CN114538589A

CN114538589A - Method for enriching terpenoid in fermentation liquor and cellulose-based flocculant used by method

Info

Publication number: CN114538589A
Application number: CN202210181242.XA
Authority: CN
Inventors: 王鹏; 马国良; 郑之明; 王丽; 王晗; 赵根海
Original assignee: Hefei Institutes of Physical Science of CAS
Current assignee: Hefei Institutes of Physical Science of CAS
Priority date: 2022-02-25
Filing date: 2022-02-25
Publication date: 2022-05-27
Anticipated expiration: 2042-02-25
Also published as: CN114538589B

Abstract

A method for enriching terpenoids in fermentation liquor and a cellulose-based flocculant used by the method comprise the following steps of 1: centrifuging fermentation liquor containing terpenoid obtained by a microbial fermentation production method to obtain supernatant; step 2: adjusting the pH value of the supernatant to 4.8-6.0, and stirring at the rotation speed of 200-; then adding 0.1-1.0% by mass of cellulose-based flocculant into the mixture, stirring the mixture for 10-30min at the rotating speed of 30-60r/min, adding diatomite according to the mass ratio of 0.25-1.00%, mixing the mixture until the mixture is uniformly suspended, and then centrifuging the mixture to collect flocculating precipitate; and step 3: extracting heptaene menadione in the flocculation precipitation by using absolute ethyl alcohol or butyl acetate solution to obtain heptaene menadione ethanol solution or heptaene menadione butyl acetate solution. The enrichment rate of the terpenoid in the fermentation liquor is up to more than 80%.

Description

Method for enriching terpenoid in fermentation liquor and cellulose-based flocculant used by method

Technical Field

The invention relates to a method for enriching terpenoids in fermentation liquor and a cellulose-based flocculant used by the method, and belongs to the technical field of biological fermentation.

Background

Terpenoids are compounds derived from pentamethylene dihydroxy acid and having isoprene unit (C5 unit) as basic structural unit in molecular skeleton, and derivatives thereof. These oxygen-containing derivatives may be alcohols, aldehydes, ketones, carboxylic acids, esters, and the like. Terpenoids are widely found in nature, are the main components of essences, resins, pigments, etc. constituting certain plants, and are indispensable raw materials in the cosmetic and food industries.

Terpenoids are mainly produced in two ways at present, the first way is direct extraction from plants, and the other way is preparation by microbial fermentation, for example, terpenoids with medicinal value and health care function such as vitamin K2, squalene, paclitaxel, lycopene, etc. can be prepared by microbial fermentation.

Taking vitamin K2 as an example, the main production strain of vitamin K2 is Bacillus subtilis, VK2 with the strain yield of 60% -80% exists in the supernatant of fermentation liquor after fermentation, but VK2 has a small content in the supernatant of fermentation liquor and is difficult to extract and enrich, and most of the prior methods rely on macroporous resin and reverse silica gel to be matched with organic reagents for enrichment extraction, so that the process has the defects of large consumption of the organic reagents, high use cost of the reverse silica gel, difficult cleaning of waste liquid and the like, and is difficult to apply in large scale in industrial production. Researches find that the flocculant is effective in extracting and enriching trace valuable substances in the supernatant of fermentation liquor, has the characteristics of small dosage, no pollution, low cost, easy treatment of waste liquid and the like, and is widely applied to the industries of sewage treatment, biological pharmacy, paper pulp production, skin care, metal processing and the like at present.

Depending on the composition, flocculants are broadly classified into three types, inorganic flocculants, organic flocculants, and biological flocculants. With the research on solid-liquid separation, researchers began to prepare novel flocculants by using natural organic molecules such as cellulose and starch as substrates. Cellulose is a renewable natural polymer, which is abundant, widely available and very readily available in nature. It has the characteristics of good biocompatibility, easy biodegradation and the like. Cellulose is a fibrous high molecular polymer with three-dimensional structural characteristics, has a large specific surface area, contains a large number of holes in the structure, and contains a plurality of hydrophilic hydroxyl groups in the molecule, so that the cellulose has certain performance of adsorbing small organic molecules and heavy metal ions. However, researches show that when natural cellulose is directly used as an adsorbent, the adsorption capacity and selectivity are very low, because a large number of hydroxyl groups exist in the structure of a cellulose polymer, hydrogen bonds are widely formed between molecular chains and in the molecular chains, and the special structure influences the reaction activity of the cellulose polymer. Therefore, many researches can directly modify cellulose and prepare the cellulose-based flocculant through oxidation reaction, etherification reaction, esterification reaction, graft copolymerization reaction and other modes, so that a functional group is directly introduced to a cellulose skeleton to serve as a binding site of suspended substances and colloidal particles, and the extraction and enrichment efficiency is enhanced. Modified cellulose-based flocculants have been widely used in water treatment and liquid extraction studies. The flocculant is mainly classified into four types, namely a nonionic type, a cationic type, an anionic type and an amphoteric type according to different groups carried by the flocculant. The main action mechanism comprises: has the functions of neutralization, adsorption bridging, net catching, rolling, sweeping and the like. The reason is that most solutions have colloidal properties, and the flocculation process is the process of destabilization, coagulation, flocculation and sedimentation of substances and suspended matters in colloidal particle state in the solutions under the action of a flocculating agent.

