Preparation method of oil-tea camellia seed oil with nut aroma

Abstract

The application discloses a preparation method of oil-tea camellia seed oil with nut aroma, and comprises the following steps: 1) pretreating oil-tea camellia seed shells and oil-tea camellia fruits to obtain a fermentation substrate; 2) preparing a fermentation medium containing the oil-tea camellia seed shells and the oil-tea camellia fruits; 3) yeast fermentation: inoculating yeast into the fermentation medium to perform fermentation, the fermentation time is 36-42 hours, and a fermentation product is obtained; 4) enzymatic fermentation: adding glucose oxidase into the fermentation product to perform enzymatic hydrolysis and deglucose treatment, the enzymatic hydrolysis and deglucose treatment is accompanied by continuous fermentation of the yeast, the enzymatic hydrolysis and deglucose treatment time is 24-30 hours, and a fermentation enzymolysis liquid is obtained; and 5) cold pressing: drying and steaming the fermentation enzymolysis liquid and then performing cold pressing to obtain the oil-tea camellia seed oil. The method of first adopting yeast fermentation and then adopting glucose oxidase enzymolysis makes the content of tetramethylpyrazine in the prepared oil-tea camellia seed oil reach 2120 ug / mL, gives the oil-tea camellia seed oil very strong nut aroma, and improves the flavor quality of the oil-tea camellia seed oil.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of camellia seed oil flavoring preparation, and particularly relates to a preparation method of camellia seed oil with nut aroma. BACKGROUND

[0002] Camellia seed is the seed of a camellia oleifera abel plant, and the camellia seed oil extracted from the camellia seed has a similar fatty acid composition to olive oil. In particular, the camellia seed oil prepared by a hot pressing method has the characteristics of rich aroma and high nutritional value, and is favored by consumers in China.

[0003] Key flavor substances in the camellia seed oil are aldehydes, esters, heterocyclic compounds, phenols and terpenes, etc. Aldehydes have a relatively high content and are the main volatile flavor components, which endow the camellia seed oil with unique green, fruity and floral aroma. Esters endow the camellia seed oil with sweet and honey aroma. Pyrazines, a kind of heterocyclic compound, are the main flavor substances of the camellia seed oil, which provide the camellia seed oil with a unique aroma of baking and nuts, which is relatively rare in the camellia seed oil.

[0004] At present, the main flavoring methods for the camellia seed oil are frying and baking processes. The camellia seed is fried or baked, and is treated by adjusting moisture and the like, and the pressing process conditions are controlled. The cold degumming process is used for the pressed crude oil, and the product with rich aroma is obtained after winterization. The principle of the method is that Strecker degradation, peroxidation of fatty acids and Maillard reaction of oil at high temperature can lead to the generation of pyrazines. The process can greatly contribute to the aroma components of the camellia seed oil, but the high temperature in the process can cause adverse effects on the color, acid value, peroxide value and content of benzopyrene of the camellia seed oil.

[0005] In order to avoid the high temperature conditions of frying and baking on the oil tea seed oil, the microbial fermentation method gradually becomes the research focus. For example, Wu Qian Di, Wu Mianhua, Shen Yi, Wang Xi, Luo Aimin published the paper "Research on the mixed fermentation of Bacillus, yeast and Rhizopus quorum to improve the content of tetramethylpyrazine" in "Brewing Science and Technology", which discloses that the fermentation conditions are: 1-2d, 30℃, 90%RH; 3-4d, 37℃, 95%RH; 5-6d, 45℃, 95%RH; 7d, 55℃, 95%RH; 8d, 65℃, 95%RH. Through Bacillus pure strain fermentation (A); inoculating yeast and Bacillus at the same time (B); inoculating yeast first, then inoculating Bacillus (C); inoculating yeast and Rhizopus quorum first, then inoculating Bacillus fermentation (D), the contents of tetramethylpyrazine, acetoin and ammonia, the change of reducing sugar and acidity after fermentation are measured. The results show that the content of tetramethylpyrazine in D group is the highest, which is 881.5ug / g, the content of tetramethylpyrazine in C group is lower than that in D group, which is 493.9ug / g, the contents of tetramethylpyrazine in A group and B group are lower than that in C group, which are 293.6ug / g and 150.1ug / g respectively. This study lays a foundation for improving the content of tetramethylpyrazine in liquor through multi-strain synergistic fermentation.

[0006] Although the mixed fermentation of Bacillus, yeast and Rhizopus quorum can improve the content of tetramethylpyrazine to a certain extent in the research, there are still the following problems:

[0007] 1) The fermentation time of the process is 8d, the fermentation time is long, and the application to the oil tea seed oil flavoring process makes the preparation period of the oil tea seed oil long and the preparation efficiency low;

[0008] 2) In the fermentation process, the pyrazine precursor acetoin will gradually accumulate with the extension of the fermentation time, and the high content of acetoin in the fermentation liquid in the middle and late stages of fermentation has an adverse effect on the activity and survival rate of the bacteria, which seriously reduces the production rate of pyrazine substances, resulting in low content of tetramethylpyrazine prepared, and when it is added to the oil tea seed oil, the oil tea seed oil has weak nutty aroma and not strong nutty flavor. SUMMARY

[0009] The technical problem to be solved by the present application is to overcome the shortcomings of the prior art and provide a preparation method of oil tea seed oil with simple process, short fermentation time and strong nutty aroma.

[0010] The technical scheme adopted by the present application to solve the technical problem is:

[0011] A method for preparing oil-tea camellia seed oil with nut aroma, comprising the following steps: 1) pretreating oil-tea camellia seed shell and oil-tea camellia fruit to obtain fermentation substrate; 2) preparing fermentation medium containing oil-tea camellia seed shell and oil-tea camellia fruit; 3) yeast fermentation: inoculating yeast into the fermentation medium for fermentation, the fermentation time being 36-42 h to obtain fermentation product; 4) enzymatic fermentation: adding glucose oxidase into the fermentation product for enzymatic desugaring treatment, the enzymatic desugaring treatment being accompanied by continuous fermentation of the yeast, and the enzymatic desugaring treatment time being 24-30 h to obtain fermentation enzymatic hydrolysate; and 5) cold pressing: drying, steaming and then cold pressing the fermentation enzymatic hydrolysate to obtain oil-tea camellia seed oil.

[0012] Further, the specific operation steps of the step 1) are as follows: placing the oil-tea camellia seed shell and the oil-tea camellia fruit in an oven with a temperature of 35-50 ℃ for drying for 3-5 h, taking out, mixing the dried oil-tea camellia seed shell and the oil-tea camellia fruit at a shell content of 6-8%, and crushing with a crusher to 80-120 mesh to obtain the fermentation substrate, which is placed in a dryer for standby.

[0013] Further, the specific operation steps of the step 2) are as follows: stirring uniformly 100 g of the fermentation substrate, 1 g of KH2PO4, 0.5 g of MgSO4·7H2O and 1 g of (NH4)2SO4, and sterilizing at 121 ℃ for 35 min.

[0014] Further, the specific operation steps of the step 3) are as follows: first preparing yeast bacterial suspension with a concentration of 1×10 8 CFU / mL, then inoculating the yeast bacterial suspension into the fermentation medium at an inoculation amount of 0.5-1%, and then placing it at 35-40 ℃ for fermentation for 36-42 h to obtain the fermentation product.

[0015] Preferably, the fermentation time is 36 h.

