chromaticity of the resulting PHA pellets.

[0119] The above extraction method of the present invention is suitable for a variety of PHA products, and PHA can be short-chain PHA or medium-chain PHA. 3A large number of experiments have been carried out at the fermentation level, and it has strong value for industrial scale-up.

[0120] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention. Industrial Applicability

[0121] The present invention provides a PHA extraction method for adjusting the color of PHA materials, as well as its products and applications. The extraction method comprises: performing solid-liquid separation on a halophilic bacteria fermentation broth to obtain a concentrated bacterial liquid; adjusting the pH of the concentrated bacterial liquid to acidic, adding a composite enzyme preparation 1 thereto, and adjusting the average particle size of the resulting emulsion to 1 to 10 μm under mechanical action, then adjusting the pH to alkaline, adding a composite enzyme preparation 2 thereto, and continuing mechanical action to obtain a wall-breaking liquid. The aqueous phase extraction method of the present invention has mild conditions and is suitable for large-scale continuous production. The special enzyme preparation and specific equipment cooperate with each other to reduce various impurities that cause yellowing, so that the color of the PHA pellets obtained by further processing is low. Moreover, during the thermoplastic processing process, it can effectively inhibit the reduction of molecular weight, inhibit the increase in MFR, and improve the toughness of various molded bodies. The risk of dissolution in a solvent environment in subsequent applications is low, and it has good economic value and application prospects.

Claims

1. A PHA extraction method for adjusting the color of a PHA material, characterized in that: include: The halophilic bacteria fermentation liquid is subjected to solid-liquid separation to obtain a concentrated bacterial liquid; The pH of the concentrated bacterial liquid is adjusted to acidic, and the first complex enzyme preparation is added thereto, and the mixture is mixed under mechanical action so that the average particle size of the obtained emulsion is 1 to 10 μm. The pH is then adjusted to alkaline, and the second complex enzyme preparation is added thereto, and the cell wall is broken by mechanical action to obtain a broken cell liquid.

2. The PHA extraction method for adjusting the color of the PHA material according to claim 1, characterized in that: The mechanical action is carried out in a high-speed mechanical mixing device, which includes a colloid mill, an ultrafine pulverizer, a homogenizing emulsification pump or a high-pressure homogenizer; And / or, the composite enzyme preparation one comprises two or more of lysozyme, nuclease, snail enzyme and glucanase; preferably, the composite enzyme preparation one is mainly composed of snail enzyme and lysozyme; further preferably, the composite enzyme preparation one comprises 4 to 6 parts of snail enzyme and 2 to 4 parts of lysozyme by weight; And / or, the second complex enzyme preparation includes two or more of alkaline protease, amylase, peptidase and nuclease; preferably, in terms of weight, the second complex enzyme preparation is mainly composed of alkaline protease and peptidase; further preferably, the second complex enzyme preparation includes 3 to 6 parts of alkaline protease and 3 to 4 parts of peptidase.

3. The PHA extraction method for adjusting the color of the PHA material according to claim 1 or 2, characterized in that: The addition amount of the composite enzyme preparation 1 is 0.01% to 0.1% (w / v) of the volume of the feed solution during enzymolysis; And / or, the addition amount of the second composite enzyme preparation is 0.01% to 0.1% (w / v) of the volume of the feed solution during enzymatic hydrolysis; preferably, the pH is adjusted to alkaline by adjusting the pH value to 8.0 to 12.0, preferably 9.0 to 11.

0.

4. The PHA extraction method for adjusting the color of a PHA material according to any one of claims 1 to 3, characterized in that: include: The cell-breaking liquid is subjected to solid-liquid separation to obtain a crude PHA product, and the crude PHA product is subjected to enzymatic hydrolysis and purification using a composite enzyme preparation three under alkaline conditions to obtain an enzymatic hydrolysis purified liquid; Preferably, the complex enzyme preparation three includes two or more of phospholipase, lipase, phosphatase, glycosidase, nuclease and polysaccharide hydrolase.

5. The PHA extraction method for adjusting the color of PHA material according to claim 4, characterized in that: The addition amount of the composite enzyme preparation 3 is 0.01% to 0.1% (w / v) of the volume of the feed solution during enzymolysis; The temperature during enzymatic purification is 30°C to 80°C, preferably 40°C to 60°C; The pH value during enzymatic purification is 8.5 to 13.0, preferably 10.0 to 12.

0.

6. The PHA extraction method for adjusting the color of the PHA material according to claim 4 or 5, characterized in that: include: Adding a surfactant to the enzymatic hydrolysis purification solution for mixing and stirring, and performing solid-liquid separation, washing and drying on the obtained liquid to obtain PHA; Preferably, the surfactant is mainly composed of anionic surfactants; the anionic surfactant is preferably one or more of sodium dodecylbenzene sulfonate, sodium dodecyl sulfate, sodium octadecyl sulfate, etc.; More preferably, the amount of surfactant added is 0 to 5% (w / v), preferably 0.02% to 0.5% (w / v), of the volume of the feed solution during enzymatic purification.

7. The PHA extraction method for adjusting the color of a PHA material according to any one of claims 1 to 6, characterized in that: include: (1) taking a halophilic bacteria fermentation liquid and performing solid-liquid separation to obtain a concentrated bacterial liquid with an impurity removal rate of ≥90%; (2) After heating the concentrated bacterial solution obtained in step (1), adding the composite enzyme preparation 1, mixing with a high-speed mechanical mixer for 3 to 30 minutes, stirring at a constant temperature, and adjusting the pH, and then adding the composite enzyme preparation 2, mixing with a high-speed mechanical mixer for 3 to 30 minutes, stirring at a constant temperature, and obtaining a cell-broken solution; (3) performing solid-liquid separation on the broken liquid obtained in step (2) to obtain a crude PHA product with an impurity removal rate of ≥70%; (4) using the composite enzyme preparation 3 to hydrolyze and purify the crude PHA obtained in step (3), and then adding a surfactant thereto for mixing and stirring; (5) The liquid obtained in step (4) is subjected to solid-liquid separation, washing, and drying to obtain pure PHA.

8. The PHA extraction method for adjusting the color of PHA material according to claim 7, characterized in that: include: In step (1), the solid-liquid separation can be carried out by centrifugation or filtration, preferably centrifugation; in step (1), the solid content of the concentrated bacterial liquid is 13-30%, preferably 15-25%; and / or, in step (2), the temperature is raised to 30°C to 95°C, preferably 40°C to 80°C, and more preferably 45°C to 70°C; in step (2), stirring is continued during heat preservation in each link, the stirring speed is 100 to 1000 rpm / min, and the stirring time is 30 to 90 min; And / or, in step (3), the solid-liquid separation is carried out by centrifugation or filtration; And / or, in step (4), the stirring speed required for enzymatic purification is 100-1000 rpm / min, and the stirring time is 10-60 min; after adding the surfactant, the stirring speed required is 100-1000 rpm / min, and the stirring time is 10-60 min.

9. A PHA powder obtained by the PHA extraction method for adjusting the color of a PHA material according to any one of claims 1 to 8.

10. A PHA pellet or PHA molded body, obtained by melt-processing the PHA powder according to claim 9.

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