CN108893235A - A kind of method and flavouring Lycium chinense wine improving fermented type fructus lycii wine aroma - Google Patents

Abstract

The present invention relates to a kind of methods for improving fermented type fructus lycii wine aroma, belong to brewed wine technical field.The method includes the following steps：Fructus lycii mashing, add carotenoid degrading enzyme, additive amount 3%-5%, in 60 DEG C of reaction 3-5h, after filtering and removing slag, Chinese wolfberry clear juice is taken, 40-60ml/L pectase, the liquid sulfurous acid that 40-60ml/L concentration is 6% is added, it is 20-25% with white granulated sugar sugar addition, pH is 3.3-3.5, and inoculation 2-3% yeast activated liquid ferments at 25-28 DEG C is less than 4g/L, fermentation ends to residual sugar.The carotenoid degrading enzyme is CCTCC NO by deposit number：Ku Teshi bacillus (Kurthia sp) NXUGQ15 of M2017524, which ferments, to be prepared.Method for increasing aroma of the present invention can increase the volatilization fragrance component of Lycium chinense wine, significantly improve the quality of Lycium chinense wine.

Description

A kind of method and flavouring Lycium chinense wine improving fermented type fructus lycii wine aroma

Technical field

The present invention relates to a kind of methods for improving fermented type fructus lycii wine aroma, and are related to flavouring Lycium chinense wine obtained, belong to Brewed wine technical field.

Background technique

It is well known that fructus lycii has nourishing liver and kidney, benefiting shrewd head, soreness of waist and knee joint, dizziness and tinnitus, Heat Diabetes, blood are treated Empty sallow effect, therefore, Lycium chinense wine is made as health liquor in fructus lycii by people.Lycium chinense wine preparation method has infusion method and fermentation at present Two kinds of method.Infusion method is generally formed whole grain fructus lycii white wine or yellow rice wine are brewed, and wine degree is higher, and nutritional ingredient can not be abundant Dissolution.Fermentation method Lycium chinense wine is also less to have oxygen participation because not being related to high-temperature heating process, not only maintains in fructus lycii substantially Natural nutrition ingredient, and be more conducive to absorption of human body through everfermentation, be a kind of extraordinary health-care nutritive fruit wine. But fermented type Lycium chinense wine, due to being influenced by factors such as raw material and processing technologys, quality is irregular, the mouthfeel of product is fragrant Gas is generally insufficient, needs to improve.

Publication number:101323823 invention《The brewing method of Lycium chinense wine》A kind of brewing method of Lycium chinense wine is disclosed, it will Diastatic fermentation distiller's yeast and distiller's yeast is added in the fructus lycii of broken deseeding and the starchy material after curing, is sent out in the fermenter Ferment；Then the mash fermented is squeezed, wine juice is clarified, sterilization, ageing, blends and bottles after filtering.Using shallow lake Silty raw material as fermentation carbon source, brew pure fermentation Lycium chinense wine, mainly solve the problems, such as be：It reduces and is produced into the maximum extent This.

Publication number:1513970 invention《A kind of fermented wolfberry fruit grape and brew method》Disclose a kind of fermentation fructus lycii Wine and fermentating wine blend preparation method, and this method is separately fermented using fructus lycii and grape as raw material, then carry out preparation tune The process matched.It is with fresh fructus lycii or dry wolfberry are broken pulp (fruit juice) fermentation to be made fermentation Lycium chinense wine is made；White grape or Red grape is made fruit juice (pulp) fermentation and is brewed into fermentating wine through broken.Ferment Lycium chinense wine and fermentating wine according to (1%-99%)：

The ratio of (99%-1%) blend proportion after be tuned into different types of pol, through Overheating Treatment, cold treatment, filtering, Bottling, sterilization, are made fermented wolfberry fruit grape.The fermented wolfberry fruit grape that the method is brewed, Lycium berry and grape fragrance is harmonious, mouthfeel Plentiful coordination, fructus lycii taste is strong, and typicalness is prominent.

Publication number:1104248 invention《A kind of brew method of Lycium chinense wine》A kind of brew method of Lycium chinense wine is disclosed, This method includes featured raw material, squeezing, fermentation, filtering, sterilizing, it is characterised in that fresh fructus lycii is featured, squeezes into wolfberry juice Afterwards, after sodium sulfite vulcanization is added, yeast is added, after adding glucose to pol to be 22 ° of BX, sealing hair in investment pressurized tank Ferment, fermentation temperature are (20-25) DEG C, and the time is after 5-8 days, and adding glucose to pol again is 18 ° of BX, is carried out again close Seal ferment, fermentation temperature are (15-20) DEG C, and when fermentation to tank bottom precipitating has pomace and yeast, wine liquid tentatively to clarify, pol is It after 5 ° of BX or less, after medlar liquid pomace, yeast mixt are stirred, heat in tank, then is cooled down, by fructus lycii mixture Suitable sodium sulfite is added in filtering resulting medlar liquid and adds honey clarification, filters again, filtered fluid is store for filtering In in bucket, changes bucket and once carry out filling pasteurization afterwards within storage 2-3 months under being 10 DEG C or so in temperature, that is, be brewed into fructus lycii The medlar liquid of above-mentioned brew is put into a certain amount of fructus lycii soak, is configured to the Lycium chinense wine of different wine degree by wine.

Publication number:1782060 invention《A kind of brew method of Lycium chinense wine》The brew method of disclosed Lycium chinense wine：Fresh Chinese holly Qi or dry wolfberry are sorted, clean, if dry wolfberry is also impregnated 12-24 hours with the water of 2-5 times of its weight, are then crushed At wolfberry fruit syrup, alcoholic strength is adjusted to 20-25% (v/v) with the deodorizing alcohol of 95% (v/v), is impregnated 10-20 days, during immersion often It beat circulation 1-4 times, after isolate soaking wine；Fresh fructus lycii pomace after separation adds the soft water of 0.5-2 times of fresh fructus lycii weight； Dry wolfberry pomace adds the soft water of 2-5 times of dry wolfberry weight, with sucrose sugar addition to 210-230g/L, with citric acid or tartaric acid Total acid is adjusted to 6.0-8.0g/L, adjusts SO₂To 80-100mg/L, add pectase 0.2-0.5g/L, adds dry ferment 0.15- 0.3g/L, control temperature are fermented at 18-30 DEG C, the time 5-7 days, when specific gravity is down to 1000g/L or less, analyze residual sugar < 4g/L Isolate fermented wine；Soaking wine and fermented wine are mixed, then be through ageing, allotment, lower glue, filtering, degerming, bottling, packaging Finished product.

Publication number:1265420 invention《A kind of brew method of Lycium chinense wine》The brew method of disclosed Lycium chinense wine：It will divide Blanching in the fructus lycii merging citric acid and sodium sulfite mixed liquor chosen, the fructus lycii after blanching first after crushing, add soft water It impregnates, fructus lycii and soft water are with 7:3 (volume ratio) tinnings are stood after adjusting composition with sulfur dioxide and pectinase treatment, inoculation hair Ferment isolates fermented wine stored for future use, sorted broken 95% deodorization edible wine of fructus lycii merging after primary fermentation and post-fermentation It impregnates 30 days or so and separates in essence, soaking wine is made, soaking wine and fermented wine are pressed 1:4 (volume ratios) blend allotment, storage one After the section time, filters through lower glue, bottle after aseptic filtration.

