CN109266624B - Preparation method of carotenoid degrading enzyme - Google Patents
Preparation method of carotenoid degrading enzyme Download PDFInfo
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Abstract
The invention relates to a preparation method of carotenoid degrading enzyme, belonging to the technical field of microbial fermentation. The preparation method comprises inoculating 2% Kurthia sp seed solution in liquid culture medium, fermenting at 30-35 deg.C and pH of 3.0-3.5 at 140r/min for 8-10h to obtain fermentation liquid; centrifuging the fermentation liquor at 4 ℃ and 10000r/min for 100-150min, and taking the supernatant to obtain the crude enzyme liquid of the carotenoid degrading enzyme. The Kurthia sp is NXUGQ15, and the preservation number is CCTCC NO: and M2017524. The carotenoid degrading enzyme produced by the preserved strain has the enzyme activity of 8.87U/mL of the crude enzyme liquid, and the wolfberry residue or wolfberry pulp is treated by the carotenoid degrading enzyme, so that the volatile aroma components of the wolfberry wine can be increased, and the quality of the wolfberry wine can be improved.
Description
Technical Field
The invention relates to a preparation method of carotenoid degrading enzyme, belonging to the technical field of microbial fermentation.
Background
As is well known, the medlar has the efficacies of nourishing liver and kidney, replenishing vital essence and improving eyesight, and treating soreness and pain of waist and knees, dizziness and tinnitus, internal heat and thirst and blood deficiency and chlorosis, so people make medlar wine as health care wine. The existing methods for preparing the medlar wine comprise a soaking method and a fermentation method. The soaking method is generally to soak whole-grain medlar with white spirit or yellow wine, the alcohol content is higher, and the nutrient components can not be fully dissolved out. The fermentation method of the medlar wine does not involve a high-temperature heating process, and oxygen is less involved, so that the medlar wine not only basically keeps natural nutritional components in medlar, but also is more beneficial to human body to absorb after fermentation, and is a very good nutritional health-care fruit wine. However, the fermented medlar wine is affected by the factors such as raw materials, processing technology and the like, so that the quality is uneven, the taste and the aroma of the product are generally insufficient, and the quality needs to be improved.
101323823 discloses a brewing method of fructus Lycii wine, which comprises adding saccharifying fermentation distiller's yeast and yeast into fructus Lycii crushed and deseeded and cooked starch material, fermenting in a fermentation tank; squeezing the fermented mash, clarifying the wine juice, sterilizing, brewing, blending, filtering and bottling. The pure fermentation medlar wine is brewed by adopting starchy raw materials as a carbon source for fermentation, and mainly solves the problems that: the production cost is reduced to the maximum extent.
1513970 patent publication No. describes a blending method of fermented fructus Lycii wine and fermented grape wine, which uses fructus Lycii and grape as raw materials, and separately ferments them, and then prepares them. Fresh or dried fructus Lycii is crushed to make fruit pulp (fruit juice) and fermented to make fermented fructus Lycii wine; crushing white grape or red grape to obtain fruit juice (fruit pulp), and fermenting to obtain fermented wine. The fermented medlar wine and the fermented grape wine are prepared according to the following weight percentage (1-99%): (99% -1%) blending, blending to different sugar degrees, heat treating, cooling, filtering, bottling, and sterilizing to obtain fermented fructus Lycii wine. The fermented medlar wine brewed by the method has harmonious fragrance of medlar and grape, rich and harmonious taste, rich medlar flavor and outstanding typicality.
1104248 discloses a method for brewing Chinese wolfberry wine, which comprises selecting raw materials, squeezing, fermenting, filtering, sterilizing, and is characterized in that fresh Chinese wolfberry is selected, squeezed into Chinese wolfberry juice, sodium sulfite is added for vulcanization, yeast is added, glucose is added until the sugar degree is 22 degrees BX, the mixture is put into a pressure tank for sealed fermentation at the fermentation temperature of (20-25) DEG C for 5-8 days, glucose is added again until the sugar degree is 18 degrees BX, sealed fermentation is carried out again until the fermentation temperature is (15-20) DEG C, when fruit residues and yeast are precipitated at the bottom of the tank and the wine is primarily clarified, the sugar degree is below 5 degrees BX, the mixture of the fruit residues and the yeast in the tank is stirred, heated, cooled, the Chinese wolfberry mixture is filtered, a proper amount of sodium sulfite is added into the filtered liquid for clarification, filtering again, storing the filtrate in barrel, storing at about 10 deg.C for 2-3 months, changing barrel once, filling, pasteurizing, and brewing to obtain fructus Lycii wine, and adding a certain amount of fructus Lycii soaking solution into the brewed fructus Lycii liquid to obtain fructus Lycii wine with different alcohol content.
1782060 invention, a brewing method of medlar wine, comprising the following steps: sorting and cleaning fresh or dried fructus Lycii, soaking in 2-5 times of water for 12-24 hr, crushing into fructus Lycii pulp, adjusting alcohol content to 20-25% (v/v) with 95% (v/v) deodorized alcohol, soaking for 10-20 days, circulating for 1-4 times every day during soaking, and separating out the soaking liquor; adding soft water 0.5-2 times of fresh fructus Lycii weight into the separated fresh fructus Lycii residue; adding soft water 2-5 times the weight of dried fructus Lycii into dried fructus Lycii residue, adjusting sugar degree to 230g/L with sucrose, adjusting total acid to 6.0-8.0g/L with citric acid or tartaric acid, and adjusting SO2Adding 0.2-0.5g/L pectase, adding 0.15-0.3g/L dry yeast, fermenting at 18-30 deg.C for 5-7 days, and analyzing residual sugar content to be less than 4g/L to separate out fermented wine when specific gravity is reduced to below 1000 g/L; mixing the soaking wine and the fermented wine, aging, blending, adding glue, filtering, sterilizing, bottling, and packaging to obtain the final product.
