Synergistic fermentation and low-temperature extraction method of floral perfume as well as product and application thereof

Abstract

The invention provides a synergistic fermentation and low-temperature extraction method of floral perfume and a product and application thereof, the method is carried out in a membrane bioreactor, and a hollow fiber membrane module divides the internal space of the membrane bioreactor into a membrane module external space and a membrane module internal space; adding roses and water into the external space of the membrane module, sterilizing, inoculating pichia pastoris, and performing first-stage fermentation; inoculating lactobacillus plantarum into the inner space of the membrane component, and performing second-stage fermentation; and after fermentation is finished, carrying out flash extraction, and collecting fractions to obtain the floral perfume. According to the method, a physical-biological dual-regulation synergistic fermentation system is constructed, direct competition and metabolic inhibition among strains are avoided, stable and ordered metabolic flow from the aroma precursor to the target ester substance is ensured, degradation and dissipation of thermosensitive components are avoided through flash extraction, and the concentration and aroma fidelity of the target product are improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bio-chemical engineering and perfume technology, and specifically relates to a synergistic fermentation and low-temperature extraction method of floral perfume, as well as products and applications thereof. BACKGROUND

[0002] Natural plant perfumes have irreplaceable value in the fields of food, daily chemicals, and medicine, etc. The use of microbial fermentation technology to transform the aroma precursor substances in plant raw materials is an important technical means to produce or enhance natural perfumes. This method converts the sugar glycosides and amino acids with no or weak aroma in the raw materials into alcohol, aldehyde, and ester compounds with typical aroma notes through specific metabolic pathways of microorganisms.

[0003] In the prior art, multiple functional strains are sometimes required to work synergistically to achieve complex aroma transformation. However, when mixed fermentation is used, strains with different physiological characteristics will inevitably compete for nutrients and space in the same limited environment, and there will also be cross-inhibition effects between their metabolic products, which directly limits the overall metabolic flow efficiency and stability from the substrate to the final target product. Even if simple sequential fermentation is used, due to the lack of precise monitoring and connection of the reaction process at each stage, the timing of the intervention of the subsequent strain is often not optimal, resulting in insufficient substrate conversion and the inability to achieve efficient biological catalytic cascade reaction.

[0004] In the product extraction step after fermentation, the traditional process usually involves transferring the fermentation broth to a separate distillation equipment. In this material transfer step, the highly volatile aroma components are extremely easy to escape and be lost due to exposure to an open or semi-open environment. More importantly, the most widely used thermal extraction method such as water vapor distillation has an operating temperature close to 100℃. Such high temperature conditions will cause a large number of heat-sensitive aroma substances (especially esters and aldehydes) to undergo hydrolysis or chemical deterioration, which not only directly causes the loss of target products, but also destroys the original aroma characteristics of the products, significantly reducing the aroma fidelity.

[0005] In addition, existing conventional extraction techniques, such as one-time water vapor distillation, usually aim to completely recover all volatile substances, and the final product is a single fraction with mixed components and single characteristics. This method cannot selectively separate different aroma components according to their volatility differences, i.e., it cannot collect different components that determine the head, body, and tail notes of the aroma in steps and segments. This results in insufficient aroma layering of the final product and limits its flexibility and value as high-quality, differentiated perfume materials in subsequent fine perfumery applications. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application aims to provide a method for synergistic fermentation and low-temperature extraction of floral fragrance and products and applications thereof, which constructs a biological catalytic cascade reaction through physical separation and metabolite triggering, solves the problem of strain competition, avoids the degradation and escape of heat-sensitive components through in-situ low-temperature extraction, and improves the concentration and aroma fidelity of the target product.

[0007] To achieve the object of the present application, the following technical solutions are adopted:

[0008] In a first aspect, the present application provides a method for synergistic fermentation and low-temperature extraction of floral fragrance, which is carried out in a membrane bioreactor, wherein a hollow fiber membrane assembly divides the internal space of the membrane bioreactor into an external space of the membrane assembly and an internal space of the membrane assembly.

[0009] Rose flowers and water are added to the external space of the membrane assembly, sterilized, inoculated with Pichia pastoris, and subjected to first-stage fermentation; Lactobacillus plantarum is inoculated into the internal space of the membrane assembly, and subjected to second-stage fermentation; after the fermentation is completed, flash extraction is carried out, and the distillate is collected to obtain the floral fragrance.