The research provides theoretical reference for treating fermentation liquor by using a flocculation technology, but the invention of flocculation and sedimentation of fermentation supernatant of bacillus subtilis by using a flocculating agent is few at present, and in addition, the treatment of VK2 fermentation liquor by using flocculation and extraction processes is also rarely reported. Recently, lauryl methacrylate is grafted to the surface of cellulose microcrystals to prepare a cellulose flocculant for treating bacillus natto fermentation liquor, the flocculation enrichment effect of the flocculant on VK2 in the supernatant of the fermentation liquor is researched to overcome the defects of high organic reagent consumption, high cost, easy environmental pollution and the like in the traditional separation and purification technology of VK2 fermentation liquor, and the specific enrichment of the flocculant prepared in the invention is researched to provide a basis for efficient separation and purification and industrial production of VK2 and naphthoquinone compounds.

Disclosure of Invention

The invention aims to provide a method for enriching terpenoids in fermentation liquor and a cellulose-based flocculant used by the method.

In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a cellulose-based flocculant, the method of preparation comprising the steps of:

step 1: mixing a sodium hydroxide solution and a urea solution to obtain a sodium hydroxide-urea mixed solution;

step 2: dissolving cellulose microcrystals in a pre-frozen sodium hydroxide-urea mixed solution to obtain a cellulose solution with the concentration of 20g/L-100 g/L;

and step 3: adding the cellulose solution into a reaction vessel, introducing nitrogen at the temperature of 60-80 ℃ for 20-40min, then adding an ammonium persulfate aqueous solution, and reacting for 20-40 min; then adding lauryl methacrylate, heating to 90-100 ℃, and reacting for 1.5-2.5h to obtain a product;

and 4, step 4: and washing the product by using an ethanol solution, finally washing by using deionized water, and grinding into powder after freeze drying to obtain the cellulose-based flocculant.

The preferable technical scheme is as follows: the mass fraction of the sodium hydroxide solution is 5-10%, the mass fraction of the urea solution is 11-20%, and the volume ratio of the sodium hydroxide solution to the urea solution is 1: 0.8-1.2.

The preferable technical scheme is as follows: the concentration of the ammonium persulfate aqueous solution is 1.5-2.5mg/ml, and the volume ratio of the ammonium persulfate aqueous solution to the cellulose solution is 1-3: 20-40 parts of; the ratio of lauryl methacrylate to cellulose solution is 2-6 g: 20-40 ml.

In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a method for enriching terpenoids in a fermentation broth, comprising the steps of:

step 1: centrifuging fermentation liquor containing terpenoid obtained by a microbial fermentation production method to obtain supernatant;

step 2: adjusting the pH value of the supernatant to 4.8-6.0, and stirring at the rotation speed of 200-; then adding 0.1-1.0% by mass of cellulose-based flocculant into the mixture, stirring the mixture for 10-30min at the rotating speed of 30-60r/min, adding diatomite according to the mass ratio of 0.25-1.00%, mixing the mixture until the mixture is uniformly suspended, and then centrifuging the mixture to collect flocculating precipitate; the cellulose-based flocculant is the cellulose-based flocculant according to any one of claims 1 to 3;

and 3, step 3: extracting heptaene menadione in the flocculation precipitation by using absolute ethyl alcohol or butyl acetate to obtain an ethanol solution of the heptaene menadione or a butyl acetate solution of the heptaene menadione.

The preferable technical scheme is as follows: the fermentation liquid comprises bacillus natto thallus, a fermentation culture medium, thallus metabolites and heptamenadione, and the content of the heptamenadione in the fermentation liquid is 30 mg/L-80 mg/L.

