A method for extracting aroma components of raw materials using Oenococcus oeni and its application

Abstract

The invention provides an application of Oenococcus oeni in extracting aroma components from a raw material and an extraction method, belonging to the technical field of perfume processing. The application of Oenococcus oeni in extracting aroma components from a raw material and the extraction method mainly comprise preparing an enzyme preparation, enzymatically hydrolyzing a raw material with the enzyme preparation to obtain an enzymatic hydrolyzate, and then extracting aroma components from the enzymatic hydrolyzate by using a simultaneous distillation and extraction method; culturing Oenococcus oeni to prepare a cell disrupted liquid of the Oenococcus oeni, separating the cell disrupted liquid into a solid and a liquid to obtain a solid, and resuspending the solid to obtain an enzyme preparation; crushing the raw material and mixing it with a buffer solution to obtain a mixed liquid, and then adding the enzyme preparation for enzymatic hydrolysis to obtain an enzymatic hydrolyzate; and extracting aroma components from the enzymatic hydrolyzate by using a simultaneous distillation and extraction method to obtain an aroma component extract. The invention can effectively extract aroma components from the raw material, and in particular has a significant extraction effect on linalool, ionone, benzyl alcohol, and 2-phenylethanol components.

Description

Technical Field

[0001] The present invention belongs to the technical field of spice processing, and specifically relates to a method for extracting aroma components of raw materials by utilizing Oenococcus oeni and an application thereof. Background Art

[0002] Aroma is a key quality indicator for grapes and wine. Grape varieties can be categorized by aroma type, including rose, strawberry, and neutral. Aroma components in grape berries are classified by molecular structure, including terpenes, norisoprenoids, aliphatic compounds, and aromatic compounds. The distribution of aromatic compounds in grape berries follows the order peel > flesh > juice. Grape peel is an important bioresource, but its current utilization in my country remains limited to simple, crude processing, with grape peels often used as fertilizer, feed, or even disposed of as waste, resulting in low value. Therefore, fully utilizing this resource is crucial.

[0003] Tea has many health benefits and is one of the most popular beverages. Improving the aroma components of tea is an important means to enhance the sensory quality of tea.

[0004] β-glucosidase is a key enzyme for the degradation of glycoside-bound aroma compounds. It can hydrolyze the non-reducing glycosidic bond bound to the end of the substrate and release glucose and the corresponding ligand.

[0005] In recent years, there have been many studies on β-glucosidase from microorganisms, such as Aspergillus niger, yeast, and Oenococcus oeni. The β-glucosidase in Aspergillus niger poses a food safety risk and is restricted in food processing; the activity of β-glucosidase in yeast is low and is mainly concentrated in the cell; while the β-glucosidase in Oenococcus oeni has more advantages in stress environments, and the hydrolytic activity of β-glucosidase depends on the corresponding aglycone structure.

[0006] Research on existing technologies has found that the activity of β-glucosidase in Oenococcus oeni varies among strains and is affected by the chemical structure of glycosides. Different strains are selective in the hydrolysis of glycosides, resulting in different aroma characteristics. There are currently few reports on the use of Oenococcus oeni to extract aroma components. Summary of the Invention

[0007] In view of this, the present invention provides an extraction method and application thereof for extracting aroma components of raw materials using Oenococcus oeni, wherein Oenococcus oeni can extract aroma from raw materials.

[0008] The present invention is achieved in that:

[0009] A first aspect of the present invention provides a method for extracting aroma components from a raw material using Oenococcus oeni, comprising the following steps:

[0010] S1: culturing Oenococcus oeni, preparing a cell lysate of the Oenococcus oeni, performing solid-liquid separation on the cell lysate to obtain a solid, and resuspending the solid to prepare an enzyme preparation;

[0011] S2: crushing the raw material and mixing it with a buffer solution to obtain a mixed solution, and then adding the enzyme preparation in S1 to perform enzymolysis to obtain an enzymatic hydrolyzate;

[0012] S3: extracting aroma components from the enzymatic hydrolysate in S2 by a simultaneous distillation extraction method to prepare an aroma component extract.

[0013] Here, resuspension is to resuspend the solid obtained by centrifugation or sedimentation using an appropriate buffer or culture medium.

[0014] On the basis of the above technical solution, the method of extracting aroma components of raw materials using Oenococcus oeni can be further improved as follows:

[0015] Among them, the strain number of the Oenococcus oeni is CICC6066 in the China Industrial Microbiology Culture Collection Center.

[0016] The method for culturing Oenococcus oeni described in S1 comprises the following steps:

[0017] Step 1: Inoculate Oenococcus oeni onto solid culture medium and culture at 25-30°C until a single colony grows;

[0018] Step 2: inoculating the single colony into a liquid culture medium and culturing at 25-30° C. for 48-72 hours to prepare a seed solution;

[0019] Step 3: inoculating the liquid culture medium with an inoculum amount of 0.5-2% by volume of the seed solution;

[0020] Step 4: Cultivate at a temperature of 25-30°C until the OD value is 1.4-1.6 to prepare a bacterial suspension;

[0021] Step 5: Collect the bacteria, wash them with a NaCl solution with a mass fraction of 0.85-0.90%, and set aside.

[0022] The solid culture medium is ATB solid culture medium; the liquid culture medium is ATB liquid culture medium. OD stands for optical density, also known as absorbance. Absorbance is a method of identifying or measuring a substance using its specific absorption spectrum. The substance being measured absorbs some light, and its concentration is calculated from the absorbance.

[0023] Among them, the solid culture medium is prepared by adding about 1.5% to 2.0% agar to the liquid culture medium, heating it to 100°C to dissolve it, and cooling and solidifying it at 40°C to make it into a solid state;

[0024] The method for preparing the cell disruption solution described in S1 comprises the following steps:

[0025] Step 1: Suspend the bacteria in 1× PBS buffer;

[0026] Step 2: The above solution is ultrasonically disrupted in an ice bath to prepare a cell disruption solution.

