Method for removing thalli from high-viscosity pullulan fermentation liquor

Abstract

The invention belongs to the technical field of biochemical engineering separation, and discloses a method for removing thalli from high-viscosity pullulan fermentation liquor. The method comprises the following four steps: pretreatment of fermentation liquor, addition of a filter aid, dead end filtration and washing filtration. When the method provided by the invention is used for treating the high-viscosity pullulan fermentation liquor, thalli can be effectively removed, and the method has the characteristics of low pullulan retention rate, high yield and low energy consumption, and has a wide application prospect in the production of pullulan.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical separation and discloses a method for removing bacteria from a high-viscosity pullulan fermentation broth. Background Art

[0002] Pullulan, also known as pullulan, pullulan, or pullulan, is a macromolecular extracellular polysaccharide produced by the fermentation of budding Aureobasidium pullulans. It possesses numerous excellent properties, including good solubility, adhesion, plasticity, stability, and safety, making it a safe and harmless product. It has broad application prospects in the pharmaceutical, food, petroleum, and chemical industries. Pullulan is typically produced through microbial fermentation. However, after the fermentation process, in addition to the target product, the fermentation broth contains byproducts such as bacterial cells, pigment molecules, miscellaneous proteins and polysaccharides, as well as impurities such as small organic molecules and inorganic salts remaining in the culture medium. Therefore, the fermentation broth must be cleaned to obtain impurity-free pullulan.

[0003] The first step in product impurity removal is to remove bacterial cells from the fermentation broth. Centrifugal sterilization is usually used, which has high energy consumption and large polysaccharide loss, making it unsuitable for industrial production. Alternatively, filtration sterilization is used, which can cause the clogging of the filter membrane, resulting in the retention of Rulan polysaccharide, which also leads to polysaccharide loss and reduces the product yield. Summary of the Invention

[0004] To address these shortcomings, this invention provides a method for removing microorganisms from high-viscosity pullulan fermentation broth. This method removes polymalic acid, decomposes proteins, and flocculates the microorganisms into clusters rather than allowing them to settle. After adding a filter aid, dead-end filtration is then used to remove microorganisms from the fermentation broth. This method achieves a high microorganism removal rate, reduces pullulan retention, and offers simple operation, high throughput, and low energy consumption.

[0005] To achieve the above objectives, the present invention provides the following technical solutions: In a first aspect, the present invention provides a method for removing bacteria from a high-viscosity pullulan fermentation broth, the method comprising the following steps: S1. Fermentation broth pretreatment: adjusting the pH value of the high-viscosity pullulan fermentation broth to 1-6; heating and maintaining the fermentation broth at 60-121°C for 20-30 minutes; stopping heating and waiting until the fermentation broth temperature is ≤55°C, adding acidic protease and flocculant in an amount of 0.01%-0.04%, mixing, and then standing for 0.5-3 hours; diluting the fermentation broth with deionized water to obtain a diluted fermentation broth; S2: adding filter aids to the diluted fermentation broth obtained in S1, and mixing thoroughly to obtain fermentation broth with added filter aids; S3 dead-end filtration: The fermentation broth with the filter aid added obtained in S2 is sterilized by dead-end filtration at 0.05-0.8 MPa and 30-50°C to obtain a fermentation broth clear liquid and filter residue free of bacterial cells; S4 filtration washing: adding filtration washing solvent into the filter assembly containing the filter residue described in S3 to perform filtration washing on the filter residue.

[0006] The percentage sign "%" involved in the present invention refers to the mass volume percentage, that is, the number of grams of solute contained in 100 mL of solution.

[0007] The present invention pre-treats the fermentation broth before sterilization. By adjusting the pH and heating (60-121°C), the fermentation broth's bacterial cells are inactivated. Polymalic acid, a byproduct produced during the fermentation of Aureobasidium pullulans, is decomposed under high-temperature, acidic conditions. Some proteins in the fermentation broth form complexes with pullulan molecules. These complexes have molecular weights much greater than those of individual polysaccharide molecules and are retained by the filter cake or membrane during filtration, resulting in polysaccharide loss. By adding an appropriate amount of acidic protease at a certain temperature, the proteins in the fermentation broth are completely or partially decomposed into small peptide fragments, releasing the protein-bound pullulan and reducing clogging of the filter cake and membrane by the complexes. Simultaneously, a low dose of flocculant is added to cause the bacteria to agglomerate rather than flocculate and settle, and the viscosity of the fermentation broth is reduced by dilution. After the pre-treatment and the addition of a filter aid, the fermentation broth forms a loosely structured filter cake during dead-end filtration, effectively removing bacteria while increasing permeation flux and significantly reducing pullulan retention.