[0010] The present application constructs two physically isolated but metabolically connected reaction regions in a single reactor by setting a hollow fiber membrane assembly, triggers the subsequent fermentation stage in combination with the concentration monitoring of specific signal metabolites, and constitutes a synergistic fermentation system under the physical-biological dual regulation. Pichia pastoris hydrolyzes aroma precursors to generate phenethyl alcohol, and Lactobacillus plantarum converts phenethyl alcohol into phenethyl acetate. The fermentation system avoids direct competition and metabolic inhibition between the two functional strains, ensures that each strain performs directional biochemical conversion in the optimal physiological window period, and thus ensures that the metabolic flow from aroma precursors to target ester substances is stable and orderly. Through flash extraction, the degradation and escape of heat-sensitive components are avoided, and the concentration and aroma fidelity of the target product are improved.

[0011] Preferably, the pore size of the hollow fiber membrane is 0.1-0.2 μm, for example, it can be 0.1 μm, 0.11 μm, 0.12 μm, 0.13 μm, 0.14 μm, 0.15 μm, 0.16 μm, 0.17 μm, 0.18 μm, 0.19 μm or 0.2 μm, etc.

[0012] Other specific point values within the above numerical ranges can also be selected, which will not be described here.

[0013] The hollow fiber membrane of the present application has a specific size of membrane pore size, effectively traps Pichia pastoris (cell size 2-4 μm) and Lactobacillus plantarum (cell size 1-5 μm), and at the same time allows small molecule metabolites to pass freely.

[0014] Preferably, the rose and water are used in a ratio of (50~80) g:1 L.

[0015] Specific point values in (50~80) can be 50, 52, 55, 57, 60, 63, 65, 68, 70, 75, or 80, etc.

[0016] Preferably, the sterilization temperature is 110~130℃, for example, it can be 110℃, 112℃, 115℃, 117℃, 120℃, 123℃, 125℃, 128℃, or 130℃, etc.; the time is 20~30 min, for example, it can be 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, or 30 min, etc.

[0017] Other specific point values in the above numerical ranges can be selected, which will not be repeated here.

[0018] Preferably, the inoculation amount of Pichia pastoris is 8~10 vol%, for example, it can be 8 vol%, 8.2 vol%, 8.5 vol%, 8.7 vol%, 9 vol%, 9.3 vol%, 9.5 vol%, 9.8 vol%, or 10 vol%, etc.

[0019] Preferably, the viable cell count of the Pichia pastoris seed liquid is (1~3)×10 9 CFU / mL.

[0020] Specific point values in (1~3) can be 1, 1.2, 1.5, 1.7, 2, 2.3, 2.5, or 2.8, etc.

[0021] Preferably, the temperature of the first-stage fermentation is 28~30℃, for example, it can be 28℃, 28.2℃, 28.5℃, 28.7℃, 29℃, 29.3℃, 29.5℃, 29.8℃, or 30℃, etc.

[0022] Other specific point values in the above numerical ranges can be selected, which will not be repeated here.

[0023] Preferably, it further comprises the step of monitoring the concentration of metabolites reaching a threshold before inoculating Lactobacillus plantarum.

[0024] Preferably, the metabolite is acetic acid.

[0025] Preferably, the threshold value of the metabolite concentration is 0.08-0.12 g / L, for example, can be 0.08 g / L, 0.085 g / L, 0.09 g / L, 0.095 g / L, 0.10 g / L, 0.105 g / L, 0.11 g / L, 0.115 g / L or 0.12 g / L, etc.

[0026] Preferably, the inoculation amount of the Lactobacillus plantarum is 3-5 vol%, for example, can be 3 vol%, 3.2 vol%, 3.5 vol%, 3.7 vol%, 4 vol%, 4.3 vol%, 4.5 vol%, 4.8 vol% or 5 vol%, etc.

[0027] Preferably, the viable cell count of the Lactobacillus plantarum seed liquid is (1-3) x 10 8 CFU / mL.

[0028] The specific point value in (1-3) can be 1, 1.2, 1.5, 1.7, 2, 2.3, 2.5 or 2.8, etc.

[0029] Preferably, the temperature of the second stage fermentation is 35-37℃, for example, can be 35℃, 35.2℃, 35.5℃, 35.7℃, 36℃, 36.3℃, 36.5℃, 36.8℃ or 37℃, etc.; the time is 24-36 h, for example, can be 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h, 31 h, 32 h, 33 h, 34 h, 35 h or 36 h, etc.

[0030] Other specific point values in the above numerical ranges can be selected, which will not be described here one by one.

[0031] Preferably, the flash extraction is carried out in situ.

[0032] The present application completes the fermentation and extraction process in the same closed reactor in situ, which eliminates the loss of high volatile aroma components during the material transfer process.