Publication number:1077744 invention《Process for preparation of fermentation Chinese wolfberry fruit wine》The preparation method of disclosed fermentation Chinese wolfberry fruit wine：It will Fructus lycii is rinsed through impurity elimination, and hot dipping mentions, and is crushed, and filtering adds auxiliary material, sterilizes, cooling, prior fermentation, purified treatment, filtering, ageing, Altar processed, allotment, the processes such as filtering and fermentation Chinese wolfberry fruit wine is made, concrete technology is：a：Fructus lycii impurity elimination is rinsed, with 65 DEG C -75 DEG C hot water extraction 1.5-2.5 hours, its residue is placed again into 65 DEG C of -75 DEG C of hot water after crushing filtering, extracts 0.8-1.2 It carries out filtering for second after hour, b：Residue after a secondary filters is crushed, its filter residue, c are abandoned after filtering again：In b 20% white sugar auxiliary material, d is added in the filtrate of item：Solution made from c is sterilized 20-25 seconds at a temperature of 95 DEG C -100 DEG C, it is cold But to 65 DEG C -70 DEG C, it is put into wine jar, continues to be cooled to 30 DEG C -32 DEG C, e：By solution made from d under the conditions of 28 DEG C -30 DEG C It carries out prior fermentation 6-8 days, then carries out later stage fermentation 9-11 days at room temperature, f：Solution made from e is purified Processing is added gelatin (being added in the ratio of 10mL solution 0.008g gelatin), filters within lower glue 14-16 days, g：It will be molten made from f Liquid ageing 3-5 months, altar, placed 10-20 days under the conditions of 50 DEG C -60 DEG C, places 5-7 under the conditions of subzero 2 DEG C -44 DEG C It, filtering, filtrate is stored 1 month, h：Flavoring agent is added in filtrate made from g and tonic is deployed, i：H are made Solution filtering, sterilizing, as finished product.

Although Catotenoids From Lycium Barbarum content is very rich, after undergoing microbial fermentation, carotenoid in Lycium chinense wine Content is not high, not soluble in water mainly due to carotenoid, is greatly removed when fermentation ends pomace separates, one Part is degraded in brewing process and is lost.

Oneself has centuries for the time-honored Traditional Brand of lycium barbarum, cultural advantage history, and Zhongning County is State Council's name " township of Chinese fructus lycii ", lycium barbarum quilt《Chinese Pharmacopoeia》Be classified as the homologous food of medicine food, this be other any provinces and regions all without Method analogy.

National No. 1 file clearly proposes that " reinforcing new raw-food material, integration of drinking and medicinal herbs food development and application " " adds within 2017 Strong modern biotechnology and fortification technical research, excavating exploitation has the function of the food of health care ", it is called in response to country, Development function health food develops Ningxia advantage characteristic fructus lycii resource, promotes the quality of health lycium chinense, finds a kind of raising hair The method of ferment type fructus lycii wine aroma, is the technical problems to be solved by the invention.

Summary of the invention

To solve the above problems, the present invention provides a kind of method for improving fermented type fructus lycii wine aroma, use is isolated One plant of carotenoid degradation bacteria, collect its produced carotenoid degrading enzyme, handle wolfberry fruit syrup, not using carotenoid Stablize, the degradable characteristic for generating drop isoprenoid volatile compound such as light, oxygen, heat, biological enzyme generates it a large amount of C₉-、C₁₀-、C₁₃And C₁₅It drops isoprenoid compound (norisoprenoids), these substances are that have volatile virtue Aroma compounds, these compounds have positive contribution because sense organ threshold value is lower, to food.The carotenoid that fructus lycii is rich in It include mainly beta carotene, luteole, luteole acid dipalmitate, and wherein most with luteole acid dipalmitate, About the 77.5% of carotenoid total amount.It is most of to be lost in fructus lycii slag due to not soluble in water but in brewed wine, it makes At very big waste.The present invention uses the produced degrading enzyme of microorganism, utilizes to the carotenoid in wolfberry fruit syrup, makes institute It degrades containing a large amount of carotenoid, plays the role of increasing wine aroma, improve the quality of Lycium chinense wine.

Traditional fermentation process is since Catotenoids From Lycium Barbarum is not soluble in water, and a large amount of carotenoid is by fructus lycii slag band It walks, method for increasing aroma of the present invention can degrade carotenoid in fructus lycii slag, make to generate drop isoprenoid aroma substance, Neng Gouti High aroma.Improve fructus lycii wine aroma, improves Lycium chinense wine quality, innovated fructus lycii wine brewage technology.

Drop isoprenoid compound (norisoprenoids) is a kind of special by having for carotenoid degradation generation The substance of fragrance, and the substance have lower odor threshold, thus on a small quantity exist can to the flavor of food generate compared with Big organoleptic effects.Drop isoprenoid compound containing 9,10,11 and 13 carbon atoms in food is mostly by carotenoid Degradation generates, such as dorinone, alpha, beta-lonone, dihydroactinidiolide, all has good flavor.Isoprenoid drops Compound is one of main aroma-producing substance of the freshs fruit of vegetables such as grape, and influences the important compound of fruit wine flavouring essence quality.Fruit There is explored drop isoprenoid compound in wine aroma ingredient：Alpha, beta-lonone, isophorone, limonene, safranal, Geranyl acetone, dihydro-β-ionone, dorinone, dihydroactinidiolide, methyl heptenone, β-cyclocitral, 2- heptan Olefine aldehydr, dihydro jasmone, α-cyclocitral, isogeraniol, 2,5,6- trimethyl -4- teracrylic acid -one, 2,4- nonadienal, 3- Decanone, 2- nonenyl aldehyde, 2,19 kinds of drop pentadiene class compounds such as 2,6- trimethyl annulenones, odor characteristic are shown in Table 3.

Improvement of the present invention to Lycium chinense wine technique increases the flavor substance of Lycium chinense wine using the degradation of carotenoid.It should The Lycium chinense wine of method brewing, increases drop isoprene kind compound content and type, they are mainly β-purple after testing Rowland ketone, 2,2,6- trimethylcyclohexanones (TCH), isophorone, geranyl acetone, dihydro-β-ionone, geraniol, hiding Safflower aldehyde, β-cyclocitral, limonene, pseudoionone, dihydroactinidiolide.And aldehyde C-9, amyl valerate, certain herbaceous plants with big flowers acid second Ester.The taste threshold value of these substances is low, plays a good role for Lycium chinense wine sensory evaluation.

The Lycium chinense wine carbonyl compound content increases, and relative peak area accounts for the 5.78% of total ingredient, higher than old technology 4.23%；It is 3- undecyl ketone, dorinone, alpha, beta-lonone, β-cyclocitral, safranal and two that wherein content is higher Hydrogen-alpha, beta-lonone, the higher alpha, beta-lonone 3.136% of relative amount, β-cyclocitral 3.615%, it is special that they have Fruit aroma.

Ester type compound relative peak area be 14.08%, higher than the 11.03% of prior art, be in Lycium chinense wine content compared with More aromatic substances.The 13.68% of total ingredient is accounted for, higher than the 10.73% of prior art, larger, certain herbaceous plants with big flowers is contributed to fructus lycii wine aroma Acetoacetic ester and amyl valerate are also important catabolite, the fragrance with pears perfume (or spice) and banana；Dihydroactinidiolide, acetone are fragrant Leaf ester is one of intermediate product and the higher esters of content of carotenoid degradation, and fruit and flowery odour are prominent.

A method of fermented type fructus lycii wine aroma is improved, is included the following steps：

Fresh fructus lycii mashing, collects the bacterium by fermented and cultured bacterium solution for Ku Teshi bacillus NXUGQ15 (Kurthia sp) Degrading enzyme is added in wolfberry fruit syrup and digests by the carotenoid degrading enzyme of generation, and catabolite is made to drop isoprenoid It dissolves in wolfberry fruit syrup, then Chinese wolfberry skin slag separates, and takes fermentation of clear juice, filters to take wine, obtains flavouring Lycium chinense wine.