[0027] The ultrasonic crushing conditions are as follows: ultrasonic power is 150-200w, and ultrasonic treatment time is 15-30min.

[0028] The 1× PBS buffer solution is a 0.01 M PBS buffer solution, and the temperature of the ice bath is 0°C.

[0029] The preparation method of the enzyme preparation described in S1 is to suspend the solid in a NaCl solution with a mass fraction of 0.85-0.90% to prepare the enzyme preparation.

[0030] Among them, the solid can be suspended in a NaCl solution with a mass fraction of 0.85-0.90% to prepare an enzyme preparation; the solid can also be suspended in a NaCl solution with a mass fraction of 0.85-0.90% with the same volume as the bacterial suspension to prepare an enzyme preparation.

[0031] The specific steps of S2 include:

[0032] Step 1: crush the raw materials and pass through a 40-mesh sieve;

[0033] Step 2: Mix the raw material and the buffer at a mass volume ratio of 1 to (10 to 20) g / mL, and mix the enzyme preparation to the mixed solution at a volume ratio of 1 to (5 to 10);

[0034] Step 3: Stirring the mixture under the conditions of an enzymatic hydrolysis temperature of 30 to 50° C. and an enzymatic hydrolysis pH of 4 to 6, so that the enzymatic hydrolysis time of the mixed solution is 50 to 150 minutes.

[0035] The buffer solution is citrate buffer solution, and the conditions are as follows: the enzymatic hydrolysis time is 100 min, the enzymatic hydrolysis temperature is 40° C., and the enzymatic hydrolysis pH is 5.

[0036] Furthermore, the simultaneous distillation and extraction method in S3 comprises the following steps:

[0037] Step 1: adding the enzymatic hydrolysate in S2 to zeolite and heating to boiling; Step 2: taking an organic solvent of 1 / 3 to 1 / 5 of the volume of the enzymatic hydrolysate, heating it to 40 to 60° C., and simultaneously distilling and extracting for 2 to 4 hours;

[0038] Step 3: Add 3-5% of anhydrous sodium sulfate by weight of the organic phase to the organic solvent and let it stand for 1-2 hours to obtain an extract containing aroma components;

[0039] Step 4: Concentrate the extract at 40-50° C. to 1 / 2-1 / 10 of its volume, then add an equal volume of anhydrous ethanol and concentrate at 40-50° C. to 1 / 2-1 / 10 of its volume to obtain an aroma component extract.

[0040] The organic solvent is any one of ether, pentane, dichloromethane, and methylene chloride. Preferably, the organic solvent is methylene chloride.

[0041] A second aspect of the present invention provides an application of using Oenococcus oeni to extract aroma components of raw materials, wherein the above-mentioned Oenococcus oeni is used in extracting aroma components of raw materials.

[0042] On the basis of the above technical solution, the application of the present invention of using Oenococcus oeni to extract aroma components of raw materials can also be improved as follows:

[0043] Furthermore, the raw material includes any one of grape peel and tea leaves.

[0044] Preferably, the grape variety is "Rose Fragrance" grapes, and the tea variety is Tieguanyin tea; the grape peel preparation method comprises the steps of pressing grape fruits through an air bag to obtain grape pomace, naturally drying the grape pomace and removing grape seeds to obtain grape peel.

[0045] The method for preparing tea samples comprises the following steps:

[0046] (1) crushing Tieguanyin tea leaves through a grinder;

[0047] (2) Pass through a 40-mesh sieve and store for later use.

[0048] Furthermore, the aroma components include linalool, ionone, benzyl alcohol, and 2-phenylethanol.

[0049] Compared with the prior art, the beneficial effects of the application of Oenococcus oeni in extracting aroma components from raw materials and the extraction method provided by the present invention are as follows: by culturing Oenococcus oeni, a cell disruption solution of Oenococcus oeni is prepared, the cell disruption solution is separated into solid and liquid to obtain a solid, and the solid is resuspended to prepare an enzyme preparation; the raw material is then crushed and mixed with a buffer solution to prepare a mixed solution, and then the enzyme preparation is added for enzymolysis to prepare an enzymatic hydrolyzate; finally, the aroma components of the enzymatic hydrolyzate are extracted by simultaneous distillation and extraction to prepare an aroma component extract. The aroma components in the raw material can be effectively extracted by Oenococcus oeni, especially the extraction effect on linalool, ionone, benzyl alcohol, and 2-phenylethanol components is obvious. The extraction method of the present invention for extracting aroma components from raw materials using Oenococcus oeni is simple to operate, has low equipment requirements, and is easy to implement. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0051] Figure 1 This is a flow chart of a method for extracting aroma components from raw materials using Oenococcus oeni;

[0052] Figure 2 This is the localization map of β-glucosidase activity of Oenococcus oeni CICC 6066;

[0053] Figure 3 This is the standard curve of p-nitrophenol;

[0054] Figure 4 This is the localization map of β-glucosidase activity of Oenococcus oeni CICC 6057;

[0055] Figure 5 Schematic diagram of the simultaneous distillation and extraction apparatus;

[0056] Figure 6 This is the total ion current of aroma components in grape skin;

[0057] Figure 7 This is the total ion current diagram of aroma components in tea;

[0058] Figure 8 This is a graph showing the differences in aroma component content in grape skin;

[0059] Figure 9 This is a diagram showing the differences in aroma component content in tea; DETAILED DESCRIPTION

[0060] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0061] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention as claimed, but rather merely represents selected embodiments of the present invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are also within the scope of protection of the present invention.

[0062] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, it does not require further definition or explanation in subsequent drawings.

[0063] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise" and the like to indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as limiting the present invention.