[0033] Preferably, the pressure of the flash extraction is -0.04--0.09 MPa, for example, can be -0.04 MPa, -0.045 MPa, -0.05 MPa, -0.055 MPa, -0.06 MPa, -0.065 MPa, -0.07 MPa, -0.075 MPa, -0.08 MPa, -0.085 MPa or -0.09 MPa, etc.; the temperature is 25-40℃, for example, can be 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 32℃, 35℃, 37℃ or 40℃, etc.

[0034] Other specific point values within the above-mentioned numerical ranges can be selected, which will not be repeated here.

[0035] The application can extract aroma substances at a temperature below 45 DEG C, thereby avoiding chemical degradation or structural deterioration of heat-sensitive esters, aldehydes and other key aroma components to the maximum extent, and ensuring the aroma fidelity of the final product.

[0036] Preferably, the flash extraction includes a first-stage flash extraction and a second-stage flash extraction.

[0037] Preferably, the first-stage flash extraction has a pressure of -0.04 to -0.06 MPa, for example, -0.04 MPa, -0.042 MPa, -0.045 MPa, -0.047 MPa, -0.05 MPa, -0.053 MPa, -0.055 MPa, -0.058 MPa or -0.06 MPa, etc., and a temperature of 25 to 30 DEG C, for example, 25 DEG C, 25.5 DEG C, 26 DEG C, 26.5 DEG C, 27 DEG C, 27.5 DEG C, 28 DEG C, 28.5 DEG C, 29 DEG C, 29.5 DEG C or 30 DEG C, etc.

[0038] Preferably, the second-stage flash extraction has a pressure of -0.08 to -0.09 MPa, for example, -0.08 MPa, -0.081 MPa, -0.082 MPa, -0.083 MPa, -0.084 MPa, -0.085 MPa, -0.086 MPa, -0.087 MPa, -0.088 MPa, -0.089 MPa or -0.09 MPa, etc., and a temperature of 35 to 40 DEG C, for example, 35 DEG C, 35.5 DEG C, 36 DEG C, 36.5 DEG C, 37 DEG C, 37.5 DEG C, 38 DEG C, 38.5 DEG C, 39 DEG C, 39.5 DEG C or 40 DEG C, etc.

[0039] Other specific point values within the above-mentioned numerical ranges can be selected, which will not be repeated here.

[0040] The application adopts sequential segmented flash extraction technology, and controls the pressure and temperature in a programmed manner, so as to realize step-by-step and segmented collection according to the volatility difference of different aroma components, and obtain fractions with different aroma characteristics, thereby increasing the flexibility of blending.

[0041] The first-stage flash extraction enriches aldehydes, ketones and short-chain esters with lower boiling points and the strongest volatility in the fermentation liquor, and constitutes the top note part of the aroma; the second-stage flash extraction enriches main aroma components such as phenethyl acetate with medium and high boiling points, and constitutes the body note part of the aroma.

[0042] Preferably, the floral fragrance comprises the first-stage flash extraction fraction and / or the second-stage flash extraction fraction.

[0043] Preferably, the floral fragrance comprises the first-stage flash extraction fraction and the second-stage flash extraction fraction.

[0044] Preferably, the mass ratio of the first-stage flash extraction fraction and the second-stage flash extraction fraction is 1:(5~30).

[0045] Specific point values in the range of (5~30) can be 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25 or 30, etc.

[0046] Other specific point values in the above-mentioned numerical ranges can be selected, which will not be repeated here.

[0047] Preferably, the flash extraction is carried out under pulse treatment.

[0048] Preferably, the power of the pulse treatment is 100~350 W, for example, it can be 100 W, 120 W, 150 W, 170 W, 200 W, 230 W, 250 W, 280 W, 300 W or 350 W, etc.; the working time is 2~8 s, for example, it can be 2 s, 2.5 s, 3 s, 3.5 s, 4 s, 4.5 s, 5 s, 5.5 s, 6 s, 6.5 s, 7 s, 7.5 s or 8 s, etc.; the intermittent time is 2~8 s, for example, it can be 2 s, 2.5 s, 3 s, 3.5 s, 4 s, 4.5 s, 5 s, 5.5 s, 6 s, 6.5 s, 7 s, 7.5 s or 8 s, etc.

[0049] Other specific point values in the above-mentioned numerical ranges can be selected, which will not be repeated here.

