Method for preparing oil-tea camellia seed oil by alkaline protease and endogenous protease in cooperation

Abstract

The application discloses a method for preparing oil-tea camellia seed oil by alkaline protease and endogenous protease synergistic demulsification, which comprises the following steps: 1) collecting emulsion; 2) extracting endogenous protease; 3) alkaline protease and endogenous protease synergistic demulsification: adding distilled water and alkaline protease into the emulsion for primary enzymolysis, the adding amount of the alkaline protease is 0.5-2% of the weight of the emulsion, the primary enzymolysis time is T1, and 60min<=T1<75min, adding endogenous protease according to 0.6-1.4% of the weight of the emulsion for secondary enzymolysis, the secondary enzymolysis time is T2, and 15min<=T2<=35min, the total time of the primary enzymolysis and the secondary enzymolysis is less than 100min, and oil emulsion is obtained; 4) enzyme inactivation and centrifugation, and oil-tea camellia seed oil is obtained. The alkaline protease and the endogenous protease are used for synergistic demulsification, which can not only improve the demulsification rate and the extraction rate of the oil-tea camellia seed oil, but also inhibit the generation of bitter amino acids, so that the prepared oil-tea camellia seed oil does not contain bitterness, and the flavor and quality of the oil-tea camellia seed oil are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of edible oil processing, and particularly relates to a method for preparing oil-tea camellia seed oil by alkaline protease and endogenous protease in cooperation. BACKGROUND

[0002] Oil-tea camellia is a unique woody edible oil tree species in China, and has a history of cultivation and utilization of more than 2000 years. It is called as one of the four woody oil plants in the world together with olive, oil palm and coconut, and is called as one of the four woody oil plants in China together with Chinese tallow tree, tung tree and walnut. The oil content of oil-tea camellia seed is between 20% and 35%, and the oil-tea camellia seed oil is a woody plant edible oil with high nutritional value and certain health care function. The oil-tea camellia seed oil is easy to be absorbed by human body, and is good for preventing heart disease, blood vessel sclerosis and treating hypertension, and is praised as “Oriental olive oil”.

[0003] At present, common extraction methods of the oil-tea camellia seed oil include organic solvent extraction, physical pressing and aqueous enzymatic extraction. Among them, the aqueous enzymatic extraction is a process of adding enzymes for destroying the structure of plant cell wall or oil body in water as an extraction medium to extract oil in cells, and then separating the oil from the protein and carbohydrate according to the difference in solubility of oil, water, protein and carbohydrate. The aqueous enzymatic extraction has the advantages of effectively recovering the protein (or its hydrolysis product) and carbohydrate in raw materials and removing toxic or anti-nutritional factors in some oil materials during the extraction of oil. However, a large amount of emulsion is produced in the process of the aqueous enzymatic extraction, and the extracted oil enters the emulsion, which makes it difficult to separate the free oil, so that the oil extraction rate is not high, the economic benefit is reduced, and the oil needs to be recovered after proper demulsification.

[0004] A common demulsification method is to add alkaline protease to the emulsion. For example, patent application No. CN201510589650.9 discloses a demulsification treatment method for extracting tea seed oil by aqueous enzymatic extraction. The method adopts a centrifugal method to collect the emulsion produced in the process of extracting tea seed oil by aqueous enzymatic extraction, and uses alkaline protease to destroy the stability of the emulsion to obtain clear oil, so as to improve the oil extraction rate of tea seed oil and improve the economic benefit. By selecting appropriate reaction conditions, the demulsification rate can reach 92.1% by using the method, which solves the technical problem of demulsification for the industrial application of tea seed.

[0005] The above method uses alkaline protease to demulsify the emulsion extracted by aqueous enzymatic extraction, which can improve the demulsification rate to a certain extent. However, it is found in actual application that the demulsification with alkaline protease produces bitterness in the demulsification process, so that the oil-tea camellia seed oil prepared contains bitterness, which not only reduces the taste of the finished product, but also reduces the extraction rate of the oil-tea camellia seed oil prepared when the demulsification produces bitterness. Therefore, how to solve the bitterness produced by alkaline protease demulsification and improve the extraction rate of the oil-tea camellia seed oil has become a problem to be solved. SUMMARY

[0006] The technical problem solved by the present application is to overcome the shortcomings of the prior art, and provide a method for preparing oil tea seed oil by alkali protease and endogenous protease synergistic demulsification, which has simple process and can effectively improve the extraction rate and reduce the bitterness.

[0007] The technical solution adopted by the present application to solve the technical problem is: comprising the following steps: 1) collecting the emulsion produced in the process of extracting fresh fruit oil tea seed oil by aqueous enzymatic method; 2) extracting endogenous protease from tea seed cake; 3) alkali protease and endogenous protease synergistic demulsification: first adding distilled water and alkali protease to the emulsion for primary enzymolysis, the addition amount of alkali protease is 0.5-2% of the weight of the emulsion, the primary enzymolysis time is T1, and 60min≤T1<75min, then adding endogenous protease according to 0.6-1.4% of the weight of the emulsion for secondary enzymolysis, the secondary enzymolysis time is T2, and 15min≤T2≤35min, and the total time of the primary enzymolysis and the secondary enzymolysis is less than 100min, to obtain oil emulsion; 4) enzyme inactivation and centrifugation to obtain finished oil tea seed oil

[0008] The present application first uses alkali protease to perform primary enzymolysis treatment on the emulsion produced in the process of extracting oil tea seed oil by aqueous enzymatic method, which can preliminarily destroy the stability of the emulsion, realize the preliminary demulsification of the emulsion, and make part of the free oil gradually separated from the emulsion; with the extension of the enzymolysis time, bitter amino acids are generated in the enzymolysis process of the alkali protease, in order to control the content of the bitter amino acids which form the main bitterness below the threshold value that can be tasted, the present application adds endogenous protease to the primary enzymolysis liquid for secondary enzymolysis at the primary enzymolysis T1(60min≤T1<75min), the addition of the endogenous protease in the critical time period(60min≤T1<75min) not only can synergize with the alkali protease to further destroy the stability of the emulsion, make the residual oil in the emulsion effectively separated out, improve the extraction rate of the oil tea seed oil, but also can timely and effectively inhibit the generation of the bitter amino acids which form the main bitterness in the demulsification process due to the enzymolysis of the alkali protease, reduce the generation rate of the bitter amino acids, make the content of the bitter amino acids in the whole demulsification process always below the threshold value, and ensure that the oil tea seed oil does not contain bitterness, improve the flavor and quality of the oil tea seed oil.