[0008] Preferably, the diafiltration in S4 is reverse diafiltration; the diafiltration solvent is water, has a pH value of 6.0-7.5, and a temperature 0-10°C higher than the filtration temperature; and the amount of the diafiltration solvent is 1-7 times the volume of the filter assembly chamber.

[0009] Dead-end filtration involves placing raw water upstream of a membrane. Driven by a pressure differential, water and particles smaller than the membrane pores pass through the membrane, while particles larger than the pores are retained. This pressure differential can be achieved by applying pressure to the water side or by applying a vacuum to the filtrate side. With dead-end filtration, as filtration time increases, retained particles form a fouling layer on the membrane surface, increasing filtration resistance. At constant operating pressure, the membrane's permeability decreases. Periodic cleaning of the fouling layer or replacement of the membrane is necessary.

[0010] The reverse filtration of the present invention is to introduce the filtration solvent from the other end of the membrane module chamber (the end corresponding to the feed port), apply a certain pressure, and make the filtration liquid flow through the filter cake in the opposite direction of the feed direction. Figure 1After backwashing, low-pressure flushing (0.05-0.8 MPa) can be performed to further recover pullulan adsorbed and retained by the filter cake while reducing impact on the membrane. Backwashing can recover pullulan retained in the filter residue, further reducing pullulan loss.

[0011] Since the binding density of the filter residue gradually increases along the filtration direction, the forward filtration flux will be low and the target product recovery effect will be poor. The reverse flushing of the filter residue improves the recovery rate of the retained material and the filtration flux of the filter residue filtration, further reducing the loss of the product during the extraction process.

[0012] Preferably, the pH value of the high-viscosity pullulan fermentation broth is adjusted to 2-4 in S1.

[0013] Preferably, in S1, the fermentation liquid temperature is heated and maintained at 121° C. for 20 min.

[0014] Preferably, the fermentation broth is diluted with deionized water in S1, and the volume ratio of deionized water to the fermentation broth is 1-3:1.

[0015] More preferably, in S1, the pH value of the high-viscosity pullulan fermentation broth is adjusted to 3.

[0016] More preferably, the volume ratio of deionized water to the fermentation broth is 1:1.

[0017] Preferably, the heating is stopped in S1 and when the temperature of the fermentation liquid drops to 40-55°C, a flocculant and an acid protease are added, mixed and then allowed to stand for 0.5-2 hours.

[0018] Preferably, the flocculant in S1 is chitosan; the amount of the acidic protease added is 0.01%-0.1%, and the flocculant is chitosan.

[0019] Preferably, the filter aid in S2 is added in an amount of 0.2%-10% by mass volume.

[0020] Preferably, the filter aid in S2 is a single filter aid or a composite filter aid; the filter aid is an organic filter aid or an inorganic filter aid.

[0021] Filter aid is an auxiliary substance used to reduce filtration resistance and improve filtration efficiency during the filtration process.

[0022] Organic filter aids are organic substances used to improve filtration efficiency and cleanliness during filtration operations. They typically have a fibrous structure and can form a three-dimensional network on the filter medium, thereby intercepting particulate impurities in the liquid and achieving solid-liquid separation. They include but are not limited to activated carbon and cellulose.

[0023] The inorganic filter aid is mainly made of natural minerals or inorganic compounds, has good adsorption and chemical stability, and includes but is not limited to diatomaceous earth, perlite, and white clay.

[0024] More preferably, the filter aid is added in an amount of 0.5%-1.2% by mass volume.

[0025] More preferably, the filter aid in S2 is one of 325 mesh red diatomaceous earth, 400 mesh perlite, 200 mesh white diatomaceous earth, 325 mesh activated carbon, an equal volume mixture of 200 mesh perlite and 800 mesh red diatomaceous earth, or an equal volume mixture of 200 mesh red diatomaceous earth and 400 mesh white diatomaceous earth.

[0026] Preferably, the fermentation broth to which the filter aid is added in S3 is sterilized by dead-end filtration at 0.2 MPa and 40°C.

[0027] Preferably, the filter membrane used for the dead-end filtration in S3 is an organic membrane or an inorganic membrane, the membrane pore size is 0.1-4 μm, and the membrane assembly type is a flat plate type; the material of the organic membrane is one of polyethylene, polyester, nylon or polypropylene; the weaving method of the organic membrane is plain weave; the inorganic membrane is a ceramic membrane, a metal membrane or a metal and ceramic composite membrane; the material of the ceramic membrane is one of aluminum oxide, cobalt oxide, silicon oxide, aluminum silicate or silicon carbide; the metal membrane is one of silver film, nickel film or titanium film.

