A method for nondestructive reduction of toxic components in ginkgo nut kernels and products prepared therefrom and uses thereof

Abstract

The application discloses a method for lossless reduction of toxic components in ginkgo nuts and products and applications prepared by the method. The method comprises the following steps: removing the middle seed coat and inner seed coat of raw ginkgo nuts to obtain raw ginkgo whole kernel; boiling the raw ginkgo whole kernel with water to obtain boiled ginkgo whole kernel; cooling the boiled ginkgo whole kernel and water boiling liquid to room temperature; adding alpha-amylase to the boiled ginkgo whole kernel and water boiling liquid; performing constant temperature water bath after adding the alpha-amylase, filtering out the boiled ginkgo whole kernel to obtain water bath extraction liquid and attenuated boiled ginkgo whole kernel; and recovering 4'-O-methyl pyridoxine and 4'-O-methyl pyridoxine-5'-glucoside in the water bath extraction liquid. The method effectively removes MPN and MPNG in ginkgo nuts, is simple in process operation, is high in safety, and uses only water and heat treatment. The low-toxicity boiled ginkgo whole kernel obtained by the method can be used as raw material of various ginkgo foods, and is particularly suitable for ginkgo foods that need to be cooked with whole kernel.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of food engineering, and particularly relates to a detoxification method for ginkgo fruit kernels and products prepared therefrom and applications thereof. BACKGROUND

[0002] Ginkgo biloba L. is a single species of Ginkgo L. in Ginkgoaceae of Gymnospermae. Ginkgo biloba L. can be traced back to 345 million years ago, and is one of the oldest surviving plants in the world, known as the “living fossil”. Ginkgo biloba L. has a long history in China. Since ancient times, there have been more than 10 names for Ginkgo biloba L. “Shatangzhi, Huafengche” recorded in “Shanglin Fu” is a tree planted in the imperial gardens of the Han Dynasty, and “Che” is the earliest name for Chinese Ginkgo. In addition, there are also names such as “Gongsun Tree”, “White Fruit”, “Duck Feet”, “Spiritual Eye”, “Sacred Fruit”, “Buddha Finger Fruit”, “Moth Tree” and “Bodhi Tree”. Ginkgo biloba L. is mainly planted in Zhejiang, Anhui, Jiangsu, Shandong, Guangxi, Guizhou, Henan and Hubei, etc. in China. At present, Ginkgo biloba L. is widely planted in the world, in addition to China, including Japan, North Korea, South Korea, Canada, New Zealand, Australia, the United States, France and Russia, etc. Ginkgo biloba L. is an important plant with food value, medicinal value, ecological value, cultural value, social value and scientific research value.

[0003] The dried mature fruits of Ginkgo biloba L. are commonly known as white fruits, and the white fruits are light yellow or orange yellow. The medicinal use of white fruits was first recorded in the book “Yao Yong Ben Cao” by Wu Rui during the reign of Emperor Wenzong of Yuan Dynasty. It is recorded in “Ben Cao Gang Mu” by Li Shizhen that “white fruits, when cooked, can warm the lungs, benefit qi, stop cough, shrink urine and stop leukorrhea. When raw, they can reduce phlegm and kill insects”. It is also recorded in the 2020 edition of “Chinese Pharmacopoeia” that white fruits “have the effects of astringing the lung, stopping asthma, and stopping leukorrhea and shrinking urine”. Modern research has shown that in addition to containing nutrients such as starch, protein, fat, vitamins and minerals, white fruits also contain a variety of active ingredients such as ginkgo terpene lactones, ginkgo flavones and ginkgo polysaccharides, etc., and have functions such as antioxidant, anti-tumor, improving immunity, reducing blood lipids, reducing blood pressure and inhibiting bacteria and killing viruses. In view of the nutritional and medicinal value of ginkgo fruits, the National Health Commission has included ginkgo fruits in the list of “food with medicinal value”.

[0004] However, although ginkgo has high nutritional and medical value, there are still reports of poisoning and even death caused by eating ginkgo. In order to prevent adverse events, only limited consumption of ginkgo can be taken in daily life, such as not more than 6 or 10. This kind of estimation without theoretical basis cannot meet the demand of people for this kind of food with high nutritional and medical value, and greatly limits the development of ginkgo food processing industry, leading to ginkgo oversupply, economic loss of ginkgo farmers and ginkgo processing enterprises, and affecting the healthy development of ginkgo industry.

