Preparation method of a hawthorn extract for cigarettes and application thereof in cigarettes

Abstract

The application discloses a preparation method of a hawthorn extract for cigarettes and application of the hawthorn extract in cigarettes, and the preparation method comprises the following steps: mixing and sterilizing rutin with water; inoculating Pseudomonas acidovorans ZY-03, Bacillus subtilis SMXP-58, Micrococcus ZY02 and Pichia membranacei into culture media respectively to form seed liquids; inoculating the seed liquids of the Pseudomonas acidovorans ZY-03 and the Bacillus subtilis SMXP-58 into the rutin solution to obtain a fermentation product 1; grinding and crushing hawthorn and mixing the hawthorn with water and heating, then filtering and mixing the hawthorn with the fermentation product 1 to obtain a mixed solution; mixing the four seed liquids and inoculating the mixed solution into the mixed solution; after fermentation, the mixed solution is extracted by refluxing with an organic solvent, centrifuged, rotary evaporated, and added with an organic reagent to obtain the hawthorn extract for cigarettes. The rutin and the hawthorn are mixed and fermented by using microorganisms, the fermentation effect is improved, flavonoids such as rutin and macromolecular substances in the hawthorn are degraded and converted into aroma components, and the quality of the hawthorn extract for cigarettes is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbiological technology, in particular to a preparation method of a hawthorn extract for tobacco and its application in cigarettes. BACKGROUND

[0002] Hawthorn, also known as shanlihong and red fruit, contains various aroma components and rich trace elements, mainly including flavonoids, organic acids, triterpenoids and other chemical components, and is currently recognized as one of the high-quality raw materials for processed health foods, and is widely used in the food, health product, daily chemical, and tobacco industries.

[0003] At present, the traditional processing method of hawthorn extract for tobacco is continuous extraction with low ethanol to obtain hawthorn tincture. In addition to aroma components, the product obtained by this method mainly contains sugars and pectin substances, and glycoside aroma precursors are difficult to release, which to some extent limits the application of hawthorn extract for tobacco. In order to improve the aroma amount, some enzyme preparations or microorganisms are used for further fermentation of hawthorn, but most of them focus on the degradation of pectin and cellulose, and still cannot meet the demand for further improving the quality of hawthorn extract for tobacco. Flavonoids, as the main active components in hawthorn (such as quercetin and rutin), directly added to tobacco products can combine with proteins and glycoproteins in oral saliva, making them lose the lubricating effect on the oral cavity, and causing the contraction of the epithelial tissue of the tongue surface, resulting in dry mouth, tongue surface astringency, and the feeling of unclean oral cavity. The glycoprotein produced by the cleavage of flavonoid glycosides can supplement the content of glycoprotein in oral saliva, providing a comfortable and clean aftertaste, and a mellow and sweet smoking taste. Under the action of oxidase, glycoprotein is degraded to generate alcohol and furan, and flavonoid glycosides are degraded to generate polyquinone, furanone, and pyranone. There is no good research on the degradation of flavonoids to improve the quality of hawthorn extract for tobacco in the prior art, and therefore, it is necessary to further study the preparation and application of hawthorn extract for tobacco. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a preparation method of hawthorn extract for tobacco and its application in cigarettes, which has the effects of increasing smoke permeability, softening smoke, and sweetening when applied to cigarette products.

[0005] The technical problem to be solved by the present application is solved by the following technical scheme:

[0006] A preparation method of hawthorn extract for tobacco, comprising the following steps:

[0007] (1) mixing rutin and water in a mass ratio of 1: (1-7), sterilizing at 121℃ for 15 min;

[0008] (2) Inoculate the Pseudomonas paraficca ZY-03 into YPD liquid medium, Bacillus subtilis SMXP-58, Micrococcus ZY02, and Pichia membranacei respectively into LB medium, and ferment at 30℃, 150r / min for 24-48h to form seed liquid;