[0028] More preferably, the membrane pore size is 0.5-3 μm.

[0029] In a second aspect, the present invention also provides a fermentation broth clear solution prepared according to the above method for removing bacteria from a high-viscosity pullulan fermentation broth.

[0030] The fermentation liquid obtained by the method provided by the present invention has a high pullulan content and no bacterial residue, thereby effectively improving the recovery rate of pullulan.

[0031] In a third aspect, the present invention further provides the use of the above-mentioned method for removing bacteria from a high-viscosity pullulan fermentation broth in extracting pullulan. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 Schematic diagram of the reverse filtration method of the present invention; Figure 2 Schematic diagram of dead-end filtration in the present invention. DETAILED DESCRIPTION

[0034] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0035] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0036] The high-viscosity pullulan fermentation broth used in the embodiments of the present invention (hereinafter referred to as "fermentation broth") is: Aureobasidium pullulans ( Aureobasidium pullulans CGMCC No. 7055 was inoculated into the fermentation medium and then fermented at a variable temperature for 96 hours (the first 24 hours were at 32°C, followed by the remaining 72 hours at 28°C). This produced a high-viscosity pullulan fermentation broth with a pH of 4.93, a pullulan content of 66.5 g / L, and a protein content of 8 g / L.

[0037] Aureobasidium pullulans ( Aureobasidium pullulans CGMCC No. 7055 was obtained through screening and mutagenesis of a laboratory-preserved strain of Aureobasidium pullulans TKPM 10017, which produces low amounts of pigment. It was deposited with the General Microbiology Center of the China Culture Collection Administration on December 25, 2012. It is disclosed in the patent "A Liquid Mulch Film and Its Preparation Method" (CN2014107209592).

[0038] Example 1 1. Effects of low pH and heating on pullulan and protein during pretreatment 500 mL of fermentation broth was taken respectively, and the pH of the fermentation broth was adjusted to 2.0, 3.0 and 4.0 using 0.1 M HCl solution. The fermentation broth with adjusted pH was then heated to 121°C and maintained for 20 min. After the fermentation, the polysaccharide content and protein content in the fermentation broth were detected, and the polysaccharide recovery rate (%) and protein removal rate (%) were calculated. The results are shown in Table 1.

[0039] Polysaccharide recovery rate (%) = (polysaccharide content after treatment / original polysaccharide content) × 100; Wherein: original polysaccharide content refers to the pullulan content in the fermentation broth before treatment; treated polysaccharide content refers to the pullulan content in the fermentation broth after treatment.

[0040] Protein removal rate (%) = (1-protein content after treatment / original protein content) × 100; Wherein: original protein content refers to the protein content in the fermentation broth before treatment; post-treatment protein content refers to the protein content in the fermentation broth after treatment.

[0041] Table 1 Fermentation broth pretreatment effect

[0042] 2. Effect of acid protease on pullulan and protein during pretreatment The fermentation broth was taken and the pH was adjusted to 3.0 using HCl. The fermentation broth was then heated to 121°C and maintained for 20 min. When the temperature dropped to 45°C, 0.01% acid protease was added and maintained for 1 h to obtain pretreatment solution 1.

[0043] Pullulan fermentation broth was taken and the pH was adjusted to 3.0 using HCl. The fermentation broth was then heated to 121°C and maintained for 20 min. When the temperature dropped to 50°C, 0.05% acid protease was added and maintained for 1.5 h to obtain pretreatment solution 2.

[0044] Pullulan fermentation broth was adjusted to pH 3.0 using HCl, and then heated to 121°C and maintained for 20 min. When the temperature dropped to 55°C, 0.1% acid protease was added and maintained for 2 h to obtain pretreatment solution 3.

[0045] The polysaccharide content and protein content in pretreatment solutions 1-3 were detected, and the polysaccharide recovery rate (%) and protein removal rate (%) were calculated. The results are shown in Table 2.

[0046] Table 2 Fermentation broth pretreatment effect

[0047] 3. Effect of flocculants on pullulan and protein during pretreatment The fermentation broth was adjusted to pH 2.0 using HCl, and then heated to 121°C and maintained for 20 min. When the temperature dropped to 40°C, 0.01% acid protease and 0.01% chitosan flocculant were added and maintained for 0.5 h to obtain pretreated solution 1.

[0048] Pullulan fermentation broth was adjusted to pH 3.0 using HCl, and then heated to 121°C and maintained for 20 min. When the temperature dropped to 45°C, 0.05% acid protease and 0.02% chitosan flocculant were added and maintained for 1.0 h to obtain pretreated solution 2.