1265420 invention, a brewing method of medlar wine, comprising the following steps: placing the sorted medlar into a mixed solution of citric acid and sodium sulfite for blanching, crushing the blanched medlar, adding soft water for soaking, canning the medlar and the soft water at a volume ratio of 7:3, treating with sulfur dioxide and pectinase, adjusting the components, standing, inoculating for fermentation, separating fermented wine for storage after pre-fermentation and post-fermentation, placing the sorted and crushed medlar into 95% deodorized edible alcohol for soaking for about 30 days for separation to prepare soaking wine, blending the soaking wine and the fermented wine according to a volume ratio of 1:4, storing for a period of time, filtering by glue, sterilizing, filtering and bottling.
1077744, the invention discloses a preparation method of medlar fermented wine, which comprises the following steps: the medlar is subjected to impurity removal and washing, hot extraction, crushing, filtration, auxiliary material addition, sterilization, cooling, early fermentation, purification treatment, filtration, aging, jar making, blending, filtration and other processes to prepare the medlar fermented wine, and the specific process is as follows: a: removing impurities from the wolfberry fruits, washing, leaching for 1.5-2.5 hours by using hot water at 65-75 ℃, crushing, filtering, putting the remainder into the hot water at 65-75 ℃ again, leaching for 0.8-1.2 hours, and then filtering for the second time, wherein b: crushing the residues obtained after the secondary filtration in the item a, filtering again and then discarding filter residues, and c: adding 20% of white sugar auxiliary materials into the filtrate of the item b, and d: sterilizing the solution prepared in the step c at the temperature of 95-100 ℃ for 20-25 seconds, cooling to 65-70 ℃, putting into a wine jar, continuously cooling to 30-32 ℃, and e: and (3) carrying out early fermentation on the solution prepared in the step (d) for 6-8 days at the temperature of 28-30 ℃, and then carrying out later fermentation for 9-11 days at room temperature, wherein f: purifying the solution prepared in item e, adding gelatin (according to the proportion of 0.008g gelatin in 10mL solution), filtering for 14-16 days, and filtering, g: aging the solution prepared in the step f for 3-5 months, pouring the solution into a jar, standing the solution at 50-60 ℃ for 10-20 days, standing the solution at minus 2-44 ℃ for 5-7 days, filtering, storing the filtrate for 1 month, and h: adding flavoring agents and tonics into the filtrate obtained in the step g for blending, wherein the ratio of (i): and (5) filtering the solution prepared in the step h, and sterilizing to obtain a finished product.
From the disclosure of the prior art we found that: the enzyme commonly used in the production of the medlar wine is only pectinase, the aim is mainly to carry out enzymolysis on pulp so as to improve the juice yield and finally improve the wine yield, and the application of other enzymes in the fermentation of the medlar wine is not reported. After microbial fermentation, because the carotenoid is insoluble in water, a large part of the carotenoid is removed when the pomace is separated after the fermentation is finished, and a part of the carotenoid is degraded and lost in the brewing process, so that the carotenoid content in the wolfberry wine is not high finally.
The traditional brand and cultural advantage history of the Ningxia wolfberry fruit with a long history have been hundreds of years, the Zhongning county is named as the county of Chinese wolfberry fruit in the state institute, the Ningxia wolfberry fruit is listed as a food and drug homologous food by the Chinese pharmacopoeia, and the food and drug homologous food cannot be compared with other provinces.
The invention provides a preparation method of carotenoid degrading enzyme, which is a technical problem to be solved by the invention, and aims to respond to the national call, develop functional health food, develop a characteristic medlar resource with Ningxia superiority, improve the quality of medlar health wine and find a preparation method of carotenoid degrading enzyme, wherein the technical problem is clearly provided by the No. 1 document in 2017, and the preparation method is to enhance the development and application of new food raw materials and medicinal and edible food, enhance the research of modern biology and nutrition strengthening technology, and excavate and develop food with health care function.
Disclosure of Invention
In order to solve the problems, the invention provides a preparation method of carotenoid degrading enzyme, which adopts a strain of carotenoid degrading bacteria obtained by separation, and utilizes the characteristics of carotenoid instability and easy degradation by light, oxygen, heat, biological enzyme and the like to generate isoprenoid aroma compounds to ensure that the carotenoid degrading enzyme generates a large amount of C9-、C10-、C13-and C15Norisoprenoids (norrisopridines), which are volatile aromatic compounds that have a positive contribution to food products because of their low sensory threshold. The rich carotenoid of the medlar mainly comprises beta-carotene, zeaxanthin and zeaxanthin dipalmitate, and the maximum content of the zeaxanthin dipalmitate is about 77.5 percent of the total carotenoid. But most of the water is not dissolved in the medlar residues when brewing the wine, thereby causing great waste. The carotenoid degrading enzyme can degrade the carotenoid insoluble in the medlar residues or medlar pulp, so that a large amount of carotenoid contained in the medlar wine is degraded, a large amount of aromatic compounds are generated in the medlar wine, the effect of increasing the wine fragrance is achieved, and the quality of the medlar wine is improved. Of course, the carotenoid of the invention is not limited to the aroma improvement of the fermented medlar wine, and can also be used in other food, feed, cosmetics and other fields.
In the traditional fermentation process, because the carotenoid in the medlar is insoluble in water, and a large amount of carotenoid is taken away by medlar residues, the carotenoid degrading enzyme prepared by the method can degrade the carotenoid in the medlar residues or pulp, so that isoprene-reduced aroma substances are generated, and the bouquet can be obviously improved. Improves the aroma of the Chinese wolfberry wine, improves the quality of the Chinese wolfberry wine and innovating the brewing process of the Chinese wolfberry wine.