[0016] Further, the specific operation steps of the step 4) are as follows: diluting the fermentation product with water at a material-liquid ratio of 1:1, adjusting the system pH to 5.5 with 20% sodium citrate buffer solution, then adding glucose oxidase for enzymatic desugaring treatment, the addition amount of the glucose oxidase being 0.5-1% of the weight of the fermentation product, the enzymatic desugaring treatment being accompanied by continuous fermentation of the yeast, and the enzymatic desugaring treatment time being 30 h to obtain the fermentation enzymatic hydrolysate.

[0017] Further, the enzyme activity of the glucose oxidase is 231 U / mg.

[0018] Further, the fermentation enzymatic hydrolysate is placed at 60 ℃ for drying until the moisture content is 8-10%, then passed through a 2 mm sieve, then steamed to obtain tea seed cake, and then pressed to obtain oil-tea camellia seed oil with nut aroma.

[0019] Further, the temperature of the steaming is 90 DEG C, the time of the steaming is 30 min, the temperature of the pressing is 65 DEG C, and the time of the pressing is 23 min.

[0020] The preparation method of the oil tea seed oil with nut aroma has the following beneficial effects:

[0021] (1) The process of the present application is simple, and the method of using yeast fermentation and then using glucose oxidase enzymolysis and accompanying yeast fermentation can effectively improve the content of tetramethylpyrazine in the oil tea seed oil under the synergistic action of yeast and glucose oxidase, so that the content of tetramethylpyrazine in the prepared oil tea seed oil reaches 2120 ug / mL, and the oil tea seed oil is endowed with very rich nut aroma, and the flavor quality of the oil tea seed oil is improved.

[0022] (2) The whole fermentation process of the present application for preparing the oil tea seed oil is only 66-72 h, which effectively shortens the fermentation time during the microbial fermentation for increasing aroma, improves the fermentation efficiency, and thus greatly shortens the preparation period of the oil tea seed oil and effectively improves the preparation efficiency of the oil tea seed oil.

[0023] (3) The present application is green, efficient, safe and reliable, and the oil tea seed oil is synergistically flavored by microbial fermentation and enzymolysis treatment, and no chemical reagent harmful to human body is used in the whole preparation process, so that the environment and production are not polluted, and the present application does not use high temperature, so that the content of benzopyrene in the oil tea seed oil can be effectively reduced, and the prepared oil tea seed oil meets the standard of national standard GB / T11765-2018. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Fig. 1 is a process schematic diagram of the preparation method of the oil tea seed oil with nut aroma of the present application.

[0025] Figure 2 Fig. 3 is a trend graph of the change of the cell viability with time in the yeast fermentation process.

[0026] Figure 3 Fig. 5 is a trend graph of the change of the survival rate of the bacteria with time in the yeast fermentation process.

[0027] Figure 4 Fig. 7 is a trend graph of the change of the synthesis rate of tetramethylpyrazine with time in the fermentation liquid in the yeast fermentation process.

[0028] Figure 5 Fig. 9 is a comparison graph of the change of the cell viability with time in the yeast fermentation and the yeast fermentation combined with glucose oxidase desugarization.

[0029] Figure 6 Fig. 11 is a comparison graph of the change of the survival rate of the bacteria with time in the yeast fermentation and the yeast fermentation combined with glucose oxidase desugarization.

[0030] Figure 7 Figure 1 is a comparison chart of the synthesis rate of tetramethylpyrazine over time for the fermentation of yeast alone and the combined glucose oxidase deglucose treatment of yeast. DETAILED DESCRIPTION

[0031] The present application is further described below in conjunction with the accompanying drawings and examples, but these specific embodiments do not limit the scope of protection of the present application in any way.

[0032] The yeast used in the embodiments of the present application can be routinely purchased, specifically active dry yeast, and purchased from Angel Yeast Co., Ltd.

[0033] Example 1

[0034] A preparation method of oil tea seed oil with nut aroma, the process flow chart is as shown in Figure 1 The preparation method comprises the following steps:

[0035] 1) Pretreatment of oil tea seed shell and oil tea fruit: the oil tea seed shell and oil tea fruit are placed in an oven at a temperature of 40℃ and dried for 4h, then after being taken out, the dried oil tea seed shell and oil tea fruit are mixed at a shell content of 7%, and are crushed to 100 mesh by a crusher to obtain a fermentation substrate, which is placed in a dryer for standby;

[0036] 2) Preparation of fermentation medium containing oil tea seed shell and oil tea fruit: 100g of the fermentation substrate, 1g of KH2PO4, 0.5g of MgSO4·7H2O, and 1g of (NH4)2SO4 are stirred uniformly and then sterilized at 121℃ for 35min;

[0037] 3) Yeast fermentation: a yeast bacterial suspension with a concentration of 1.0×10 8 CFU / mL is inoculated into the fermentation medium at an inoculation amount of 0.7%, and then is placed at 35℃ for fermentation for 36h to obtain a fermentation product;

[0038] The preparation steps of the yeast bacterial suspension with a concentration of 1.0×10 8 CFU / mL are as follows:

[0039] a) The yeast (specifically, active dry yeast) stored at 4℃ is inoculated on YPD solid medium (20.0g of glucose, 20.0g of peptone, 10.0g of yeast extract powder, 20.0g of agar, 1000mL of tap water, pH is adjusted to 5.8, and sterilized by high-pressure steam at 121℃ for 20min) under sterile operation, and is incubated at 30℃ for 36-48h;

[0040] b) picking single colony inoculated in 100 mL YPD liquid medium (glucose 20.0 g, peptone 20.0 g, yeast extract 10.0 g, tap water 1000 mL, pH adjusted to 5.8, 121 ℃ high pressure steam sterilization 20 min) for full activation, continuous passage for 3 times, and inoculated in 200 mL YPD liquid medium at a proportion of 2%, 30 ℃ for 20 h, diluted with sterile normal saline, and adjusted to 1.0 x 10 8 CFU / mL;

[0041] 4) Enzymatic hydrolysis fermentation: after dilution with water at a ratio of 1:1, the pH of the fermentation product was adjusted to 5.5 with 20% sodium citrate buffer solution, then glucose oxidase with an enzyme activity of 231 U / mg was added for enzymatic hydrolysis and degsugar treatment, the addition amount of glucose oxidase was 0.8% of the weight of the fermentation product, and the enzymatic hydrolysis and degsugar treatment was accompanied by the continuous fermentation of yeast, and the enzymatic hydrolysis and degsugar treatment time was 30 h, to obtain a fermentation enzymatic hydrolysis liquid;

[0042] 5) Cold pressing: the fermentation enzymatic hydrolysis liquid was dried at 60 ℃ to a moisture content of 8% to 10%, passed through a 2 mm sieve, then steamed at a temperature of 90 ℃ and a pressure of 4 MPa for 30 min, pressed into a tea seed cake, and then pressed at a temperature of 65 ℃ for 23 min, filtered, to obtain oil tea seed oil with nutty aroma; the finished oil tea seed oil was subjected to edible oil national standard detection, and the results were: acid value 0.5 mg / g, peroxide value 0.074 g / 100 g, moisture and volatile matter content 0.03%, insoluble impurities 0.02%, all meeting the standards of "GB / T 11765-2018 Oil Tea Seed Oil".