[0049] Pullulan fermentation broth was adjusted to pH 4.0 using HCl, and then heated to 121°C and maintained for 20 min. When the temperature dropped to 50°C, 0.1% acid protease and 0.04% chitosan flocculant were added and maintained for 1.5 h to obtain pretreated solution 3.

[0050] The polysaccharide content and protein content in pretreatment solutions 1-3 were detected, and the polysaccharide recovery rate (%) and protein removal rate (%) were calculated. The results are shown in Table 3.

[0051] Table 3 Fermentation broth pretreatment effect

[0052] The experimental results show that during the pretreatment process, only by adjusting the pH value and heating treatment, some proteins in the fermentation broth can be preliminarily removed, and the protein removal rate is between 36% and 40%. On this basis, 0.01% to 0.1% acid protease is added to decompose the protein, and then a flocculant is added to agglomerate the bacteria. The results show that the entire pretreatment process has little effect on pullulan, with almost no loss. The addition of flocculants can help remove some proteins, and the overall protein removal rate is between 60% and 100%.

[0053] Example 2 This embodiment provides a method for removing bacteria from a high-viscosity pullulan fermentation broth, which specifically comprises the following steps.

[0054] (1) Fermentation broth pretreatment: Take the fermentation broth and adjust the pH value to 3 using 0.1 M HCl solution; then heat the fermentation broth to 121°C and maintain it for 20 min; stop heating and wait until the temperature of the fermentation broth drops to 40°C, then add 0.01% acid protease and 0.01% chitosan flocculant and maintain it for 0.5 h; add water equal to the volume of the fermentation broth to dilute it.

[0055] (2) Adding filter aid: Add 0.5% 325 mesh red diatomaceous earth to the diluted fermentation broth obtained in step 1, mix thoroughly, and obtain fermentation broth with added filter aid.

[0056] (3) Dead-end filtration: The fermentation broth with filter aid added obtained in step 2 was subjected to dead-end filtration at a filtration pressure of 0.2 MPa and 30°C using a polypropylene plain filter membrane with a pore size of 0.5-1 μm. The filtration time was 70 min to obtain the fermentation broth clear liquid and filter residue with bacterial cells removed.

[0057] (4) Filtration: Add 7 times the amount of deionized water (pH value of 6.0-6.5) than that in the equipment compartment into the filter assembly containing the filter residue, and perform reverse filtration on the filter residue. The operating pressure during filtration is 0.2 MPa and the temperature is 30°C.

[0058] Example 3 This embodiment provides a method for removing bacteria from a high-viscosity pullulan fermentation broth, which specifically comprises the following steps.

[0059] (1) Fermentation broth pretreatment: Take the fermentation broth and adjust the pH value to 3 using 0.1 M HCl solution; then heat the fermentation broth to 121°C and maintain it for 20 min; stop heating and wait until the temperature of the fermentation broth drops to 45°C, then add 0.01% acid protease and 0.01% chitosan flocculant and maintain it for 1 h; add water equal to the volume of the fermentation broth to dilute it.

[0060] (2) Adding filter aid: Add 0.8% 400-mesh perlite to the diluted fermentation broth obtained in step 1 and mix thoroughly to obtain fermentation broth with added filter aid.

[0061] (3) Dead-end filtration: The fermentation broth with filter aid added obtained in step 2 was subjected to dead-end filtration at a filtration pressure of 0.2 MPa and 40°C using a polypropylene plain filter membrane with a pore size of 1-1.5 μm. The filtration time was 60 min to obtain the fermentation broth clear liquid and filter residue with bacterial cells removed.

[0062] (4) Filtration: Add deionized water (pH value is 6.5-7.0) three times that of the equipment compartment into the filter assembly containing the filter residue, and perform reverse filtration on the filter residue. The operating pressure during filtration is 0.2 MPa and the temperature is 40°C.

[0063] Example 4 This embodiment provides a method for removing bacteria from a high-viscosity pullulan fermentation broth, which specifically comprises the following steps.

[0064] (1) Fermentation broth pretreatment: Take the fermentation broth and adjust the pH value to 3 using 0.1 M HCl solution; then heat the fermentation broth to 121°C and maintain it for 20 min; stop heating and wait until the temperature of the fermentation broth drops to 40°C, then add 0.05% acid protease and 0.02% chitosan flocculant and maintain it for 0.5 h; add water equal to the volume of the fermentation broth to dilute it.