Norisoprenoid compounds (norrisopridines) are substances with special fragrance generated by carotenoid degradation, and the substances have a low olfactory threshold, so that the small amount of norisoprenoid compounds can have a large sensory effect on the flavor of food. The food contains norisoprenoid compounds with 9, 10, 11 and 13 carbon atoms, which are mostly generated by degrading carotenoid, such as beta-damascenone, beta-ionone, dihydroactinidiolide and the like, and all have good flavor. The isoprenoid compound is one of main aroma-producing substances of fresh fruits and vegetables such as grapes and is also an important compound influencing the aroma quality of fruit wine. The detected norisoprene compounds in the aroma components of the fruit wine comprise: 19 types of norpentadiene compounds such as beta-ionone, isophorone, limonene, saffron aldehyde, geranylacetone, dihydro-beta-ionone, beta-damascenone, dihydroactinidiolide, methyl heptenone, beta-cyclocitral, 2-heptenal, dihydrojasmone, alpha-cyclocitral, isogeraniol, 2,5, 6-trimethyl-4-hepten-3-one, 2, 4-nonadienal, 3-decanone, 2-nonenal, 2, 6-trimethylcycloenone and the like, and the aroma characteristics are shown in Table 3.
The invention improves the process of the medlar wine, and increases the flavor substances of the medlar wine by degrading the carotenoid. The enzyme prepared by the invention is used for brewing medlar wine, the content and the variety of isoprenoid compounds are increased through detection, and the isoprenoid compounds mainly comprise beta-ionone, 2, 6-Trimethylcyclohexanone (TCH), isophorone, geranylacetone, dihydro-beta-ionone, geraniol, saffron aldehyde, beta-cyclocitral, limonene, pseudoionone and dihydro actinidiolide. And nonanal, amyl valerate, ethyl decanoate. The substances have low taste threshold value, and play a good role in sensory evaluation of the medlar wine.
The fructus Lycii wine has increased content of carbonyl compounds, and relative peak area of 5.78% of total components, which is higher than 4.23% of that of the original process; the high content of the components is 3-undecanone, beta-damascenone, beta-ionone, beta-cyclocitral, safranal and dihydro-beta-ionone, and the high content of the components is 3.136% of the beta-ionone and 3.615% of the beta-cyclocitral, which have special fruit fragrance.
The relative peak area of the ester compound is 14.08 percent and is higher than 11.03 percent of the prior art, and the ester compound is an aromatic substance with a high content in the medlar wine. The fragrance of the medlar wine is greatly contributed, ethyl decanoate and amyl valerate are also important degradation products and have the fragrance of pear and banana; the dihydroactinidiolide and the geranyl acetone ester are intermediate products for degrading carotenoid, are one of esters with higher content, and have prominent fruit and flower fragrance.
A method for preparing carotenoid degrading enzyme comprises the following steps:
preparation of bacterial liquid of Kurthe's rod
Liquid medium (g/L): 3 parts of sodium nitrate, 1 part of dipotassium phosphate, 0.5 part of magnesium sulfate, 0.5 part of potassium chloride, 0.01 part of ferric sulfate, 30 parts of sucrose, 6.7 parts of YNB synthetic medium (amino-free yeast nitrogen source medium) and 15 parts of beta-carotene; pH 3.2.
② enlargement culture
Culturing slant Cuteus (Kurthia sp) NXUGQ151 → 10mL liquid culture medium 35-37 deg.C, culturing at 130r/min for 10-12h → 100mL liquid culture medium 35-37 deg.C, culturing at 130r/min for 10-12h → 3000mL liquid culture medium 35-37 deg.C, culturing at 130r/min for 10-12h → bacterial liquid concentration reaches 10%6cfu/ml → Kurthia sp seed fluid;
③ fermentation
Liquid culture medium → inoculation of seed liquid of Kurthia sp for 2% → 30-35 deg.C, pH 3.0-3.5, 140r/min for fermentation for 8-10h → fermentation liquid;
preparation of carotenoid degrading enzyme
Fermentation broth → 4 ℃, centrifugation at 10000r/min for 100-.
The preparation method of the carotenoid degradation enzyme can further comprise the following steps: and (3) freezing, drying and concentrating the crude enzyme solution, and primarily purifying by using a dialysis method to obtain the carotenoid degrading enzyme.
The Kurthia sp is NXUGQ15, and the preservation number is CCTCC NO: m2017524;
the invention also provides a carotenoid degrading enzyme, the enzyme activity is high, the enzyme activity of the crude enzyme solution is 8.87U/mL, and the enzyme production is good.
Isolation and obtaining of the Kurthia strains
Separating strains capable of degrading carotenoid from the medlar juice, screening a Kurthia sp strain NXUGQ15(Kurthia sp) through ultraviolet mutagenesis, degrading the carotenoid in medlar pulp by using carotenoid degrading enzyme generated by the strain, improving the medlar wine brewing process and improving the medlar wine quality. The strain is obtained by separating Zhanghui from fructus Lycii juice of planting base of Ningxia Bairui source fructus Lycii Gmbycis.
Kurthella strain NXUGQ15(Kurthia sp), with a collection number of CCTCC NO: m2017524, deposited in the chinese type culture collection (wuhan) on 21.9.2017, address: wuhan university. The strain can degrade beta-carotene, and has optimal growth temperature of 35-37 deg.C and pH of 2-3; the temperature of the degrading enzyme produced by the strain is 70-90 ℃, and the optimal pH value of the enzyme reaction is 1-3. The carotenoid degrading enzyme produced by the strain has short time and high enzyme activity, and the enzyme activity of the crude enzyme solution is 8.87U/mL, which is superior to that of the developed strain GQ-16.