The method for improving fermented type fructus lycii wine aroma, includes the following steps：Fructus lycii mashing, addition carotenoid drop Enzyme is solved, additive amount 3%-5% takes Chinese wolfberry clear juice after 60 DEG C of reaction 3-5h, filtering and removing slag, 40-60ml/L pectin is added Enzyme, the liquid sulfurous acid that 40-60ml/L concentration is 6% are 20-25%, pH 3.3-3.5, inoculation with white granulated sugar sugar addition 2-3% yeast activated liquid ferments at 25-28 DEG C is less than 4g/L, fermentation ends to residual sugar.

The activated yeast liquid and preparation method thereof includes the following steps：

Slant tube yeast takes one ring → 25-28 DEG C of 10mL malt juice liquid medium culture 24-48h → 50mL50% wheat 25-28 DEG C of culture 24-48h of fluid nutrient medium of+50% wolfberry juice mass percent of bud juice → 10mL is taken to be seeded in 100mL fructus lycii Culture 24-48h → whole is poured into and cultivates 24-48h → acquisition bacterium solution in 1000mL wolfberry juice at 22-25 DEG C at 25-28 DEG C in juice Concentration reaches 10⁷The bacterium solution of cfu/ml is to get yeast activated liquid → stand-by.

The preparation method of the carotenoid degrading enzyme, includes the following steps：

1. the preparation of Ku Teshi bacillus liquid

Fluid nutrient medium (g/L)：Sodium nitrate 3, dipotassium hydrogen phosphate 1, magnesium sulfate 0.5, potassium chloride 0.5, ferric sulfate 0.01, sugarcane Sugared 30, YNB synthetic media (no amino yeast nitrogen culture medium) 6.7,15'-dioxygenase；PH=3.2.

2. expanding culture

Take slant tube Ku Teshi bacillus (Kurthia sp) NXUGQ151 ring → 35-37 DEG C of 10mL fluid nutrient medium, It is cultivated under 130r/min and cultivates 10-12h → 3000mL liquid under 35-37 DEG C of 10-12h → 100mL fluid nutrient medium, 130r/min 10-12h → bacterial concentration is cultivated under 35-37 DEG C of culture medium, 130r/min reaches 10⁶Cfu/ml → Ku Teshi bacillus seed liquor；

3. fermenting

Fluid nutrient medium → -35 DEG C, pH=3.0-3.5,140r/min of 2% → 30 DEG C of inoculation Ku Teshi bacillus seed liquor hairs Ferment 8-10h → fermentation liquid；

4. prepared by carotenoid degrading enzyme

Fermentation liquid → 4 DEG C, 10000r/min centrifugation 100-150min → take supernatant → thick enzyme of carotenoid degrading enzyme Liquid.

The preparation method of the carotenoid degrading enzyme can also include：Crude enzyme liquid is chilled, dry, concentration And using after dialysis preliminary purification, carotenoid degrading enzyme is obtained.

The Ku Teshi bacillus is (Kurthia sp) NXUGQ15, and deposit number is CCTCC NO：M2017524；

The method for improving fermented type fructus lycii wine aroma can also include measurement flavor substance and sensory evaluation.

The separation and acquisition of the Ku Teshi bacillus strain

The strain of the isolated carotenoid that can degrade from wolfberry juice, and go out Ku Teshi bar through Uv-induced screening Bacteria strain NXUGQ15 (Kurthia sp), using the carotenoid degrading enzyme of this plant of bacterium generation, the class in wolfberry fruit syrup of degrading is recklessly Radish element, improves fructus lycii wine brewage technology, improves Lycium chinense wine quality.The bacterium starting strain is by Zhang Huiling from Ningxia Berry source fructus lycii It is isolated in the wolfberry juice of limited liability company planting base.

Ku Teshi bacillus strain NXUGQ15 (Kurthia sp), deposit number CCTCC NO：M2017524, in 2017 9 The moon is preserved in China typical culture collection center (Wuhan), address on the 21st：The Chinese Wuhan Wuhan University.The bacterium can degrade Beta carotene, optimum growth temp are 35-37 DEG C, pH=2-3；Degrading enzyme tolerable temperature caused by the bacterium is 70-90 DEG C, Best enzyme reaction pH=1-3.The bacterium produces that the carotenoid degrading enzyme time is short, and enzyme activity is high, the enzyme activity 8.87U/m L of crude enzyme liquid, excellent In bacterium germination GQ-16 out.

The identification of the Ku Teshi bacillus strain

Preliminary Identification：After fluid nutrient medium culture 48h, microscopy is carried out using microscope, in oily its form under the microscope. The result shows that bacterial strain NXUGQ15 is Gram-positive bacillus, no gemma.

Molecular biology identification：It is analyzed using 16S-23S rDNA ISR polymorphism and sequence, constructs its systematic evolution tree See attached drawing 2.By systematic evolution tree it is found that be based on 16rDNA region sequence phylogenetic tree, bacterial strain NXUGQ15 (Kurthia sp) with Zuo Shi Al Kut Salmonella is got together, and illustrates bacterial strain NXUGQ15 (Kurthia sp) and Zuo Shi Al Kut Salmonella is same species, i.e. bacterium Strain NXUGQ15 (Kurthia sp) is Al Kut Salmonella.

Degradation of the Ku Teshi bacillus to carotenoid

Bacterial strain NXUGQ15 (Kurthia sp) is inoculated into fluid nutrient medium, the β-that luteole is sole carbon source respectively Carrotene is the fluid nutrient medium of sole carbon source, luteole acid dipalmitate is to carry out in the fluid nutrient medium of sole carbon source Breeding metabolism.It takes 5ml fermentation liquid in 20ml ml headspace bottle after 48h, carries out solid-phase headspace microextraction and GC-MS measurement.Determination condition For：

The solid phase microextraction of sample

It takes sample Lycium chinense wine 8ml in 20ml ml headspace bottle, 2.0g sodium chloride is added, and 8 μ L sec-n-octyl alcohol solution are added, it is permanent 40 DEG C of balance 10min on warm magnetic stirring apparatus, insertion CAR/DVB/PDMS fiber head 40 DEG C of absorption 15min, GC desorb 5min, use It is analyzed in GC-MS.

Gaschromatographic mass spectrometry operating condition

Chromatographic condition is：Chromatographic column is DB-5MS (30m × 0.25mm × 0.25 μm), and carrier gas He, volume flow is 1mL/min, injector temperature are 250 DEG C.Temperature programming：40 DEG C of holding 3min, rise to 120 DEG C with the heating rate of 5 DEG C/min, 230 DEG C are risen to the heating rate of 8 DEG C/min again, keeps 10min.Filament flow is 0.20mA.Mass Spectrometry Conditions are：EI ionization Source, electron energy 70eV, detector voltage 350V.Scanning range is 20~450AMU, and ion source temperature is 200 DEG C.

Present invention simultaneously provides flavouring Lycium chinense wines made from above-mentioned preparation method.

Beneficial effect

The degradation bacteria Ku Teshi bacillus strain NXUGQ15 of the mutagenic obtained medlar carotenoid that can degrade of the present invention (Kurthiasp) (deposit number CCTCC NO：M2017524).Compared to the ability that bacterium germination out improves degradation carotenoid, and send out The ferment production carotenoid degrading enzyme time is short, and enzyme activity is high, the enzyme activity 8.87U/mL of crude enzyme liquid, and producing enzyme is preferable.