[0065] (2) Adding filter aid: Add 1.0% 200 mesh white diatomaceous earth to the diluted fermentation broth obtained in step 1, mix thoroughly, and obtain fermentation broth with added filter aid.

[0066] (3) Dead-end filtration: The fermentation broth with filter aid added obtained in step 2 was subjected to dead-end filtration at a filtration pressure of 0.2 MPa and 40°C using a polypropylene plain filter membrane with a pore size of 1-1.5 μm. The filtration time was 65 min to obtain the fermentation broth clear liquid and filter residue with bacterial cells removed.

[0067] (4) Filtration: Add deionized water (pH value is 6.5-7.0) three times that of the equipment compartment into the filter assembly containing the filter residue, and perform reverse filtration on the filter residue. The operating pressure during filtration is 0.2 MPa and the temperature is 50°C.

[0068] Example 5 This embodiment provides a method for removing bacteria from a high-viscosity pullulan fermentation broth, which specifically comprises the following steps.

[0069] (1) Fermentation broth pretreatment: Take the fermentation broth and adjust the pH value to 3 using 0.1 M HCl solution; then heat the fermentation broth to 121°C and maintain it for 20 min; stop heating and wait until the temperature of the fermentation broth drops to 45°C, then add 0.05% acid protease and 0.04% chitosan flocculant and maintain it for 1 h; add water equal to the volume of the fermentation broth to dilute it.

[0070] (2) Adding filter aid: Add 1.2% of a composite filter aid (the composite filter aid is a combination of 200 mesh red diatomaceous earth and 400 mesh white diatomaceous earth in a volume ratio of 1:1) to the diluted fermentation broth obtained in step 1, and mix thoroughly to obtain a fermentation broth with added filter aid.

[0071] (3) Dead-end filtration: The fermentation broth with filter aid added obtained in step 2 was subjected to dead-end filtration at a filtration pressure of 0.2 MPa and 45°C using a polyester plain filter membrane with a pore size of 1-3 μm. The filtration time was 40 min to obtain the fermentation broth clear liquid and filter residue with bacterial cells removed.

[0072] (4) Filtration: Add deionized water (pH value is 6.5-7.0) three times that of the equipment compartment into the filter assembly containing the filter residue, and perform reverse filtration on the filter residue. The operating pressure during filtration is 0.2 MPa and the temperature is 50°C.

[0073] Example 6 This embodiment provides a method for removing bacteria from a high-viscosity pullulan fermentation broth, which specifically comprises the following steps.

[0074] (1) Fermentation broth pretreatment: Take the fermentation broth and adjust the pH value to 3 using 0.1 M HCl solution; then heat the fermentation broth to 100°C and maintain it for 25 min; stop heating and wait until the temperature of the fermentation broth drops to 45°C, then add 0.1% acid protease and 0.04% chitosan flocculant and maintain it for 1 h; add water equal to the volume of the fermentation broth to dilute it.

[0075] (2) Adding filter aid: Add 1.0% 325 mesh activated carbon to the diluted fermentation broth obtained in step 1 and mix thoroughly to obtain fermentation broth with added filter aid.

[0076] (3) Dead-end filtration: The fermentation broth with filter aid added obtained in step 2 was subjected to dead-end filtration at a filtration pressure of 0.2 MPa and 45°C using a polyester plain filter membrane with a pore size of 2-3 μm. The filtration time was 35 min to obtain the fermentation broth clear liquid and filter residue with bacterial cells removed.

[0077] (4) Filtration: Add deionized water (pH value is 6.5-7.0) three times that of the equipment compartment into the filter assembly containing the filter residue, and perform reverse filtration on the filter residue. The operating pressure during filtration is 0.2 MPa and the temperature is 50°C.

[0078] Example 7 This embodiment provides a method for removing bacteria from a high-viscosity pullulan fermentation broth, which specifically comprises the following steps.

[0079] (1) Fermentation broth pretreatment: Take the fermentation broth and adjust the pH value to 3 using 0.1 M HCl solution; then heat the fermentation broth to 100°C and maintain it for 25 min; stop heating and wait until the temperature of the fermentation broth drops to 45°C, then add 0.1% acid protease and 0.04% chitosan flocculant and maintain it for 1 h; add water equal to the volume of the fermentation broth to dilute it.

[0080] (2) Adding filter aid: Add 0.8% of a composite filter aid (the composite filter aid is a combination of 200-mesh perlite and 800-mesh red diatomaceous earth in a volume ratio of 1:1) to the diluted fermentation broth obtained in step 1, and mix thoroughly to obtain a fermentation broth with added filter aid.