[0050] The power of the pulse treatment in the first-stage flash extraction is 100~200 W, for example, it can be 100 W, 110 W, 120 W, 130 W, 140 W, 150 W, 160 W, 170 W, 180 W, 190 W or 200 W, etc.; the working time is 2~3 s, for example, it can be 2 s, 2.1 s, 2.2 s, 2.3 s, 2.4 s, 2.5 s, 2.6 s, 2.7 s, 2.8 s, 2.9 s or 3 s, etc.; the intermittent time is 5~8 s, for example, it can be 5 s, 5.2 s, 5.5 s, 5.7 s, 6 s, 6.3 s, 6.5 s, 6.8 s, 7 s, 7.5 s or 8 s, etc.

[0051] The power of the pulse treatment in the second-stage flash extraction is 250-350 W, for example, 250 W, 260 W, 270 W, 280 W, 290 W, 300 W, 310 W, 320 W, 330 W, 340 W or 350 W, etc.; the working time is 5-8 s, for example, 5 s, 5.2 s, 5.5 s, 5.7 s, 6 s, 6.3 s, 6.5 s, 6.8 s, 7 s, 7.5 s or 8 s, etc.; and the intermittent time is 2-3 s, for example, 2 s, 2.1 s, 2.2 s, 2.3 s, 2.4 s, 2.5 s, 2.6 s, 2.7 s, 2.8 s, 2.9 s or 3 s, etc.

[0052] Other specific point values in the above numerical ranges can be selected, which will not be repeated here.

[0053] The present application realizes efficient extraction of aroma substances at low temperature by applying pulse treatment in stages in flash extraction.

[0054] Preferably, the hollow fiber membrane assembly is prepared by a method comprising the following steps:

[0055] Polyvinylidene fluoride, polyvinylpyrrolidone and solvent are mixed to form a casting solution; the casting solution is extruded through a spinneret by dry-wet phase inversion spinning technology, passes through an air section, and then enters a coagulation bath for solidification and molding to obtain a hollow fiber membrane primary fiber; the bundle of hollow fiber membrane primary fibers is bundled together, and the two ends are sealed with epoxy resin to obtain the hollow fiber membrane assembly.

[0056] Preferably, the mass ratio of polyvinylidene fluoride, polyvinylpyrrolidone and solvent is (15-20):(5-8):(72-80).

[0057] The specific point value in (15-20) can be 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5 or 20, etc.

[0058] The specific point value in (5-8) can be 5, 5.2, 5.5, 5.7, 6, 6.3, 6.5, 6.8, 7, 7.5 or 8, etc.

[0059] The specific point value in (72-80) can be 72, 73, 74, 75, 76, 77, 78, 79 or 80, etc.

[0060] Other specific point values in the above numerical ranges can be selected, which will not be repeated here.

[0061] Preferably, the solvent comprises any one or a combination of at least two of N-methyl-2-pyrrolidone, N,N-dimethylacetamide, dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, dichloromethane or trichloromethane.

[0062] Preferably, the mixing is performed under a dry nitrogen atmosphere.

[0063] Preferably, the mixing is performed by stirring.

[0064] Preferably, the mixing is performed at a temperature of 55-65℃, for example, 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 64℃ or 65℃, etc., for a time period of 7-9 h, for example, 7 h, 7.2 h, 7.5 h, 7.7 h, 8 h, 8.3 h, 8.5 h, 8.8 h or 9 h, etc.

[0065] Preferably, the air section has a length of 10-20 cm, for example, 10 cm, 11 cm, 12 cm, 13 cm, 14 cm, 15 cm, 16 cm, 17 cm, 18 cm, 19 cm or 20 cm, etc.

[0066] Preferably, the coagulation bath is water.

[0067] Preferably, the coagulation bath has a temperature of 22-27℃, for example, 22℃, 22.5℃, 23℃, 23.5℃, 24℃, 24.5℃, 25℃, 25.5℃, 26℃, 26.5℃ or 27℃, etc.

[0068] Other specific point values within the above-mentioned numerical ranges can also be selected, which are not listed here.

[0069] In a second aspect, the present application provides a floral fragrance prepared by the method of the first aspect.

[0070] In a third aspect, the present application provides use of the floral fragrance of the second aspect in the preparation of a cigarette.

[0071] Compared with the prior art, the present application has the following beneficial effects:

[0072] The application sets up two physically isolated but metabolically connected reaction zones in a single reactor by setting up a hollow fiber membrane module, combined with concentration monitoring of specific signal metabolites to trigger subsequent fermentation stages, constituting a synergistic fermentation system under the dual regulation of physical-biological. Pichia pastoris hydrolyzes aroma precursor to produce phenylethanol, and Lactobacillus plantarum converts phenylethanol into phenylethyl acetate, avoiding direct competition and metabolic inhibition between the two functional strains, ensuring that each strain performs directional biochemical conversion in the optimal physiological window period, thereby ensuring stable and orderly metabolic flow from aroma precursors to target ester substances. Through flash extraction, degradation and escape of heat-sensitive components are avoided, and the concentration and aroma fidelity of the target product are improved. DETAILED DESCRIPTION

[0073] In order to further illustrate the technical means adopted by the present application and its effects, the technical solutions of the present application will be further described below in combination with preferred embodiments of the present application, but the present application is not limited in the scope of the embodiments.