[0009] (3) Mix the seed liquid of Pseudomonas paraficca ZY-03 and the seed liquid of Bacillus subtilis SMXP-58 according to the mass ratio (9-12):(8-11), and inoculate 10-15% of the total inoculation amount into rutin solution, and ferment at 25-40℃ for 72-96h to obtain fermentation product 1 after the fermentation is completed;

[0010] (4) Grind the hawthorn dry into 40-100 mesh, mix the hawthorn dry with water according to the mass ratio 1:10, heat at 60℃ for 90min, filter, mix the filtrate with water sterilized at 121℃ for 15min under sterile conditions with fermentation product 1 according to the mass ratio 10:1-30:1 to become mixed liquid;

[0011] (5) Mix the seed liquid of Pseudomonas paraficca ZY-03, the seed liquid of Pichia membranacei, the seed liquid of Micrococcus ZY02, and the seed liquid of Bacillus subtilis SMXP-58 according to the following mass ratio (35-55):(20-30):(10-20):(25-45), and inoculate 2-6% of the total inoculation amount into the mixed liquid, and ferment at 25-40℃, 120-180r / min for 48-96h;

[0012] (6) Mix the mixed liquid after fermentation with organic solvent, and reflux extract at 70-80℃ for 2-4h, wherein the volume ratio of the mixed liquid to the organic solvent is 1:1-1.5:1, and the organic reagent is composed of ethyl acetate, ethanol, and n-hexane according to the mass ratio (20-30):(50-65):(5-10), centrifuge after the extraction, take the supernatant, rotary evaporate at 60℃, 60-100mbar to the density 1.09±0.01, add ethanol according to the volume ratio 1:1, and place at -22℃ for 24h to obtain tobacco hawthorn extract by centrifugation and adding 30% propylene glycol.

[0013] Preferably, in the above technical solution, in step (1), the mass ratio of rutin to water is 1:3.

[0014] Preferably, in the above technical solution, the Pseudomonas parafulva ZY-03 is preserved in the China General Microbiological Culture Collection Center (CGMCC) with a preservation number of CGMCC No. 24422; the Bacillus subtilis SMXP-58 is preserved in the China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC NO: M 2016083; the Micrococcaceae Pribram ZY02 is preserved in the China General Microbiological Culture Collection Center (CGMCC) with a preservation number of CGMCC NO. 21313; and the Pichia membranifuciens CGMCC 2.4059.

[0015] Preferably, in the above technical solution, the LB liquid medium comprises 10 g / L of NaCl, 10 g / L of protein peptone, and 5 g / L of yeast extract powder, and is prepared after high-pressure sterilization at 121℃ for 15 min; and the YPD liquid medium comprises 10 g / L of yeast powder, 20 g / L of protein peptone, and 20 g / L of glucose, and is prepared after high-pressure sterilization at 121℃ for 15 min.

[0016] Preferably, in the above technical solution, in step (2), the seed liquid is cultured at 30℃ and 150 r / min for 24-48 h, and the OD value is 1.2-2.0. 600 Preferably, in the above technical solution, in step (2), the seed liquid is cultured at 30℃ and 150 r / min for 24-48 h, and the OD value is 1.2-2.0.

[0017] Preferably, in the above technical solution, step (3) is specifically as follows: the Pseudomonas parafulva ZY-03 seed liquid and the Bacillus subtilis SMXP-58 seed liquid are mixed at a mass ratio of 1:1, inoculated into the rutin solution at a total inoculation amount of 10%, and fermented at 30℃ for 84 h, to obtain the fermentation product 1.

[0018] Preferably, in the above technical solution, in step (5), the Pseudomonas parafulva ZY-03 seed liquid, the Pichia membranifuciens seed liquid, the Micrococcaceae Pribram ZY02 seed liquid, and the Bacillus subtilis SMXP-58 seed liquid are mixed at a mass ratio of 4:2:1:3, inoculated into the mixed liquid at a total inoculation amount of 4%, and fermented at 30℃ and 180 r / min for 96 h.