[0081] (3) Dead-end filtration: The fermentation broth with filter aid added obtained in step 2 was subjected to dead-end filtration at a filtration pressure of 0.2 MPa and 50°C using a polyester plain filter membrane with a pore size of 1-2 μm. The filtration time was 82 min, and the fermentation broth clear liquid and filter residue with bacterial cells removed were obtained.

[0082] (4) Filtration: Add deionized water (pH value is 6.5-7.0) three times that of the equipment compartment into the filter assembly containing the filter residue, and perform reverse filtration on the filter residue. The operating pressure during filtration is 0.2 MPa and the temperature is 50°C.

[0083] Comparative Example 1 This comparative example uses a traditional method for removing bacteria from a high-viscosity pullulan fermentation broth, which specifically comprises the following steps.

[0084] (1) Fermentation broth pretreatment: Add 1% aluminum oxide to the fermentation broth, stir it, and let it stand for 8 h to obtain the pretreated fermentation broth.

[0085] (2) Cross-flow filtration: The pretreated fermentation broth was cross-flow filtered using a 1.4 μm ceramic membrane at a filtration pressure of 0.2 MPa and room temperature for 126 min to obtain the fermentation clear liquid with bacterial cells removed.

[0086] Comparative Example 2 This comparative example provides a method for removing bacteria from a high-viscosity pullulan fermentation broth, which specifically comprises the following steps.

[0087] (1) Fermentation broth pretreatment: Take the fermentation broth and adjust the pH value to 3 using 0.1 M HCl solution; then heat the fermentation broth to 100°C and maintain it for 20 min; stop heating and wait until the temperature of the fermentation broth drops to 45°C, then add 0.01% acid protease and 0.01% chitosan flocculant and maintain it for 1 h; add water equal to the volume of the fermentation broth to dilute it.

[0088] (2) Cross-flow filtration: The diluted fermentation broth was cross-flow filtered using a 1-3 μm ceramic membrane at a filtration pressure of 0.2 MPa and 40°C for 131 min to obtain the fermentation broth clear liquid and filter residue after removing bacterial cells.

[0089] Comparative Example 3 This comparative example provides a method for removing bacteria from a high-viscosity pullulan fermentation broth, which specifically comprises the following steps.

[0090] (1) Fermentation broth pretreatment: Take the fermentation broth and adjust the pH value to 3 using 0.1 M HCl solution; then heat the fermentation broth to 100°C and maintain it for 25 min; stop heating and wait until the temperature of the fermentation broth drops to 45°C, add 0.04% chitosan flocculant and maintain it for 1 h; add water equal to the volume of the fermentation broth to dilute it.

[0091] (2) Adding filter aid: Add 0.8% of a composite filter aid (the composite filter aid is a combination of 200-mesh perlite and 800-mesh red diatomaceous earth in a volume ratio of 1:1) to the diluted fermentation broth obtained in step 1, and mix thoroughly to obtain a fermentation broth with added filter aid.

[0092] (3) Dead-end filtration: The fermentation broth with filter aid added obtained in step 2 was subjected to dead-end filtration at a filtration pressure of 0.2 MPa and 40°C using a polyester plain filter membrane with a pore size of 1-2 μm. The filtration time was 82 min, and the fermentation broth clear liquid and filter residue with bacterial cells removed were obtained.

[0093] The bacterial cell content and protein content of the fermentation broth supernatants obtained in Examples 2-7 and Comparative Examples 1-3, as well as the polysaccharide content in the filter residue, the average permeate flux during filtration, and the energy consumption of the entire operation were measured. The bacterial cell removal rate, polysaccharide retention rate, and protein removal rate were calculated. The results are shown in Table 4.

[0094] Table 4 Filtering effect

[0095] From the above results, it can be seen that the method provided by the present invention can remove more than 98% of the bacteria in the original fermentation broth, with excellent bacteria removal effect, and the polysaccharide retention rate is between 6-9%. Compared with the comparative example, the polysaccharide retention rate is significantly reduced, and the average permeation flux is between 124.65-205.69 L / m 2 Within the range of 1.5 h, the fermentation broth can be quickly separated. Protein removal efficiency is negatively correlated with polysaccharide retention, indicating that protein removal during pretreatment helps reduce polysaccharide retention. While dead-end filtration and cross-flow filtration offer similar bacterial removal rates, the polysaccharide retention rate of dead-end filtration is significantly lower than that of cross-flow filtration. Average permeate flux, product yield, and energy consumption are significantly superior to those of cross-flow filtration.