The preferable technical scheme is as follows: in the step 1, the rotation speed of the centrifugation is 1000-.

The preferable technical scheme is as follows: in step 2, the rotation speed of centrifugation is 800-.

Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:

the enrichment rate of the terpenoid in the fermentation liquor is up to more than 80%.

Drawings

FIG. 1 scanning electron micrograph of cellulose.

FIG. 2 is a scanning electron microscope image of the cellulose-based flocculant prepared by the present invention.

FIG. 3 is a graph of flocculant elution from absolute ethanol.

FIG. 4 is a graph showing concentration detection of menatetrenone (MK7) enriched in a flocculated precipitate using HPLC.

FIG. 5 shows the enrichment ratio of flocculant on VK3 and squalene.

Detailed Description

The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will be apparent to those skilled in the art from the present disclosure.

Please refer to fig. 1-5. It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are provided for a better understanding of the present invention, and are not intended to limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were all purchased from a conventional biochemical laboratory unless otherwise specified.

The detection method of heptamenadione (MK7) adopts high performance chromatography.

The method for detecting the content of the protein by the high performance chromatography comprises the following steps: heptarenylnaphthoquinone (MK7) enriched in the flocculent precipitate was extracted using a mixed solution of n-hexane/isopropanol/n-butanol (1: 2: 1). The high performance liquid chromatography detection experiment of heptaene menadione (MK7) adopts a mobile phase system of methanol/dichloromethane (4: 1), the detection wavelength is 248nm, the flow rate is 1mL/min, and the column temperature is 37 ℃.

And (4) calculating the flocculation enrichment rate. And respectively calculating the content of menadione (MK7) in the flocculated and settled product and the residual amount of menadione (MK7) in the supernatant of the flocculated fermentation liquor, and finally calculating the enrichment rate.

The formula for calculating the enrichment rate in the step (5) is as follows:

the enrichment ratio (enrichment ratio) ═ m0-m1)/m0 × 100%;

in the formula, m0 represents the total mass of menadione (MK7) in the supernatant of the fermentation liquid before flocculation and m1 represents the residual mass of menadione (MK7) in the supernatant of the fermentation liquid after flocculation and the unit is mg.

Example 1: method for enriching terpenoid in fermentation liquor and cellulose-based flocculant used by method

Preparing a cellulose flocculant (MCC-g-LAM) by using cellulose microcrystals and lauryl methacrylate, flocculating heptaene menadione (MK7) in the supernatant of bacillus subtilis fermentation liquor by using a cellulose-based flocculant (MCC-g-LAM), and then extracting the heptaene menadione (MK7) enriched in the flocculation sediment by using absolute ethyl alcohol to calculate the enrichment ratio.

Firstly, dissolving cellulose microcrystals (3g) by using a pre-frozen 5 wt% NaOH/11 wt% urea solution at the temperature of-20 ℃ to obtain a transparent cellulose solution, adding the transparent cellulose solution into a four-neck flask with a nitrogen introducing device, a thermometer, a stirring paddle and a condenser, introducing nitrogen for stirring for 40min at the temperature of 70 ℃, then adding a certain amount of ammonium persulfate aqueous solution (3mg/ml) for reacting for 30min, wherein the water phase of the whole reaction system is not more than 40 ml. Lauryl methacrylate (4g) was added and the reaction was allowed to proceed for about 2 hours at a temperature of 95 ℃ to form a gum. After the reaction is finished, the colloidal product is added with water: repeatedly washing with anhydrous ethanol (30:70), washing with deionized water, freeze drying, grinding into powder, sealing, and storing as experimental flocculant. 5 wt% NaOH/11 wt% urea means a mixture of 1: 1.

the flocculation capacity of microcrystalline cellulose (MCC) and a cellulose-based flocculant (MCC-g-LAM) was tested using a Bacillus subtilis fermentation broth. Firstly, taking six 200ml of bacillus subtilis fermentation liquor with the concentration of heptamenadione (MK7) detected, placing the bacillus subtilis fermentation liquor into a brown beaker, taking three parts of fermentation liquor as a group of parallel experiments, adjusting the pH value of the fermentation liquor to 5, and stirring for 20 min. Then 50mg of anhydrous CaCl2 was added and stirred for 10 min. Adding equal amounts of microcrystalline cellulose and MCC-g-LAM into different beakers, stirring the suspension at 250rpm for 60min, and slowly mixing at 50rpm for 30 min; finally, 1g of diatomaceous earth was added as a filter aid and the suspension was centrifuged in a centrifuge at 1500rpm for 5min, leaving a precipitate.