[0019] Preferably, in the above technical solution, in step (6), the ethyl acetate, the ethanol, and the n-hexane are mixed at a mass ratio of 6:10:1.

[0020] A hawthorn extract for cigarettes is prepared according to the preparation method.

[0021] Preferably, in the above technical solution, the hawthorn extract for cigarettes is diluted with water at a mass ratio of 1:(50-500), and then 0.5-1 uL / branch is added to conventional cigarettes or heated cigarettes; or the obtained hawthorn extract for cigarettes is diluted with water at a mass ratio of 1:(5000-50000), and is sprayed on tobacco at 10-15%, and is placed for 2-6 h.

[0022] The above technical solution of the present application has the following beneficial effects:

[0023] The present application utilizes specific combination of microorganisms to ferment rutin and hawthorn, which can improve the fermentation effect, degrade rutin and other flavonoids and macromolecular substances in hawthorn into aroma components, and improve the quality of hawthorn extract for cigarettes. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present application will now be described in detail. It should be noted that the relative arrangement of components and steps, numerical expressions, and numerical values set forth in these embodiments are not limiting to the scope of the present application unless otherwise specifically stated.

[0025] Rutin is a sophora flower bud extract rutin (≥95%) purchased from the Internet.

[0026] The Pichia membranifuciens is purchased from the China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO.2.4059.

[0027] The Pseudomonas parafulva ZY-03 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.24422.

[0028] The Bacillus subtilis SMXP-58 is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO:M 2016083.

[0029] The Micrococcaceae Pribram ZY02 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC NO.21313.

[0030] The Pichia membranifuciens is purchased from the China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO.2.4059.

[0031] LB liquid medium was composed of NaCl 10 g / L, peptone 10 g / L, yeast extract 5 g / L, and prepared by high pressure sterilization at 121℃ for 15 min.

[0032] YPD liquid medium was composed of yeast extract 10 g / L, peptone 20 g / L, and glucose 20 g / L, and prepared by high pressure sterilization at 121℃ for 15 min.

[0033] Example 1

[0034] (1) Rutin was added to 3 times of water (mass ratio) and sterilized at 121℃ for 15 min.

[0035] (2) Pseudomonas ZY-03 was inoculated into YPD liquid medium, and Bacillus subtilis SMXP-58 was inoculated into LB liquid medium, and fermented at 30℃, 150 r / min for 24-48 h to prepare seed liquid with OD 600 value of about 2.0.

[0036] (3) According to the mass ratio of Pseudomonas ZY-03 to Bacillus subtilis SMXP-58 1:1, 10% of the total inoculation amount was inoculated into the rutin solution, and fermented at 30℃ for 84 h. After fermentation, fermentation product 1 was obtained.

[0037] (4) Hawthorn was ground to 40-100 mesh, and hawthorn and water were mixed at a mass ratio of 1:10, heated at 60℃ for 90 min, filtered, and the filtrate was sterilized at 121℃ for 15 min. Under sterile conditions, the sterilized filtrate and fermentation product 1 were mixed at a mass ratio of 20:1 to become a mixed liquid.

[0038] (5) Pseudomonas ZY-03 was inoculated into YPD medium, and Bacillus subtilis SMXP-58, Micrococcus ZY02, and Pichia membranacei were inoculated into LB medium, and fermented at 30℃, 150 r / min for 24 h to prepare seed liquid with OD 600 value of about 2.0.

[0039] (6) According to the mass ratio of Pseudomonas ZY-03, Pichia membranacei, Micrococcus ZY02, and Bacillus subtilis SMXP-58 4:2:1:3, 4% of the total inoculation amount was inoculated into the mixed liquid, and fermented at 30℃, 180 r / min for 96 h.

[0040] (7) The mixed solution after fermentation is uniformly mixed with ethyl acetate, ethanol and n-hexane, and extracted at 80°C for 3h, wherein the volume ratio of the mixed solution to the solvent is 1:1, the mass ratio of ethyl acetate, ethanol and n-hexane is 30:50:5, the supernatant is obtained by centrifugation, rotary evaporation is performed at 60°C and 60mbar until the density is 1.09, 1:1 of ethanol by volume is added, the supernatant is obtained by centrifugation after being placed at -22°C for 24h, and 30% propylene glycol is added to obtain the hawthorn extract for cigarettes.