[0064] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Thus, a feature identified as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, "plurality" means two or more, unless otherwise specifically defined.

[0065] like Figure 1 FIG. 1 is a flow chart of a method for extracting aroma components from raw materials using Oenococcus oeni according to a first aspect of the present invention, comprising the following steps:

[0066] S1: culturing Oenococcus oeni, preparing a cell lysate of the Oenococcus oeni, performing solid-liquid separation of the cell lysate to obtain a solid, and resuspending the solid to prepare an enzyme preparation;

[0067] S2: crushing the raw material and mixing it with a buffer solution to obtain a mixed solution, and then adding the enzyme preparation in S1 for enzymatic hydrolysis to obtain an enzymatic hydrolyzate;

[0068] S3: extracting aroma components from the enzymatic hydrolysate in S2 by a simultaneous distillation extraction method to prepare an aroma component extract.

[0069] Among them, in the above technical solution, the strain number of Oenococcus oeni in the China Industrial Microbiological Culture Collection Center is CICC 6066.

[0070] Among them, in the above technical solution, the method for culturing Oenococcus oeni in S1 includes the following steps:

[0071] Step 1: Inoculate Oenococcus oeni onto solid culture medium and culture at 25-30°C until a single colony grows;

[0072] Step 2: Inoculate a single colony into liquid culture medium and culture at 25-30°C for 48-72 hours to prepare seed solution;

[0073] Step 3: inoculate the liquid culture medium at an inoculum rate of 0.5-2% of the volume fraction of the seed solution;

[0074] Step 4: Cultivate at a temperature of 25-30°C until the OD value is 1.4-1.6 to prepare a bacterial suspension;

[0075] Step 5: Collect the bacteria, wash them with a NaCl solution with a mass fraction of 0.85-0.90%, and set aside.

[0076] Among them, the optimal OD value is 1.5; ATB medium is acidic tomato medium.

[0077] Among them, in the above technical solution, the method for preparing the cell lysis solution in S1 comprises the following steps:

[0078] Step 1: Suspend the bacteria in 1× PBS buffer;

[0079] Step 2: The above solution is ultrasonically disrupted in an ice bath to prepare a cell disruption solution.

[0080] The ultrasonic crushing conditions are as follows: ultrasonic power is 150-200w, and ultrasonic treatment time is 15-30min.

[0081] Among them, the optimal ultrasonic power is 180w, and the optimal ultrasonic treatment time is 25min.

[0082] Among them, in the above technical solution, the preparation method of the enzyme preparation in S1 is to suspend the solid in a NaCl solution with a mass fraction of 0.85-0.90% to prepare the enzyme preparation.

[0083] Among them, in the above technical solution, the specific steps of S2 include:

[0084] Step 1: crush the raw materials and pass through a 40-mesh sieve;

[0085] Step 2: Mix the raw material and the buffer at a mass volume ratio of 1 to (10 to 20) g / mL, and mix the enzyme preparation to the mixed solution at a volume ratio of 1 to (5 to 10);

[0086] The optimal mixing ratio of raw materials and buffer is 15g / mL, and the optimal mixing volume ratio of enzyme preparation and mixed solution is 5;

[0087] Step 3: Stirring the mixture under the conditions of an enzymatic hydrolysis temperature of 30 to 50° C. and an enzymatic hydrolysis pH of 4 to 6, so that the enzymatic hydrolysis time of the mixed solution is 50 to 150 minutes.

[0088] The optimal temperature for enzymatic hydrolysis is 40℃, the optimal pH value for enzymatic hydrolysis stirring is 5, and the optimal enzymatic hydrolysis time for the mixed solution is 80min.

[0089] Furthermore, in the above technical solution, the method of simultaneous distillation and extraction in S3 comprises the following steps:

[0090] Step 1: Add the enzymatic hydrolysate in S2 to zeolite and heat to boiling. Step 2: Take 1 / 3 to 1 / 5 of the volume of the enzymatic hydrolysate and heat the organic solvent to 40 to 60°C, and distill and extract for 2 to 4 hours.

[0091] Step 3: Add 3-5% of anhydrous sodium sulfate by weight of the organic phase to the organic solvent and let it stand for 1-2 hours to obtain an extract containing aroma components;

[0092] Step 4: Concentrate the extract at 40-50° C. to 1 / 2-1 / 10 of its volume, then add an equal volume of anhydrous ethanol and concentrate at 40-50° C. to 1 / 2-1 / 10 of its volume to obtain an aroma component extract.

[0093] like Figure 2-9 As shown, the second aspect of the present invention provides a first embodiment of the application of using Oenococcus oeni to extract aroma components of raw materials. In this embodiment, the above-mentioned Oenococcus oeni is used to extract aroma components of raw materials.

[0094] Furthermore, in the above technical solution, the raw material includes any one of grape peel and tea leaves.

[0095] Furthermore, in the above technical solution, the aroma components include linalool, ionone, benzyl alcohol, and 2-phenylethanol.

[0096] Experimental part

[0097] 1. Preparation of experimental materials:

[0098] 1. Oenococcus oeni CICC 6066, China Industrial Microbiology Culture Collection Management Center;

[0099] 2. Oenococcus oeni CICC 6057, China Industrial Microbiology Culture Collection Center;

[0100] 3. Acidic tomato (ATB) medium (glucose 10 g / L; peptone 10 g / L;

[0101] Yeast extract powder 5g / L; MgSO4·7H2O 2g / L; MnSO4·4H2O 0.05g / L;

[0102] Tomato extract powder 25 g / L; cysteine ​​hydrochloride 0.5 g / L), Shanghai Ruichu Biotechnology Co., Ltd.

[0103] 4. 1× PBS buffer (NaCl 140 mmol / L; KCl 2.7 mmol / L; Na2HPO4 10 mmol / L; KH2PO4 1.8 mmol / L; pH = 7.4), Thermo Fisher Scientific;

[0104] 5. Citrate buffer, Shanghai MacLean Biochemical Technology Co., Ltd.