[0096] Therefore, a pretreatment method is adopted, which first adjusts the pH value and heat treatment, and then adds acidic protease and flocculants to remove part or all of the protein and put the bacteria into a clumping state (non-flocculated state), which is convenient for the subsequent removal of bacteria. At the same time, it can reduce the blockage of the filter membrane and filter cake. Combined with the dead-end filtration method, the retention rate of pullulan can be significantly reduced.

[0097] Example 8 This embodiment provides a method for removing bacteria from a high-viscosity pullulan fermentation broth, which specifically comprises the following steps.

[0098] (1) Fermentation broth pretreatment: Take pullulan fermentation broth and adjust the pH value to 3 using 0.1 M HCl solution; then heat the fermentation broth to 60°C and maintain it for 30 min; stop heating and wait until the temperature of the fermentation broth drops to 45°C, then add 0.1% acid protease and 0.04% chitosan flocculant and maintain it for 1 h; add water equal to the volume of the fermentation broth to dilute it.

[0099] (2) Adding filter aid: Add 0.8% of a composite filter aid (the composite filter aid is a combination of 200-mesh perlite and 800-mesh red diatomaceous earth in a volume ratio of 1:1) to the diluted fermentation broth obtained in step 1, and mix thoroughly to obtain a fermentation broth with added filter aid.

[0100] (3) Dead-end filtration: The fermentation broth with filter aid added obtained in step 2 was subjected to dead-end filtration at a filtration pressure of 0.2 MPa and 40°C using a polyester plain filter membrane with a pore size of 1-3 μm. The filtration time was 70 min to obtain the fermentation broth clear liquid and filter residue with bacterial cells removed.

[0101] (4) Filtration: Add 5 times the amount of deionized water (pH value of 7.0-7.5) as that in the equipment compartment into the filter assembly containing the filter residue, and perform reverse filtration on the filter residue. The operating pressure during filtration is 0.2 MPa and the temperature is 40°C.

[0102] Example 9 This embodiment provides a method for removing bacteria from a high-viscosity pullulan fermentation broth, which specifically comprises the following steps.

[0103] (1) Fermentation broth pretreatment: Take pullulan fermentation broth and adjust the pH value to 3 using 0.1 M HCl solution; then heat the fermentation broth to 60°C and maintain it for 30 min; stop heating and wait until the temperature of the fermentation broth drops to 45°C, then add 0.1% acid protease and 0.04% chitosan flocculant and maintain it for 1 h; add water equal to the volume of the fermentation broth to dilute it.

[0104] (2) Adding filter aid: Add 0.8% of a composite filter aid (the composite filter aid is a combination of 200-mesh perlite and 800-mesh red diatomaceous earth in a volume ratio of 1:1) to the diluted fermentation broth obtained in step 1, and mix thoroughly to obtain a fermentation broth with added filter aid.

[0105] (3) Dead-end filtration: The fermentation broth with filter aid added obtained in step 2 was subjected to dead-end filtration at a filtration pressure of 0.2 MPa and 40°C using a 1-3 μm polyester plain filter membrane for 70 min to obtain the fermentation broth clear liquid and filter residue with bacterial cells removed.

[0106] (4) Filtration: Add 5 times the amount of deionized water (pH value is 7.0-7.5) than that in the equipment compartment into the filter assembly containing the filter residue, and perform forward filtration on the filter residue. The operating pressure during filtration is 0.2 MPa and the temperature is 40°C.

[0107] The polysaccharide content in the fermentation supernatants of Examples 8 and 9, the polysaccharide content in the filtrates obtained after diafiltration, the average permeate flux and energy consumption during diafiltration were measured, and the polysaccharide recovery rate in the filtrates was calculated. The results are shown in Table 5.

[0108] Polysaccharide recovery rate in the filtrate (%) = (polysaccharide content in the filtrate / polysaccharide content lost after treatment) × 100; Wherein: the polysaccharide content in the filtrate refers to the pullulan content in the filtrate obtained after washing and filtration; the polysaccharide content lost after treatment = the pullulan content in the fermentation liquid before treatment - the pullulan content in the fermentation liquid after treatment after dead-end filtration.

[0109] Table 5 Filtration effect

[0110] The results show that reverse diafiltration can significantly improve the recovery rate of polysaccharides, reduce energy consumption, and maintain efficient filtration performance, and is suitable for the diafiltration treatment of pullulan polysaccharide fermentation broth.

[0111] In summary, the sterilization method provided by the present invention performs well in removing bacteria, and has high filtration efficiency and low energy consumption. It is an effective method for removing bacteria from high-viscosity pullulan fermentation broth. At the same time, the fermentation broth clear liquid has a high pullulan content and very little bacteria content. The subsequent extraction of pullulan using the pullulan is helpful to improve the pullulan recovery rate. If the protein is completely decomposed in the pretreatment stage, there is no need to perform protein removal operation when subsequently extracting pullulan.