[0074] If a specific technology or condition is not specified in the embodiments, it is carried out according to the technology or condition described in the literature in the art or according to the product manual. If the reagent or instrument used is not specified by the manufacturer, it is a conventional product that can be commercially available through a regular channel.

[0075] Pichia pastoris GS115 was inoculated in YPD liquid medium and cultured at 29℃ with 210 rpm shaking for 22 h to prepare Pichia pastoris seed liquid, and the viable cell count was 1×10 9 CFU / mL. Lactobacillus plantarum ATCC8014 was inoculated in MRS liquid medium and statically cultured at 36℃ for 21 h to prepare Lactobacillus plantarum seed liquid, and the viable cell count was 1×10 8 CFU / mL.

[0076] Under a dry nitrogen atmosphere, polyvinylidene fluoride, polyvinylpyrrolidone and N,N-dimethylacetamide were mixed in a mass ratio of 18:7:76, stirred at 60℃ for 8 h to form a casting solution. The casting solution was extruded through a spinneret using dry-wet phase inversion spinning technology, passed through a 15 cm air section, and then entered a 25℃ deionized water coagulation bath to solidify and form a hollow fiber membrane primary fiber. 150 hollow fiber membrane primary fibers were cut and bundled together, and the two ends were sealed with epoxy resin to obtain the hollow fiber membrane module, with a membrane pore size of 0.1 μm. The hollow fiber membrane module was soaked in 75 vol% ethanol aqueous solution for storage, and washed with sterile deionized water to neutral before use.

[0077] Example 1

[0078] The present embodiment provides a method for extracting a floral perfume, which comprises:

[0079] The hollow fiber membrane module is placed in the membrane bioreactor, which divides the internal space of the membrane bioreactor into the external space of the membrane module and the internal space of the membrane module. 65 g of rose flowers and 1 L of water are added to the external space of the membrane module, sterilized at 120℃ for 25 min, and then inoculated with 9 vol% of Pichia pastoris seed liquid to perform the first-stage fermentation at 29℃. When the acetic acid concentration reaches 0.10 g / L, 4 vol% of Lactobacillus plantarum seed liquid is inoculated into the internal space of the membrane module to perform the second-stage fermentation at 36℃ for 30 h.

[0080] After the fermentation is completed, two-stage flash extraction is performed in situ: (1) the first-stage flash extraction is performed at a pressure of -0.05 MPa and a temperature of 28℃, during which pulse treatment is additionally applied at a power of 150 W, an operation time of 2.5 s and an intermittent time of 6 s, and the first fraction is collected. (2) the second-stage flash extraction is performed at a pressure of -0.085 MPa and a temperature of 38℃, during which pulse treatment is additionally applied at a power of 300 W, an operation time of 7 s and an intermittent time of 2.5 s, and the second fraction is collected.

[0081] Example 2

[0082] The present embodiment provides a method for extracting a floral perfume, which comprises:

[0083] The hollow fiber membrane module is placed in the membrane bioreactor, which divides the internal space of the membrane bioreactor into the external space of the membrane module and the internal space of the membrane module. 80 g of rose flowers and 1 L of water are added to the external space of the membrane module, sterilized at 130℃ for 20 min, and then inoculated with 10 vol% of Pichia pastoris seed liquid to perform the first-stage fermentation at 28℃. When the acetic acid concentration reaches 0.08 g / L, 5 vol% of Lactobacillus plantarum seed liquid is inoculated into the internal space of the membrane module to perform the second-stage fermentation at 35℃ for 36 h.

[0084] After the fermentation is completed, two-stage flash extraction is performed in situ: (1) the first-stage flash extraction is performed at a pressure of -0.06 MPa and a temperature of 25℃, during which pulse treatment is additionally applied at a power of 200 W, an operation time of 2 s and an intermittent time of 8 s, and the first fraction is collected. (2) the second-stage flash extraction is performed at a pressure of -0.08 MPa and a temperature of 40℃, during which pulse treatment is additionally applied at a power of 250 W, an operation time of 8 s and an intermittent time of 2 s, and the second fraction is collected.