Identification of the Kurthia strains
Preliminary identification: after culturing in a liquid medium for 48 hours, the shape was observed under an oil microscope by microscopic examination. The results show that the strain NXUGQ15 is a gram-positive rod-shaped bacterium without spores.
Molecular biological identification: the 16S-23S rDNA ISR polymorphism and sequence analysis is adopted to construct the phylogenetic tree shown in figure 2. According to phylogenetic trees, based on a 16rDNA region sequence phylogenetic tree, the strain NXUGQ15(Kurthia sp) and the Kurthia zoffii come together, which indicates that the strain NXUGQ15(Kurthia sp) and the Kurthia zoffii are the same species, namely the strain NXUGQ15(Kurthia sp) is the Kurthia.
Degradation of carotenoids by Kurthia sp
The strain NXUGQ15(Kurthia sp) is respectively inoculated into a liquid culture medium with zeaxanthin as a unique carbon source, a liquid culture medium with beta-carotene as a unique carbon source and a liquid culture medium with zeaxanthin dipalmitate as a unique carbon source for reproduction and metabolism. And after 48 hours, taking 5ml of fermentation liquor in a 20ml headspace bottle, and carrying out solid phase headspace microextraction and GC-MS (gas chromatography-mass spectrometry) determination. The measurement conditions were:
solid phase microextraction of samples
Taking 8ml of medlar wine sample, putting the medlar wine sample into a 20ml headspace bottle, adding 2.0g of sodium chloride, adding 8 mu L of 2-octanol solution, balancing for 10min at 40 ℃ on a constant temperature magnetic stirrer, inserting a CAR/DVB/PDMS fiber head for adsorption for 15min at 40 ℃, and desorbing for 5min by GC for GC-MS analysis.
Working conditions of gas chromatography-mass spectrometry
The chromatographic conditions are as follows: the chromatographic column was DB-5MS (30m × 0.25mm × 0.25 μm), the carrier gas was He, the volume flow was 1mL/min, and the injection port temperature was 250 ℃. Temperature programming: keeping the temperature at 40 ℃ for 3min, increasing the temperature to 120 ℃ at the temperature increasing speed of 5 ℃/min, increasing the temperature to 230 ℃ at the temperature increasing speed of 8 ℃/min, and keeping the temperature for 10 min. The filament flow was 0.20 mA. The mass spectrum conditions are as follows: EI ionization source, electron energy 70eV, detector voltage 350V. The scanning range is 20-450 AMU, and the ion source temperature is 200 ℃.
Advantageous effects
The invention obtains a degrading bacterium Kurthia sp strain NXUGQ15(Kurthia sp) capable of degrading the lycium barbarum carotenoid by mutagenesis (the preservation number is CCTCC NO: M2017524). Compared with the starting strain, the capability of degrading carotenoid is improved, the time for producing carotenoid degrading enzyme by fermentation is short, the enzyme activity is high, the enzyme activity of the crude enzyme solution is 8.87U/mL, and the enzyme production is better.
Innovating new process of Chinese wolfberry wine
The invention innovates the fermentation process of the medlar wine, treats medlar residues or medlar pulp by using carotenoid degrading enzyme produced by a preserved strain, can increase volatile aroma components of the medlar wine, improves the quality of the medlar wine and improves the market competitiveness of the medlar wine. Meanwhile, the waste generated by fermenting the medlar wine is reduced, the waste is reduced, the difficulty of subsequent treatment is reduced, the energy is saved, the environment is protected, and the popularization value is high.
Drawings
FIG. 1-Process flow diagram of the process for preparing carotenoid degrading enzymes according to the invention;
FIG. 2 is a phylogenetic tree diagram of the bacterium Kurthia according to the invention;
FIG. 3 is a graph showing sensory evaluation results of a medlar wine aromatized with a carotenoid degrading enzyme according to the present invention;
in the figure: the wine sample 1 is the aroma-enhanced medlar wine obtained by treating medlar residues by utilizing Kurthia sp and then putting the medlar residues back into medlar wine; the wine sample 2 is the aroma-enhanced medlar wine obtained by degrading medlar residues under high pressure and putting the medlar residues back into the medlar wine; the wine sample 3 is the aroma-enhanced medlar wine obtained by using the carotenoid degrading enzyme to enhance aroma.
Detailed Description
The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.
Example 1
Isolation and obtaining of Kurthia strains
Separating strains capable of degrading carotenoid from the medlar juice, screening a Kurthia sp strain NXUGQ15(Kurthia sp) through ultraviolet mutagenesis, degrading the carotenoid in medlar pulp by using carotenoid degrading enzyme generated by the strain, improving the medlar wine brewing process and improving the medlar wine quality. The strain is obtained by separating Zhanghui from fructus Lycii juice of planting base of Ningxia Bairui source fructus Lycii Gmbycis.
Kurthella strain NXUGQ15(Kurthia sp), with a collection number of CCTCC NO: m2017524, deposited in the chinese type culture collection (wuhan) on 21.9.2017, address: wuhan university. The strain can degrade beta-carotene, and has optimal growth temperature of 35-37 deg.C and pH of 2-3; the temperature of the degrading enzyme produced by the strain is 70-90 ℃, and the optimal pH value of the enzyme reaction is 1-3. The carotenoid degrading enzyme produced by the strain has short time and high enzyme activity, and the enzyme activity of the crude enzyme solution is 8.87U/m L, which is superior to that of the developed strain GQ-16.
Identification of the Kurthia strains
Preliminary identification: after culturing in a liquid medium for 48 hours, the shape was observed under an oil microscope by microscopic examination. The results show that the strain NXUGQ15 is a gram-positive rod-shaped bacterium without spores.