[0005] To change this situation, it is necessary to identify the toxic components in Ginkgo biloba L. and take appropriate measures to remove them to provide safe raw materials for Ginkgo food processing. Summarizing the research at home and abroad, the toxic components in Ginkgo biloba L. are likely to be cyanogenic glycosides, ginkgolic acids, and 4'-O-methylpyridoxine (MPN, commonly known as ginkgotoxin). Research on toxic substances in Ginkgo biloba L. has first focused on cyanide. Cyanide is a highly toxic chemical substance. When it enters the human body, cyanide ions bind to the ferric iron in the oxidative cytochrome oxidase in the mitochondria of cells, causing cells to be unable to breathe normally, leading to tissue hypoxia, and thus causing the human body to be in a state of internal suffocation. Whether cyanide exists in Ginkgo biloba L. has been a controversial issue. At present, whether cyanide exists in Ginkgo biloba L. still needs to improve the determination method and increase the precision for further confirmation. Ginkgolic acids are urusholic acid compounds, including ginkgo acid, bilobal, and biloban. Ginkgolic acid compounds are found in Ginkgo biloba leaves, Ginkgo biloba outer seed coat, and Ginkgo biloba kernels, with the highest content in the outer seed coat, followed by Ginkgo biloba leaves, and the least in Ginkgo biloba kernels. Current research shows that ginkgolic acids have a strong sensitizing effect rather than a toxic effect. However, because ginkgolic acids have a certain sensitizing effect and are present in high concentrations in Ginkgo biloba leaves, the content of ginkgolic acids in Ginkgo biloba leaf extract (EGB 761, containing 24% flavonoid glycosides and 6% terpene trilactones) is required to be less than 5 ppm. Therefore, both Ginkgo biloba leaf products and Ginkgo biloba fruit products must strictly control the content of ginkgolic acids to ensure the safety of Ginkgo-related products. According to the first record of Ginkgo biloba poisoning, two Japanese boys died from Ginkgo biloba poisoning. The characteristics of Ginkgo biloba poisoning are loss of consciousness, clonic or tonic convulsions, and vomiting. After World War II, when food was in short supply, the first peak of Ginkgo biloba poisoning occurred in the 1940s and 1950s. However, it was not until 1985 that Keiji et al. first isolated ginkgotoxin from Ginkgo biloba by using multi-step separation combined with animal experiments, confirming that it was the main toxin causing acute poisoning symptoms. Keiji et al. further identified the compound as 4'-O-methylpyridoxine (ginkgotoxin, MPN) by high-performance liquid chromatography-mass spectrometry (HPLC-MS) (Keiji, W. et al. Chem Pharm Bull, 1985, 33: 3555-3557; Keiji, W. et al. Chem Pharm Bull, 1988, 36: 1779-1782). In 2000, Scott et al. first detected the glycoside form of MPN, 4'-O-methylpyridoxine-5'-glucoside (ginkgotoxin-5'-glucoside, MPNG), in Ginkgo biloba. MPN and MPNG were detected simultaneously in Ginkgo biloba.In 2011, Kobayashi et al. conducted a toxicity experiment on MPN and MPNG, and the results of the study showed that in the acute toxicity experiment of mice, the lethal dose of MPN was 0.2 mmol / kg, and the lethal dose of MPNG was 0.8 mmol / kg, and the toxicity of MPNG was smaller than that of MPN (Daisuke, K. et al. Food Chemistry, 2011, 126: 1198-1202). At present, MPN and MPNG are considered to be the main toxic substances in ginkgo nuts.