[0043] Example 2

[0044] The difference between this example and Example 1 is that the fermentation time in step 3) is 42 h, and the finished oil tea seed oil is subjected to edible oil national standard detection, and the results are: acid value 0.48 mg / g, peroxide value 0.069 g / 100 g, moisture and volatile matter content 0.02%, insoluble impurities 0.02%, all meeting the standards of "GB / T 11765-2018 Oil Tea Seed Oil" for first grade oil tea seed oil by aqueous enzymatic method.

[0045] Example 3

[0046] The difference between this example and Example 1 is that the enzymatic hydrolysis and degsugar treatment time in step 4) is 24 h, and the finished oil tea seed oil is subjected to edible oil national standard detection, and the results are: acid value 0.58 mg / g, peroxide value 0.078 g / 100 g, moisture and volatile matter content 0.03%, insoluble impurities 0.02%, all meeting the standards of "GB / T 11765-2018 Oil Tea Seed Oil" for first grade oil tea seed oil by aqueous enzymatic method.

[0047] Comparative Example 1

[0048] A preparation method of oil-tea camellia seed oil, comprising the following steps: placing oil-tea camellia seed shells and oil-tea camellia fruits in an oven with a temperature of 40℃ for drying for 4h, then mixing the dried oil-tea camellia seed shells and oil-tea camellia fruits with a shell content of 7%, crushing the mixture by using a crusher and then passing the mixture through a 2mm sieve, then steaming the mixture at a temperature of 90℃ and a pressure of 4MPa for 30min, pressing the mixture into a tea seed cake, then pressing the tea seed cake at a temperature of 65℃ for 23min, and filtering to obtain the oil-tea camellia seed oil.

[0049] Comparative Example 2

[0050] A preparation method of oil-tea camellia seed oil, comprising the following steps: mixing oil-tea camellia seed shells and oil-tea camellia fruits with a shell content of 7%, then placing the mixture in a drum seed roaster for frying at 150-180℃ until the mixture turns yellow, then placing the fried mixture of the oil-tea camellia seed shells and the oil-tea camellia fruits in a presser for pressing at 150℃, and filtering to obtain the oil-tea camellia seed oil.

[0051] Comparative Example 3

[0052] The difference between the present comparative example and Example 1 lies in that, in step 1), the oil-tea camellia fruits are directly placed in an oven with a temperature of 40℃ for drying for 4h, and then the dried oil-tea camellia fruits are crushed by using a crusher to 100 mesh to obtain a fermentation substrate, which is placed in a dryer for standby use. The other steps are the same as those in Example 1.

[0053] Comparative Example 4

[0054] The difference between the present comparative example and Example 1 lies in that, no glucose oxidase is added for treatment, and the specific operation steps are as follows.

[0055] 1) Oil-tea camellia seed shell and oil-tea camellia fruit pretreatment: placing oil-tea camellia seed shells and oil-tea camellia fruits in an oven with a temperature of 40℃ for drying for 4h, then mixing the dried oil-tea camellia seed shells and oil-tea camellia fruits with a shell content of 7%, crushing the mixture by using a crusher to 100 mesh to obtain a fermentation substrate, and placing the fermentation substrate in a dryer for standby use;

[0056] 2) Preparation of fermentation medium containing oil-tea camellia seed shells and oil-tea camellia fruits: stirring uniformly 100g of the fermentation substrate, 1g of KH2PO4, 0.5g of MgSO4·7H2O and 1g of (NH4)2SO4, and then sterilizing at 121℃ for 35min;

[0057] 3) Yeast fermentation: first preparing a yeast bacterial suspension with a concentration of 1×10 8 CFU / mL, then inoculating the yeast bacterial suspension into the fermentation medium with an inoculation amount of 0.7%, and then placing the mixture in a 35℃ environment for fermentation for 66h to obtain a fermentation product;

[0058] 4) Cold pressing: the fermentation product is dried at 60°C to a moisture content of 8% to 10%, passed through a 2 mm sieve, then steamed at a temperature of 90°C and a pressure of 4 MPa for 30 min, pressed into a tea seed cake, then pressed at a temperature of 65°C for 23 min, filtered, and the oil tea seed oil is obtained.

[0059] Comparative Example 5

[0060] The difference between this comparative example and Example 1 is that no yeast fermentation treatment is used, and the specific operation steps are as follows:

[0061] A method for preparing oil tea seed oil with nut aroma, comprising the following steps:

[0062] 1) Pretreatment of oil tea seed shells and oil tea fruits: the oil tea seed shells and oil tea fruits are dried in an oven at a temperature of 40°C for 4 h, then mixed after being taken out, and ground to 100 mesh using a grinder to obtain an enzymatic substrate, which is placed in a dryer for use;

[0063] 2) Enzymatic hydrolysis: the enzymatic substrate is diluted with water at a ratio of 1:1, then the pH of the system is adjusted to 5.5 using a 20% sodium citrate buffer solution, then glucose oxidase with an enzyme activity of 231 U / mg is added for enzymatic hydrolysis and deglucosylation treatment, the addition amount of the glucose oxidase is 0.8% of the weight of the fermentation substrate, and the enzymatic hydrolysis and deglucosylation treatment time is 30 h, to obtain a fermentation enzymatic hydrolysate;

[0064] 3) Cold pressing: the fermentation enzymatic hydrolysate is dried at 60°C to a moisture content of 8% to 10%, passed through a 2 mm sieve, then steamed at a temperature of 90°C and a pressure of 4 MPa for 30 min, pressed into a tea seed cake, then pressed at a temperature of 65°C for 23 min, filtered, and the oil tea seed oil is obtained.

[0065] Comparative Example 6

[0066] The difference between this comparative example and Example 1 is that Bacillus subtilis is used for fermentation in step 3), and Bacillus subtilis is used as the enzyme hydrolysis and fermentation strain in step 4). The specific operation steps of step 3) are as follows:

[0067] First, a Bacillus subtilis bacterial suspension with a concentration of 1 x 10 8 CFU / mL is prepared, then the Bacillus subtilis bacterial suspension is inoculated into the fermentation medium at an inoculation amount of 0.7%, then the fermentation is carried out at 35°C for 36 h, and the fermentation product is obtained;

[0068] wherein the concentration of the Bacillus subtilis bacterial suspension is 1 x 10 8The preparation steps of the Bacillus subtilis bacterial suspension with CFU / mL are as follows: a) Bacillus subtilis activation: inoculate Bacillus subtilis into a slant medium (10.00 g of proteose peptone, 3.00 g of yeast extract powder, 5.00 g of NaCl, 3.00 g of glucose, 20 g of agar powder, 1000 mL of tap water, pH adjusted to 7, sterilized at 121 ℃ for 20 min), and cultivate at 32 ℃ for 12 h;

[0069] b) pick a single colony and inoculate into 100 mL of LB liquid medium (10.00 g of proteose peptone, 3.0 g of yeast extract powder, 5.00 g of NaCl, 1000 mL of tap water, pH adjusted to 6.2-6.4, sterilized at 121 ℃ for 20 min), fully activate, continuously subculture for 3 times, inoculate into LB liquid medium at a proportion of 2%, expand culture at 37 ℃ for 6 h, dilute with sterile normal saline, and adjust the bacterial liquid concentration to 1.0 x 10 8 CFU / mL.