[0105] 2. Experimental part:

[0106] 1. Sample preparation;

[0107] Example 1:

[0108] (1) Inoculate Oenococcus oeni CICC 6066 onto ATB solid culture medium plates and place them in a 25°C constant temperature incubator for static culture;

[0109] (2) After the strain has grown, a single colony is selected and transferred to ATB liquid medium. The culture temperature is 25°C and the culture temperature is 48 hours to prepare a seed solution. The seed solution is inoculated into ATB liquid medium at a volume fraction of 1% and the culture temperature is 25°C until the OD value is 1.5-1.6 to prepare a bacterial suspension.

[0110] (3) The bacterial suspension was centrifuged at 12,000 rpm for 5 min at 4°C to collect the cells, which were then washed with 0.85% NaCl solution and resuspended in the same volume of 1× PBS buffer as the bacterial suspension. The cells were then sonicated at 200 W for 30 min in an ice bath using an ultrasonic cell disruptor to obtain a cell disrupted solution.

[0111] (4) The cell disruption solution was centrifuged at 4°C, 12,000 rpm, for 5 min to collect the cell residues, i.e., the precipitate. The precipitate was suspended in a 0.85% NaCl solution with the same volume as the bacterial suspension to obtain the enzyme preparation.

[0112] β-glucosidase activity detection method:

[0113] Enzyme activity assay reaction system (3 mL): Mix 0.5 mL of treated sample (e.g., bacterial supernatant, cell lysate, etc.) with 0.5 mL of citrate phosphate buffer-pNPG solution (pH 5.0, pNPG concentration 5 mmol / L). Incubate in a 37°C water bath for 1 h. Immediately after completion of the reaction, add 2 mL of 1.0 mol / L Na2CO3 solution to terminate the reaction. Centrifuge the solution (4°C, 12,000 rpm, 5 min), and measure the absorbance of the supernatant at 400 nm.

[0114] One unit of β-glucosidase activity is defined as the amount of pNPG (4-nitrophenyl-β-D-pyranoglucopyranoside) produced per minute (μmol / (g·min)) per gram of bacterial cells under the above reaction conditions. Dry cell weight (g / L) = A600nm / 2.6168, where A600nm is the absorbance of the bacterial suspension at 600nm and 2.6168 is a constant coefficient.

[0115] Comparative Example 1:

[0116] (1) Inoculate Oenococcus oeni CICC 6057 onto ATB solid culture medium plates and place them in a 25°C constant temperature incubator for static culture;

[0117] (2) After the strain has grown, a single colony is selected and transferred to ATB liquid medium. The culture temperature is 25°C and the culture temperature is 48 hours to prepare a seed solution. The seed solution is inoculated into ATB liquid medium at a volume fraction of 1% and the culture temperature is 25°C until the OD value is 1.5-1.6 to prepare a bacterial suspension.

[0118] (3) The bacterial suspension was centrifuged at 12,000 rpm for 5 min at 4°C to collect the cells, which were then washed with 0.85% NaCl solution and resuspended in the same volume of 1× PBS buffer as the bacterial suspension. The cells were then sonicated at 200 W for 30 min in an ice bath using an ultrasonic cell disruptor to obtain a cell disrupted solution.

[0119] (4) The cell disruption solution was centrifuged at 4°C, 12,000 rpm, for 5 min to collect the cell residues, i.e., the precipitate. The precipitate was suspended in a 0.85% NaCl solution with the same volume as the bacterial suspension to obtain the enzyme preparation.

[0120] Example 2:

[0121] 1. A method for preparing a grape peel sample comprises the following steps:

[0122] (1) "Rose Fragrance" grapes are squeezed through airbags to obtain grape pomace and juice;

[0123] (2) taking fresh grape pomace and drying it naturally to remove the grape seeds to obtain dried grape peels;

[0124] (3) Crush the dried peel and pass it through a 40-mesh sieve for later use.

[0125] 2. The method for preparing tea samples comprises the following steps:

[0126] (1) crushing Tieguanyin tea leaves through a grinder;

[0127] (2) Pass through a 40-mesh sieve and store for later use.

[0128] Example 3:

[0129] (1) Weigh 10 g of the grape peel prepared in Example 2, add 200 mL of a citrate buffer solution with a pH value of 4.0, and preheat in a 30°C water bath for 5 min. Then, add 20 mL of the enzyme preparation prepared in Example 1, and place in a water bath with a magnetic stirrer for constant temperature enzymolysis. The enzymolysis time is 50 min, the enzymolysis temperature is 30°C, and the enzymolysis pH is 4, to obtain a sample solution after enzymolysis;

[0130] (2) Using the simultaneous distillation extraction method, the sample solution after enzymatic hydrolysis was placed on the light phase side, and 50 mL of dichloromethane was weighed and placed on the heavy phase side. Figure 5 , add zeolite to the light phase side and heat to boiling, keep the heavy phase side heated in a water bath at 60℃, and distill and extract for 2h;

[0131] (3) After completion, add 2.5 g of anhydrous sodium sulfate to the dichloromethane bottle and let it stand for 2 hours;

[0132] (4) The sample prepared in step (3) was concentrated to 5 mL by rotary evaporation in a water bath at 40°C under normal pressure, an equal volume of anhydrous ethanol was added, and the mixture was further concentrated to 1 mL by rotary evaporation in a water bath at 40°C under normal pressure to obtain an aroma component extract.