[0041] Application Example 1

[0042] The hawthorn extract for cigarettes obtained in Example 1 is recorded as T1 and diluted with water according to a mass ratio of 1:500, and then 1uL / branch is added to a conventional cigarette.

[0043] Comparative Example 1

[0044] (1) The hawthorn dry is ground to 40-100 mesh, the hawthorn dry is mixed with water according to a mass ratio of 1:10, heated at 60°C for 90min, filtered, the filtrate is sterilized at 121°C for 15min, and the sterilized filtrate is mixed with the fermentation product 1 under sterile conditions according to a mass ratio of 20:1 to become a mixed solution.

[0045] (2) The mixed solution is uniformly mixed with ethyl acetate, ethanol and n-hexane, and extracted at 80°C for 3h, wherein the volume ratio of the mixed solution to the solvent is 1:1, the mass ratio of ethyl acetate, ethanol and n-hexane is 30:50:5, the supernatant is obtained by centrifugation, rotary evaporation is performed at 60°C and 60mbar until the density is 1.09, 1:1 of ethanol by volume is added, the supernatant is obtained by centrifugation after being placed at -22°C for 24h, and 30% propylene glycol is added to obtain the hawthorn extract for cigarettes.

[0046] (3) The hawthorn extract for cigarettes obtained is recorded as CK1 and diluted with water according to a mass ratio of 1:500, and then 1uL / branch is added to a conventional cigarette.

[0047] Comparative Example 2

[0048] (1) The rutin is added to 3 times of water (mass ratio) and sterilized at 121°C for 15min.

[0049] (2) The hawthorn dry is ground to 40-100 mesh, the hawthorn dry is mixed with water according to a mass ratio of 1:10, heated at 60°C for 90min, filtered, the filtrate is sterilized at 121°C for 15min, and the sterilized filtrate is mixed with the rutin solution under sterile conditions according to a mass ratio of 20:1 to become a mixed solution.

[0050] (3) The mixed solution is uniformly mixed with ethyl acetate, ethanol and n-hexane, and extracted at 80°C for 3h, wherein the volume ratio of the mixed solution to the solvent is 1:1, the mass ratio of ethyl acetate, ethanol and n-hexane is 30:50:5, the supernatant is obtained by centrifugation, rotary evaporation is performed at 60°C and 60mbar until the density is 1.09, 1:1 of ethanol is added according to the volume ratio, and the supernatant is obtained by centrifugation after being placed at -22°C for 24h, and 30% propylene glycol is added to obtain the hawthorn extract for cigarettes.

[0051] (4) The obtained hawthorn extract for cigarettes, recorded as CK2, is diluted with water according to a mass ratio of 1:500, and then 1uL / branch is added to a conventional cigarette.

[0052] Comparative Example 3

[0053] (1) Rutin is added to 3 times of water (mass ratio), and sterilized at 121°C for 15min.

[0054] (2) The hawthorn is ground to 40-100 mesh, mixed with water according to a mass ratio of 1:10, heated at 60°C for 90min, filtered, and the filtrate is sterilized at 121°C for 15min. The sterilized filtrate and the rutin solution are mixed under sterile conditions according to a mass ratio of 20:1 to become a mixed solution.

[0055] (3) Pichia pastoris is inoculated into LB medium, fermented at 30°C and 150r / min for 24h to prepare a seed solution with an OD 600 value of about 2.0.

[0056] (4) Pichia pastoris is inoculated into the mixed solution at a total inoculation amount of 4%, and fermented at 30°C and 180r / min for 96h.