[0009] Further, the specific operation steps of the step 1) are as follows: the oil tea seed powder is mixed with distilled water to prepare a homogenate at a material-liquid ratio of 1:1-7, 2-3% of the oil tea seed powder by weight is added to the homogenate to carry out enzymolysis at a temperature of 55℃ for 3h, and then the enzyme is inactivated at 100℃ for 10min, and after cooling, centrifugation is carried out to obtain a heavy solid phase (cake), a water phase (containing soluble starch and protein) and a mixed phase (oil tea seed oil-protein-water emulsion phase), and the mixed phase is collected to obtain an emulsion.

[0010] Preferably, the material-liquid ratio of the oil tea seed powder to the distilled water is 1:5.

[0011] Further, the preparation method of the oil tea seed powder is as follows: freshly picked oil tea fruits are selected, color selection is carried out, and the green shells of the oil tea fruits are removed for standby; the fruits after removing impurities are put into a shelling machine for shelling, and then are crushed in a crusher for standby; the crushed oil tea seeds are dried, the drying temperature is 100-150℃, and the drying time is 20-40min.

[0012] Preferably, the drying temperature is 140℃, and the drying time is 30min.

[0013] Further, in the step 1), the enzyme activity of the neutral protease is 50000U / g, the centrifugal speed is 4000r / min-10000r / min, the centrifugal time is 10min-30min, and the water content of the oil tea seed powder is 3-8%.

[0014] Preferably, the water content of the oil tea seed powder is 4%.

[0015] Further, the specific operation steps of the step 2 are as follows:

[0016] a) the defatted oil tea seed cake meal is mixed with distilled water at a material-liquid ratio of 1:1-3, and sterilized at 121℃ for 20min;

[0017] b) an acetic acid bacteria bacterial suspension with a concentration of 8.0×10 7 CFU / mL and a yeast bacterial suspension with a concentration of 8.0×10 7 CFU / mL are prepared;

[0018] c) the acetic acid bacteria bacterial suspension and the yeast bacterial suspension are mixed at a volume ratio of 1:1-5 to obtain a mixed bacterial suspension;

[0019] d) the mixed bacterial suspension is inoculated into the sterilized oil tea seed cake meal at an inoculation amount of 1.0-2.5%, and then is placed at a temperature of 30-35℃ for liquid state fermentation for 3-5d to obtain a fermentation product for standby;

[0020] e) using the fermentation product to prepare a crude enzyme solution of endogenous protease, adjusting the pH of the crude enzyme solution to 4.5 with 0.08 mol / L NaOH to precipitate the impurities, centrifuging to obtain a transparent supernatant, and subjecting the supernatant to ultrafiltration and spray drying to obtain the endogenous protease.

[0021] Preferably, the ratio of the oil-tea camellia seed cake to distilled water is 1:2, the volume ratio of the acetic acid bacteria suspension to the yeast bacteria suspension is 1:4, the inoculation amount of the acetic acid bacteria suspension and the yeast bacteria suspension is 1.5%, and the temperature of the liquid fermentation is 33°C.

[0022] Further, the preparation of the acetic acid bacteria suspension in step b) is as follows: inoculating the acetic acid bacteria into HS flat plate culture medium in a streaking manner to obtain single colonies (the composition of the HS flat plate culture medium is: glucose 2%, peptone 0.5%, yeast extract powder 0.5%, citric acid 0.115%, disodium hydrogen phosphate dodecahydrate 0.68%, 0.1% natamycin, 1.8% agar, and the rest is sterile water, and the pH is adjusted to 6.0 with acetic acid), and then inoculating the single colonies into HS culture medium (the composition of the HS culture medium is: glucose 2%, peptone 0.5%, yeast extract powder 0.5%, citric acid 0.115%, disodium hydrogen phosphate dodecahydrate 0.68%, and the rest is sterile water, and the pH is adjusted to 6.0 with acetic acid), and incubating at 30°C for 24 hours to obtain a seed liquid; using the dilution plate method to count the acetic acid bacteria in the seed liquid, diluting with sterile distilled water, and determining that the final concentration of the acetic acid bacteria suspension is 8.0×10 7 CFU / mL, ready for use;

[0023] The preparation of the yeast bacteria suspension is as follows: inoculating the yeast bacteria into wort agar medium (the composition of the wort agar medium is: wort extract 2%, glucose 2%, peptone 0.1%, agar 1.5%, and the rest is sterile water) to obtain single colonies; then inoculating the single colonies into yeast extract powder peptone glucose medium (the composition of the yeast extract powder peptone glucose medium is: glucose 2%, peptone 2%, yeast extract powder 1%, and the rest is sterile water), and incubating at 30°C for 24 hours to obtain a seed liquid; using the dilution plate method to count the yeast bacteria in the seed liquid, diluting with sterile distilled water, and determining that the final concentration of the yeast bacteria suspension is 8.0×10 7 CFU / mL, ready for use.

[0024] Further, the preparation step of the crude enzyme solution in step e) is: adding 4 DEG C deionized water to the fermentation product at a volume ratio of 1:2, mixing, stirring at a speed of 10000 rpm for 1 min, standing for 40 s, then continuing to stir at a speed of 10000 rpm for 1 min, repeating the operation for 5 times, and then standing for 10 min at 4 DEG C to fully leach the endogenous protease; then centrifuging the leaching liquid at 8000 rpm for 10 min, and retaining the supernatant, which is the crude enzyme solution.

[0025] Further, the distilled water in step 3) is added to the emulsion at a material-liquid ratio of 1-1:3, the temperature of the first enzymolysis is 40-60 DEG C, the pH is 8-9, the temperature of the second enzymolysis is 60-70 DEG C, and the pH is 3-5.