[0070] Comparative Example 7

[0071] The difference between this comparative example and Example 1 is that step 3) is mixed fermentation of yeast and Bacillus subtilis, and the strain for enzyme hydrolysis fermentation in step 4) is yeast and Bacillus subtilis. The specific operation steps of step 3) are as follows:

[0072] First, prepare a yeast bacterial suspension with a concentration of 1 x 10 8 CFU / mL / mL, and a Bacillus subtilis bacterial suspension with a concentration of 1 x 10 8 CFU / mL / mL (the specific preparation method is the same as that of the corresponding bacterial suspension in Example 1 and Comparative Example 5), then mix the yeast bacterial suspension and the Bacillus subtilis bacterial suspension at a volume ratio of 1:1, inoculate the mixed bacterial suspension into the fermentation medium at an inoculation amount of 0.7%, and then ferment at 35 ℃ for 36 h to obtain a fermentation product.

[0073] Comparative Example 8

[0074] The difference between this comparative example and Example 1 is that step 4) is water washing to remove sugar, and the specific operation steps are as follows:

[0075] First, filter the fermentation product, collect the filtrate and the residue; then add water to the residue at a solid-liquid ratio of 1:2 and wash for 2 times, filter after each washing; after the washing is completed, combine all the filtrate and the residue, then let the combined filtrate stand to separate into layers, take the upper layer oil sample and add it to the combined residue to obtain a mixed liquid, then dry the mixed liquid at 60 ℃ until the moisture content is 8%-10%, pass through a 2 mm sieve, then steam at a temperature of 90 ℃ and a pressure of 4 MPa for 30 min, press into a tea seed cake, then press at a temperature of 65 ℃ for 23 min, filter, and obtain camellia seed oil.

[0076] The content of tetramethylpyrazine and the content of benzopyrene in the oil-tea camellia seed oil prepared in Examples 1 and 2 and Comparative Examples 1-8 were determined, and the nut flavor of the oil-tea camellia seed oil was sensory evaluated. The strength of the nut flavor can be divided into five grades, from weak to strong, in turn: none, relatively strong, strong, very strong. The results are shown in Table 1.

[0077] The determination method of the content of tetramethylpyrazine is as follows:

[0078] Sample preparation: weigh 10 g of sample, add 0.1 g of anhydrous CaCl2, 30 mL of 60% ethanol, and ultrasonically treat at 25°C for 20 min, and centrifuge at 8000 r / min for 10 min. Take the supernatant through a 0.22 μm filter membrane, and measure.

[0079] Chromatographic conditions: Agilent 8860 type high performance liquid chromatograph; Agilent Poroshell 120 EC-C18 chromatographic column (4.6 mm x 150 mm, 4 μm); mobile phase is methanol: ultrapure water (add 0.8% sodium acetate) = 42:58, column temperature is 30°C, detection wavelength is 278 nm, flow rate is 1.0 mL / min, injection volume is 5 μL.

[0080] Preparation of standard curve of tetramethylpyrazine: dissolve tetramethylpyrazine standard substance in the mobile phase to make the solution concentration 5 mg / mL. Then dilute with the mobile phase to 5 concentration gradients (200 μg / mL, 500 μg / mL, 1000 μg / mL, 1500 μg / mL, 4000 μg / mL).

[0081] Tetramethylpyrazine standard curve: y = 19453.32x - 1.245, R 2 = 0.9995, detection limit: 0.07 μg / mL.

[0082] Benzopyrene refers to GB 5009.27-2016.

[0083] Table 1 Content of tetramethylpyrazine and benzopyrene in oil-tea camellia seed oil prepared in Examples 1-2 and Comparative Examples 1-8 and sensory evaluation results of nut flavor

[0084]

[0085]

[0086] As can be seen from Table 1, the oil-tea camellia seed oil prepared by Comparative Example 1 and Comparative Example 2 respectively adopts cold-pressing and hot-pressing method, and no yeast fermentation and glucose oxidase enzymolysis desugaring treatment are adopted in the preparation process, and the oil-tea camellia seed oil prepared has no nut aroma, and the content of tetramethylpyrazine is only 205 ug / mL and 218 ug / mL respectively; while the oil-tea camellia seed oil prepared by Example 1 and Example 2 of the present application adopts yeast fermentation and glucose oxidase enzymolysis desugaring treatment has very strong nut aroma, and the content of tetramethylpyrazine reaches 2120 ug / mL and 2154 ug / mL respectively.

[0087] Compared with Comparative Example 1 and Comparative Example 2, the method of yeast fermentation combined with glucose oxidase desugaring treatment adopted in the present application can significantly increase the content of tetramethylpyrazine in the oil-tea camellia seed oil, and the content of tetramethylpyrazine in the oil-tea camellia seed oil can impart very strong nut aroma to the oil-tea camellia seed oil, thereby making the prepared oil-tea camellia seed oil have very strong nut aroma; and the content of benzopyrene in the oil-tea camellia seed oil prepared by Example 1 and 2 is significantly lower than that of Comparative Example 1 and 2, which also indicates that the method of yeast fermentation combined with glucose oxidase desugaring treatment can also effectively reduce the formation of benzopyrene in the preparation process of the oil-tea camellia seed oil.

[0088] Compared with Example 1, Comparative Example 3 does not add oil-tea camellia seed shell in the preparation process of the oil-tea camellia seed oil, and the content of tetramethylpyrazine in the oil-tea camellia seed oil prepared is also significantly lower than that of Example 1, but significantly higher than that of Comparative Example 1-2 and Comparative Example 4-8, which indicates that using oil-tea camellia seed shell and oil-tea camellia fruit as fermentation raw materials together can effectively increase the content of tetramethylpyrazine in the oil-tea camellia seed oil, so that the oil-tea camellia seed oil has strong nut aroma, and the reason is that the oil-tea camellia seed shell can also provide part of the carbon source for subsequent yeast fermentation, so that the yeast fermentation is more complete and sufficient, thereby increasing the content of tetramethylpyrazine.

[0089] The content of tetramethylpyrazine in the oil-tea camellia seed oil prepared by not adding glucose oxidase in the process of preparing the oil-tea camellia seed oil in Comparative Example 4 is not very different from that in Comparative Example 1, but the content of tetramethylpyrazine is significantly lower than that in Example 1, and the method of combining yeast fermentation and glucose oxidase enzymolysis can significantly increase the content of tetramethylpyrazine by 86.9% compared with the method of only using yeast fermentation, and can impart very strong nut aroma to the oil-tea camellia seed oil. The reason is that: during the whole period of yeast fermentation, there are two ways of generation and degradation of acetoin (i.e. generation of tetramethylpyrazine), and the generation amount of acetoin is greater than the degradation amount during 0-36h of fermentation, and the accumulation amount of acetoin in the fermentation broth increases continuously, so the content of acetoin in the fermentation broth is higher and higher, when the fermentation is carried out to 36h, the concentration of acetoin reaches the concentration that has a slight toxic effect on the growth of yeast, and with the continuous extension of the fermentation time to 48h, the degree of the toxic effect will be more and more serious, which will cause the survival rate of yeast to decrease more obviously, and will greatly reduce the synthesis rate of tetramethylpyrazine, so that the content of tetramethylpyrazine increases very slowly during the subsequent fermentation period;

[0090] Therefore, by adding glucose oxidase for enzymolysis and desugaring treatment after 36h of fermentation, the sugar content will gradually decrease during the subsequent enzymolysis and desugaring treatment, and the yeast is inhibited to a certain extent during the subsequent fermentation, so that the generation amount of acetoin is reduced, and due to the existence of the reaction of acetoin converting into tetramethylpyrazine, the reaction will gradually consume the content of accumulated acetoin in the fermentation broth, which can effectively reduce the accumulation amount of acetoin and reduce the toxic effect of acetoin on the yeast, and also can increase the synthesis rate of tetramethylpyrazine continuously, prolong the time for the synthesis rate of tetramethylpyrazine to reach the maximum value, and thus can effectively increase the content of tetramethylpyrazine, so that the content of tetramethylpyrazine in the oil-tea camellia seed oil prepared in Example 1 is significantly higher than that in Comparative Example 4.