[0057] (5) The mixed solution after fermentation is uniformly mixed with ethyl acetate, ethanol and n-hexane, and extracted at 80°C for 3h, wherein the volume ratio of the mixed solution to the solvent is 1:1, the mass ratio of ethyl acetate, ethanol and n-hexane is 30:50:5, the supernatant is obtained by centrifugation, rotary evaporation is performed at 60°C and 60mbar until the density is 1.09, 1:1 of ethanol is added according to the volume ratio, and the supernatant is obtained by centrifugation after being placed at -22°C for 24h, and 30% propylene glycol is added to obtain the hawthorn extract for cigarettes.

[0058] (6) The obtained hawthorn extract for cigarettes, recorded as CK3, is diluted with water according to a mass ratio of 1:500, and then 1uL / branch is added to a conventional cigarette.

[0059] Comparative Example 4

[0060] (1) Rutin is added to 3 times of water (mass ratio), and sterilized at 121°C for 15min.

[0061] (2) The hawthorn dry is ground to 40-100 mesh, mixed with water at a mass ratio of 1:10, heated at 60°C for 90 min, filtered, and the filtrate is sterilized at 121°C for 15 min. The sterilized filtrate is mixed with the rutin solution under sterile conditions at a mass ratio of 20:1 to become a mixed solution.

[0062] (3) The micrococcus ZY02 and Pichia membranifaciens are inoculated into LB medium at 30°C and 150 r / min for 24 h to prepare seed liquid with OD 600 of about 2.0.

[0063] (4) The micrococcus ZY02 and Pichia membranifaciens are inoculated into the mixed solution at a mass ratio of 1:2 and a total inoculation amount of 4% at 30°C and 180 r / min for 96 h.

[0064] (5) The fermented mixed solution is uniformly mixed with ethyl acetate, ethanol, and n-hexane, and extracted at 80°C for 3 h. The volume ratio of the mixed solution to the solvent is 1:1, the mass ratio of ethyl acetate, ethanol, and n-hexane is 30:50:5. The supernatant is obtained by centrifugation, and the density is reduced to 1.09 by rotary evaporation at 60°C and 60 mbar. Ethanol is added at a volume ratio of 1:1, and the supernatant is obtained by centrifugation after being placed at -22°C for 24 h. The tobacco hawthorn extract is obtained by adding 30% propylene glycol.

[0065] (6) The obtained tobacco hawthorn extract is recorded as CK4, diluted with water at a mass ratio of 1:500, and then 1 uL / branch is added to a conventional cigarette.

[0066] Comparative Example 5

[0067] (1) Rutin is added to 3 times water (mass ratio) and sterilized at 121°C for 15 min.

[0068] (2) Bacillus subtilis SMXP-58 is inoculated into LB medium at 30°C and 150 r / min for 24 h to prepare seed liquid with OD 600 of about 2.0.

[0069] (3) Bacillus subtilis SMXP-58 is inoculated into the rutin solution at a total inoculation amount of 10% at 30°C for 84 h to obtain fermentation product 1.

[0070] (4) The hawthorn dry is ground to 40-100 mesh, mixed with water at a mass ratio of 1:10, heated at 60°C for 90 min, filtered, and the filtrate is sterilized at 121°C for 15 min. The sterilized filtrate is mixed with the fermentation product 1 under sterile conditions at a mass ratio of 20:1 to become a mixed solution.

[0071] (5) Bacillus subtilis SMXP-58, Micrococcus ZY02, Pichia membranacei were inoculated into LB medium at 30℃, 150r / min, and fermented for 24h to prepare seed liquid with OD 600 value of about 2.0.

[0072] (6) Bacillus subtilis SMXP-58, Micrococcus ZY02, Pichia membranacei were inoculated into mixed liquid at 30℃, 180r / min for 96h, according to the mass ratio of 3:1:2 and total inoculation amount of 4%.

[0073] (7) The fermented mixed liquid was uniformly mixed with ethyl acetate, ethanol and n-hexane, and extracted at 80℃ for 3h, wherein the volume ratio of mixed liquid to solvent was 1:1, the mass ratio of ethyl acetate, ethanol and n-hexane was 30:50:5, the supernatant was obtained by centrifugation, and the density was 1.09 by rotary evaporation at 60℃ and 60mbar, then 1:1 volume ratio of ethanol was added, and the supernatant was obtained by centrifugation at-22℃ for 24h, and 30% propylene glycol was added to obtain tobacco hawthorn extract.