Molecular biological identification: the 16S-23S rDNA ISR polymorphism and sequence analysis is adopted to construct the phylogenetic tree shown in figure 2. According to phylogenetic trees, based on a 16rDNA region sequence phylogenetic tree, the strain NXUGQ15(Kurthia sp) and the Kurthia zoffii come together, which indicates that the strain NXUGQ15(Kurthia sp)) and the Kurthia zoffii are the same species, namely the strain NXUGQ15(Kurthia sp) is the Kurthia.
Degradation of carotenoids by Kurthia sp
The strain NXUGQ15(Kurthia sp) is respectively inoculated into a liquid culture medium with zeaxanthin as a unique carbon source, a liquid culture medium with beta-carotene as a unique carbon source and a liquid culture medium with zeaxanthin dipalmitate as a unique carbon source for reproduction and metabolism. And after 48 hours, taking 5ml of fermentation liquor in a 20ml headspace bottle, and carrying out solid phase headspace microextraction and GC-MS (gas chromatography-mass spectrometry) determination. The measurement conditions were:
solid phase microextraction of samples
Taking 8ml of medlar wine sample, putting the medlar wine sample into a 20ml headspace bottle, adding 2.0g of sodium chloride, adding 8 mu L of 2-octanol solution, balancing for 10min at 40 ℃ on a constant temperature magnetic stirrer, inserting a CAR/DVB/PDMS fiber head for adsorption for 15min at 40 ℃, and desorbing for 5min by GC for GC-MS analysis.
Working conditions of gas chromatography-mass spectrometry
The chromatographic conditions are as follows: the chromatographic column was DB-5MS (30m × 0.25mm × 0.25 μm), the carrier gas was He, the volume flow was 1mL/min, and the injection port temperature was 250 ℃. Temperature programming: keeping the temperature at 40 ℃ for 3min, increasing the temperature to 120 ℃ at the temperature increasing speed of 5 ℃/min, increasing the temperature to 230 ℃ at the temperature increasing speed of 8 ℃/min, and keeping the temperature for 10 min. The filament flow was 0.20 mA. The mass spectrum conditions are as follows: EI ionization source, electron energy 70eV, detector voltage 350V. The scanning range is 20-450 AMU, and the ion source temperature is 200 ℃.
Breeding of Kurthia strains
(1) The inventor separates and identifies 1 strain which can degrade carotenoid and is a Kurthia sp strain GQ-16 from the medlar juice in 2016 (1 month). In order to improve the degradation capability of the strain for degrading carotenoid, the strain is subjected to mutagenesis, and the mutagenesis is detailed as follows:
(2) induced Kurthia sp GQ-16
The Kurthia sp GQ-16 is used as the initial strain to carry out mutation breeding, so as to improve the enzyme production activity and shorten the fermentation time.
The experiments were as follows:
ultraviolet mutagenesis:
taking a ring of original strains, inoculating the original strains into a liquid culture medium, and culturing for 5-6h at 35 ℃ to the middle stage of logarithmic phase. Diluting the bacterial suspension to a concentration of 105CFU/mL. The optimal irradiation time and irradiation distance are obtained by taking the lethality rate of 80% as a condition through a preliminary experiment. Uniformly coating the bacterial suspension on a liquid culture medium, placing the liquid culture medium under an ultraviolet lamp of 30W at a distance of 25cm, irradiating for 5min, after irradiation treatment, wrapping the culture medium with black cloth, culturing for 8-12h at 35-37 ℃, screening 20 strains with fast growth and good colony morphology, then screening 5 strains with excellent performance through fermentation performance test experiments, respectively named as NXUGQ15, NXUGQ46, NXUGQ7, NXUGQ18 and NXUGQ39, respectively extracting each enzyme to ferment the Chinese wolfberry wine, and determining the fermentation performance by measuring the fermentation time. The shorter the fermentation time, the better the fermentation performance, and the results are shown in Table 1.
Microwave mutagenesis:
placing 5mL of bacterial suspension in a culture dish with the diameter of 9cm, adopting a domestic microwave oven with the maximum power of 700W and the pulse power of 2450MHz to irradiate for 5s through a pre-experiment under the condition of 80% of fatality rate, rapidly cooling on ice for 5s, repeating the step, uniformly coating 0.5mL of the treatment solution on a solid culture medium, wrapping the culture medium by using black cloth, and culturing for 8-12h at the temperature of 35-37 ℃. Screening 20 strains which grow fast and have good colony morphology, then screening 5 strains with excellent performance through a fermentation performance test experiment, respectively naming the strains as W-NXUGQ-5, W-NXUGQ-26, W-NXUGQ-1, W-NXUGQ-18 and W-NXUGQ-35, respectively extracting each enzyme to ferment the medlar wine, and determining the fermentation performance by measuring the fermentation time. The shorter the fermentation time, the better the fermentation performance, and the results are shown in Table 2.
Definition of enzyme activity: the amount of carotenoid decomposed by 1g of enzyme powder or 1mL of enzyme solution at 50 ℃ and pH 3.5 for 1h is one enzyme activity unit (U). The enzyme activity calculation formula of the crude enzyme solution is as follows:
in the formula: y is the mass of carotenoid degraded by enzyme action, mg; n is the sample dilution factor; 2 1/2 taking reaction liquid when measuring enzyme activity; t is the time taken for the reaction.
The results of the effect of UV mutagenesis treatment and microwave mutagenesis treatment on the enzyme production (carotenoid-degrading enzyme) of the strain are shown in tables 1 and 2, respectively.