[0091] The content of tetramethylpyrazine in the oil-tea camellia seed oil prepared by not adding yeast fermentation in the process of preparing the oil-tea camellia seed oil in Comparative Example 5 is not very different from that in Comparative Example 1, but the content of tetramethylpyrazine is significantly lower than that in Example 1, and the method of combining yeast fermentation and glucose oxidase enzymolysis can significantly increase the content of tetramethylpyrazine by 85% compared with the method of only using glucose oxidase enzymolysis, and can impart very strong nut aroma to the oil-tea camellia seed oil. The reason is that: only during the fermentation period of yeast, a large amount of acetoin can be generated, and then a large amount of acetoin can be converted into tetramethylpyrazine, and only by adding glucose oxidase, a large amount of acetoin cannot be generated, and then a large amount of acetoin cannot be converted into tetramethylpyrazine, so that the content of tetramethylpyrazine in the oil-tea camellia seed oil prepared is very low, and there is no significant difference between Comparative Example 1.

[0092] The content of tetramethylpyrazine in the oil-tea camellia seed oil prepared by the fermentation of Bacillus subtilis in the preparation process of the oil-tea camellia seed oil is 910 ug / mL, and the content of tetramethylpyrazine is significantly lower than that in Example 1, resulting in that the degree of richness of nut aroma of the oil-tea camellia seed oil is weaker than that in Example 1, and the oil-tea camellia seed oil does not have very rich nut aroma, indicating that the yeast fermentation combined with glucose oxidase enzymolysis desugaring treatment can produce more tetramethylpyrazine than the Bacillus subtilis fermentation combined with glucose oxidase enzymolysis desugaring treatment, and has a better effect of increasing the rich nut aroma of the oil-tea camellia seed oil.

[0093] The content of tetramethylpyrazine in the oil-tea camellia seed oil prepared by the fermentation of Bacillus subtilis in the preparation process of the oil-tea camellia seed oil is 910 ug / mL, and the content of tetramethylpyrazine is significantly lower than that in Example 1, resulting in that the degree of richness of nut aroma of the oil-tea camellia seed oil is weaker than that in Example 1, and the oil-tea camellia seed oil does not have very rich nut aroma, indicating that the yeast fermentation combined with glucose oxidase enzymolysis desugaring treatment can produce more tetramethylpyrazine than the Bacillus subtilis fermentation combined with glucose oxidase enzymolysis desugaring treatment, and has a better effect of increasing the rich nut aroma of the oil-tea camellia seed oil.

[0094] The content of tetramethylpyrazine in the oil-tea camellia seed oil prepared by the fermentation of Bacillus subtilis in the preparation process of the oil-tea camellia seed oil is 910 ug / mL, and the content of tetramethylpyrazine is significantly lower than that in Example 1, resulting in that the degree of richness of nut aroma of the oil-tea camellia seed oil is weaker than that in Example 1, and the oil-tea camellia seed oil does not have very rich nut aroma, indicating that the yeast fermentation combined with glucose oxidase enzymolysis desugaring treatment can produce more tetramethylpyrazine than the Bacillus subtilis fermentation combined with glucose oxidase enzymolysis desugaring treatment, and has a better effect of increasing the rich nut aroma of the oil-tea camellia seed oil.

[0095] In summary, in the process of preparing the oil-tea camellia seed oil with very rich nut flavor, any one of the lack of fermentation or desugaring treatment or the replacement of the fermentation strain and the desugaring method will affect the content of tetramethylpyrazine, and further affect the degree of richness of nut aroma of the oil-tea camellia seed oil. The yeast fermentation and the glucose oxidase enzymolysis desugaring treatment in the present application interact with each other and jointly affect the content of tetramethylpyrazine. Only when the two treatment methods are combined, the content of tetramethylpyrazine can be effectively increased, and the prepared oil-tea camellia seed oil is given very rich nut flavor.

[0096] Experimental Example 1: Study on the changes of the contents of acetoin and tetramethylpyrazine in the fermentation broth, cell viability and cell survival rate during the yeast fermentation process

[0097] Fermentation medium and yeast suspension were prepared according to steps 1-3 of Example 1. The yeast suspension was then inoculated into the fermentation medium at an inoculum size of 0.7%, and fermented at 35°C. The content of acetoin and tetramethylpyrazine, cell viability, and cell survival rate in the fermentation broth were measured every 6 hours. The increments of acetoin and tetramethylpyrazine every 6 hours, as well as the synthesis rate of tetramethylpyrazine at 12 hours and subsequent time points, were calculated. The corresponding increments of acetoin and tetramethylpyrazine content are shown in Table 2. Cell viability, cell survival rate, and the synthesis rate of tetramethylpyrazine are shown in Table 2. Figures 2-4 As shown.

[0098] The method for determining the content of acetoin is as follows:

[0099] Sample preparation: Weigh 10g of sample, add 0.1g of anhydrous CaCl2 and 30mL of 60% ethanol, sonicate at 25℃ for 20min, and centrifuge at 8000r / min for 10min. Take the supernatant and filter it through a 0.22μm filter membrane for analysis.

[0100] Chromatographic conditions: Agilent 8860 high-performance liquid chromatograph; Agilent Poroshell 120 EC-C18 column (4.6 mm × 150 mm, 4 μm). Mobile phase: methanol: ultrapure water (with 0.05% trifluoroacetic acid) = 3:7; column temperature: 35℃; detection wavelength: 280 nm; flow rate: 1.0 mL / min; injection volume: 5 μL.

[0101] Plotting the standard curve for acetoin: Dissolve the acetoin standard in the mobile phase to a concentration of 20 mg / mL. Dilute with the mobile phase to five concentration gradients (1.0 mg / mL, 2.0 mg / mL, 4 mg / mL, 8 mg / mL, 10 mg / mL).

[0102] Standard curve for β-chromosome marriage: y = 178.216x - 2.354, R 2 =0.9999, detection limit: 7.21 μg / mL.

[0103] The cell viability assay was performed as follows: 1 mL of fermentation broth was thoroughly mixed with 9 mL of sterile water and incubated at 28°C for 12 h. 1 mL of the culture was then inoculated into 1 L of YPD liquid medium and activated at 28°C for 24–48 h. The OD was adjusted with sterile physiological saline. 600The value (absorbance of yeast bacterial solution at 600 nm) was added to a 96-well culture plate (100 μL was added to each well). Then 20 μL / well of MTT (0.5 g of tetramethyl azo salt was accurately weighed, and distilled water was added to 100 mL, and it was stored in a refrigerator at 4°C in the dark for standby use.) was added, and it was placed in a 37°C incubator for 4 h. After the incubation, the supernatant in each culture well was carefully aspirated, and then 100 μL of dimethyl sulfoxide was added to each well and shaken for 10 min. The absorbance A of each well was determined on an enzyme marker. 490 (wavelength 490 nm).