[0074] (8) The obtained tobacco hawthorn extract was diluted with water according to the mass ratio of 1:500, and then 1uL / branch was added to the conventional cigarette.

[0075] Comparative Example 6

[0076] The conventional cigarette without adding any tobacco hawthorn extract was recorded as CK0.

[0077] Results:

[0078] When the sensory quality of tobacco hawthorn extract was evaluated, 13 indexes including aroma quality, aroma amount, permeability, odor, concentration, strength, delicacy, cohesiveness, irritation, dryness, freshness, sweetness and aftertaste were scored, and the total score was calculated.

[0079] Table 1 Sensory evaluation results

[0080]

[0081] Compared with the cigarette CK0 without adding any cigarette hawthorn extract, several cigarette hawthorn extracts all have improved cigarette sensory, and can improve aroma quality and sweetness; CK1 improves aroma quality, delicacy and sweetness, but aroma amount and aftertaste decrease; CK2 improves aroma quality, aroma amount and concentration, but irritability increases and aftertaste decreases; CK3 improves aroma quality, aroma amount and sweetness, but does not improve permeability and delicacy; CK4 and CK5 improve aroma quality, aroma amount, permeability, sweetness and aftertaste, but the improvement is smaller than that of the examples; example T1 significantly improves aroma quality, aroma amount, permeability, delicacy, freshness, sweetness and aftertaste of smoke, greatly improves sensory quality of smoke, and further improves the quality of cigarette hawthorn extract.

[0082] CK1 is a cigarette hawthorn extract prepared without adding rutin and without any fermentation treatment, and other steps are the same as those of the examples, and GC-MS analysis is performed on the cigarette hawthorn extract, and main aroma components are compared as shown in Table 2.

[0083] Table 2 Main aroma components (μg / mL)

[0084]

[0085] The results show that the prepared cigarette hawthorn extract of the present application significantly increases 5-methylfurfural, 2H-pyran-2,6(3H)-dione, 2,4-dihydroxy-2,5-dimethyl-3(2H)-furan-3-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one, 5-methyl-2(3H)-furanone, 5-hydroxymethylfurfural and other aroma components, and produces 2,3-dihydrobenzofuran, tetrahydrobenzofuranone, 5-acetyldihydro-2(3H)-furanone, phenethyl alcohol, phenylacetaldehyde and other aroma substances, so that the cigarette aroma amount is larger and the smoke is more abundant.

[0086] Although the present application has been disclosed as above with examples, it is not intended to limit the present application, and any person skilled in the art can make various selections and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application is defined by the claims and equivalent forms thereof.

Claims

1. A method for preparing a hawthorn extract for tobacco use, characterized in that, Includes the following steps: (1) Mix rutin and water at a mass ratio of 1:(1-7) and sterilize at 121℃ for 15 min; (2) Inoculate Pseudomonas paraxanthizobium ZY-03 into YPD liquid medium, and inoculate Bacillus subtilis SMXP-58, Micrococcus ZY02 and Pichia pastoris into LB liquid medium respectively. Ferment at 30℃ and 150r / min for 24-48h to form seed liquid; (3) Mix the seed liquid of Pseudomonas paraxanthizobium ZY-03 and the seed liquid of Bacillus subtilis SMXP-58 at a mass ratio of (9-12): (8-11), and inoculate 10-15% of the total inoculum into the rutin solution. Ferment at 25-40℃ for 72-96h. After fermentation, fermentation product 1 is obtained. (4) Grind the dried hawthorn into 40-100 mesh, mix the dried hawthorn with water at a mass ratio of 1:10, heat at 60℃ for 90 min, filter, sterilize the filtrate at 121℃ for 15 min, and mix the sterilized filtrate with fermentation product 1 at a mass ratio of 10:1-30:1 under aseptic conditions to form a mixture. (5) Mix the seed liquids of Pseudomonas paraxanthii ZY-03, Pichia pastoris, Micrococcus ZY02, and Bacillus subtilis SMXP-58 in the following mass ratio (35-55): (20-30): (10-20): (25-45), and inoculate 2-6% of the total amount into the mixture. Ferment at 120-180 r / min for 48-96 h at 25-40℃. (6) The fermented mixture is mixed with an organic solvent and refluxed at 70-80℃ for 2-4 hours. The volume ratio of the mixture to the organic solvent is 1:1-1.5:

1. The organic reagent is composed of ethyl acetate, ethanol, and n-hexane in a mass ratio of (20-30):(50-65):(5-10). After extraction, the mixture is centrifuged and the supernatant is collected. The supernatant is then evaporated at 60℃ and 60-100mbar to a density of 1.09±0.

01. Ethanol is added at a volume ratio of 1:1 and the mixture is placed at -22℃ for 24 hours. The supernatant is then centrifuged and 30% propylene glycol is added to obtain the hawthorn extract for tobacco. The Pseudomonas paraxanthii ( Pseudomonas parafulva ZY-03, deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 24422; the described Bacillus subtilis ( Bacillus subtilis SMXP-58, is currently deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M 2016083; the micrococcus ( Micrococcaceae Pribram ZY02, is currently deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.21313; the described *Pichia pastoris* ( Pichia membranifuciens CGMCC NO. 2.4059.

2. The method for preparing a hawthorn extract for tobacco use according to claim 1, characterized in that, In step (1), the mass ratio of rutin to water is 1:

3.

3. The method for preparing a hawthorn extract for tobacco use according to claim 1, characterized in that, The LB liquid culture medium comprises: 10 g / L NaCl, 10 g / L peptone, and 5 g / L yeast extract, prepared by autoclaving at 121°C for 15 min; the YPD liquid culture medium comprises: 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose, prepared by autoclaving at 121°C for 15 min.

4. The method for preparing a hawthorn extract for tobacco use according to claim 1, characterized in that, In step (2), the culture conditions for the seed culture are: 30℃, 150r / min, fermentation for 24-48h, to prepare OD. 600 Seed liquid with a value of 1.2-2.

0.

5. The method for preparing a hawthorn extract for tobacco use according to claim 1, characterized in that, Step (3) is as follows: Mix the seed liquid of Pseudomonas paraxanthizobium ZY-03 and the seed liquid of Bacillus subtilis SMXP-58 at a mass ratio of 1:1, and inoculate 10% of the total inoculum into the rutin solution. Ferment at 30°C for 84 hours. After fermentation, fermentation product 1 is obtained.

6. The method for preparing a hawthorn extract for tobacco use according to claim 1, characterized in that, In step (5), the seed liquids of Pseudomonas paraxanthioides ZY-03, Pichia pastoris, Micrococcus ZY02, and Bacillus subtilis SMXP-58 were mixed in the following mass ratio of 4:2:1:3, and 4% of the total inoculum was inoculated into the mixture. Fermentation was carried out at 30°C and 180r / min for 96h.

7. The method for preparing a hawthorn extract for tobacco use according to claim 1, characterized in that, In step (6), ethyl acetate, ethanol, and n-hexane are used in a mass ratio of 6:10:

1.

8. The application of a hawthorn extract for tobacco use in cigarettes, characterized in that, The hawthorn extract for tobacco is prepared by any one of the preparation methods described in claims 1-7.

9. The application according to claim 8, characterized in that, Dilute the hawthorn extract for tobacco with water at a mass ratio of 1:(50-500), and then add 0.5-1uL / cigarette to regular or heated cigarettes; or, dilute the obtained hawthorn extract for tobacco with water at a mass ratio of 1:(5000-50000), spray it onto the tobacco at a concentration of 10-15%, and let it stand for 2-6 hours.

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