[0026] Preferably, the distilled water in step 3) is added to the emulsion at a material-liquid ratio of 1:1, the temperature of the first enzymolysis is 55 DEG C, the pH is 9, the temperature of the second enzymolysis is 65 DEG C, and the pH is 4.

[0027] Further, the enzyme activity of the alkaline protease is 5000 U / g, and the addition amount is 1%, and the enzyme activity of the endogenous protease is 5000 U / g, and the addition amount is 1%.

[0028] Further, the time of the first enzymolysis is 60 min, and the time of the second enzymolysis is 15 min.

[0029] Further, the specific operation steps of step 4) are: deactivating the enzyme at 100 DEG C for 10 min, and then centrifuging at 8000 r / min for 10 min.

[0030] The method has the advantages that:

[0031] (1) The process is simple, the emulsion is prepared by water enzymatic extraction of oil tea seeds using neutral protease, then the emulsion is preliminarily broken using alkaline protease, and then the endogenous protease and alkaline protease are used for synergistic demulsification, under the combined action of the endogenous protease and alkaline protease, the demulsification rate and the extraction rate of oil tea seed oil reach 98% and 99.5% respectively, which can effectively improve the demulsification rate and the extraction rate of oil tea seed oil.

[0032] (2) The alkaline protease and endogenous protease synergistic demulsification method for preparing oil-tea camellia seed oil can effectively inhibit the generation of bitter amino acids (valine and arginine) caused by alkaline protease enzymolysis in the demulsification process in time, so that the content of valine and arginine, which contribute most to bitterness, is kept below the threshold value in the whole demulsification process, the problem of bitterness caused by alkaline protease in the preparation of oil-tea camellia seed oil is solved, and the oil-tea camellia seed oil prepared by the alkaline protease demulsification method does not contain bitterness, the flavor and quality of the oil-tea camellia seed oil are improved. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 — the process flow chart of the alkaline protease and endogenous protease synergistic demulsification method for preparing oil-tea camellia seed oil;

[0034] Figure 2 — the influence diagram of alkaline protease enzymolysis time on valine content;

[0035] Figure 3 — the influence diagram of alkaline protease enzymolysis time on arginine content;

[0036] Figure 4 — the influence diagram of one-time enzymolysis time on the extraction rate of the prepared finished oil-tea camellia seed oil;

[0037] Figure 5 — the influence of total enzymolysis time on the extraction rate of the finished oil-tea camellia seed oil

[0038] Figure 6 — the influence diagram of total enzymolysis time on valine content;

[0039] Figure 7 — the influence diagram of total enzymolysis time on arginine content;

[0040] Figure 8 — the total diagram of the influence of total enzymolysis time on valine content when only alkaline protease demulsification and alkaline protease and endogenous protease synergistic demulsification are adopted;

[0041] Figure 9 — the total diagram of the influence of total enzymolysis time on arginine content when only alkaline protease demulsification and alkaline protease and endogenous protease synergistic demulsification are adopted;

[0042] Figure 10 — the influence diagram of different endogenous protease addition amounts on the increase value of valine;

[0043] Figure 11 — the influence diagram of different endogenous protease addition amounts on the increase value of arginine.

[0044] Figure 12Figure of the effect of different amounts of added endogenous protease on the extraction rate of oil tea seed oil. DETAILED DESCRIPTION

[0045] The application will be further described in conjunction with the accompanying drawings and examples, but these specific embodiments do not limit the protection scope of the application in any way.

[0046] Example 1

[0047] A method for preparing oil tea seed oil by synergistic demulsification of alkaline protease and endogenous protease, the process flow chart is shown as Figure 1 The method comprises the following steps:

[0048] 1) Collecting emulsion produced in the process of extracting fresh fruit oil tea seed oil by aqueous enzymatic method:

[0049] S1. Selecting freshly picked oil tea fresh fruit after removing impurities, performing color selection, removing the green shell of the oil tea fresh fruit, and reserving;

[0050] S2. The fresh fruit after removing impurities enters the sheller for shelling, and then is crushed in the crusher, and is reserved;

[0051] S3. The crushed oil tea seeds are dried, the drying temperature is 140℃, the drying time is 30min, and the moisture content of the oil tea seed powder obtained after drying is 4%;

[0052] S4. The dried oil tea seed powder is mixed with distilled water at a material-to-liquid ratio of 1:5 to prepare a homogenate, 3% of the oil tea seed powder by weight is added to the homogenate, a neutral protease (CAS No. 9068-59-1, a protease disclosed in the prior art) with an enzyme activity of 50000 U / g, and the homogenate is enzymolyzed at a temperature of 55℃ for 3h, then the enzyme is inactivated at 100℃ for 10min, and after cooling, it is centrifuged at 10000r / min for 10min to obtain a heavy solid phase (dregs), an aqueous phase (containing soluble starch and protein), and a mixed phase (oil tea seed oil-protein-water emulsion phase), and the mixed phase is collected to obtain an emulsion; the content of amino acids in the emulsion is detected and whether it has a bitter taste is tasted, the detection method of amino acids refers to the literature “Preparation of low bitter peptides from wheat protein and mechanism of bitter removal”, the determination of oil extraction rate = weight of oil in emulsion / weight of oil in oil tea seed powder x 100, the results show that the content of valine in the emulsion obtained after neutral protease enzymolysis is 0.15mg / g, the content of arginine is 0.1mg / g, there is no bitter taste, and the extraction rate is 55.2%.