[0133] Example 4:

[0134] (1) Weigh 10 g of the grape peel prepared in Example 2, add 200 mL of a pH 4.0 citrate buffer solution, and preheat in a 30°C water bath for 5 min. Then, add 20 mL of the enzyme preparation prepared in Example 1, and place in a water bath with a magnetic stirrer for constant temperature enzymolysis. The enzymolysis time is 100 min, the enzymolysis temperature is 40°C, and the enzymolysis pH is 5, to obtain a sample solution after enzymolysis;

[0135] (2) Using the simultaneous distillation extraction method, the sample solution after enzymatic hydrolysis was placed on the light phase side, and 50 mL of dichloromethane was weighed and placed on the heavy phase side. Figure 5 , add zeolite to the light phase side and heat to boiling, keep the heavy phase side heated in a water bath at 60℃, and distill and extract for 2h;

[0136] (3) After completion, add 2.5 g of anhydrous sodium sulfate to the dichloromethane bottle and let it stand for 2 hours;

[0137] (4) The sample prepared in step (3) was concentrated to 5 mL by rotary evaporation in a water bath at 40°C under normal pressure, an equal volume of anhydrous ethanol was added, and the mixture was further concentrated to 1 mL by rotary evaporation in a water bath at 40°C under normal pressure to obtain an aroma component extract.

[0138] Embodiment 5:

[0139] (1) Weigh 10 g of the grape peel prepared in Example 2, add 200 mL of a citrate buffer solution with a pH value of 4.0, and preheat in a 30°C water bath for 5 min. Then, add 20 mL of the enzyme preparation prepared in Example 1, and place in a water bath with a magnetic stirrer for constant temperature enzymolysis. The enzymolysis time is 150 min, the enzymolysis temperature is 50°C, and the enzymolysis pH is 6, to obtain a sample solution after enzymolysis;

[0140] (2) Using the simultaneous distillation extraction method, the sample solution after enzymatic hydrolysis was placed on the light phase side, and 50 mL of dichloromethane was weighed and placed on the heavy phase side. Figure 5 , add zeolite to the light phase side and heat to boiling, keep the heavy phase side heated in a water bath at 60℃, and distill and extract for 2h;

[0141] (3) After completion, add 2.5 g of anhydrous sodium sulfate to the dichloromethane bottle and let it stand for 2 hours;

[0142] (4) The sample prepared in step (3) was concentrated to 5 mL by rotary evaporation in a water bath at 40°C under normal pressure, an equal volume of anhydrous ethanol was added, and the mixture was further concentrated to 1 mL by rotary evaporation in a water bath at 40°C under normal pressure to obtain an aroma component extract.

[0143] Comparative Example 2:

[0144] (1) Weigh 10 g of the grape peel prepared in Example 2, add 200 mL of a citrate buffer solution with a pH value of 4.0, and preheat in a 30°C water bath for 5 min. Then, add 20 mL of the enzyme preparation prepared in Comparative Example 1, and place in a water bath with a magnetic stirrer for constant temperature enzymolysis. The enzymolysis time is 100 min, the enzymolysis temperature is 40°C, and the enzymolysis pH is 5, to obtain a sample solution after enzymolysis;

[0145] (2) Using the simultaneous distillation extraction method, the sample solution after enzymatic hydrolysis was placed on the light phase side, and 50 mL of dichloromethane was weighed and placed on the heavy phase side. Figure 5 , add zeolite to the light phase side and heat to boiling, keep the heavy phase side heated in a water bath at 60℃, and distill and extract for 2h;

[0146] (3) After completion, add 2.5 g of anhydrous sodium sulfate to the dichloromethane bottle and let it stand for 2 hours;

[0147] (4) The sample prepared in step (3) was concentrated to 5 mL by rotary evaporation in a water bath at 40°C under normal pressure, an equal volume of anhydrous ethanol was added, and the mixture was further concentrated to 1 mL by rotary evaporation in a water bath at 40°C under normal pressure to obtain an aroma component extract.

[0148] Example 6:

[0149] (1) Weigh 10 g of tea leaves prepared in Example 2, add 200 mL of a pH 4.0 citrate buffer solution, and preheat in a 30°C water bath for 5 min. Then, add 20 mL of the enzyme preparation prepared in Example 1, and place in a water bath with a magnetic stirrer for constant temperature enzymolysis. The enzymolysis time is 100 min, the enzymolysis temperature is 40°C, and the enzymolysis pH is 5, to obtain a sample solution after enzymolysis.

[0150] (2) Using the simultaneous distillation extraction method, the sample solution after enzymatic hydrolysis was placed on the light phase side, and 50 mL of dichloromethane was weighed and placed on the heavy phase side. Figure 5 , add zeolite to the light phase side and heat to boiling, keep the heavy phase side heated in a water bath at 60℃, and distill and extract for 2h;

[0151] (3) After completion, add 2.5 g of anhydrous sodium sulfate to the dichloromethane bottle and let it stand for 2 hours;

[0152] (4) The sample prepared in step (3) was concentrated to 5 mL by rotary evaporation in a water bath at 40°C under normal pressure, an equal volume of anhydrous ethanol was added, and the mixture was further concentrated to 1 mL by rotary evaporation in a water bath at 40°C under normal pressure to obtain an aroma component extract.

[0153] Comparative Example 3:

[0154] (1) Weigh 10 g of tea leaves prepared in Example 2, add 200 mL of citrate buffer with a pH value of 4.0, and preheat in a 30°C water bath for 5 min. Then, add 20 mL of the enzyme preparation prepared in Comparative Example 1, and place in a water bath with a magnetic stirrer for constant temperature enzymolysis. The enzymolysis time is 100 min, the enzymolysis temperature is 40°C, and the enzymolysis pH is 5 to obtain a sample solution after enzymolysis.