Lycium chinense wine new process is innovated

The present invention has innovated the zymotechnique of Lycium chinense wine, and the carotenoid degrading enzyme produced using preservation of bacteria strain handles fructus lycii Slurry, can increase the volatilization fragrance component of Lycium chinense wine, significantly improve the quality of Lycium chinense wine, improve the Lycium chinense wine market competitiveness. Reduce the waste that fermentation Lycium chinense wine generates simultaneously, reduces waste, reduce the difficulty of subsequent processing, energy conservation and environmental protection has Very high promotional value.

Detailed description of the invention

Fig. 1-present invention improves the method and process flow chart of fermented type fructus lycii wine aroma；

Fig. 2-Ku Teshi bacillus phyletic evolution tree graph of the present invention；

The sensory evaluation result figure of the method flavouring Lycium chinense wine of Fig. 3-three kinds of raising fermented type fructus lycii wine aromas；

In figure：Wine sample 1 is to put back to that Lycium chinense wine is made in Lycium chinense wine after handling fructus lycii slag using Ku Teshi bacillus；Wine sample 2 is It is put back to using high pressure degradation fructus lycii slag and Lycium chinense wine is made in Lycium chinense wine；Wine sample 3 is Lycium chinense wine made from method for increasing aroma of the present invention.

Specific embodiment

The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention It is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, it is not intended to limit the present invention Range, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this Under the premise of invention spirit and scope, to the various changes or change of material component and dosage progress in these embodiments It belongs to the scope of protection of the present invention.

Embodiment 1

The separation and acquisition of Ku Teshi bacillus strain

The strain of the isolated carotenoid that can degrade from wolfberry juice, and go out Ku Teshi bar through Uv-induced screening Bacteria strain NXUGQ15 (Kurthia sp), using the carotenoid degrading enzyme of this plant of bacterium generation, the class in wolfberry fruit syrup of degrading is recklessly Radish element, improves fructus lycii wine brewage technology, improves Lycium chinense wine quality.The bacterium starting strain is by Zhang Huiling from Ningxia Berry source fructus lycii It is isolated in the wolfberry juice of limited liability company planting base.

Ku Teshi bacillus strain NXUGQ15 (Kurthia sp), deposit number CCTCC NO：M2017524, in 2017 9 The moon is preserved in China typical culture collection center (Wuhan), address on the 21st：The Chinese Wuhan Wuhan University.The bacterium can degrade Beta carotene, optimum growth temp are 35-37 DEG C, pH=2-3；Degrading enzyme tolerable temperature caused by the bacterium is 70-90 DEG C, Best enzyme reaction pH=1-3.The bacterium produces that the carotenoid degrading enzyme time is short, and enzyme activity is high, the enzyme activity 8.87U/m L of crude enzyme liquid, excellent In bacterium germination GQ-16 out.

The identification of the Ku Teshi bacillus strain

Preliminary Identification：After fluid nutrient medium culture 48h, microscopy is carried out using microscope, in oily its form under the microscope. The result shows that bacterial strain NXUGQ15 is Gram-positive bacillus, no gemma.

Molecular biology identification：It is analyzed using 16S-23S rDNA ISR polymorphism and sequence, constructs its systematic evolution tree See attached drawing 2.By systematic evolution tree it is found that be based on 16rDNA region sequence phylogenetic tree, bacterial strain NXUGQ15 (Kurthia sp) with Zuo Shi Al Kut Salmonella is got together, and illustrates bacterial strain NXUGQ15 (Kurthia sp) and Zuo Shi Al Kut Salmonella is same species, i.e. bacterium Strain NXUGQ15 (Kurthia sp) is Al Kut Salmonella.

Degradation of the Ku Teshi bacillus to carotenoid

Bacterial strain NXUGQ15 (Kurthia sp) is inoculated into fluid nutrient medium, the β-that luteole is sole carbon source respectively Carrotene is the fluid nutrient medium of sole carbon source, luteole acid dipalmitate is to carry out in the fluid nutrient medium of sole carbon source Breeding metabolism.It takes 5ml fermentation liquid in 20ml ml headspace bottle after 48h, carries out solid-phase headspace microextraction and GC-MS measurement.Determination condition For：

The solid phase microextraction of sample

It takes sample Lycium chinense wine 8ml in 20ml ml headspace bottle, 2.0g sodium chloride is added, and 8 μ L sec-n-octyl alcohol solution are added, it is permanent 40 DEG C of balance 10min on warm magnetic stirring apparatus, insertion CAR/DVB/PDMS fiber head 40 DEG C of absorption 15min, GC desorb 5min, use It is analyzed in GC-MS.

Gaschromatographic mass spectrometry operating condition

Chromatographic condition is：Chromatographic column is DB-5MS (30m × 0.25mm × 0.25 μm), and carrier gas He, volume flow is 1mL/min, injector temperature are 250 DEG C.Temperature programming：40 DEG C of holding 3min, rise to 120 DEG C with the heating rate of 5 DEG C/min, 230 DEG C are risen to the heating rate of 8 DEG C/min again, keeps 10min.Filament flow is 0.20mA.Mass Spectrometry Conditions are：EI ionization Source, electron energy 70eV, detector voltage 350V.Scanning range is 20~450AMU, and ion source temperature is 200 DEG C.

The breeding of Ku Teshi bacillus strain

(1) inventor separated and identifies from wolfberry juice in January, 2016, had obtained the 1 plant of carotenoid that can degrade Strain is Ku Teshi bacillus strain GQ-16.In order to improve the degradation capability of its carotenoid of degrading, which is lured Become, sees below：

(2) mutagenesis Al Kut Salmonella GQ-16

Using Al Kut Salmonella GQ-16 as starting strain, mutagenic and breeding is carried out, when purpose improves inulinase-producing activity shortening fermentation Between.

It tests as follows：

Ultraviolet mutagenesis：

One ring of starting strain is taken, is linked into fluid nutrient medium, cultivates 5-6h under the conditions of 35 DEG C, until logarithmic phase mid-term.It will It is 10 that bacteria suspension, which is diluted to concentration,⁵CFU/m L.With lethality 80% be condition by preliminary experiment, obtain best irradiation time with Irradiation distance.Bacteria suspension is spread evenly across on fluid nutrient medium, distance 25cm under the ultraviolet lamp of 30W is placed in, irradiates 5min, After radiation treatment, with black cloth package culture medium under the conditions of 35 DEG C -37 DEG C, 8-12h is cultivated, screening grows fast and colonial morphology Good 20 plants of bacterial strain, then pass through fermenting property test experiments, and excellent 5 plants of bacterial strain of screenability are respectively designated as NXUGQ15, NXUGQ46, NXUGQ7, NXUGQ18, NXUGQ39 extract each enzyme respectively and carry out fermentation of medlar wine, pass through measurement Fermentation time come identify its fermenting property height.Fermentation time is shorter, and indicating that fermenting property is better the results are shown in Table 1.

Microwave irradiation：

5m L bacteria suspension is taken in the culture dish of diameter 9cm, with lethality 80% is condition by preliminary experiment, selects maximum Power 700W, pulse power 2450MHz household microwave oven are irradiated 5s, are quickly cooled down 5s on ice, repeat this step, take It states treatment fluid 0.5m L to be spread evenly across on solid medium, with black cloth package culture medium under the conditions of 35 DEG C -37 DEG C, cultivates 8- 12h.Screening grows fast and good colonial morphology 20 plants of bacterial strain, then passes through fermenting property test experiments, and screenability is excellent 5 plants of bacterial strain, be respectively designated as W-NXUGQ-5, W-NXUGQ-26, W-NXUGQ-1, W-NXUGQ-18, W-NXUGQ-35, respectively It extracts each enzyme and carries out fermentation of medlar wine, its fermenting property height is identified by measurement fermentation time.Fermentation time is shorter, table Showing that fermenting property is better the results are shown in Table 2.