TABLE 1 UV mutagenesis treatment
| | NXUGQ | 15 | NXUGQ46 | NXUGQ 7 | NXUGQ 18 | NXUGQ39 | Growth promoting bacteria GQ-16 |
| Enzyme activity U/mL | 8.87 | 7.90 | 8.46 | 8.84 | 8.12 | 6.32 | |
| Time of fermentation | 8h | 13h | 10h | 12h | 11h | 10h |
TABLE 2 microwave mutagenesis treatment
| Numbering | W-NXUGQ-5 | W-NXUGQ-26 | W-NXUGQ-1 | W-NXUGQ-18 | W-NXUGQ-35 | Growth promoting bacteria GQ-16 |
| Enzyme activity U/mL | 7.87 | 7.89 | 7.46 | 8.05 | 8.12 | 6.32 |
| Time of fermentation | 11h | 8h | 10h | 10h | 14h | 10h |
As can be seen from the above tables, NXUGQ15 is the most preferable among 5 strains subjected to ultraviolet mutagenesis, and W-NXUGQ-5 is the most preferable among 5 strains subjected to microwave mutagenesis,
the two strains are respectively used for stability test for 15 times, and comparison shows that NXUGQ15 is relatively stable, compared with the original strain, the carotenoid degradation capability is improved, the time for producing carotenoid degradation enzyme by fermentation is short, the enzyme activity is high, the enzyme activity of crude enzyme liquid is 8.87U/mL, and the enzyme production is better. The strain was selected and finally designated NXUGQ15, a strain of Kurthia sp, NXUGQ15(Kurthia sp), and was deposited.
Example 2
A method for preparing carotenoid degrading enzyme comprises the following steps:
preparation of bacterial liquid of Kurthe's rod
Liquid medium (g/L): 3 parts of sodium nitrate, 1 part of dipotassium phosphate, 0.5 part of magnesium sulfate, 0.5 part of potassium chloride, 0.01 part of ferric sulfate, 30 parts of sucrose, 6.7 parts of YNB synthetic medium (amino-free yeast nitrogen source medium) and 15 parts of beta-carotene; pH 3.2.
② enlargement culture
Taking slant Cultibacter (Kurthia sp) NXUGQ151 ring → 10mL liquid culture medium 35-37 ℃, culturing 11h → 100mL liquid culture medium 35-37 ℃, culturing 11h → 3000mL liquid culture medium 35-37 ℃ at 130r/min, culturing 11h → bacteria liquid concentration reaches 10h6cfu/ml → Kurthia sp seed fluid;
③ fermentation
Liquid culture medium → inoculation of the seed liquid of the Bacillus clarkii, 2% → 33 ℃, and fermentation at a pH of 3.3 and 140r/min for 9h → fermentation liquid;
preparation of carotenoid degrading enzyme
The fermentation liquid → 4 ℃, 10000r/min centrifugation for 120min → supernatant liquid → crude enzyme liquid of carotenoid degradation enzyme; and (3) freezing, drying and concentrating the crude enzyme solution, and primarily purifying by using a dialysis method to obtain the carotenoid degrading enzyme.
The Kurthia sp is NXUGQ15, and the preservation number is CCTCC NO: and M2017524.
The carotenoid degrading enzyme of the invention is used for improving the fragrance of the medlar wine according to the following steps: pulping fructus Lycii, adding carotenoid degrading enzyme with an addition amount of 4%, reacting at 60 deg.C for 4 hr, filtering to remove residue, collecting fructus Lycii clear juice, adding 50ml/L pectase and liquid sulfurous acid with a concentration of 50ml/L and 6%, adjusting sugar degree to 22% with white sugar, adjusting pH to 3.3-3.5, inoculating 2% yeast activating solution, fermenting at 25-28 deg.C until residual sugar is less than 4g/L, fermenting, and aging at low temperature to obtain the flavored fructus Lycii wine.
The preparation method of the yeast activation liquid comprises the following steps:
taking a ring of slant test tube yeast → 10mL of malt wort liquid culture medium for culturing at 25-28 ℃ for 36h → 50mL of liquid culture medium for culturing at 25-28 ℃ of 50% malt wort and 50% medlar juice in percentage by mass for culturing at 25-28 ℃ for 36h → taking 10mL of slant test tube yeast for inoculating in 100mL of medlar juice for culturing at 25-28 ℃ for 36h → pouring all the slant test tube yeast into 1000mL of medlar juice for culturing at 22-25 ℃ for 36h → obtaining the concentration of bacteria liquid reaching 107Bacteria of cfu/mlAnd (4) obtaining yeast activation liquid → for standby.
The medlar wine prepared in example 2 was subjected to flavor determination and sensory evaluation. The results are shown in Table 3, Table 4 and FIG. 3.
Example 3
A method for preparing carotenoid degrading enzyme comprises the following steps:
preparation of bacterial liquid of Kurthe's rod
Liquid medium (g/L): 3 parts of sodium nitrate, 1 part of dipotassium phosphate, 0.5 part of magnesium sulfate, 0.5 part of potassium chloride, 0.01 part of ferric sulfate, 30 parts of sucrose, 6.7 parts of YNB synthetic medium (amino-free yeast nitrogen source medium) and 15 parts of beta-carotene; pH 3.2.
② enlargement culture
Culturing slant Cuteus (Kurthia sp) NXUGQ151 ring → 10mL liquid culture medium 35-37 deg.C, culturing at 130r/min for 10h → 100mL liquid culture medium 35-37 deg.C, culturing at 130r/min for 10h → 3000mL liquid culture medium 35-37 deg.C, culturing at 130r/min for 10h → the concentration of the liquid reaches 10%6cfu/ml → Kurthia sp seed fluid;
③ fermentation
Liquid culture medium → inoculation of the seed liquid of the Kurthia sp at 2% → 30-32 deg.C, pH 3.0-3.2, fermenting at 140r/min for 10h → fermentation liquid;
preparation of carotenoid degrading enzyme
Fermentation liquor → 4 ℃, centrifugation at 10000r/min for 100min → supernatant fluid → crude enzyme liquid of carotenoid degrading enzyme.
The Kurthia sp is NXUGQ15, and the preservation number is CCTCC NO: and M2017524.