[0155] (2) Using the simultaneous distillation extraction method, the sample solution after enzymatic hydrolysis was placed on the light phase side, and 50 mL of dichloromethane was weighed and placed on the heavy phase side. Figure 5 , add zeolite to the light phase side and heat to boiling, keep the heavy phase side heated in a water bath at 60℃, and distill and extract for 2h;

[0156] (3) After completion, add 2.5 g of anhydrous sodium sulfate to the dichloromethane bottle and let it stand for 2 hours;

[0157] (4) The sample prepared in step (3) was concentrated to 5 mL by rotary evaporation in a water bath at 40°C under normal pressure, an equal volume of anhydrous ethanol was added, and the mixture was further concentrated to 1 mL by rotary evaporation in a water bath at 40°C under normal pressure to obtain an aroma component extract.

[0158] Comparative Example 4:

[0159] (1) Weigh 10 g of the grape peel prepared in Example 2, add 200 mL of pH 4.0 citrate buffer, and preheat in a 30°C water bath for 5 minutes to prepare a sample solution;

[0160] (2) Using the simultaneous distillation extraction method, place the sample solution on the light phase side and weigh 50 mL of dichloromethane on the heavy phase side. Figure 5 , add zeolite to the light phase side and heat to boiling, keep the heavy phase side heated in a water bath at 60℃, and distill and extract for 2h;

[0161] (3) After completion, add 2.5 g of anhydrous sodium sulfate to the dichloromethane bottle and let it stand for 2 hours;

[0162] (4) The sample prepared in step (3) was concentrated to 5 mL by rotary evaporation in a water bath at 40°C under normal pressure, an equal volume of anhydrous ethanol was added, and the mixture was further concentrated to 1 mL by rotary evaporation in a water bath at 40°C under normal pressure to obtain an aroma component extract.

[0163] Comparative Example 5:

[0164] (1) Weigh 10 g of the tea leaves prepared in Example 2, add 200 mL of pH 4.0 citrate buffer, and preheat in a 30°C water bath for 5 min to prepare a sample solution;

[0165] (2) Using the simultaneous distillation extraction method, place the sample solution on the light phase side and weigh 50 mL of dichloromethane on the heavy phase side. Figure 5 , add zeolite to the light phase side and heat to boiling, keep the heavy phase side heated in a water bath at 60℃, and distill and extract for 2h;

[0166] (3) After completion, add 2.5 g of anhydrous sodium sulfate to the dichloromethane bottle and let it stand for 2 hours;

[0167] (4) The sample prepared in step (3) was concentrated to 5 mL by rotary evaporation in a water bath at 40°C under normal pressure, an equal volume of anhydrous ethanol was added, and the mixture was further concentrated to 1 mL by rotary evaporation in a water bath at 40°C under normal pressure to obtain an aroma component extract.

[0168] 2. Experimental Test

[0169] The aroma components prepared in Examples 3 to 5 and Comparative Example 2, Example 6 and Comparative Example 3, and Comparative Examples 4 to 5 were detected.

[0170] Detection method: Gas chromatography-mass spectrometry combined with internal standard method was used to analyze the composition and content of aroma compounds in the prepared samples. Figure 6 、 Figure 7 .

[0171] The specific effects are as follows:

[0172] The aroma components of grape peel after enzymatic hydrolysis in Example 3 are shown in Table 1. After enzymatic hydrolysis, a total of 35 aroma components were detected, with a total content of 105.37 μg / g (i.e., 105.37 μg of aroma components were extracted per gram of grape peel), including 12 terpenes, 5 norisoprenoids, 14 aliphatic compounds, and 4 aromatic compounds. The contents of 19 aroma components were increased to varying degrees. The difference in the content of each category of aroma components in the grape peels of Example 3 and Comparative Example 4 without enzymatic hydrolysis is shown in Table 1. Figure 8 The total amount of aroma components after enzymatic hydrolysis increased by 7.32% compared with that before enzymatic hydrolysis, among which the content of terpene compounds increased by 39.03% and the content of aromatic compounds increased by 35.27%.

[0173] The aroma components of grape peel after enzymatic hydrolysis in Example 4 are shown in Table 1. After enzymatic hydrolysis, a total of 37 aroma components were detected, with a total content of 129.3 μg / g (i.e., 129.3 μg of aroma components were extracted per gram of grape peel), including 13 terpenes, 5 norisoprenoids, 15 aliphatic compounds, and 4 aromatic compounds. The contents of 24 aroma components were increased to varying degrees. The difference in the content of each category of aroma components in the grape peel of Example 4 and Comparative Example 4 without enzymatic hydrolysis is shown in Table 1. Figure 8The total amount of aroma components after enzymatic hydrolysis increased by 31.70% compared with that before enzymatic hydrolysis, among which the content of terpene compounds increased by 85.02%; the content of norisoprene compounds increased by 26.61%; the content of dihydroactinol in aliphatic compounds increased significantly, which was 3.18 times that of unhydrolyzed compounds; the content of aromatic compounds increased by 88.30%.

[0174] The aroma components of grape peel after enzymatic hydrolysis in Example 5 are shown in Table 1. After enzymatic hydrolysis, a total of 36 aroma components were detected, with a total content of 115.07 μg / g (i.e., 115.07 μg of aroma components were extracted per gram of grape peel), including 13 terpenes, 4 norisoprenoids, 15 aliphatic compounds, and 4 aromatic compounds. The contents of 20 aroma components were increased to varying degrees. The difference in the content of each category of aroma components in the grape peels of Example 5 and Comparative Example 4 without enzymatic hydrolysis is shown in Table 1. Figure 8 The total amount of aroma components after enzymatic hydrolysis increased by 17.20% compared with that before enzymatic hydrolysis, among which the content of terpenes increased by 63.92% and the content of aromatic compounds increased by 61.82%.