Enzyme activity definition：Under the conditions of 50 DEG C, 3.5 pH, the amount that 1h decomposes carotenoid is one for 1g enzyme powder or 1m L enzyme solution A enzyme-activity unit (U).The enzyme activity calculation formula of crude enzyme liquid is as follows：

In formula：Y is the quality .mg of enzyme effect degradation carotenoid；N is sample extension rate；2 be when measuring enzyme activity The 1/2 of reaction solution is taken；T is to react time .h used.

The influence result of strain enzyme-producing (carotenoid degrading enzyme) is distinguished in ultraviolet mutagenesis processing and microwave irradiation processing It is shown in Table 1 and table 2.

The processing of 1 ultraviolet mutagenesis of table

Number

NXUGQ 15

NXUGQ46

NXUGQ 7

NXUGQ 18

NXUGQ39

Bacterium germination GQ-16 out

Enzyme activity U/mL

8.87

7.90

8.46

8.84

8.12

6.32

Fermentation time

8h

13h

10h

12h

11h

10h

The processing of 2 microwave irradiation of table

Number

W-NXUGQ-5

W-NXUGQ-26

W-NXUGQ-1

W-NXUGQ-18

W-NXUGQ-35

Bacterium germination GQ-16 out

Enzyme activity U/mL

7.87

7.89

7.46

8.05

8.12

6.32

Fermentation time

11h

8h

10h

10h

14h

10h

As can be seen from the above two tables, preferably, microwave irradiation handles 5 plants to NXUGQ 15 in 5 plants of bacterium of ultraviolet mutagenesis processing W-NXUGQ-5 is best in bacterium,

It is carried out stability test 15 times with two plants of bacterium respectively, by comparing finding that NXUGQ 15 is more stable, compared to setting out Bacterium improves the ability of degradation carotenoid, and the production carotenoid degrading enzyme time of fermenting is short, and enzyme activity is high, the enzyme activity of crude enzyme liquid 8.87U/mL, producing enzyme are preferable.The strain is selected, NXUGQ15, i.e. Ku Teshi bacillus strain NXUGQ15 are finally named as (Kurthia sp), and carry out culture presevation.

Embodiment 2

The method for improving fermented type fructus lycii wine aroma, includes the following steps：Fructus lycii mashing, addition carotenoid drop Enzyme is solved, additive amount 4% takes Chinese wolfberry clear juice after 60 DEG C of reaction 4h, filtering and removing slag, 50ml/L pectase is added, The liquid sulfurous acid that 50ml/L concentration is 6%, is 22%, pH 3.3-3.5 with white granulated sugar sugar addition, and 2% yeast of inoculation is living Change liquid to be fermented at 25-28 DEG C to residual sugar less than 4g/L, fermentation ends.

Flavor substance measurement and sensory evaluation are carried out to Lycium chinense wine prepared by embodiment 2.It the results are shown in Table 3, table 4 and attached drawing 3。

The activated yeast liquid and preparation method thereof includes the following steps：

Slant tube yeast takes one ring → 25-28 DEG C of 10mL malt juice liquid medium culture 36h → 50mL50% malt 25-28 DEG C of culture 36h of fluid nutrient medium of+50% wolfberry juice mass percent of juice → 10mL is taken to be seeded in 100mL wolfberry juice 36h → whole is cultivated at 25-28 DEG C pour into 1000mL wolfberry juice culture 36h → acquisition bacterial concentration at 22-25 DEG C reach 10⁷The bacterium solution of cfu/ml is to get yeast activated liquid → stand-by.

The preparation method of the carotenoid degrading enzyme, includes the following steps：

1. the preparation of Ku Teshi bacillus liquid

Fluid nutrient medium (g/L)：Sodium nitrate 3, dipotassium hydrogen phosphate 1, magnesium sulfate 0.5, potassium chloride 0.5, ferric sulfate 0.01, sugarcane Sugared 30, YNB synthetic media (no amino yeast nitrogen culture medium) 6.7,15'-dioxygenase；PH=3.2.

2. expanding culture

Take slant tube Ku Teshi bacillus (Kurthia sp) 1 ring of NXUGQ15 → 35-37 DEG C of 10mL fluid nutrient medium, It is cultivated under 130r/min and cultivates 11h → 3000mL fluid nutrient medium under 35-37 DEG C of 11h → 100mL fluid nutrient medium, 130r/min 35-37 DEG C, culture 11h → bacterial concentration reaches 10 under 130r/min⁶Cfu/ml → Ku Teshi bacillus seed liquor；

3. fermenting

Fluid nutrient medium → 2% → 33 DEG C of seed liquor of inoculation Ku Teshi bacillus, pH=3.3,140r/min fermentation 9h → hair Zymotic fluid；

4. prepared by carotenoid degrading enzyme

Fermentation liquid → 4 DEG C, 10000r/min centrifugation 120min → take supernatant → carotenoid degrading enzyme crude enzyme liquid；It will Crude enzyme liquid is chilled, it is dry, be concentrated and using after dialysis preliminary purification, obtain carotenoid degrading enzyme.

The Ku Teshi bacillus is (Kurthia sp) NXUGQ15, and deposit number is CCTCC NO：M2017524.

Embodiment 3

The method for improving fermented type fructus lycii wine aroma, includes the following steps：Fructus lycii mashing, addition carotenoid drop Enzyme is solved, additive amount 3% takes Chinese wolfberry clear juice after 60 DEG C of reaction 3h, filtering and removing slag, 40ml/L pectase is added, and 40ml/L is dense The liquid sulfurous acid that degree is 6% is 20%, pH 3.3 with white granulated sugar sugar addition, is inoculated with 2% yeast activated liquid at 25-28 DEG C Lower fermentation to residual sugar is less than 4g/L, fermentation ends.

The activated yeast liquid and preparation method thereof includes the following steps：

Slant tube yeast takes one ring → 25-28 DEG C of 10mL malt juice liquid medium culture for 24 hours → 50mL50% malt 25-28 DEG C of fluid nutrient medium of+50% wolfberry juice mass percent of juice culture for 24 hours → take 10mL to be seeded in 100mL wolfberry juice → all pour into 1000mL wolfberry juice and cultivate at 22-25 DEG C for 24 hours → is cultivated for 24 hours at 25 DEG C to obtain bacterial concentration and reach 10⁷The bacterium solution of cfu/ml is to get yeast activated liquid → stand-by.

The preparation method of the carotenoid degrading enzyme, includes the following steps：

1. the preparation of Ku Teshi bacillus liquid

Fluid nutrient medium (g/L)：Sodium nitrate 3, dipotassium hydrogen phosphate 1, magnesium sulfate 0.5, potassium chloride 0.5, ferric sulfate 0.01, sugarcane Sugared 30, YNB synthetic media (no amino yeast nitrogen culture medium) 6.7,15'-dioxygenase；PH=3.2.