[0053] 2) Extracting endogenous protease from tea seed cake:

[0054] a) Mixing the defatted oil tea seed cake and distilled water at a material-to-liquid ratio of 1:2, sterilizing at 121℃ for 20min;

[0055] b) preparing an acetic acid bacteria suspension with a concentration of 8.0 x 10 7 CFU / mL (as the prior art acetic acid bacteria AS1.41, commercially available) : inoculate acetic acid bacteria onto HS plate medium in a streak manner to obtain single colonies (HS plate medium: glucose 2%, peptone 0.5%, yeast extract 0.5%, citric acid 0.115%, disodium hydrogen phosphate dodecahydrate 0.68%, 0.1% natamycin, 1.8% agar, the rest being sterile water, pH adjusted to 6.0 with acetic acid); then inoculate 2 rings of single colonies into 50 mL of HS medium (HS medium: glucose 2%, peptone 0.5%, yeast extract 0.5%, citric acid 0.115%, disodium hydrogen phosphate dodecahydrate 0.68%, the rest being sterile water, pH adjusted to 6.0 with acetic acid), incubate at 30°C for 24 h as a seed liquid; count the acetic acid bacteria in the seed liquid by the dilution plate method, inoculate at a ratio of about 1 x 10 4 CFU / mL, and incubate at 30°C for 7 d after inoculation; after the incubation, count by the dilution plate method, dilute with sterile distilled water, and control the final concentration of the acetic acid bacteria suspension at 8.0 x 10 7 CFU / mL;

[0056] b) preparing a yeast suspension with a concentration of 8.0 x 10 7 CFU / mL (as the prior art Angel active dry yeast for brewing, commercially available): transfer Angel active dry yeast for brewing to malt agar medium to obtain single colonies (malt agar medium: malt extract 2%, glucose 2%, peptone 0.1%, agar 1.5%, the rest being sterile water); inoculate 4 rings of single colonies into 50 mL of yeast extract peptone glucose medium (yeast extract peptone glucose medium: glucose 2%, peptone 2%, yeast extract 1%, the rest being sterile water), incubate at 30°C for 24 h as a seed liquid; count the yeast in the seed liquid by the dilution plate method, dilute with sterile distilled water, and control the final concentration of the yeast suspension at 8.0 x 10 7 CFU / mL;

[0057] c) mixing the acetic acid bacteria suspension and the yeast suspension at a volume ratio of 1:4 to obtain a mixed bacteria suspension;

[0058] d) inoculating the mixed bacteria suspension into sterilized oil-tea camellia seed cake at an inoculation amount of 1.5%, and then liquid fermenting at a temperature of 33°C for 5 d to obtain a fermentation product, for standby;

[0059] e) adding deionized water at 4°C to the fermentation product at a volume ratio of 1:2, mixing at a speed of 10000 rpm for 1 min, continuing to stir at a speed of 10000 rpm for 1 min after standing for 40 s, and repeating the cycle 5 times and then standing at 4°C for 10 min to fully leach the endogenous protease; then centrifuging the leachate at 8000 rpm for 10 min, and retaining the supernatant, which is the crude enzyme solution of the endogenous protease;

[0060] The pH value of the crude enzyme solution was adjusted to 4.5 with 0.08 mol / L NaOH, and the solution was incubated at 50°C for 6 h with a stirring speed of 70 r / min; the incubated slurry was centrifuged at room temperature at 3000 rpm for 5 min, and the precipitate was discarded to obtain the supernatant of the hyaloplasm hydrolysate; the supernatant of the hyaloplasm hydrolysate was ultrafiltered with an 8 kDa ultrafiltration membrane to obtain a filtrate with a molecular weight of less than 8 kDa, and concentrated by a rotary evaporator, and finally spray-dried by a spray dryer to obtain the endogenous protease;

[0061] 3) Alcalase and endogenous protease synergistic demulsification: first, distilled water and Alcalase enzyme with an enzyme activity of 5000 U / g (i.e., Alcalase enzyme, consisting of 41% water, 50% glycerol and 9% subtilisin, Novozymes, Denmark, a protease disclosed in the prior art) were added to the emulsion, and a first enzymolysis was performed at a temperature of 55°C and a pH of 9, the ratio of emulsion to distilled water was 1:1, the amount of Alcalase enzyme added was 1% of the weight of the emulsion, and the first enzymolysis was performed for 60 min; then, endogenous protease with an enzyme activity of 5000 U / g (enzyme activity detection method refers to SB / T 10317) was added at 1% of the weight of the emulsion, and a second enzymolysis was performed at a temperature of 65°C and a pH of 4, the second enzymolysis time was 15 min, and the total time of the first and second enzymolysis was 75 min, to obtain an oily emulsion;

[0062] 4) enzyme inactivation and centrifugation: the specific operation steps of step 4) are as follows: the oily emulsion was inactivated at 100°C for 10 min, and then centrifuged at 8000 r / min for 10 min to obtain the finished oil tea seed oil. The finished oil tea seed oil was subjected to edible oil national standard detection, and the results were as follows: acid value 0.35 mg / g, peroxide value 0.029 g / 100 g, moisture and volatile matter content 0.04%, insoluble impurities 0.03%, which met the standards of the first grade oil tea seed oil by aqueous enzymatic method in “GB / T 11765-2018 Oil Tea Seed Oil”.

[0063] Example 2

[0064] The difference between this embodiment and embodiment 1 is that the time of the first enzymolysis is 70 min, and the time of the second enzymolysis is 15 min. The prepared finished camellia seed oil is subjected to national standard detection of edible oil, and the results are as follows: acid value 0.36 mg / g, peroxide value 0.031 g / 100 g, moisture and volatile content 0.041%, insoluble impurities 0.032%, which all meet the standards of the first grade camellia seed oil by aqueous enzymatic method in <GB / T 11765-2018 Camellia Seed Oil>.

[0065] Example 3

[0066] The difference between this embodiment and embodiment 1 is that the time of the first enzymolysis is 60 min, and the time of the second enzymolysis is 35 min. The prepared finished camellia seed oil is subjected to national standard detection of edible oil, and the results are as follows: acid value 0.35 mg / g, peroxide value 0.030 g / 100 g, moisture and volatile content 0.04%, insoluble impurities 0.035%, which all meet the standards of the first grade camellia seed oil by aqueous enzymatic method in <GB / T 11765-2018 Camellia Seed Oil>.

[0067] Comparative Example 1

[0068] The difference between this comparative example and embodiment 1 is that step 2) is removed, and only alkaline protease is used for demulsification of the prepared emulsion, and other steps are the same as those of embodiment 1. The specific demulsification operation steps are as follows:

[0069] Alkaline protease demulsification: distilled water and alkaline protease with an enzyme activity of 5000 U / g are added to the emulsion, and enzymolysis is carried out at a temperature of 55°C and a pH of 9. The ratio of the emulsion to the distilled water is 1:1, the addition amount of the alkaline protease is 2% of the weight of the emulsion, and the enzymolysis time is 75 min, to obtain an oily emulsion.