[0175] Comparative Example 2 is different from Example 4 in that the strains used to prepare the enzyme preparation are different, and all other aspects are the same. The aroma components of the grape peel after enzymatic hydrolysis are shown in Table 1. A total of 36 aroma components were detected, but the aroma component content was significantly different from that of Example 4, at 97.07 μg / g (i.e., 97.07 μg of aroma components were extracted per gram of grape peel). Figure 8 As shown in the figure, there is no significant difference in the aroma component content of the sample after enzymatic hydrolysis using the β-glucosidase preparation of Oenococcus oeni CICC 6057 compared with that of the sample without enzymatic hydrolysis, indicating that the effect of extracting aroma components by the β-glucosidase of Oenococcus oeni CICC 6057 is worse than that of the β-glucosidase of Oenococcus oeni CICC 6066.

[0176] Table 1 Composition and content of aroma components in grape peel

[0177]

[0178]

[0179] Note: “—” means not detected.

[0180] The aroma components of tea leaves after enzymatic hydrolysis in Example 6 are shown in Table 2. After enzymatic hydrolysis, a total of 26 aroma components were detected, with a total content of 23.98 μg / g (i.e., 23.98 μg of aroma components were extracted per gram of tea leaves), including 11 terpenes, 3 norisoprenes, 8 aliphatic groups, and 4 aromatic groups. The contents of 19 aroma components were increased to varying degrees. The difference in the content of each category of aroma components in the tea leaves without enzymatic hydrolysis in Example 6 and Comparative Example 5 is shown in Table 2. Figure 9The total amount of aroma components after enzymatic hydrolysis increased by 46.58% compared with that before enzymatic hydrolysis, among which the content of terpene compounds increased by 48.51%; the content of norisoprene compounds increased by 87.83%; the content of aliphatic compounds increased by 74.29%; and the content of aromatic compounds increased by 23.55%.

[0181] Comparative Example 3 is compared with Example 6, except that the strains used to prepare the enzyme preparation are different, and all other aspects are the same. The aroma components of tea leaves after enzymatic hydrolysis are shown in Table 2. A total of 26 aroma components were detected, with a total content of 17.19 μg / g (i.e., 17.19 μg of aroma components were extracted per gram of tea leaves). Figure 9 As shown in the figure, the contents of various aroma components are basically the same as those before enzymatic hydrolysis, which is roughly the same as the enzymatic hydrolysis results of grape skin. Therefore, it is once again shown that the effect of extracting aroma components by β-glucosidase of Oenococcus oeni CICC 6057 is worse than that of β-glucosidase of Oenococcus oeni CICC 6066.

[0182] Table 2 Composition and content of aroma components in tea

[0183]

[0184]

[0185] Note: “—” means not detected.

[0186] The experimental results show that the enzyme preparation prepared by Oenococcus oeni CICC 6066 is effective in extracting aroma components, especially when the enzymatic hydrolysis temperature is 40℃ and the pH is 5.

[0187] Aroma components such as linalool, ionone, benzyl alcohol, and 2-phenylethanol, as key aroma compounds in grape peels and tea, contribute significantly to the overall aroma. The data in Tables 1 and 2 also indicate that the enzyme preparation prepared from Oenococcus oeni CICC 6066 is particularly effective in extracting linalool, ionone, benzyl alcohol, and 2-phenylethanol.

[0188] 3. Experimental Results

[0189] With reference to Example 1, we can draw the following conclusions:

[0190] During the preparation of enzyme preparation of Oenococcus oeni CICC 6066, the β-glucosidase activity in different parts was different. There were differences in the β-glucosidase activity in the unbroken part (cell supernatant, cell body) and the ultrasonically broken part (cell broken liquid, broken supernatant, cell residue). Figure 2 As shown, the cell residues have higher enzyme activity, so the cell residues are selected as enzyme preparations.

[0191] Plotting of pNP (p-nitrophenol) standard curve: Weigh 13.91 mg of p-nitrophenol and dilute to 100 mL with deionized water to obtain a pNP solution with a concentration of 1 mmol / L. Draw 20, 40, 60, 80, and 100 μL of the solution, dilute to 1 mL with deionized water. The corresponding pNP concentrations are 20, 40, 60, 80, and 100 μmol / L, respectively. Add 2 mL of 1.0 mol / L Na2CO3 solution and use deionized water plus Na2CO3 solution as a blank control. Measure the absorbance at 400 nm and plot a standard curve expressing the linear relationship between absorbance and pNP concentration. Figure 3 .

[0192] With reference to Example 1 and Comparative Example 1, it can be concluded that:

[0193] During the preparation of enzyme preparation of Oenococcus oeni CICC 6057, the β-glucosidase activity in different parts was different. There were differences in the β-glucosidase activity of the unbroken part (cell supernatant, cell body) and the ultrasonically broken part (cell broken liquid, broken supernatant, cell residue). Figure 4 As shown, the enzyme activity distribution is generally consistent with that of β-glucosidase from Oenococcus oeni CICC 6066, so the cell residues were also selected as the enzyme preparation.

[0194] Depend on Figure 2 and Figure 4 It can be seen that the β-glucosidase activity of the cell residues of Oenococcus oeni CICC 6066 is not much different from that of the cell residues of Oenococcus oeni CICC 6057.

[0195] With reference to Examples 1 to 6 and Comparative Examples 1 to 5, it can be concluded that:

[0196] The experimental results show that the enzyme preparation prepared by Oenococcus oeni CICC 6066 is effective in extracting aroma components, especially when the enzymatic hydrolysis temperature is 40℃ and the pH is 5.

[0197] Aroma components such as linalool, ionone, benzyl alcohol, and 2-phenylethanol, as key aroma compounds in grape peels and tea, contribute significantly to the overall aroma. The data in Tables 1 and 2 also indicate that the enzyme preparation prepared from Oenococcus oeni CICC 6066 is particularly effective in extracting linalool, ionone, benzyl alcohol, and 2-phenylethanol.

[0198] Strain Description

[0199] 1. Introduction to the strain

[0200] 1. Strain name: Oenococcus oeni

[0201] 2. Strain number: CICC 6066

[0202] 3. Biological hazard level: Category 4

[0203] 2. Storage conditions

[0204] Freeze-dried cultures and test tube slant surfaces at 2°C to 8°C. Vial cultures should be frozen at -18°C to -33°C.