2. expanding culture

Take slant tube Ku Teshi bacillus (Kurthia sp) NXUGQ151 ring → 35-37 DEG C of 10mL fluid nutrient medium, It is cultivated under 130r/min and cultivates 10h → 3000mL fluid nutrient medium under 35-37 DEG C of 10h → 100mL fluid nutrient medium, 130r/min 35-37 DEG C, culture 10h → bacterial concentration reaches 10 under 130r/min⁶Cfu/ml → Ku Teshi bacillus seed liquor；

3. fermenting

Fluid nutrient medium → -32 DEG C, pH=3.0-3.2,140r/min of 2% → 30 DEG C of inoculation Ku Teshi bacillus seed liquor hairs Ferment 10h → fermentation liquid；

4. prepared by carotenoid degrading enzyme

Fermentation liquid → 4 DEG C, 10000r/min centrifugation 100min → take supernatant → carotenoid degrading enzyme crude enzyme liquid.

The Ku Teshi bacillus is (Kurthia sp) NXUGQ15, and deposit number is CCTCC NO：M2017524.

Embodiment 4

The method for improving fermented type fructus lycii wine aroma, includes the following steps：Fructus lycii mashing, addition carotenoid drop Enzyme is solved, additive amount 5% takes Chinese wolfberry clear juice after 60 DEG C of reaction 5h, filtering and removing slag, 60ml/L pectase is added, The liquid sulfurous acid that 60ml/L concentration is 6%, is 25%, pH 3.3-3.5 with white granulated sugar sugar addition, and 3% yeast of inoculation is living Change liquid to be fermented at 27-28 DEG C to residual sugar less than 4g/L, fermentation ends.

The activated yeast liquid and preparation method thereof includes the following steps：

Slant tube yeast takes one ring → 27-28 DEG C of 10mL malt juice liquid medium culture 48h → 50mL50% malt 27-28 DEG C of culture 48h of fluid nutrient medium of+50% wolfberry juice mass percent of juice → 10mL is taken to be seeded in 100mL wolfberry juice 48h → whole is cultivated at 27-28 DEG C pour into 1000mL wolfberry juice culture 48h → acquisition bacterial concentration at 24-25 DEG C reach 10⁷The bacterium solution of cfu/ml is to get yeast activated liquid → stand-by.

The preparation method of the carotenoid degrading enzyme, includes the following steps：

1. the preparation of Ku Teshi bacillus liquid

Fluid nutrient medium (g/L)：Sodium nitrate 3, dipotassium hydrogen phosphate 1, magnesium sulfate 0.5, potassium chloride 0.5, ferric sulfate 0.01, sugarcane Sugared 30, YNB synthetic media (no amino yeast nitrogen culture medium) 6.7,15'-dioxygenase；PH=3.2.

2. expanding culture

Take slant tube Ku Teshi bacillus (Kurthia sp) 1 ring of NXUGQ15 → 36-37 DEG C of 10mL fluid nutrient medium, It is cultivated under 130r/min and cultivates 12h → 3000mL fluid nutrient medium under 36-37 DEG C of 12h → 100mL fluid nutrient medium, 130r/min 36-37 DEG C, culture 12h → bacterial concentration reaches 10 under 130r/min⁶Cfu/ml → Ku Teshi bacillus seed liquor；

3. fermenting

Fluid nutrient medium → -35 DEG C, pH=3.4-3.5,140r/min of 2% → 34 DEG C of inoculation Ku Teshi bacillus seed liquor hairs Ferment 10h → fermentation liquid；

4. prepared by carotenoid degrading enzyme

Fermentation liquid → 4 DEG C, 10000r/min centrifugation 150min → take supernatant → carotenoid degrading enzyme crude enzyme liquid, will Crude enzyme liquid is chilled, it is dry, be concentrated and using after dialysis preliminary purification, obtain carotenoid degrading enzyme.

The Ku Teshi bacillus is (Kurthia sp) NXUGQ15, and deposit number is CCTCC NO：M2017524.

Test example

Flavor substance measurement

The extraction of isoprenoid compound is dropped：Fragrance enrichment is carried out using headspace solid-phase microextraction (HS-SPME) method, is taken One 20mL ml headspace bottle, with Sterile pipette plus 8mL wolfberry juice to be analyzed or Lycium chinense wine sample and 2.0g NaCl, in 40 DEG C of perseverances 10min is balanced on warm magnetic stirring apparatus, 40 DEG C of absorption 15min, GC desorption 5min of insertion CAR/DVB/PDMS fiber head are used for GC-MS analysis^[40]。

Gaschromatographic mass spectrometry operating condition

Chromatographic condition is：Chromatographic column is DB-5MS (30m × 0.25mm × 0.25 μm), temperature programming：40 DEG C of holding 3min, 120 DEG C are risen to the heating rate of 5 DEG C/min, then rises to 230 DEG C with the heating rate of 8 DEG C/min, keeps 10min.Carrier gas is He, volume flow 1mL/min, injector temperature are 250 DEG C.Mass Spectrometry Conditions are：EI ionization source, electron energy 70eV, lamp Silk flow is 0.20mA.Detector voltage is 350V.Scanning range is 20~450AMU, and ion source temperature is 200 DEG C.

Analysis method：

(1) analysis method of carotenoid

It is calculated before processing by standard curve respectively and the content of each single factor test treated carotenoid.Substitute into following formula In calculate the degradation rates of all kinds of carrotene.

Degradation rate (%)=[(content after content-processing before handling)/content before handling] × 100%

(2) analysis method of isoprenoid drops

According to the structure feature of compound, it is compared with the map of NIST spectrum library Plays compound, according to ion stream Figure determines its relative amount.

Compared with method for increasing aroma of the present invention is volatilized fragrance component with Lycium chinense wine prepared by other two method, sent out with traditional handicraft The Lycium chinense wine of ferment is control：

Method I：Using NXUGQ15 (Kurthia sp) bacterium direct fermentation fructus lycii slag, carotenoid is made to degrade, slag is returned It puts in Lycium chinense wine, dissolves in catabolite drop isoprenoid in wine, filter to take wine.

Method II (the method for the present invention)：Using NXUGQ15 (Kurthia sp) bacterium, by fermented and cultured bacterium solution, collecting should Degrading enzyme is added in fructus lycii slag and digests by the carotenoid degrading enzyme that bacterium generates, then in fructus lycii slag playback Lycium chinense wine, It dissolves in catabolite drop isoprenoid in wine, filters to take wine.

Method III：Fructus lycii slag is used in 121 DEG C of sterilizing 20min HIGH PRESSURE TREATMENTs, carotenoid in fructus lycii slag is made to degrade, Then it is dipped in 0.5-1h in Lycium chinense wine again, makes to drop in isoprenoid dissolution wine, filters to take wine.

Lycium chinense wine with traditional handicraft fermentation is control, and method I, method II, method III is respectively adopted and carries out Lycium chinense wine Flavouring, Lycium chinense wine obtained mainly volatilizees fragrance component and relative amount comparison result is shown in Table 3：

Mainly volatilize fragrance component and relative amount in 3 different process Lycium chinense wine of table

Note：" nd " expression is not detected.

By upper table it is concluded that：By the main volatilization fragrance of the Lycium chinense wine after method I, method II, III flavouring of method Ingredient is much higher than the Lycium chinense wine of traditional handicraft preparation, and increases nine Carotenoids aroma substances, wherein method II, That is the Lycium chinense wine of the method for the present invention preparation is especially prominent.