Example 4
A method for preparing carotenoid degrading enzyme comprises the following steps:
preparation of bacterial liquid of Kurthe's rod
Liquid medium (g/L): 3 parts of sodium nitrate, 1 part of dipotassium phosphate, 0.5 part of magnesium sulfate, 0.5 part of potassium chloride, 0.01 part of ferric sulfate, 30 parts of sucrose, 6.7 parts of YNB synthetic medium (amino-free yeast nitrogen source medium) and 15 parts of beta-carotene; pH 3.2.
② enlargement culture
Taking slant Cultibacter (Kurthia sp) NXUGQ151 ring → 10mL liquid culture medium 36-37 ℃, culturing for 12h → 100mL liquid culture medium 36-37 ℃, culturing for 12h → 3000mL liquid culture medium 36-37 ℃ at 130r/min → culturing for 12h → bacteria liquid concentration reaches 106cfu/ml → Kurthia sp seed fluid;
③ fermentation
Liquid culture medium → inoculation of seed liquid of Kurthia sp at 2% → 34-35 deg.C, pH 3.4-3.5, fermenting at 140r/min for 10h → fermentation liquid;
preparation of carotenoid degrading enzyme
And (3) centrifuging the fermentation liquor → 4 ℃ at 10000r/min for 150min → taking the supernatant → the crude enzyme liquid of the carotenoid degrading enzyme, freezing, drying and concentrating the crude enzyme liquid, and preliminarily purifying by a dialysis method to obtain the carotenoid degrading enzyme.
The Kurthia sp is NXUGQ15, and the preservation number is CCTCC NO: and M2017524.
Test examples
Flavor determination
Extraction of norisoprene compounds: performing fragrance enrichment by using a headspace solid phase microextraction (HS-SPME) method, taking a 20mL headspace bottle, adding 8mL of Chinese wolfberry juice or Chinese wolfberry wine sample to be analyzed and 2.0g of NaCl by using a sterile pipette, balancing for 10min on a constant-temperature magnetic stirrer at 40 ℃, inserting a CAR/DVB/PDMS fiber head for adsorption for 15min at 40 ℃, and performing GC desorption for 5min for GC-MS analysis.
Working conditions of gas chromatography-mass spectrometry
The chromatographic conditions are as follows: the column was DB-5MS (30 m.times.0.25 mm.times.0.25 μm), temperature programmed: keeping the temperature at 40 ℃ for 3min, increasing the temperature to 120 ℃ at the temperature increasing speed of 5 ℃/min, increasing the temperature to 230 ℃ at the temperature increasing speed of 8 ℃/min, and keeping the temperature for 10 min. The carrier gas is He, the volume flow is 1mL/min, and the injection port temperature is 250 ℃. The mass spectrum conditions are as follows: EI ionization source, electron energy is 70eV, and filament flow is 0.20 mA. The detector voltage is 350V. The scanning range is 20-450 AMU, and the ion source temperature is 200 ℃.
The analysis method comprises the following steps:
(1) method for analyzing carotenoid
The carotenoid content before and after each single factor treatment was calculated from the standard curve, respectively. Substituting the degradation rate into the following formula to calculate the degradation rate of each carotene.
The degradation rate (%) < percent [ (content before treatment-content after treatment)/content before treatment ]. times.100%
(2) Method for analyzing norisoprene compound
According to the structural characteristics of the compound, the compound is compared with the spectra of standard compounds in an NIST spectral library, and the relative content of the compound is determined according to an ion flow diagram.
Comparing the volatile aroma components of the medlar wine prepared by the carotenoid degrading enzyme aroma-improving method with those prepared by the other two methods, taking the medlar wine fermented by the traditional process as a reference:
the method I comprises the following steps: fermenting fructus Lycii residue with NXUGQ15(Kurthia sp) to degrade carotenoid, separating the residue into fructus Lycii wine, dissolving the degraded product norisoprene compound in the wine, and filtering to obtain wine.
Method II (inventive method): adopting NXUGQ15(Kurthia sp) bacteria, fermenting to culture bacteria liquid, collecting carotenoid degrading enzyme produced by the bacteria, adding the degrading enzyme into fructus Lycii pulp for enzymolysis, fermenting to prepare fructus Lycii wine, dissolving the degraded product of norisoprene compound in the wine, filtering, and collecting wine.
Method III: sterilizing fructus Lycii residue at 121 deg.C for 20min under high pressure to degrade carotenoid in fructus Lycii residue, soaking in fructus Lycii wine for 0.5-1h to dissolve isoprenoid compound in the wine, and filtering to obtain wine.
The medlar wine fermented by the traditional process is used as a reference, the fragrance of the medlar wine is enhanced by respectively adopting a method I, a method II and a method III, and the comparison results of the main volatile aroma components and the relative contents of the prepared medlar wine are shown in a table 3:
TABLE 3 Main volatile aroma components and relative contents in fructus Lycii wine prepared by different processes
Note: "nd" indicates no detection.
From the above table it can be concluded that: the medlar wine flavored by the method I, the method II and the method III has the main volatile fragrance components far higher than those of the medlar wine prepared by the traditional process, and nine carotenoid fragrance substances are added, wherein the method II, namely the medlar wine prepared by the method is particularly prominent.