[0085] Example 3

[0086] The present embodiment provides a method for extracting a floral perfume, which comprises:

[0087] The hollow fiber membrane module is placed in the membrane bioreactor, which divides the internal space of the membrane bioreactor into the external space of the membrane module and the internal space of the membrane module. 50 g of rose and 1 L of water are added to the external space of the membrane module, sterilized at 110°C for 30 min, then inoculated with 8 vol% of Pichia pastoris seed liquid, and the first-stage fermentation is carried out at 30°C. When the acetic acid concentration reaches 0.12 g / L, 3 vol% of Lactobacillus plantarum seed liquid is inoculated into the internal space of the membrane module, and the second-stage fermentation is carried out at 37°C for 24 h.

[0088] After the fermentation is completed, two-stage flash extraction is carried out in situ: (1) the first-stage flash extraction is carried out at a pressure of -0.04 MPa and a temperature of 30°C, and the first fraction is collected during the period of auxiliary pulse treatment with a power of 100 W, a working time of 3 s and an intermittent time of 5 s. (2) The second-stage flash extraction is carried out at a pressure of -0.09 MPa and a temperature of 35°C, and the second fraction is collected during the period of auxiliary pulse treatment with a power of 350 W, a working time of 5 s and an intermittent time of 3 s.

[0089] Example 4

[0090] The present embodiment provides a method for extracting a floral perfume, which comprises:

[0091] The hollow fiber membrane module is placed in the membrane bioreactor, which divides the internal space of the membrane bioreactor into the external space of the membrane module and the internal space of the membrane module. 50 g of rose and 1 L of water are added to the external space of the membrane module, sterilized at 110°C for 30 min, then inoculated with 8 vol% of Pichia pastoris seed liquid, and the first-stage fermentation is carried out at 30°C. When the acetic acid concentration reaches 0.12 g / L, 3 vol% of Lactobacillus plantarum seed liquid is inoculated into the internal space of the membrane module, and the second-stage fermentation is carried out at 37°C for 24 h.

[0092] After the fermentation is completed, two-stage flash extraction is carried out in situ: (1) the first-stage flash extraction is carried out at a pressure of -0.04 MPa and a temperature of 30°C, and the first fraction is collected during the period of auxiliary pulse treatment with a power of 100 W, a working time of 3 s and an intermittent time of 5 s. (2) The second-stage flash extraction is carried out at a pressure of -0.09 MPa and a temperature of 35°C, and the second fraction is collected during the period of auxiliary pulse treatment with a power of 350 W, a working time of 5 s and an intermittent time of 3 s.

[0093] Example 5

[0094] The present embodiment provides a method for extracting a floral perfume, which comprises:

[0095] The hollow fiber membrane module was placed in the membrane bioreactor, which divided the internal space of the membrane bioreactor into the external space of the membrane module and the internal space of the membrane module. 65 g of rose flowers and 1 L of water were added to the external space of the membrane module, sterilized at 120°C for 25 min, and then inoculated with 9 vol% of Pichia pastoris seed liquid to perform the first-stage fermentation at 29°C. When the acetic acid concentration was monitored to reach 0.10 g / L, 4 vol% of Lactobacillus plantarum seed liquid was inoculated into the internal space of the membrane module to perform the second-stage fermentation at 36°C for 30 h.

[0096] After the fermentation was completed, two-stage flash extraction was performed in situ: (1) the first-stage flash extraction was performed at a pressure of -0.05 MPa and a temperature of 28°C, during which pulse treatment was applied at a power of 300 W for 7 s and an intermittent time of 2.5 s, and the first fraction was collected.

[0097] Comparative Example 1

[0098] The present comparative example provides a method for extracting a floral fragrance, which comprises:

[0099] 65 g of rose flowers and 1 L of water were added to the membrane bioreactor, sterilized at 120°C for 25 min, and then inoculated with 9 vol% of Pichia pastoris seed liquid to perform the first-stage fermentation at 29°C. When the acetic acid concentration was monitored to reach 0.10 g / L, 4 vol% of Lactobacillus plantarum seed liquid was inoculated to perform the second-stage fermentation at 36°C for 30 h.

[0100] After the fermentation was completed, two-stage flash extraction was performed in situ: (1) the first-stage flash extraction was performed at a pressure of -0.05 MPa and a temperature of 28°C, during which pulse treatment was applied at a power of 150 W for 2.5 s and an intermittent time of 6 s, and the first fraction was collected. (2) the second-stage flash extraction was performed at a pressure of -0.085 MPa and a temperature of 38°C, during which pulse treatment was applied at a power of 300 W for 7 s and an intermittent time of 2.5 s, and the second fraction was collected.