Drop isoprene kind compound content compares in the Lycium chinense wine of three kinds of methods preparation

Lycium chinense wine with traditional handicraft fermentation is control, and method I, method II, method III is respectively adopted and carries out Lycium chinense wine Flavouring, drop isoprenoid compound is compared in Lycium chinense wine obtained, the results are shown in Table 4

Drop isoprenoid compound compares (peak area) in the Lycium chinense wine of 4 different process of table preparation

Isoprene drops	Traditional handicraft Lycium chinense wine	Method I	Method II	Method III
					Methyl heptenone	3.28	3.45	3.88	3.35
2,4- nonadienal	5.01	9.33	10.17	6.94
					Dihydro-β-ionone	0.25	3.27	4.13	1.12
Alpha, beta-lonone	0.19	4.41	7.54	6.73
					Dihydro jasmone	2.91	1.76	3.33	2.25
Safranal	1.01	2.11	2.52	1.89
					Aldehyde C-9	nd	6.48	2.13	3.32
α-cyclocitral	3.10	3.33	4.21	2.66
					β-cyclocitral	5.20	6.37	9.64	5.47
Geranyl acetone	7.88	12.59	14.86	9.84
					2,2,6- trimethylcyclohexanone	1.41	6.14	6.77	5.73
Limonene	nd	nd	2.06	1.11
					Octanoic acid	0.27	1.27	9.82	6.38
Capric acid	1.33	2.21	1.42	1.25
					Acetone geraniol ester	nd	0.01	nd	nd
Dihydroactinidiolide	0.09	2.09	3.42	1.02

As shown in Table 4:Isoprene kind compound content is dropped in Lycium chinense wine prepared by the present invention is much higher than traditional handicraft system Standby Lycium chinense wine, also above the Lycium chinense wine of high-pressure treatment process preparation.

Fragrance control is referring to table：

The odor characteristic of isoprenoid compound partially drops in 5 Lycium chinense wine of table

According to the sensory evaluation method of following table, prepared by III 3 kinds of the method for the present invention II, method I, method distinct methods Lycium chinense wine carries out sensory evaluation, judges result and draws sensory evaluation wind rose, sees attached drawing 3

6 fermentation Chinese wolfberry fruit wine sensory evaluation scores table of table

By finding Ku Teshi of the present invention to the measurement for dropping pentadiene class compound in three kinds of wine samples and in conjunction with sensory evaluation The carotenoid degrading enzyme assisted fermentation Lycium chinense wine that bacterium is extracted, fragrance and mouthfeel are best.It assists sending out using Al Kut Salmonella enzyme On the one hand the degradation rate of carotenoid not only can be improved in ferment first, to generate more drop pentadiene fragrance components.Separately On the one hand, the metabolic activity for avoiding Al Kut Salmonella itself, which generates other metabolites, influences the harmony of wine body.Secondly, biological Enzyme is low using reaction temperature, avoids and causes bad flavor to wine body because of high-temperature heating.

The present invention carries out degradation treatment, Neng Gougai using Al Kut Salmonella enzyme during fructus lycii liquor brewing, by fructus lycii slag Into the brewage process of Lycium chinense wine, fructus lycii wine aroma is improved.And can appropriateness carry out high pressure sterilization, or use Ku Teshi Bacterium handles fructus lycii slag, so that several method for increasing aroma are combined, can significantly improve the fragrance of fermentation Lycium chinense wine.It is described above Only several embodiments of the present invention are expressed for embodiment, and the description thereof is more specific and detailed, but it cannot be understood as Limitation to the scope of the patents.It should be pointed out that for those of ordinary skill in the art, not departing from present inventive concept Under the premise of, the respective embodiments described above can also make combination and improvement, naturally it is also possible to by several preparations disclosed by the invention Method is reconfigured and the increase and decrease of processing step, recombination, and these are all within the scope of protection of the present invention.

Claims (10)

1. a kind of method for improving fermented type fructus lycii wine aroma, includes the following steps：

Fructus lycii mashing, adds carotenoid degrading enzyme, and additive amount 3%-5% takes after 60 DEG C of reaction 3-5h, filtering and removing slag Chinese wolfberry clear juice, is added 40-60ml/L pectase, and the liquid sulfurous acid that 40-60ml/L concentration is 6% is adjusted with white granulated sugar Pol is 20-25%, pH 3.3-3.5, and inoculation 2-3% yeast activated liquid ferments at 25-28 DEG C is less than 4g/L, hair to residual sugar Ferment terminates；It is prepared it is characterized in that, the carotenoid degrading enzyme is fermented by Ku Teshi bacillus (Kurthia sp).

2. improving the method for fermented type fructus lycii wine aroma according to claim 1, which is characterized in that the Ku Teshi bacillus is (Kurthia sp) NXUGQ15, deposit number are CCTCC NO：M2017524.

3. improving the method for fermented type fructus lycii wine aroma according to claim 2, which is characterized in that the carotenoid drop The preparation method for solving enzyme, includes the following steps：

Take slant tube Ku Teshi bacillus (Kurthia sp) 1 ring of NXUGQ15 → 35-37 DEG C of 10mL fluid nutrient medium, 130r/ It is cultivated under min and cultivates 10-12h → 3000mL Liquid Culture under 35-37 DEG C of 10-12h → 100mL fluid nutrient medium, 130r/min 10-12h → bacterial concentration is cultivated under 35-37 DEG C of base, 130r/min reaches 10⁶Cfu/ml → Ku Teshi bacillus seed liquor；

Fluid nutrient medium → -35 DEG C, pH=3.0-3.5,140r/min fermentation 8- of 2% → 30 DEG C of inoculation Ku Teshi bacillus seed liquor 10h → fermentation liquid；

Fermentation liquid → 4 DEG C, 10000r/min centrifugation 100-150min → take supernatant → carotenoid degrading enzyme crude enzyme liquid；

The fluid nutrient medium forms as follows, unit g/L：Sodium nitrate 3, dipotassium hydrogen phosphate 1, magnesium sulfate 0.5, potassium chloride 0.5, sulphur Sour iron 0.01, sucrose 30, without amino yeast nitrogen culture medium 6.7,15'-dioxygenase；PH=3.2.

4. improving the method for fermented type fructus lycii wine aroma according to claim 3, which is characterized in that the carotenoids The preparation method of plain degrading enzyme further includes：After crude enzyme liquid is chilled, dry, concentration and utilization dialysis preliminary purification, class is obtained Carrotene degrading enzyme.

5. -4 any method for improving fermented type fructus lycii wine aroma according to claim 1, which is characterized in that the yeast is living Change liquid and preparation method thereof, includes the following steps：

Slant tube yeast takes one ring → 25-28 DEG C of 10mL malt juice liquid medium culture 24-48h → 50mL50% brewer's wort 25-28 DEG C of culture 24-48h of fluid nutrient medium of+50% wolfberry juice mass percent → 10mL is taken to be seeded in 100mL wolfberry juice Culture 24-48h → whole, which is poured into, at 25-28 DEG C cultivates 24-48h → acquisition bacterial concentration at 22-25 DEG C in 1000mL wolfberry juice Reach 10⁷The bacterium solution of cfu/ml is to get yeast activated liquid.

6. improving the method for fermented type fructus lycii wine aroma according to claim 5, which is characterized in that further include measurement flavor Matter and sensory evaluation.