Extracting heptaene menadione (MK7) in a settled product obtained by a flocculation experiment by using a certain amount of absolute ethyl alcohol, extracting residual heptaene menadione (MK7) in a supernatant of fermentation liquor by using a mixed solution of normal hexane/isopropanol/normal butanol, respectively calculating the content of heptaene menadione (MK7) in the flocculated settled product and the residual amount of heptaene menadione (MK7) in the supernatant of the flocculated fermentation liquor, and finally calculating the enrichment rate, wherein the specific calculation method is shown in formula (1).

Enrichment ratio (enrichment rate) (m0-m1)/m0 × 100% (1)

M0 in the formula (1) represents the total mass of menadione (MK7) in the fermentation liquid before flocculation and the unit of m1 represents the residual mass of menadione (MK7) in the fermentation liquid after flocculation and the unit of mg.

The flocculation enrichment rate of different flocculants on heptaene menadione (MK7) is compared in the following table.

Firstly, 50mg of vitamin K3 and 10mg of squalene are respectively added into 100ml of ultrapure water, the mixture is stirred at 250rpm for 10min and stirred into a uniform VK3 suspension solution and squalene suspension system, a certain amount of flocculant is added into the system, the mixture is stirred at 250rpm for 40min, then 1g of filter aid diatomite is added, the suspension is placed in a centrifuge and centrifuged at 1000rpm for 5min, precipitates are left, vitamin K3 and squalene enriched in the precipitates of the two systems are respectively extracted by using a mixed solution of n-hexane/isopropanol/n-butanol, and VK3 and squalene in an extraction liquid sample are measured by using high performance liquid chromatography. The high performance liquid chromatography detection experiment of VK3 adopts a mobile phase system of acetonitrile/water (70/30) solution, the detection wavelength is 265nm, the flow rate is 0.85mL/min, and the column temperature is 40 ℃. The squalene high performance liquid chromatography detection experiment adopts a mobile phase system of methanol/isopropanol (70/30), the detection wavelength is 205nm, the flow rate is 1mL/min, and the column temperature is 30 ℃. And (4) comparing the detected sample with a standard sample, and calculating the enrichment rate of the flocculant on VK3 and squalene by using a standard curve.

The experimental results are shown in fig. 5, fig. 5 (a), fig. 5 (b) show the flocculation enrichment effect of VK3 containing naphthoquinone ring before and after flocculation, and the results show that after flocculation, the enrichment rate of the flocculant on naphthoquinone ring is about 45%. Fig. 5 (c) and 5 (d) show the flocculation enrichment effect of the flocculant on squalene containing only isoprene side chains, and the result shows that after flocculation, the enrichment rate of the flocculant on isoprene side chains is about 70%.

Example 2: method for enriching terpenoid in fermentation liquor and cellulose-based flocculant used by method

The method comprises the steps of utilizing cellulose microcrystals as a substrate, grafting a certain amount of lauryl methacrylate to prepare a cellulose-based flocculant, flocculating and enriching terpenoids in fermentation liquor by using the flocculant and cellulose as media respectively, then extracting by using ethanol or ethyl acetate as a solvent for separation and purification, and finally detecting the concentrations of the terpenoids before and after the enrichment of the cellulose and the flocculant to judge whether the enrichment efficiency is improved or not.

The method mainly comprises the following steps:

(1) and (3) preparing a cellulose-based flocculant.

And preparing a mixed solution of sodium hydroxide and urea. Preparing a mixed solution of sodium hydroxide and urea according to the concentration ratio of 8 wt% NaOH to 10 wt% urea. 5 wt% NaOH/11 wt% urea means a mixture of 1: 0.8.

a cellulose solution is prepared. Dissolving 3g of cellulose microcrystals by using a mixed solution of sodium hydroxide and urea pre-frozen at the temperature of-20 ℃ to obtain a transparent cellulose solution with the concentration of 60 g/L; cellulose microcrystals are purchased from Shanghai bioengineering technology, Inc. and require dissolution with a 98% purity solution of 5 wt% NaOH/11 wt% urea.