[0101] Comparative Example 2

[0102] The present comparative example provides a method for extracting a floral fragrance, which comprises:

[0103] Into the membrane bioreactor, 65 g rose flowers and 1 L water were added, sterilized at 120 °C for 25 min, then inoculated with 9 vol% Pichia pastoris seed liquid and 4 vol% Lactobacillus plantarum seed liquid, and fermented at 32.5 °C for 54 h.

[0104] After the fermentation, two-stage flash extraction was carried out in situ: (1) the first-stage flash extraction was carried out by setting the pressure at -0.05 MPa and the temperature at 28 °C, during which pulse treatment was applied with a power of 150 W, an operation time of 2.5 s, and an intermittent time of 6 s, and the first fraction was collected. (2) the second-stage flash extraction was carried out by setting the pressure at -0.085 MPa and the temperature at 38 °C, during which pulse treatment was applied with a power of 300 W, an operation time of 7 s, and an intermittent time of 2.5 s, and the second fraction was collected.

[0105] Comparative Example 3

[0106] The present comparative example provides a method for extracting a floral fragrance, which comprises:

[0107] The hollow fiber membrane module was placed in the membrane bioreactor, which divided the internal space of the membrane bioreactor into a membrane module external space and a membrane module internal space. Into the membrane module external space, 65 g rose flowers and 1 L water were added, sterilized at 120 °C for 25 min, then inoculated with 4 vol% Lactobacillus plantarum seed liquid, and fermented at 36 °C for 30 h for the first-stage fermentation. Into the membrane module internal space, 9 vol% Pichia pastoris seed liquid was inoculated, and fermented at 29 °C for 24 h for the second-stage fermentation.

[0108] After the fermentation, two-stage flash extraction was carried out in situ: (1) the first-stage flash extraction was carried out by setting the pressure at -0.05 MPa and the temperature at 28 °C, during which pulse treatment was applied with a power of 150 W, an operation time of 2.5 s, and an intermittent time of 6 s, and the first fraction was collected. (2) the second-stage flash extraction was carried out by setting the pressure at -0.085 MPa and the temperature at 38 °C, during which pulse treatment was applied with a power of 300 W, an operation time of 7 s, and an intermittent time of 2.5 s, and the second fraction was collected.

[0109] Comparative Example 4

[0110] The present comparative example provides a method for extracting a floral fragrance, which comprises:

[0111] The hollow fiber membrane module was placed in the membrane bioreactor, which divided the internal space of the membrane bioreactor into the external space of the membrane module and the internal space of the membrane module. 65 g of rose flowers and 1 L of water were added to the external space of the membrane module, sterilized at 120℃ for 25 min, and then inoculated with 9 vol% of Pichia pastoris seed liquid. The first-stage fermentation was carried out at 29℃. When the acetic acid concentration was monitored to reach 0.10 g / L, 4 vol% of Lactobacillus plantarum seed liquid was inoculated into the internal space of the membrane module, and the second-stage fermentation was carried out at 36℃ for 30 h. After the fermentation was completed, the fermented product was taken out for atmospheric pressure steam distillation, and the distillate was collected.

[0112] Test Example

[0113] 50 mL of the second distillate obtained in Examples 1-5, the second distillate obtained in Comparative Examples 1-3, and the distillate obtained in Comparative Example 4 were taken as test samples, respectively. 20 μL of 1000 mg / L n-dodecane in dichloromethane was added to each group of test samples as an internal standard, and liquid-liquid extraction was performed with 25 mL of dichloromethane. The organic phase was separated, dried over anhydrous sodium sulfate, and then concentrated to 1 mL in a 40℃ water bath by rotary evaporation, and used for GC-MS analysis. The components were qualitatively analyzed according to the retention time, and the absolute concentrations (mg / L) of phenethyl alcohol and phenethyl acetate in each group of test samples were calculated by the ratio of the peak area of each component to the peak area of the internal standard, combined with the standard curve.

[0114] The GC-MS analysis conditions were as follows: (1) chromatographic column: DB-WAX (30 m x 0.25 mm x 0.25 μm); (2) inlet temperature: 250℃; (3) temperature program: initial temperature 50℃, hold for 2 min; increase the temperature to 230℃ at a rate of 5℃ / min, hold for 5 min; (4) carrier gas: helium, flow rate 1.0 mL / min; (5) mass spectrometry conditions: electron impact source (EI), ion source temperature 230℃, quadrupole rod temperature 150℃, scan range m / z 35-500.