7. the method for improving fermented type fructus lycii wine aroma according to claim 6, includes the following steps：

Fructus lycii mashing, adds carotenoid degrading enzyme, and additive amount 4% takes fructus lycii clear after 60 DEG C of reaction 4h, filtering and removing slag Juice, is added 50ml/L pectase, and the liquid sulfurous acid that 50ml/L concentration is 6% is 22%, pH with white granulated sugar sugar addition For 3.3-3.5, it is inoculated with 2% yeast activated liquid and is fermented at 25-28 DEG C to residual sugar less than 4g/L, fermentation ends；

The activated yeast liquid and preparation method thereof includes the following steps：

Slant tube yeast take one ring → 25-28 DEG C of 10mL malt juice liquid medium culture 36h → 50mL50% brewer's wort+ 25-28 DEG C of culture 36h of fluid nutrient medium of 50% wolfberry juice mass percent → 10mL is taken to be seeded in 25- in 100mL wolfberry juice 36h → whole is cultivated at 28 DEG C pour into 1000mL wolfberry juice culture 36h → acquisition bacterial concentration at 22-25 DEG C reach 10⁷The bacterium solution of cfu/ml is to get yeast activated liquid；

The preparation method of the carotenoid degrading enzyme, includes the following steps：

1. the preparation of Ku Teshi bacillus liquid

Fluid nutrient medium (g/L)：Sodium nitrate 3, dipotassium hydrogen phosphate 1, magnesium sulfate 0.5, potassium chloride 0.5, ferric sulfate 0.01, sucrose 30, YNB synthetic media 6.7,15'-dioxygenase；PH=3.2；

2. expanding culture

Take slant tube Ku Teshi bacillus (Kurthia sp) NXUGQ151 ring → 35-37 DEG C of 10mL fluid nutrient medium, 130r/ It is cultivated under min and cultivates 11h → 3000mL fluid nutrient medium 35-37 under 35-37 DEG C of 11h → 100mL fluid nutrient medium, 130r/min DEG C, culture 11h → bacterial concentration reaches 10 under 130r/min⁶Cfu/ml → Ku Teshi bacillus seed liquor；

3. fermenting

Fluid nutrient medium → 2% → 33 DEG C of seed liquor of inoculation Ku Teshi bacillus, pH=3.3,140r/min fermentation 9h → fermentation liquid；

4. prepared by carotenoid degrading enzyme

Fermentation liquid → 4 DEG C, 10000r/min centrifugation 120min → take supernatant → carotenoid degrading enzyme crude enzyme liquid；By thick enzyme Liquid is chilled, it is dry, be concentrated and using after dialysis preliminary purification, obtain carotenoid degrading enzyme；

The Ku Teshi bacillus is (Kurthia sp) NXUGQ15, and deposit number is CCTCC NO：M2017524.

8. the method for improving fermented type fructus lycii wine aroma according to claim 6, includes the following steps：

Fructus lycii mashing, adds carotenoid degrading enzyme, and additive amount 3% takes fructus lycii clear after 60 DEG C of reaction 3h, filtering and removing slag Juice, is added 40ml/L pectase, and the liquid sulfurous acid that 40ml/L concentration is 6% is 20%, pH with white granulated sugar sugar addition It is 3.3,2% yeast activated liquid of inoculation ferments at 25-28 DEG C is less than 4g/L, fermentation ends to residual sugar；

The activated yeast liquid and preparation method thereof includes the following steps：

Slant tube yeast take one ring → 25-28 DEG C of 10mL malt juice liquid medium culture for 24 hours → 50mL50% brewer's wort+ 25-28 DEG C of fluid nutrient medium of 50% wolfberry juice mass percent culture for 24 hours → take 10mL to be seeded in 100mL wolfberry juice 25 DEG C Lower culture → is all poured into 1000mL wolfberry juice and is cultivated at 22-25 DEG C for 24 hours → for 24 hours and obtains bacterial concentration and reach 10⁷cfu/ml Bacterium solution to get yeast activated liquid；

The preparation method of the carotenoid degrading enzyme, includes the following steps：

1. the preparation of Ku Teshi bacillus liquid

Fluid nutrient medium (g/L)：Sodium nitrate 3, dipotassium hydrogen phosphate 1, magnesium sulfate 0.5, potassium chloride 0.5, ferric sulfate 0.01, sucrose 30, YNB synthetic media 6.7,15'-dioxygenase；PH=3.2；

2. expanding culture

Take slant tube Ku Teshi bacillus (Kurthia sp) 1 ring of NXUGQ15 → 35-37 DEG C of 10mL fluid nutrient medium, 130r/ It is cultivated under min and cultivates 10h → 3000mL fluid nutrient medium 35-37 under 35-37 DEG C of 10h → 100mL fluid nutrient medium, 130r/min DEG C, culture 10h → bacterial concentration reaches 10 under 130r/min⁶Cfu/ml → Ku Teshi bacillus seed liquor；

3. fermenting

Fluid nutrient medium → -32 DEG C, pH=3.0-3.2,140r/min of 2% → 30 DEG C of inoculation Ku Teshi bacillus seed liquor fermentations 10h → fermentation liquid；

4. prepared by carotenoid degrading enzyme

Fermentation liquid → 4 DEG C, 10000r/min centrifugation 100min → take supernatant → carotenoid degrading enzyme crude enzyme liquid；

The Ku Teshi bacillus is (Kurthia sp) NXUGQ15, and deposit number is CCTCC NO：M2017524.

9. the method for improving fermented type fructus lycii wine aroma according to claim 6, includes the following steps：

Fructus lycii mashing, adds carotenoid degrading enzyme, and additive amount 5% takes fructus lycii clear after 60 DEG C of reaction 5h, filtering and removing slag Juice, is added 60ml/L pectase, and the liquid sulfurous acid that 60ml/L concentration is 6% is 25%, pH with white granulated sugar sugar addition For 3.3-3.5, it is inoculated with 3% yeast activated liquid and is fermented at 27-28 DEG C to residual sugar less than 4g/L, fermentation ends；

The activated yeast liquid and preparation method thereof includes the following steps：

Slant tube yeast take one ring → 27-28 DEG C of 10mL malt juice liquid medium culture 48h → 50mL50% brewer's wort+ 27-28 DEG C of culture 48h of fluid nutrient medium of 50% wolfberry juice mass percent → 10mL is taken to be seeded in 27- in 100mL wolfberry juice 48h → whole is cultivated at 28 DEG C pour into 1000mL wolfberry juice culture 48h → acquisition bacterial concentration at 24-25 DEG C reach 10⁷The bacterium solution of cfu/ml is to get yeast activated liquid；

The preparation method of the carotenoid degrading enzyme, includes the following steps：

1. the preparation of Ku Teshi bacillus liquid

Fluid nutrient medium (g/L)：Sodium nitrate 3, dipotassium hydrogen phosphate 1, magnesium sulfate 0.5, potassium chloride 0.5, ferric sulfate 0.01, sucrose 30, YNB synthetic media 6.7,15'-dioxygenase；PH=3.2；

2. expanding culture

Take slant tube Ku Teshi bacillus (Kurthia sp) 1 ring of NXUGQ15 → 36-37 DEG C of 10mL fluid nutrient medium, 130r/ It is cultivated under min and cultivates 12h → 3000mL fluid nutrient medium 36-37 under 36-37 DEG C of 12h → 100mL fluid nutrient medium, 130r/min DEG C, culture 12h → bacterial concentration reaches 10 under 130r/min⁶Cfu/ml → Ku Teshi bacillus seed liquor；

3. fermenting

Fluid nutrient medium → -35 DEG C, pH=3.4-3.5,140r/min of 2% → 34 DEG C of inoculation Ku Teshi bacillus seed liquor fermentations 10h → fermentation liquid；

4. prepared by carotenoid degrading enzyme

Fermentation liquid → 4 DEG C, 10000r/min centrifugation 150min → take supernatant → carotenoid degrading enzyme crude enzyme liquid, by thick enzyme Liquid is chilled, it is dry, be concentrated and using after dialysis preliminary purification, obtain carotenoid degrading enzyme；

The Ku Teshi bacillus is (Kurthia sp) NXUGQ15, and deposit number is CCTCC NO：M2017524.

10. flavouring Lycium chinense wine made from any method for improving fermented type fructus lycii wine aroma of claim 1-9.