Introducing nitrogen gas for reaction. Adding the cellulose solution into a four-neck flask with a nitrogen introducing device, a thermometer, a stirring paddle and a condenser, introducing nitrogen at 60 ℃ for 30min, adding 2ml of ammonium persulfate aqueous solution (2mg/ml), and reacting for 30min, wherein the dosage of the cellulose solution in the whole reaction system is 30 ml.

And (3) carrying out lauryl methacrylate reaction. 4g of lauryl methacrylate is added into the solution after the reaction by introducing nitrogen, the temperature is raised to 95 ℃, and the reaction is carried out for 2 hours until a colloidal substance is formed.

And (5) cleaning and drying. After the reaction is finished, the colloidal product is treated with "water: repeatedly washing with absolute ethyl alcohol (30: 70)', finally washing with deionized water, freeze-drying, grinding into powder, and sealing for storage to obtain the cellulose-based flocculant for later use.

And (3) freeze drying conditions:

(1) and (5) vacuumizing and freeze-drying the sample for about 15 hours by using a vacuum freeze dryer to obtain a dried product.

(2) Fermenting the fermentation liquor of the strain for producing terpenoid (heptaene menadione (MK7)) obtained by a microbial fermentation production method, and centrifuging to obtain fermentation supernatant.

The fermentation liquor in the step (2) basically comprises bacillus natto thallus, a fermentation culture medium (glycerol, soybean flour and corn flour clear liquid and inorganic salt), thallus metabolites, heptaene menadione (MK7) and the like; the content of menatetrenone (MK7) in the fermentation liquor is 55 mg/L.

And (3) the centrifugal equipment in the step (2) is a floor type freezing centrifugal machine, the centrifugal speed of the centrifugal machine is 2500rpm, and the centrifugal time is 15 min.

(3) Flocculation treatment

Adding a certain amount of hydrochloric acid into the fermentation supernatant prepared in the step (2) to adjust the pH value to 5.4; placing the fermentation supernatant into a photophobic stirring device at room temperature, and stirring for 7min at a rotating speed of 250 r/min; adding the cellulose-based flocculant in the step (1) into the fermentation supernatant according to the proportion of 0.5%, and stirring for 20min at the rotating speed of 50 r/min; adding filter aid diatomite according to the proportion of 0.55%, mixing until uniform suspension is realized, placing the suspension in a centrifuge, centrifuging for 6min at the rotating speed of 1500rpm, and flocculating and precipitating the receipt for later use.

(4) Extraction and enrichment of menatetrenone (MK7) in the flocculation sedimentation product.

And (3) extracting the heptaene menadione (MK7) in the flocculation sedimentation in the step (3) by using a certain amount of absolute ethyl alcohol or butyl acetate solution to obtain a heptaene menadione (MK7) ethyl alcohol solution or a heptaene menadione butyl acetate solution.

Example 3: method for enriching terpenoids in fermentation liquor and cellulose-based flocculant used by method

A cellulose-based flocculant, the method of preparation comprising the steps of:

step 2: dissolving cellulose microcrystals in a pre-frozen sodium hydroxide-urea mixed solution to obtain a cellulose solution with the concentration of 20 g/L;

and 3, step 3: adding the cellulose solution into a reaction vessel, introducing nitrogen at the temperature of 60 ℃ for 20min, then adding an ammonium persulfate aqueous solution, and reacting for 20 min; then adding lauryl methacrylate, heating to 90 ℃, and reacting for 1.5h to obtain a product;

The preferred embodiment is: the mass fraction of the sodium hydroxide solution is 5%, the mass fraction of the urea solution is 11%, and the volume ratio of the sodium hydroxide solution to the urea solution is 1: 0.8.

the preferred embodiment is: the concentration of the ammonium persulfate aqueous solution is 1.5mg/ml, and the volume ratio of the ammonium persulfate aqueous solution to the cellulose solution is 1: 20; the ratio between lauryl methacrylate and cellulose solution was 2 g: 20 ml.

A method for enriching terpenoids in a fermentation broth, comprising the steps of:

step 2: adjusting the pH value of the supernatant to 4.8, and then stirring for 50min at the rotating speed of 2000r/min in a dark condition; then adding 0.1% cellulose-based flocculant by mass into the mixture, stirring the mixture for 10min at the rotating speed of 30r/min, adding diatomite according to the mass ratio of 0.25%, mixing the mixture until the mixture is uniformly suspended, and then centrifuging the mixture to collect flocculating precipitate;

and step 3: extracting heptaene menadione in the flocculation precipitation by using absolute ethyl alcohol or butyl acetate to obtain an ethanol solution of the heptaene menadione or a butyl acetate solution of the heptaene menadione.