[0104] The total number of colonies was determined according to GB 4789.2-2016, and the survival rate of the bacteria was determined according to the method in Ouyang Dewen's "Development and Efficacy Evaluation of Immobilized Fermented Brown Rice Wine".

[0105] Table 2 Change in content of acetoin and tetramethylpyrazine in yeast fermentation broth with fermentation time

[0106]

[0107] As can be seen from Table 2, with the extension of fermentation time, the content of acetoin in the fermentation broth generally showed an increasing trend and then tended to be flat; the increase in acetoin content increased slowly after 6 h of fermentation (6-30 h), then increased significantly (30-36 h) to 8.8 mg / mL, then increased slowly (36-48 h) and remained almost unchanged (48-66 h). When the fermentation was 66 h-72 h, the increase in acetoin content decreased significantly from 11 mg / mL to 0.7 mg / mL, and then tended to be flat. This may be because in the late fermentation, the energy in the fermentation substrate was almost exhausted, resulting in a decrease in the activity and survival rate of the yeast;

[0108] The content of tetramethylpyrazine generally showed an increasing trend and then tended to be unchanged; the increase in tetramethylpyrazine content increased significantly during 0-6 h of fermentation. This may be because the energy in the fermentation substrate was very sufficient in the early fermentation, and the content of acetoin in the fermentation broth did not have a toxic effect on the cells, and the survival rate of the bacteria was also relatively high, resulting in a relatively large content of converted tetramethylpyrazine. During 6-12 h of fermentation, the increase in tetramethylpyrazine content decreased significantly. The reason may be that the content of acetoin accumulated in the fermentation broth was increasing, and the yeast was not adapted to the content of acetoin at the beginning, resulting in a significant decrease in the increase in tetramethylpyrazine. With the gradual adaptation of the yeast to the low content of acetoin, the increase in tetramethylpyrazine gradually increased during 12 h-30 h, and then the increasing trend became more significant (30-36 h). After that, the increase in tetramethylpyrazine remained almost unchanged (36-48 h), then rapidly decreased (48-54) and then tended to be flat (54-78);

[0109] Depend on Figures 2-4 It can be seen that cell viability and strain survival rate both showed a decreasing trend with the extension of fermentation time. The synthesis rate of tetramethylpyrazine showed a trend of first increasing and then decreasing. Moreover, at 36 h of fermentation, there was a sharp inflection point in both the cell viability and strain survival rate curves. The synthesis rate of tetramethylpyrazine remained almost unchanged after 36 h. Combined with the analysis of Table 2, it can be seen that because the increase in acetoin was significantly increased from 30 to 36 h of fermentation, the yeast had a greater tolerance to the low content of acetoin in the fermentation broth during this period, which also increased the content of acetoin in the fermentation broth. The synthesis rate of tetramethylpyrazine was not affected. However, when fermentation reached 36 h, the content of acetoin in the fermentation broth had a weak toxic effect on the yeast, causing cell viability and strain survival rate to show a significant inflection point from this moment.

[0110] Because the yeast is only slightly toxic after 36 hours of fermentation, with cell viability and strain survival rate close to 0.8 and 80% respectively, the synthesis rate of tetramethylpyrazine remains almost unchanged between 38 and 48 hours. However, after 48 hours of fermentation, the corresponding cell viability drops to 0.68 and the strain survival rate drops to 52.33%. At this point, the acetoin content in the fermentation broth has seriously affected the survival of the yeast, causing severe toxicity and reducing its survival rate by nearly 50%. Consequently, the synthesis rate of tetramethylpyrazine reaches its maximum at 48 hours and then drops significantly, resulting in a tetramethylpyrazine content of only 289.14 ug / mL in the fermentation broth after fermentation.

[0111] Therefore, it was considered to add glucose oxidase to remove sugar at 36 hours of fermentation, so as to reduce the content of acetoin in the fermentation broth (i.e., the accumulation of acetoin), thereby reducing the toxic effect of high acetoin content on yeast and prolonging the time for the synthesis rate of tetramethylpyrazine to reach its maximum value.

[0112] Experimental Example 2 investigated the effect of yeast fermentation time in step 3) of the present invention on the tetramethylpyrazine content in camellia seed oil. Camellia seed oil was prepared according to steps 1-5 of Example 1. The effect of different yeast fermentation times (30h, 36h, 42h, 48h and 50h) in step 3) on the tetramethylpyrazine content in camellia seed oil was studied. The results are shown in Table 3.

[0113] Table 3. Effect of yeast fermentation time on tetramethylpyrazine content in camellia seed oil.

[0114]

[0115]

[0116] As shown in the above table, in the yeast fermentation process of step 3) of the present application, when the fermentation time is 30-54h, the content of tetramethylpyrazine in the prepared oil-tea camellia seed oil generally presents a trend of first significantly increasing and then significantly decreasing, and when the fermentation time is 36-42h, the content of tetramethylpyrazine in the corresponding oil-tea camellia seed oil is all higher than 2100ug / mL, and has very strong nut aroma, therefore, preferably, the fermentation time in the yeast fermentation step is 36-42h, under the premise of ensuring the content of tetramethylpyrazine, in order to effectively shorten the fermentation time, the most preferred fermentation time is 36h.

[0117] Experimental Example 3: Study on the content of acetoin and tetramethylpyrazine in the fermentation enzyme solution and the changes of cell viability and cell survival rate in the process of adding glucose oxidase for enzymolysis deglucosylation after yeast fermentation for 36h

[0118] The fermentation product was prepared by referring to the methods of steps 1-3 of Example 1, water was added to dilute the fermentation product at a material-liquid ratio of 1:1, then a 20% sodium citrate buffer solution was used to adjust the pH of the system to 5.5, and then glucose oxidase with an enzyme activity of 231U / mg was added for enzymolysis deglucosylation treatment, and the addition amount of glucose oxidase was 0.8% of the weight of the fermentation product, and the enzymolysis deglucosylation treatment was accompanied by the continuous fermentation of yeast, and a fermentation enzyme solution was obtained, the content of acetoin and tetramethylpyrazine in the fermentation enzyme solution, the cell viability and the cell survival rate were measured every 6h, the increments of acetoin and tetramethylpyrazine every 6h were calculated, and the synthesis rate of tetramethylpyrazine at 12h and subsequent time points was calculated. And the fermentation enzyme solutions with total fermentation times (total fermentation time = yeast fermentation time before adding glucose oxidase + enzymolysis deglucosylation treatment time after adding glucose oxidase) of 42h, 48h, 54h, 60h, 66h, 72h and 78h were collected, the oil-tea camellia seed oil was prepared according to the method of step 5 of Example 1, the content of tetramethylpyrazine in the oil-tea camellia seed oil was measured, and the nut flavor was evaluated. The content of acetoin and tetramethylpyrazine and the corresponding increment results are shown in Table 4, and the cell viability, cell survival rate and synthesis rate of tetramethylpyrazine are shown in Table 5. Figures 5-7

[0119] Table 4: Changes of the content of acetoin and tetramethylpyrazine in the fermentation enzyme solution and the content of tetramethylpyrazine in the oil-tea camellia seed oil with fermentation time

[0120]

[0121]

[0122] ​As shown in Table 4, after the glucose oxidase treatment at 36 h of fermentation, the increment of acetoin content is negative when the total fermentation time is 36-42 h, and the content and increment of tetramethylpyrazine are significantly increased; when the total fermentation time is 42-60 h, the content and increment of tetramethylpyrazine are significantly increased, and when the fermentation is to 60-66 h, the content of tetramethylpyrazine is slightly increased and then almost unchanged. At each fermentation time point, the content of tetramethylpyrazine in the prepared oil tea seed oil is not significantly different from that in the fermentation enzyme solution, indicating that the treatment of step 5) of the present application does not cause the loss of tetramethylpyrazine; and the content of tetramethyl in the oil tea seed oil corresponding to the total fermentation time of 60-78 h is 2000-2200 ug / mL, which can give the oil tea seed oil a very rich nutty aroma.