[0070] Comparative Example 2

[0071] The difference between this comparative example and embodiment 1 is that only endogenous protease is used for demulsification of the emulsion, and other steps are the same as those of embodiment 1. The specific demulsification operation steps are as follows:

[0072] Endogenous protease demulsification: distilled water and endogenous protease with an enzyme activity of 5000 U / g are added to the emulsion, and enzymolysis is carried out at a temperature of 65°C and a pH of 4. The ratio of the emulsion to the distilled water is 1:1, the addition amount of the endogenous protease is 2% of the weight of the emulsion, and the enzymolysis time is 75 min, to obtain an oily emulsion.

[0073] Comparative Example 3

[0074] The difference between the present comparative example and Example 1 is that the endogenous protease used in cooperation with the alkaline protease during demulsification is replaced by flavor protease, and other steps are the same as those in Example 1. The specific demulsification operation steps are as follows:

[0075] Alkaline protease and flavor protease synergistic demulsification: first, distilled water and alkaline protease with an enzyme activity of 5000 U / g are added to the emulsion, and one-step enzymolysis is carried out at a temperature of 55°C and a pH of 9, the ratio of emulsion to distilled water is 1:1, the addition amount of alkaline protease is 1% of the weight of the emulsion, after one-step enzymolysis for 60 min, 1% of the weight of the emulsion is added to the emulsion, and the second-step enzymolysis is carried out at a temperature of 50°C and a pH of 6, the second-step enzymolysis time is 15 min, and the total time of one-step and two-step enzymolysis is 75 min, to obtain an oily emulsion.

[0076] Comparative Example 4

[0077] The difference between the present comparative example and Example 1 is that the endogenous protease used in cooperation with the alkaline protease during demulsification is replaced by acid protease, and other steps are the same as those in Example 1. The specific demulsification operation steps are as follows:

[0078] Alkaline protease and acid protease synergistic demulsification: first, distilled water and alkaline protease with an enzyme activity of 5000 U / g are added to the emulsion, and one-step enzymolysis is carried out at a temperature of 55°C and a pH of 9, the ratio of emulsion to distilled water is 1:1, the addition amount of alkaline protease is 1% of the weight of the emulsion, after one-step enzymolysis for 60 min, 1% of the weight of the emulsion is added to the emulsion, and the second-step enzymolysis is carried out at a temperature of 55°C and a pH of 3.5, the second-step enzymolysis time is 15 min, and the total time of one-step and two-step enzymolysis is 75 min, to obtain an oily emulsion.

[0079] Comparative Example 5

[0080] The difference between the present comparative example and Example 1 is that the endogenous protease used in cooperation with the alkaline protease during demulsification is replaced by trichloro sucrose, and other steps are the same as those in Example 1. The specific demulsification operation steps are as follows:

[0081] Alkaline protease and anhydrous citric acid synergistic demulsification: first, distilled water and alkaline protease with an enzyme activity of 5000 U / g are added to the emulsion, and one-step enzymolysis is carried out at a temperature of 55°C and a pH of 9, the ratio of emulsion to distilled water is 1:1, the addition amount of alkaline protease is 1% of the weight of the emulsion, after one-step enzymolysis for 60 min, 1% of the weight of the emulsion is added to the emulsion, and the second-step enzymolysis is carried out at a temperature of 65°C and a pH of 4, the second-step enzymolysis time is 15 min, and the total time of one-step and two-step enzymolysis is 75 min, to obtain an oily emulsion.

[0082] Comparative Example 6

[0083] The difference between the present comparative example and Example 1 is that the endogenous protease used in conjunction with the alkaline protease in the demulsification is replaced by anhydrous citric acid, and the other steps are the same as those in Example 1. The specific demulsification operation steps are as follows:

[0084] Alkaline protease and anhydrous citric acid demulsification: distilled water and alkaline protease with an enzyme activity of 5000 U / g were first added to the emulsion, and the first enzyme hydrolysis was carried out at a temperature of 55°C and a pH of 9. The ratio of the emulsion to distilled water was 1:1, and the addition amount of alkaline protease was 1% of the weight of the emulsion. After 60 minutes of first enzyme hydrolysis, 1% of the weight of the emulsion of anhydrous citric acid was added, and the second enzyme hydrolysis was carried out at a temperature of 65°C and a pH of 4. The second enzyme hydrolysis time was 15 minutes, and the total time of the first enzyme hydrolysis and the second enzyme hydrolysis was 75 minutes, to obtain an oil emulsion. The differences between the methods for preparing oil-tea camellia seed oil in the above-mentioned Example 1 and Comparative Examples 1-6 are shown in Table 1:

[0085] Table 1 Differences between the methods for preparing finished oil-tea camellia seed oil in Comparative Examples 1-6 and Example 1

[0086]

[0087] The extraction rate, demulsification rate, amino acid content, and bitterness of the finished oil-tea camellia seed oil prepared in Examples 1-3 and Comparative Examples 1-6 were determined (valine bitterness threshold 0.4 mg / g, arginine bitterness threshold 0.5 mg / g), and the results are shown in the following table. The extraction rate (%) = weight of finished oil-tea camellia seed oil / weight of oil in oil-tea camellia seed powder x 100, the demulsification rate (%) = weight of finished oil-tea camellia seed oil / weight of oil in emulsion x 100, and the weight of oil in oil-tea camellia seed powder and emulsion was determined by weighing.

[0088] Table 2 Determination results of various indicators of the finished oil-tea camellia seed oil prepared in Examples 1 and Comparative Examples 1-6

[0089]

[0090]

[0091] As can be seen from the detection data in Table 2, the extraction rate of the finished oil-tea camellia seed oil prepared by the alkaline protease and endogenous protease demulsification in Examples 1, 2, and 3 reached 99.5%, the demulsification rate reached 98%, and the finished oil-tea camellia seed oil prepared did not contain bitterness, thereby improving the flavor quality of the oil-tea camellia seed oil.