The preferred embodiment is: the fermentation liquid comprises bacillus natto thalli, a fermentation culture medium, thalli metabolites and heptamenadione, and the content of the heptamenadione in the fermentation liquid is 30 mg/LL.

The preferred embodiment is: in step 1, the rotation speed of centrifugation is 1000rpm, and the centrifugation time is 10 min.

The preferred embodiment is: in step 2, the rotation speed of the centrifugation is 800rpm, and the centrifugation time is 5 min.

Example 4: method for enriching terpenoid in fermentation liquor and cellulose-based flocculant used by method

step 2: dissolving cellulose microcrystals in a pre-frozen sodium hydroxide-urea mixed solution to obtain a cellulose solution with the concentration of 100 g/L;

and step 3: adding the cellulose solution into a reaction vessel, introducing nitrogen at the temperature of 80 ℃ for 40min, then adding an ammonium persulfate aqueous solution, and reacting for 40 min; then adding lauryl methacrylate, heating to 100 ℃, and reacting for 2.5h to obtain a product;

The preferred embodiment is: the mass fraction of the sodium hydroxide solution is 10%, the mass fraction of the urea solution is 20%, and the volume ratio of the sodium hydroxide solution to the urea solution is 1: 1.2.

the preferred embodiment is: the concentration of the ammonium persulfate aqueous solution is 2.5mg/ml, and the volume ratio of the ammonium persulfate aqueous solution to the cellulose solution is 3: 40; the ratio between lauryl methacrylate and cellulose solution was 6 g: 40 ml.

step 2: adjusting the pH value of the supernatant to 6.0, and then stirring for 10min at the rotating speed of 300r/min in a dark condition; then adding a cellulose-based flocculating agent with the mass of 1.0 percent of that of the mixture into the mixture, stirring the mixture for-30 min at the rotating speed of 60r/min, adding diatomite into the mixture according to the mass proportion of 1.00 percent of that of the mixture, mixing the mixture until the mixture is uniformly suspended, and then centrifuging the mixture to collect flocculating precipitate;

and step 3: extracting heptaene menadione in the flocculation precipitation by absolute ethyl alcohol or butyl acetate to obtain an ethanol solution of heptaene menadione or a butyl acetate solution of heptaene menadione.

The preferred embodiment is: the fermentation liquid comprises bacillus natto thallus, a fermentation culture medium, thallus metabolites and heptamenadione, and the content of the heptamenadione in the fermentation liquid is 80 mg/L.

The preferred embodiment is: in step 1, the rotation speed of the centrifugation is 3500rpm, and the centrifugation time is 20 min.

The preferred embodiment is: in step 2, the rotation speed of the centrifugation is 2500rpm, and the centrifugation time is 10 min.

The foregoing is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting thereof in any way, and any modifications or variations thereof that fall within the spirit of the invention are intended to be included within the scope thereof.

Claims

1. A cellulose-based flocculant, characterized in that: the preparation method comprises the following steps:

2. The cellulose-based flocculant according to claim 1, characterized in that: the mass fraction of the sodium hydroxide solution is 5-10%, the mass fraction of the urea solution is 11-20%, and the volume ratio of the sodium hydroxide solution to the urea solution is 1: 0.8-1.2.

3. The cellulose-based flocculant according to claim 1, characterized in that: the concentration of the ammonium persulfate aqueous solution is 1.5-2.5mg/ml, and the volume ratio of the ammonium persulfate aqueous solution to the cellulose solution is 1-3: 20-40 parts of; the ratio of lauryl methacrylate to cellulose solution is 2-6 g: 20-40 ml.

4. A method for enriching terpenoids in a fermentation broth, which is characterized by comprising the following steps: comprises the following steps:

5. The method for enriching terpenoids in a fermentation broth according to claim 4, wherein: the fermentation liquid comprises bacillus natto thallus, a fermentation culture medium, thallus metabolites and heptamenadione, and the content of the heptamenadione in the fermentation liquid is 30-80 mg/L.

6. The method for enriching terpenoids in a fermentation broth according to claim 4, wherein: in the step 1, the rotation speed of the centrifugation is 1000-.

7. The method for enriching terpenoids in a fermentation broth according to claim 4, wherein: in the step 2, the rotation speed of the centrifugation is 800-2500rpm, and the centrifugation time is 5-10 min.