[0123] As shown in Table 4, after the glucose oxidase treatment at 36 h of fermentation, the increment of acetoin content is negative when the total fermentation time is 36-42 h, and the content and increment of tetramethylpyrazine are significantly increased; when the total fermentation time is 42-60 h, the content and increment of tetramethylpyrazine are significantly increased, and when the fermentation is to 60-66 h, the content of tetramethylpyrazine is slightly increased and then almost unchanged. At each fermentation time point, the content of tetramethylpyrazine in the prepared oil tea seed oil is not significantly different from that in the fermentation enzyme solution, indicating that the treatment of step 5) of the present application does not cause the loss of tetramethylpyrazine; and the content of tetramethyl in the oil tea seed oil corresponding to the total fermentation time of 60-78 h is 2000-2200 ug / mL, which can give the oil tea seed oil a very rich nutty aroma. Figures 5-7 As shown in Table 4, after the glucose oxidase treatment at 36 h of fermentation, the increment of acetoin content is negative when the total fermentation time is 36-42 h, and the content and increment of tetramethylpyrazine are significantly increased; when the total fermentation time is 42-60 h, the content and increment of tetramethylpyrazine are significantly increased, and when the fermentation is to 60-66 h, the content of tetramethylpyrazine is slightly increased and then almost unchanged. At each fermentation time point, the content of tetramethylpyrazine in the prepared oil tea seed oil is not significantly different from that in the fermentation enzyme solution, indicating that the treatment of step 5) of the present application does not cause the loss of tetramethylpyrazine; and the content of tetramethyl in the oil tea seed oil corresponding to the total fermentation time of 60-78 h is 2000-2200 ug / mL, which can give the oil tea seed oil a very rich nutty aroma.

[0124] In addition, when the total fermentation time is 60 h, the synthesis rate of tetramethylpyrazine reaches the maximum value (100.83 g / mL*h), while the time when the synthesis rate of tetramethylpyrazine reaches the maximum value (5.12 g / mL*h) without the addition of glucose oxidase is 48 h, indicating that the addition of glucose oxidase not only can significantly improve the maximum value of the synthesis rate of tetramethylpyrazine, but also can prolong the time when the synthesis rate of tetramethylpyrazine reaches the maximum value from 48 h to 60 h, so that the time when the synthesis rate of tetramethylpyrazine reaches the maximum value can be postponed by 12 h, and in this period of time, the content of tetramethylpyrazine can be effectively increased, thereby the content of tetramethylpyrazine can be significantly improved. And with the continuous extension of the total fermentation time (60-78), the content of tetramethylpyrazine does not change significantly, and the synthesis rate thereof is close to 0, which may be that in the late fermentation, the energy in the fermentation substrate has been depleted, the survival rate and cell viability of the strain are close to 0, and the energy available for the production of acetoin and tetramethylpyrazine by the yeast is greatly limited, so that the contents of tetramethylpyrazine and acetoin tend to be flat.

[0125] In summary, the total fermentation time is selected to be 60-66 h, that is, the yeast fermentation is first carried out for 36 h, and then the glucose oxidase is added to enzymatically hydrolyze the sugar accompanied by the fermentation of the yeast for 30-36 h to prepare the oil tea seed oil with very strong nut aroma. In order to obtain a larger content of tetramethylpyrazine and maintain a high preparation efficiency of the oil tea seed oil, the yeast fermentation is first carried out for 36 h, and then the glucose oxidase is added to enzymatically hydrolyze the sugar accompanied by the fermentation of the yeast for 30 h to prepare the oil tea seed oil with very strong nut aroma.

[0126] Experimental Example 4 Study on the effect of yeast inoculum on the content of tetramethylpyrazine in oil tea seed oil and the change of nut aroma

[0127] The oil tea seed oil was prepared by the method of Reference Example 1, and the effects of different yeast inocula (0.1%, 0.3%, 0.5%, 0.7%, 1.0%, 1.2%) in step 3) on the content of tetramethylpyrazine in the oil tea seed oil and the nut aroma thereof were investigated, and the results are shown in Table 5:

[0128] Table 5 Effects of different yeast inocula on the content of tetramethylpyrazine in the oil tea seed oil and the nut aroma

[0129] Yeast inoculum (%) Tetramethylpyrazine content in oil-tea camellia seed oil (ug / mL) Nutty aroma 0.1 876 Lighter 0.3 921 Lighter 0.5 1621 Strong 0.7 2120 Very strong 1.0 1987 Strong 1.2 1486 Lighter

[0130] As shown in Table 5, with the increase of yeast inoculation amount, the content of tetramethylpyrazine in camellia seed oil showed a trend of first increasing (0.1-0.7%) and then decreasing (0.7-1.2%), and the intensity of the nutty aroma also showed a trend of first increasing (0.1-0.7%) and then decreasing (0.7-1.2%). Specifically, when the yeast inoculation amount was 0.5%-1%, the camellia seed oil had a strong nutty flavor, and the content of tetramethylpyrazine was highest at 0.7%, giving the camellia seed oil a very strong nutty flavor. However, when the yeast inoculation amount continued to increase to 1.2%, the camellia seed oil only had a relatively strong nutty aroma. This may be because when the yeast inoculation amount was too high, the content of other byproducts also increased, increasing the consumption of substrates. The proportion of substrates for the yeast to produce tetramethylpyrazine was relatively reduced, thus reducing its content and the intensity of the nutty aroma. Therefore, in order to make the prepared camellia seed oil have a rich or even very rich nutty aroma, the optimal yeast inoculation amount is selected to be 0.5% to 1.0%, with the optimal inoculation amount being 0.7%.

[0131] Experimental Example 5 investigated the effects of glucose oxidase addition on the tetramethylpyrazine content and nut aroma sensory properties of camellia seed oil.

[0132] Camellia seed oil was prepared according to the method in Example 1. The effects of different amounts of glucose oxidase added in step 4) (0.2%, 0.5%, 0.8%, 1.0%, 1.5%) on the tetramethylpyrazine content and sensory properties of its nutty aroma in camellia seed oil were investigated. The results are shown in Table 6.

[0133] Table 6. Effects of different glucose oxidase addition amounts on tetramethylpyrazine content and nut aroma in camellia seed oil.

[0134]

[0135] As shown in Table 6, with the increase of glucose oxidase addition, the content of tetramethylpyrazine in camellia seed oil first increases (0.2-0.8%) and then decreases (0.8-1.5%), and the intensity of the nutty aroma also shows a trend of first increasing (0.2-0.8%) and then decreasing (0.8-1.5%). Specifically, when the glucose oxidase addition is 0.5%-1%, the camellia seed oil has a rich nutty aroma, and when the glucose oxidase addition is 0.8%, the corresponding tetramethylpyrazine content is the highest, at 2120 ug / mL, giving the camellia seed oil a very rich nutty aroma. However, when the glucose oxidase addition continues to increase to 1.5%, the camellia seed oil only has a relatively rich nutty aroma. Therefore, based on the tetramethylpyrazine content and nutty aroma intensity in Table 6, the optimal addition amount of glucose oxidase is 0.8%, with a range of 0.5%–1.0%.