[0092] Compared with the comparative example 1, the extraction rate and demulsification rate of the finished oil-tea camellia seed oil prepared in the example 1 are both significantly higher than those of the comparative example 1, and the contents of valine and arginine are both significantly lower than those of the comparative example 1 and are below the threshold value, indicating that the emulsion treated by the alkaline protease and the endogenous protease after being treated by the neutral protease can effectively improve the demulsification rate and then the extraction rate of the oil-tea camellia seed oil; and the endogenous protease treatment of the enzymatic hydrolysate after the first enzymolysis by the alkaline protease can effectively inhibit the formation of bitter amino acids (valine and arginine) generated by the alkaline protease in the enzymolysis process, so that the contents of the bitter amino acids (valine and arginine) in the whole demulsification process are always below the threshold value, which can obviously improve the bitterness and ensure that the oil-tea camellia seed oil does not contain bitterness, thereby improving the flavor and quality of the oil-tea camellia seed oil.

[0093] Compared with the comparative example 1, the endogenous protease treatment of the emulsion in the demulsification process of the comparative example 2 indeed has a very obvious effect of removing bitterness, but the demulsification rate and the extraction rate are both lower than those of the comparative example 1 and are significantly lower than those of the example 1, indicating that the treatment of the emulsion by the endogenous protease alone cannot obtain the oil-tea camellia seed oil with high extraction rate and demulsification rate.

[0094] Compared with the comparative example 1, the treatment of the emulsion by the alkaline protease in the comparative examples 3-6 is followed by the treatment of the emulsion by the common flavor protease, the acid protease, the sucralose and the anhydrous citric acid respectively, and the contents of valine and arginine in the corresponding oil-tea camellia seed oil are all lower than those of the comparative example 1, and the demulsification rate and the extraction rate corresponding to the comparative examples 3-6 have no obvious difference with those of the comparative example 1, indicating that the flavor protease, the acid protease, the sucralose and the anhydrous citric acid have poor demulsification effect and cannot effectively improve the extraction rate of the oil-tea camellia seed oil, and have the effect of reducing bitterness; but compared with the example 1, the contents of valine and arginine in the oil-tea camellia seed oil corresponding to the comparative examples 3-6 are all higher than those of the example 1, and the demulsification rate and the extraction rate are both significantly lower than those of the example 1, indicating that the method of treating the emulsion by the endogenous protease and the alkaline protease in the present application not only has a better effect of inhibiting the formation rate of valine and arginine than other conventional substances for removing bitterness, but also can better remove the bitterness generated by the alkaline protease in the enzymolysis process, and can effectively improve the demulsification rate and the extraction rate of the oil-tea camellia seed oil.

[0095] Experimental example 1

[0096] According to the method of the comparative example 1, when the alkaline protease is used for the first enzymolysis, the change trends of the bitterness, the extraction rate and the content of bitter amino acids of the oil-tea camellia seed oil prepared by using the oil emulsion with the enzymolysis time of 20 min, 40 min, 60 min, 80 min and 100 min are studied, and the results are shown in Tables 3-4 and Figures 2-4

[0097] Table 3 Determination results of the bitterness of the finished oil-tea camellia seed oil prepared by the first enzymolysis time​

[0098] Assay readout 20 min 40 min 60 min 80 min 100 min Bitterness Not bitter Not bitter Not bitter Very bitter Very bitter

[0099] Table 4 Determination results of bitter amino acids in the prepared camellia seed oil by one-time enzymolysis time

[0100]

[0101] As can be seen from Table 3 and Table 4, with the extension of alkaline protease demulsification enzymolysis time (one-time enzymolysis time), the contents of bitter amino acids in the camellia seed oil all show a gradually increasing trend, and the contents of methionine, isoleucine, leucine, phenylalanine and histidine all do not reach the corresponding bitter threshold value within 100 min of enzymolysis, while the contents of valine and arginine both exceed the corresponding bitter threshold value at 80 min of enzymolysis, thereby making the camellia seed oil show a very bitter taste.

[0102] In order to further understand the relationship between alkaline protease enzymolysis time and valine and arginine, equation fitting was performed on the results of alkaline protease different enzymolysis time and valine and arginine content, and the results are shown in Figure 2 and Figure 3 .

[0103] As can be seen from Figure 2 , equation fitting is performed on the results of alkaline protease different enzymolysis time and valine content, and the equation Y=0.0663e 0.0238x , R 2 =0.9951 is obtained, indicating that the enzymolysis time and the valine content have good correlation, and the valine content is 0.4 mg / g when the enzymolysis time is 75 min.

[0104] As can be seen from Figure 3 , equation fitting is performed on the results of alkaline protease different enzymolysis time and arginine content, and the equation Y=0.0189e 0.0418x , R 2 =0.9922 is obtained, indicating that the enzymolysis time and the arginine content have good correlation.

[0105] In verification example 1, the method of comparative example 1 was referred to, and when one-time enzymolysis was performed by using alkaline protease, the enzymolysis time was 75 min, and the valine and arginine contents in the camellia seed oil under the enzymolysis time were studied.

[0106] By one-time enzymolysis by using alkaline protease, the enzymolysis time was 75 min, the valine content in the camellia seed oil was 0.4 mg / g, and the arginine content was 0.45 mg / g. It is shown that when the alkaline protease is enzymolyzed for 75 min, the content of valine just reaches the threshold value, while the content of arginine does not reach the threshold value, so that the camellia seed oil shows a slightly bitter taste.

[0107] From Figure 4 It can be seen that with the extension of the alkaline protease hydrolysis time (primary enzyme hydrolysis time), the extraction rate of oil-tea camellia seed oil presents a trend of first significantly increasing and then tending to be flat, and when reaching 60 min, the extraction rate reaches 87.3%, and with the continuous extension of the enzyme hydrolysis time, the extraction rate does not increase obviously. In order to shorten the extraction time and avoid the threshold value of valine and arginine in the enzyme hydrolysis process to improve the bitterness of alkaline protease hydrolysis, the primary enzyme hydrolysis time T1 is preferably 60 min≤T1<75 min, and most preferably 60 min.