Comparison of content of isoprenoid compounds in fructus Lycii wine prepared by three methods
Taking fructus Lycii wine fermented by conventional method as reference, flavoring fructus Lycii wine by methods I, II and III respectively, and comparing the obtained fructus Lycii wine with isoprenoid compounds, the results are shown in Table 4
TABLE 4 comparison of isoprenoid-lowering compounds in Lycium barbarum wine prepared by different processes (peak area)
| Norisoprene | Traditional Chinese wolfberry wine | Method I | Method II | Method III |
| Methyl hepteneKetones | 3.28 | 3.45 | 3.88 | 3.35 |
| 2, 4-Nondeal | 5.01 | 9.33 | 10.17 | 6.94 |
| Dihydro-beta-ionones | 0.25 | 3.27 | 4.13 | 1.12 |
| Beta-ionones | 0.19 | 4.41 | 7.54 | 6.73 |
| Dihydrojasmone | 2.91 | 1.76 | 3.33 | 2.25 |
| Saffron aldehyde | 1.01 | 2.11 | 2.52 | 1.89 |
| Nonanal | nd | 6.48 | 2.13 | 3.32 |
| Alpha-cyclocitral | 3.10 | 3.33 | 4.21 | 2.66 |
| Beta-cyclocitral | 5.20 | 6.37 | 9.64 | 5.47 |
| Geranylacetone | 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 |
| Geranyl acetone | nd | 0.01 | nd | nd |
| Dihydroactinidiolide | 0.09 | 2.09 | 3.42 | 1.02 |
It can be seen from Table 4 that the content of isoprenoid compounds in the prepared wolfberry wine is much higher than that of the wolfberry wine prepared by the traditional process and that of the wolfberry wine prepared by the high-pressure treatment process after the wolfberry pulp is treated by the carotenoid degrading enzyme of the invention, and the wolfberry wine prepared by treating the wolfberry residues by using the preservation strain.
Aroma comparison table:
TABLE 5 aroma characteristics of some of the norisoprene compounds in Lycium barbarum wine
According to the sensory evaluation method shown in the following table, the medlar wine prepared by the three different methods of the method II, the method I and the method III is subjected to sensory evaluation, and the evaluation result is drawn into a sensory evaluation rose diagram, which is shown in figure 3
TABLE 6 sensory evaluation chart for fermented wolfberry fruit wine
Through determination of the pentadiene-reducing compounds in the three wine samples and combined sensory evaluation, the carotenoid degrading enzyme extracted by the Kurthia sp. By adopting the cotteria enzyme for auxiliary fermentation, firstly, the degradation rate of the carotenoid can be improved, so that more pentadiene-reducing aroma components are generated. On the other hand, the coordination that other metabolites are generated by the own metabolic activity of the Kurthia sp to influence the wine body is avoided. And secondly, the reaction temperature of the biological enzyme is low, so that the bad flavor of the wine body caused by high-temperature heating is avoided.
According to the invention, during brewing of the medlar wine, the medlar pulp is subjected to degradation treatment by adopting Kurthia sp enzyme, so that the brewing process of the medlar wine can be improved, and the fragrance of the medlar wine can be improved. And can be moderately sterilized under high pressure, or the medlar residues are treated by adopting Kurthia sp, so that the aroma of the fermented medlar wine can be obviously improved by combining several aroma improving methods. The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the patent. It should be noted that, for those skilled in the art, combinations and modifications of the above embodiments may be made without departing from the spirit of the present invention, and it is needless to say that several preparation methods disclosed in the present invention may be recombined, and the process steps may be increased, decreased, and recombined, which are within the protection scope of the present invention.
Claims (5)
1. A method for preparing carotenoid degrading enzyme is characterized by comprising the following steps:
preparation of bacterial liquid of Kurthe's rod
② enlargement culture
Taking slant CutobacteriumKurthia sp) NXUGQ151 ring → 10mL liquid medium 35-37 deg.C, culturing at 130r/min for 10-12h → 100mL liquid medium 35-37 deg.C, culturing at 130r/min for 10-12h → 3000mL liquid medium 35-37 deg.C, culturing at 130r/min for 10-12h → bacterial liquid concentration reaches 10-6cfu/ml → Kurthia sp seed fluid;
③ fermentation
Liquid culture medium → inoculation of the seed liquid of the Kurthia sp 2% → 30 ℃ -35 ℃, pH =3.0-3.5, 140r/min fermentation for 8-10h → fermentation broth;
preparation of carotenoid degrading enzyme
The fermentation liquid → 4 ℃, centrifugation at 10000r/min for 100-;
the bacterium Kurthella is (A), (B) and (C)Kurthia sp) NXUGQ15, the preservation number is CCTCC NO: m2017524;
the liquid culture medium in the steps comprises the following components: 3g/L of sodium nitrate, 1g/L of dipotassium hydrogen phosphate, 0.5g/L of magnesium sulfate and chlorine
Potassium chloride 0.5g/L, ferric sulfate 0.01g/L, sucrose 30g/L, YNB synthetic medium 6.7g/L, beta-carotene 15 g/L; pH = 3.2.
2. The method for producing a carotenoid degrading enzyme according to claim 1, characterized by comprising the steps of: the crude enzyme solution was frozen, dried, concentrated and purified by dialysis.
3. The process for producing a carotenoid degrading enzyme according to claim 2, wherein the step of ② the enlarged cultivation is carried out, and the cultivation time at each stage is 11 hours; fermenting at 33 ℃ for 9h, wherein the pH is =3.3 during fermentation; and step (iv) preparing carotenoid degrading enzyme, wherein the centrifugation time is 120 min.
4. The process for producing a carotenoid degrading enzyme according to claim 1, wherein the step of ② the enlarged cultivation is carried out, and the cultivation time at each stage is 10 hours; fermenting at 30-32 deg.c for 10 hr with pH = 3.0-3.2; and step (iv) preparing carotenoid degrading enzyme, wherein the centrifugation time is 100 min.
5. The process for producing carotenoid degrading enzyme according to claim 2, wherein the step of ② the enlarged cultivation is carried out at a cultivation temperature of 36-37 ℃ for 12 hours at each stage; fermenting at 34-35 deg.c for 10 hr with pH = 3.4-3.5; and step (iv) preparing carotenoid degrading enzyme, wherein the centrifugation time is 150 min.
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