[0136] Experimental Example 6 Study on the Influence of Tetramethylpyrazine Content in Oil-tea Camellia Seed Oil on Nutty Aroma Sensory

[0137] According to GB / T16291.1-2012 "Sensory Analysis - Selection, Training and Monitoring of Assessors - Part 1: Guidelines for the Selection of Assessors", an oil-tea camellia seed oil nutty aroma sensory evaluation group of 10 people was established.

[0138] The oil sample to be evaluated was equally placed in a tasting cup, and the oil sample temperature was (28±2)℃. Each tasting cup should be marked with a random code composed of numbers, letters or a combination of numbers and letters, and the marker pen or label was odorless material.

[0139] Odor evaluation: Before evaluation, the oil-tea camellia seed oil container was swirled and then the lid was opened in front of the nose, and the odor was directly inhaled into the back nasal cavity to feel. The analysis technique of odor was used to detect the nutty aroma of oil-tea camellia seed oil, and identify the nutty aroma in the oil-tea camellia seed oil sample.

[0140] In the evaluation, the concentration of tetramethylpyrazine in the oil-tea camellia seed oil was evaluated from low to high to avoid causing "olfactory paralysis" or "fatigue" of the evaluators. The concentration was 0-600ug / mL, 600-1500ug / mL, 1500-2000ug / mL, 2000-2500ug / mL, and above 2500ug / mL. The nasal inhalation time should not exceed 30s, if no conclusion was determined within 30s, 3min rest was needed before evaluation, and after the evaluation was completed, the lid was immediately covered, and the evaluation form was filled out. According to the degree of aroma concentration, scoring was carried out, wherein 0-2 represented no aroma, 3-5 represented relatively rich aroma, 4-6 represented rich aroma, 6-8 represented very rich aroma, and 8-10 represented too rich aroma producing a greasy taste.

[0141] According to the evaluation results obtained by the evaluation group, the original data was summarized and collected by Excel, and the data was statistically analyzed by IBM SPSS Statistics 23.0. The following table was obtained

[0142] Table 7 Relationship between tetramethylpyrazine content in oil-tea camellia seed oil and nutty aroma concentration

[0143] Tetramethylpyrazine concentration (ug / mL) Degree of nutty aroma 0-600 None 600-1500 Lighter 1500-2000 Strong 2000-2500 Very strong Above 2500 Too strong to be pleasant

[0144] According to the results in Table 7, the relationship between the tetramethylpyrazine content and the degree of nutty aroma was obtained, which indicated that the higher the tetramethylpyrazine content, the richer the nutty aroma, and when the tetramethylpyrazine content exceeded 2500ug / mL, the oil-tea camellia seed oil produced a greasy taste, causing an adverse flavor and affecting the flavor quality of the oil-tea camellia seed oil. This experimental example can provide a sensory evaluation standard for the oil-tea camellia seed oil flavoring process prepared by the present application.

[0145] In the description of the specification, the description of the terms "embodiment", "specific embodiment", "example" or "specific example" and the like means that the specific features, materials, steps or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the application. In the specification, the illustrative description of the above terms is not necessarily directed to the same embodiment or example. Moreover, the specific features, materials, steps or characteristics described can be combined in any suitable manner in any one or more embodiments or examples. In addition, those skilled in the art can combine and combine the different embodiments or examples described in the specification and the features of the different embodiments or examples without contradiction.

[0146] The above is only the preferred embodiment of the present application, not any form of limitation on the present application, although the present application has been disclosed as above with the preferred embodiment, however, not to limit the present application, any skilled in the art, without departing from the scope of the technical scheme of the present application, can make some changes or modifications of the above disclosed technical content for equivalent embodiments with equivalent changes, but as long as it does not deviate from the technical scheme content of the present application, any simple modification, equivalent change and modification of the above embodiment according to the technical essence of the present application, still belongs to the scope of the technical scheme of the present application.

Claims

1. A method for preparing oil-tea camellia seed oil having a nutty aroma, characterized in that, The method comprises the following steps: 1) Camellia oleifera Abel seed shell, Camellia oleifera Abel fruit is placed in the oven at a temperature of 35℃-50℃ drying 3h-5h, after taking out, according to the shell rate is 6%-8% Camellia oleifera Abel seed shell and Camellia oleifera Abel fruit mixed with a pulverizer to 80 mesh-120 mesh, get fermentation substrate, placed in a dryer for standby;2) preparation of Camellia oleifera Abel seed shell and Camellia oleifera Abel fruit fermentation medium;3) yeast fermentation: first prepared concentration of 1×10 8 CFU / mL of yeast bacteria suspension, then the yeast bacteria suspension with inoculation amount of 0.5%-1% inoculation to fermentation medium, then placed in 35℃-40℃ under fermentation 36-42h, get fermentation product;4) enzymatic fermentation: to the fermentation product added glucose oxidase enzymatic deglucose treatment, enzymatic deglucose treatment also accompanied by the continued fermentation of yeast bacteria, and enzymatic deglucose treatment time is 24-30h, get fermentation enzymatic hydrolysis liquid;5) cold pressing: fermentation enzymatic hydrolysis liquid drying, steaming after cold pressing, get Camellia oleifera Abel seed oil.

2. The method for preparing camellia seed oil with a nutty aroma as described in claim 1, characterized in that: The specific operation steps of the step 2) are as follows: the fermentation substrate 100 g, KH2PO4 1 g, MgSO4·7H2O 0.5 g, (NH4)2SO4 1 g are stirred uniformly and then sterilized at 121 DEG C for 35 min.

3. The preparation method of the oil tea seed oil with nut aroma according to claim 1, characterized in that: The fermentation time in the step 3) is 36 h.

4. The preparation method of the oil tea seed oil with nut aroma according to claim 1, characterized in that: The specific operation steps of the step 4) are as follows: the fermentation product is diluted with water at a material-liquid ratio of 1:1, then a 20% sodium citrate buffer solution is added to adjust the system pH to 5.5, then glucose oxidase is added for enzymatic hydrolysis and desugaring treatment, the addition amount of the glucose oxidase is 0.5-1% of the weight of the fermentation product, the enzymatic hydrolysis and desugaring treatment is accompanied by the continuous fermentation of the yeast, and the enzymatic hydrolysis and desugaring treatment time is 30 h, to obtain a fermentation enzyme hydrolysis liquid.

5. The preparation method of the oil tea seed oil with nut aroma according to claim 4, characterized in that: The enzyme activity of the glucose oxidase is 231 U / mg.

6. The process as claimed in claim 1, wherein the oil tea seed oil having nutty aroma is prepared by the process comprising of: The specific operation steps of the step 5) are as follows: the fermentation enzyme hydrolysis liquid is placed at 60 DEG C for drying until the moisture content is 8%-10%, then the fermentation enzyme hydrolysis liquid is passed through a 2 mm sieve, then steaming is performed to obtain tea seed cakes, and then cold pressing is performed to obtain the oil tea seed oil with nut aroma.

7. The process as claimed in claim 1, wherein the oil tea seed oil having nutty aroma is prepared by the process comprising of: The steaming temperature is 90 DEG C, the steaming time is 30 min, the cold pressing temperature is 65 DEG C, and the cold pressing time is 23 min.

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