[0108] Experimental Example 2

[0109] The method of Example 1 was used to study the bitterness, extraction rate and content change trend of valine and arginine of oil-tea camellia seed oil prepared from oil emulsion with alkaline protease primary enzyme hydrolysis for 60 min and secondary enzyme hydrolysis for 15 min, 20 min, 25 min, 30 min, 35 min and 40 min corresponding to total enzyme hydrolysis time (75 min, 80 min, 85 min, 90 min, 95 min and 100 min), and the results are shown in Table 5 and Figures 5-9

[0110] Table 5 Measurement results of various indicators of finished oil-tea camellia seed oil prepared by total enzyme hydrolysis time

[0111]

[0112] It can be seen from Table 5 and Figure 5 It can be seen that when the secondary enzyme hydrolysis time is 15 min-40 min (total enzyme hydrolysis time is 75-100 min), the extraction rate of oil-tea camellia seed oil is further significantly improved on the basis of primary enzyme hydrolysis for 60 min (corresponding to total enzyme hydrolysis time for 60 min, the extraction rate is 87.3%), which shows that adding endogenous protease to the oil emulsion after primary enzyme hydrolysis for 60 min can further demulsify the oil emulsion in cooperation with alkaline protease, thereby significantly improving the extraction rate of oil-tea camellia seed oil; when the total enzyme hydrolysis time is 95 min (corresponding to the secondary enzyme hydrolysis time for 35 min), the extraction rate reaches 99.7%, and when the total enzyme hydrolysis time continues to extend to 100 min, the extraction rate does not change, and there is no significant difference between the extraction rate when the total enzyme hydrolysis time is 75 min (corresponding to the secondary enzyme hydrolysis time for 15 min) and the extraction rate when the total enzyme hydrolysis time is 95 min. In order to improve the preparation efficiency of oil-tea camellia seed oil, the secondary enzyme hydrolysis time is most preferably 15 min.

[0113] From Figure 6 and Figure 7 ​It can be seen that the contents of valine and arginine did not reach the threshold within the total enzymatic hydrolysis time of 100 min, and the content of valine only reached the threshold when the total enzymatic hydrolysis time was 100 min.

[0114] from Figure 8 It can be seen that when alkaline protease is used for a single enzymatic hydrolysis of 60 min, without the addition of endogenous protease to synergistically break the emulsion (corresponding to the blue line in the figure, data marker type is circle), the valine content reaches the threshold (0.4 mg / g) at a total enzymatic hydrolysis time of 75 min, and the camellia seed oil exhibits a slightly bitter taste. However, by using the method of this invention, by adding endogenous protease to the hydrolysate in a timely manner at the 60 min stage of the single enzymatic hydrolysis (corresponding to the orange line in the figure, data marker type is square), the valine content remains below its bitterness threshold within a total enzymatic hydrolysis time of 60-100 min, and the extraction rate is greater than or equal to 99.5%. This indicates that the addition of endogenous protease can not only further improve the extraction rate of camellia seed oil, but also effectively inhibit the valine generation rate during enzymatic hydrolysis, thereby improving the taste of camellia seed oil.

[0115] from Figure 9 It can be seen that when alkaline protease is used for a single enzymatic hydrolysis of 60 min, if no endogenous protease is added to assist in demulsification (corresponding to the blue line in the figure, data marker type is circle), the arginine content exceeds the threshold (0.5 mg / g) when the total enzymatic hydrolysis time is 80 min, and the camellia seed oil exhibits a distinct bitter taste. However, by using the method of this invention, endogenous protease is added to the hydrolysate in time at the first enzymatic hydrolysis of 60 min (corresponding to the orange line in the figure, data marker type is square), so that the arginine content remains below its bitterness threshold during the total enzymatic hydrolysis time of 60-100 min, and the extraction rate is greater than or equal to 99.5%. This indicates that the addition of endogenous protease can not only further improve the extraction rate of camellia seed oil, but also effectively inhibit the arginine generation rate during enzymatic hydrolysis, thereby improving the taste of camellia seed oil.

[0116] Experimental Example 3

[0117] The method described in Example 1 was used to study the extraction rate and the changing trends of valine and arginine content in camellia seed oil prepared from oily emulsions after secondary enzymatic hydrolysis with different amounts of endogenous protease. Specifically, the procedure involved a first enzymatic hydrolysis with alkaline protease for 60 min, followed by a second enzymatic hydrolysis with different amounts of endogenous protease for 15 min. The effects of endogenous protease additions of 0%, 0.2%, 0.6%, 1%, 1.4%, 1.8%, 2.2%, 2.6%, and 3.0% on the changes (increases) of valine and arginine content in the oily emulsion-prepared camellia seed oil were investigated. The increase was calculated as: Amino acid content in the finished camellia seed oil - Amino acid content in the first hydrolysis solution. The results are shown below.Figure 10 、 Figure 11 and Figure 12 as shown.

[0118] As can be seen from Figure 10 , with the increase of the addition amount of endogenous protease, the increase value of valine content presents a trend of significant decrease (addition amount of 0-0.6%) and then tends to be constant (0.6-3%), and the increase value of valine content and the addition amount of endogenous protease present a negative correlation, indicating that the endogenous protease can effectively inhibit the generation rate of valine.

[0119] As can be seen from Figure 11 , with the increase of the addition amount of endogenous protease, the increase value of arginine content presents a trend of significant decrease (addition amount of 0-0.6%) and then tends to be constant (0.6-3%), and the increase value of arginine content and the addition amount of endogenous protease present a negative correlation, indicating that the endogenous protease can effectively inhibit the generation rate of arginine. Figure 12 As can be seen from , with the increase of the addition amount of endogenous protease, the extraction rate presents a trend of significant increase (0-0.6%) and then gentle (0.6-3%), and when the addition amount is 0.6%, the extraction rate has reached 98.5%, and for the consideration of cost saving, the addition amount of endogenous protease is selected to be 0.6-1.4%, and the optimal one is 1%.