[0115] As shown in Table 1, from Examples 1-3, the present application sets up a hollow fiber membrane module to construct two physically isolated but metabolically connected reaction zones in a single reactor, avoiding direct competition and metabolic inhibition between the two functional strains, and ensuring stable and orderly metabolic flow from aroma precursor to target ester. As shown in Comparative Examples 1-2, without physical isolation, the two strains grow in competition and metabolic products in the same space, resulting in limited efficiency of Pichia pastoris in hydrolyzing aroma precursors to produce phenethyl alcohol and limited efficiency of Lactobacillus plantarum in converting phenethyl alcohol to phenethyl acetate. As shown in Comparative Example 3, the inoculation order of Pichia pastoris and Lactobacillus plantarum affects the biological catalytic cascade reaction and the conversion efficiency of the substrate. As shown in Comparative Example 4, the high temperature condition (about 100°C) of conventional steam distillation causes the hydrolysis of phenethyl acetate that has been generated, and the regeneration of phenethyl alcohol, resulting in the loss of target product and the concentration of raw material alcohol rising. As shown in Examples 4-5, applying a specific pulse treatment during flash extraction can effectively prevent the degradation and escape of heat-sensitive components, and improve the concentration of target product and aroma fidelity.

[0116] Table 1

[0117]

[0118] The present application is described by the above examples to illustrate a method for synergistic fermentation and low-temperature extraction of a floral fragrance and products and applications thereof, but the present application is not limited to the above examples, i.e. it does not mean that the present application must rely on the above examples to be implemented. It should be understood by those skilled in the art that any improvement on the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc. all fall within the protection scope and disclosure scope of the present application.

[0119] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above embodiments, and within the technical concept scope of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

[0120] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any suitable manner without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination manners.

Claims

1. A method of synergistic fermentation and low temperature extraction of floral perfumes, characterized in that, The method is carried out in a membrane bioreactor, and a hollow fiber membrane assembly divides the internal space of the membrane bioreactor into an external space of the membrane assembly and an internal space of the membrane assembly; Rose flowers and water are added to the external space of the membrane assembly, sterilized, inoculated with Pichia pastoris, and subjected to first-stage fermentation; Lactobacillus plantarum is inoculated into the internal space of the membrane assembly, and subjected to second-stage fermentation; after the fermentation is completed, flash extraction is carried out, and fractions are collected to obtain a floral fragrance.

2. The method of claim 1, wherein, The pore size of the hollow fiber membrane is 0.1-0.2 μm; Preferably, the use amount ratio of the rose flowers and water is (50-80) g:1 L; Preferably, the sterilization temperature is 110-130℃, and the sterilization time is 20-30 min.

3. The method according to claim 1 or 2, characterized in that, The inoculation amount of the Pichia pastoris is 8-10 vol%; Preferably, the first-stage fermentation temperature is 28-30℃.

4. The method according to any one of claims 1 to 3, characterized in that, The method further comprises a step of monitoring the concentration of a metabolite to reach a threshold value before the Lactobacillus plantarum is inoculated; Preferably, the metabolite is acetic acid; Preferably, the threshold value of the metabolite concentration is 0.08-0.12 g / L; Preferably, the inoculation amount of the Lactobacillus plantarum is 3-5 vol%; Preferably, the second-stage fermentation temperature is 35-37℃, and the second-stage fermentation time is 24-36 h.

5. The method according to any one of claims 1 to 4, characterized in that, The flash extraction is carried out in situ; Preferably, the flash extraction pressure is -0.04--0.09 MPa, and the flash extraction temperature is 25-40℃.

6. The method according to any one of claims 1 to 5, characterized in that, The flash extraction comprises first-stage flash extraction and second-stage flash extraction; The first-stage flash extraction pressure is -0.04--0.06 MPa, and the first-stage flash extraction temperature is 25-30℃; The second-stage flash extraction pressure is -0.08--0.09 MPa, and the second-stage flash extraction temperature is 35-40℃.

7. The method according to any one of claims 1 to 6, characterized in that, The flash extraction is carried out under pulse treatment; Preferably, the pulse treatment power is 100-350 W, the pulse treatment working time is 2-8 s, and the pulse treatment intermittent time is 2-8 s.

8. The method of claim 6, wherein, The flash extraction is carried out under pulse treatment; In the first-stage flash extraction, the pulse treatment power is 100-200 W, the pulse treatment working time is 2-3 s, and the pulse treatment intermittent time is 5-8 s; In the second-stage flash extraction, the pulse treatment power is 250-350 W, the pulse treatment working time is 5-8 s, and the pulse treatment intermittent time is 2-3 s.

9. A floral fragrance prepared by the method according to any one of claims 1-8.

10. Use of the floral fragrance according to claim 9 in preparing cigarettes.

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