[0112] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for removing bacteria from a high-viscosity pullulan fermentation broth, characterized in that: The method comprises the following steps: S1. Fermentation broth pretreatment: adjusting the pH value of the high-viscosity pullulan fermentation broth to 1-6; heating and maintaining the fermentation broth at 60-121°C for 20-30 minutes; stopping heating and waiting until the fermentation broth temperature is ≤55°C, adding acidic protease and flocculant in an amount of 0.01%-0.04%, mixing, and then standing for 0.5-3 hours; diluting the fermentation broth with deionized water to obtain a diluted fermentation broth; S2: adding filter aids to the diluted fermentation broth obtained in S1, and mixing thoroughly to obtain fermentation broth with added filter aids; S3 dead-end filtration: The fermentation broth with the filter aid added obtained in S2 is sterilized by dead-end filtration at 0.05-0.8 MPa and 30-50°C to obtain a fermentation broth clear liquid and filter residue free of bacterial cells; S4 filtration washing: adding filtration washing solvent into the filter assembly containing the filter residue described in S3 to perform filtration washing on the filter residue.

2. The method for removing bacteria from high-viscosity pullulan fermentation broth according to claim 1, characterized in that: The diafiltration in S4 is reverse diafiltration; the diafiltration solvent is water, has a pH value of 6.0-7.5, and a temperature 0-10°C higher than the filtration temperature; and the amount of the diafiltration solvent used is 1-7 times the volume of the filter assembly chamber.

3. The method for removing bacteria from high-viscosity pullulan fermentation broth according to claim 1, characterized in that: In said S1, the pH value of the high-viscosity pullulan fermentation broth is adjusted to 2-4; and / or In S1, the fermentation liquid temperature is heated and maintained at 121° C. for 20 min; and / or In S1, the fermentation broth is diluted with deionized water, and the volume ratio of deionized water to the fermentation broth is 1-3:

1.

4. The method for removing bacteria from high-viscosity pullulan fermentation broth according to claim 3, characterized in that: In said S1, the pH value of the high-viscosity pullulan fermentation broth is adjusted to 3; and / or The volume ratio of deionized water to the fermentation broth is 1:

1.

5. The method for removing bacteria from high-viscosity pullulan fermentation broth according to claim 1, characterized in that: In S1, heating is stopped and when the temperature of the fermentation liquid drops to 40-55° C., flocculant and acid protease are added, mixed and then allowed to stand for 0.5-2 h; the amount of acid protease added is 0.01%-0.1%, and the flocculant is chitosan.

6. The method for removing bacteria from high-viscosity pullulan fermentation broth according to claim 1, characterized in that: The amount of filter aid added in S2 is 0.2%-10%; and / or The filter aid in S2 is a single filter aid or a composite filter aid; the filter aid is an organic filter aid or an inorganic filter aid.

7. The method for removing bacteria from a high-viscosity pullulan fermentation broth according to claim 6, wherein: The amount of filter aid added in S2 is 0.5%-1.2%; and / or The filter aid in S2 is one of 325 mesh red diatomaceous earth, 400 mesh perlite, 200 mesh white diatomaceous earth, 325 mesh activated carbon, an equal volume mixture of 200 mesh perlite and 800 mesh red diatomaceous earth, or an equal volume mixture of 200 mesh red diatomaceous earth and 400 mesh white diatomaceous earth.

8. The method for removing bacteria from a high-viscosity pullulan fermentation broth according to claim 1, wherein: Preferably, the filter membrane used for the dead-end filtration in S3 is an organic membrane or an inorganic membrane, the membrane pore size is 0.1-4 μm, and the membrane assembly type is a flat plate type; the material of the organic membrane is one of polyethylene, polyester, nylon or polypropylene; the weaving method of the organic membrane is plain weave; the inorganic membrane is a ceramic membrane, a metal membrane or a metal and ceramic composite membrane; the material of the ceramic membrane is one of aluminum oxide, cobalt oxide, silicon oxide, aluminum silicate or silicon carbide; the metal membrane is one of silver film, nickel film or titanium film.

9. The fermentation liquid obtained by the method for removing bacteria from high-viscosity pullulan fermentation liquid according to any one of claims 1 to 8.

10. Use of the method for removing bacteria from a high-viscosity pullulan fermentation broth according to any one of claims 1 to 8 in extracting pullulan.

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