[0205] 3. Culture Conditions

[0206] 1. Culture medium number: CM0196 Grape Juice Medium

[0207] 2. Medium ingredients: (1) Grape juice 500.0 ml, distilled water 500.0 ml, yeast extract 0.5 g, Tween 80 0.05-0.5 ml. [Note] Grape juice was prepared by pressing. Adjust the pH to 4.5 with NaOH and incubate at 1.05 kg / cm 2 Sterilize under pressure for 15 minutes. (2) Tryptone 20g, yeast extract 5g, peptone 5g, glucose 5g, Tween 80 0.05g, tomato juice 250ml, distilled water 750ml, agar 20g, pH 5.5. 1.05kg / cm 2 Sterilize for 15 minutes.

[0208] 3. Aerobic type: aerobic

[0209] 4. Cultivation temperature: 25

[0210] IV. Precautions

[0211] 1. Conventional culture time for strains: 1-2 days for bacteria, 3 days for yeast, 5-7 days for mold, and 7-10 days for large fungi.

[0212] 2. Please transfer the test tube slant culture as soon as possible. Long-term storage is not recommended.

[0213] 3. When using for the first time, please follow the recommended conditions in this manual for resurrection culture. CICC is not responsible for any loss of bacterial viability caused by using other types of culture media or culture conditions.

[0214] 4. Users should ensure the safe storage and operation of bacteria, and contaminated waste should be sterilized under high pressure before disposal.

[0215] 5. For other information related to the strain, please refer to the CICC website (www.china-cicc.org).

[0216] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A method for extracting aroma components from raw materials using Oenococcus oeni, characterized in that: The following steps are involved: S1: Cultivating Oenococcus oeni CICC 6066, preparing a cell lysate of the Oenococcus oeni, performing solid-liquid separation on the cell lysate to obtain a solid, and resuspending the solid to prepare an enzyme preparation; S2: crushing grape peels or tea leaves and mixing them with a buffer solution to prepare a mixed solution, and then adding the enzyme preparation in S1 to perform enzymolysis to prepare an enzymatic hydrolyzate; S3: extracting aroma components from the enzymatic hydrolysate in S2 by a simultaneous distillation extraction method to prepare an aroma component extract.

2. The method for extracting aroma components from raw materials using Oenococcus oeni according to claim 1, characterized in that: The method for culturing Oenococcus oeni described in S1 comprises the following steps: Step 1: Inoculate Oenococcus oeni onto solid culture medium and culture at 25-30°C until a single colony grows; Step 2: inoculating the single colony into a liquid culture medium and culturing at 25-30° C. for 48-72 hours to prepare a seed solution; Step 3: inoculating the liquid culture medium with an inoculum amount of 0.5-2% by volume of the seed solution; Step 4: Cultivate at 25-30°C until the OD value is 1.4-1.6 to prepare a bacterial suspension; Step 5: Collect the bacteria, wash them with 0.85~0.90% NaCl solution and set aside.

3. The method for extracting aroma components from raw materials using Oenococcus oeni according to claim 1, characterized in that: The method for preparing the cell lysis solution described in S1 comprises the following steps: Step 1: Suspend the bacteria in 1× PBS buffer; Step 2: Ultrasonic disruption of the above solution in an ice bath to prepare cell disruption solution; The ultrasonic disruption conditions are as follows: ultrasonic power of 150-200 W and ultrasonic treatment time of 15-30 min.

4. The method for extracting aroma components from raw materials using Oenococcus oeni according to claim 1, characterized in that: The preparation method of the enzyme preparation described in S1 is to suspend the solid in a NaCl solution with a mass fraction of 0.85~0.90% to prepare the enzyme preparation.

5. The method for extracting aroma components from raw materials using Oenococcus oeni according to claim 1, characterized in that: The specific steps of S2 include: Step 1: crush the raw materials and pass through a 40-mesh sieve; Step 2: Mix the raw material and the buffer at a mass volume ratio of 1-(10-20) g / mL, and the enzyme preparation and the mixed solution at a volume ratio of 1-(5-10); Step 3: Stir the mixture under the conditions of enzymatic hydrolysis temperature of 30~50℃ and enzymatic hydrolysis pH of 4~6, so that the enzymatic hydrolysis time of the mixed solution is 50~150 min.

6. The method for extracting aroma components from raw materials using Oenococcus oeni according to claim 5, characterized in that: The simultaneous distillation and extraction method in S3 comprises the following steps: Step 1: adding the enzymatic hydrolysate in S2 to zeolite and heating to boiling; Step 2: taking an organic solvent of 1 / 3 to 1 / 5 of the volume of the enzymatic hydrolysate, heating it to 40 to 60°C, and simultaneously distilling and extracting for 2 to 4 hours; Step 3: Add 3-5% of the weight of the organic phase to the organic solvent with anhydrous sodium sulfate and let it stand for 1-2 hours to obtain an extract containing aroma components; Step 4: Concentrate the extract at 40-50°C to 1 / 2-1 / 10 of its volume, then add an equal volume of anhydrous ethanol and concentrate at 40-50°C to 1 / 2-1 / 10 of its volume to obtain an aroma component extract.

7. An application of extracting aroma components of raw materials using Oenococcus oeni, characterized in that: The use of Oenococcus oeni in claim 2 in extracting aroma components of raw materials.

8. The use of Oenococcus oeni for extracting aroma components from raw materials according to claim 7, characterized in that: The raw material includes any one of grape peel and tea leaves.

9. The use of Oenococcus oeni for extracting aroma components from raw materials according to claim 7, characterized in that: The aroma components include linalool, ionone, benzyl alcohol and 2-phenylethanol.

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