[0120] The above is only the preferred embodiment of the present application, and does not limit the present application in any form. Although the present application has been disclosed as above with the preferred embodiment, it is not intended to limit the present application. Any person skilled in the art can make some changes or modifications to the above disclosed technical content to make equivalent embodiments with equivalent changes, without departing from the technical solution of the present application. Any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present application are still within the scope of the technical solution of the present application.

Claims

1. A method for preparing oil-tea camellia seed oil by alkaline protease and endogenous protease synergistic demulsification, characterized in that, The method comprises the following steps: 1) collecting emulsions generated in the process of extracting fresh fruit oil tea seed oil by aqueous enzymatic method; 2) extracting endogenous protease from tea seed cake meal; 3) alkali protease and endogenous protease synergistic demulsification: first, adding distilled water and alkali protease to the emulsions for primary enzymolysis, the addition amount of alkali protease is 0.5-2% of the weight of the emulsions, the primary enzymolysis time is T1, and 60min≤T1<75min, then adding endogenous protease according to 0.6-1.4% of the weight of the emulsions for secondary enzymolysis, the secondary enzymolysis time is T2, and 15min≤T2≤35min, the total time of the primary enzymolysis and the secondary enzymolysis is less than 100min, to obtain oil emulsion; 4) enzyme inactivation and centrifugation, to obtain finished oil tea seed oil; The specific operation steps of the step 2 are: a) mixing the defatted oil tea seed cake meal and distilled water according to a solid-liquid ratio of 1:1-3, sterilizing at 121℃ for 20min; b) preparing an acetic acid bacteria cell suspension having a concentration of 8.0 x 10 7 CFU / mL and 8.0 x 10 7 CFU / mL of a yeast cell suspension; c) mixing the acetic acid bacteria suspension and the yeast bacteria suspension according to a volume ratio of 1:1-5 to obtain a mixed bacteria suspension; d) inoculating the mixed bacteria suspension into the sterilized oil tea seed cake meal at an inoculation amount of 1.0-2.5%, and then placing it in liquid state fermentation at a temperature of 30-35℃ for 3-5d to obtain a fermentation product, which is ready for use; e) preparing a crude enzyme solution of the endogenous protease by using the fermentation product, adjusting the pH of the crude enzyme solution to 4.5 by using 0.08mol / L NaOH to precipitate impure proteins, centrifuging to obtain a transparent supernatant, and then performing ultrafiltration and spray drying to obtain the endogenous protease.

2. The method for preparing camellia seed oil by synergistic demulsification of alkaline protease and endogenous protease as described in claim 1, characterized in that, The specific operation steps of the step 1) are: mixing the oil tea seed powder and distilled water according to a solid-liquid ratio of 1:1-7 to prepare a homogenate, adding neutral protease to the homogenate according to 2-3% of the weight of the oil tea seed powder, and then performing enzymolysis at a temperature of 55℃ for 3h, enzyme inactivation at 100℃ for 10min, and centrifugation after cooling to obtain a heavy solid phase, a water phase and a mixed phase, and collecting the mixed phase to obtain the emulsions.

3. The method for preparing camellia seed oil by synergistic demulsification of alkaline protease and endogenous protease as described in claim 2, characterized in that, In the step 1), the enzyme activity of the neutral protease is 50000U / g, the centrifugation speed is 4000r / min-10000r / min, the centrifugation time is 10min-30min, and the water content of the oil tea seed powder is 3-8%.

4. The method of claim 1, wherein the oil is prepared by the synergistic demulsification of alkaline protease and endogenous protease. The preparation step of the acetic acid bacteria suspension in step b) is: inoculating acetic acid bacteria on HS plate medium in streaking manner to obtain single colonies, then inoculating the single colonies into HS medium, and standing and culturing at 30°C for 24 h as seed liquid; counting the acetic acid bacteria in the seed liquid by dilution plating method, diluting with sterile distilled water, and determining that the final concentration of the acetic acid bacteria suspension is 8.0×10 7 CFU / mL, ready for use; The preparation steps of the yeast suspension are as follows: the yeast is transferred to a malt juice agar medium to obtain a single colony; then the single colony is inoculated into a yeast extract powder peptone glucose medium, and is statically cultured at 30°C for 24 hours to obtain a seed liquid; the yeast in the seed liquid is counted by a dilution coating plate method, and the yeast suspension is diluted with sterile distilled water to a final concentration of 8.0×10 7 CFU / mL, ready for use.

5. The method for preparing camellia seed oil by synergistic demulsification of alkaline protease and endogenous protease as described in claim 1, characterized in that, The preparation steps of the crude enzyme solution in the step e) are: adding 4℃ deionized water to the fermentation product according to a volume ratio of 1:2, stirring at a speed of 10000rpm for 1min, standing for 40s, and then continuously stirring at a speed of 10000rpm for 1min, repeating the operation for 5 times, and then preserving at 4℃ for 10min to fully leach the endogenous protease; then centrifuging the leaching solution at 8000rpm for 10min, and preserving the supernatant, which is the crude enzyme solution.

6. The method for preparing camellia seed oil by synergistic demulsification of alkaline protease and endogenous protease as described in claim 1, characterized in that, The distilled water in the step 3) is added to the emulsions according to a solid-liquid ratio of 1-1:3, the temperature during the primary enzymolysis is 40-60℃, the pH is 8-9, the temperature during the secondary enzymolysis is 60-70℃, and the pH is 3-5.

7. The method for preparing camellia seed oil by synergistic demulsification of alkaline protease and endogenous protease as described in claim 1, characterized in that, The enzyme activity of the alkali protease is 5000U / g, and the enzyme activity of the endogenous protease is 5000U / g.

8. The method for preparing camellia seed oil by synergistic demulsification of alkaline protease and endogenous protease as described in claim 1, characterized in that, The time of the first enzymolysis is 60 min, and the time of the second enzymolysis is 15 min.

9. The method for preparing camellia seed oil by synergistic demulsification of alkaline protease and endogenous protease as described in claim 1, characterized in that, The specific operation steps of step 4) are: centrifuging the oily emulsion at 8000 r / min for 10 min after enzyme inactivation at 100 ℃ for 10 min.

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