[0006] With the continuous occurrence of ginkgo poisoning events, ginkgo detoxification has always been a research hotspot, and there are many literature reports. The first type is to use chemical reagents such as a certain concentration of sodium carbonate or sodium bicarbonate solution to soak and wash ginkgo in the hope of removing cyanogenic glycosides in ginkgo. The second type is to use organic solvent extraction combined with physical separation methods to remove ginkgo biloba phenolic acid substances, such as the method disclosed in patent CN101785559B. It can be seen that these two methods have limitations in understanding the toxic components of ginkgo, and are not targeted at removing MPN and MPNG. In 2016, patent CN105901476A disclosed a "method for removing ginkgo toxicity based on physical and enzymatic combined method", which took MPN as one of the detoxification objects. However, this method has the following shortcomings: (1) There are still defects in the understanding of the toxic components of ginkgo, and cyanogenic glycosides are taken as one of the detoxification objects, ignoring the important toxic component MPNG; (2) Ginkgo kernels do not naturally contain MPN detoxifying enzymes, and even if there are naturally occurring MPN detoxifying enzymes, the compartmentalization of enzymes in the whole fruit makes it difficult to play a degrading role; (3) The high-temperature treatment in the first and second steps of the disclosed method inactivates the endogenous glycosidase present in ginkgo, preventing the hydrolysis of MPNG to MPN during the sample preparation process before analysis and determination, so the measured MPN content after treatment is reduced. It is not that MPN has been degraded, but MPNG cannot be converted to MPN. In fact, the total amount of MPN and MPNG has not changed (Daisuke, K. et al. Food Chemistry, 2011, 126: 1198-1202), so the purpose of removing MPN and MPNG has not been achieved; (4) The steps are complicated, repeated high-temperature treatment, high energy consumption, manual de-embryos, and low work efficiency. In 2017, patents CN107509997A and CN107568567A respectively disclosed "a method for removing toxic components of ginkgo based on endogenous glycosidase enzymolysis combined with resin adsorption and products prepared therefrom and applications thereof" and "a method for removing toxic components of ginkgo based on membrane separation and products prepared therefrom and applications thereof". Both methods efficiently remove MPN and MPNG, but both methods require ginkgo to be ground into powder or homogenized before detoxification, resulting in the final detoxification product being mainly low-toxicity ginkgo powder. Although ginkgo powder is widely used in ginkgo food, it is still inconvenient to use in many situations, such as deep-fried ginkgo and ginkgo cooking food, etc. Therefore, there is an urgent need to develop ginkgo whole kernel detoxification technology. There is currently no relevant report on ginkgo whole kernel detoxification. MPN and MPNG are easily soluble in water, and compared with ginkgo powder or ginkgo homogenate, it is more difficult to remove MPN and MPNG from whole ginkgo, which requires long-time or high-temperature treatment, especially long-time constant temperature water bath. Due to long-time soaking and high-temperature treatment, the ginkgo epidermis is prone to rupture, resulting in the loss of ginkgo nutritional and functional components.Therefore, it is necessary to solve the problem of broken ginkgo skin while ensuring efficient removal of toxic components of ginkgo. SUMMARY

[0007] The present application provides a method for reducing toxic components of ginkgo kernel without damage, which effectively avoids long-term soaking and high-temperature treatment of ginkgo detoxification, ensuring efficient removal of toxic components of ginkgo kernel while avoiding the problem of broken ginkgo skin.

[0008] Technical scheme: In order to achieve the above-mentioned purpose, the present application provides a method for reducing toxic components of ginkgo kernel without damage, comprising the following steps:

[0009] (1) After removing the middle seed coat and inner seed coat of raw ginkgo, raw ginkgo kernel is obtained;

[0010] (2) Boil the raw ginkgo kernel after adding water to obtain cooked ginkgo kernel;

[0011] (3) Cool the cooked ginkgo kernel and water cooking liquid to room temperature;

[0012] (4) Add α-amylase to the cooked ginkgo kernel and water cooking liquid;

[0013] (5) After adding α-amylase, constant temperature water bath is carried out;

[0014] (6) After water bath, filter out the cooked ginkgo kernel to obtain water bath extraction liquid and detoxified cooked ginkgo kernel;

[0015] (7) Recover MPN and MPNG in the water bath extraction liquid.

[0016] In step (2), the amount of water added during the water adding process is 10-40 times the dry weight of ginkgo kernel.

[0017] As a preferred, the amount of water added is 20 times the dry weight of ginkgo kernel, i.e. the ratio of ginkgo dry weight to water is 1g:20mL.

[0018] In step (2), the boiling time is 5-15 minutes. In step (4), the α-amylase is a high-temperature resistant α-amylase; the addition amount of the high-temperature resistant α-amylase is 40-200U / g of ginkgo dry weight. In step (5), the temperature of constant temperature water bath is 45-85℃, and the water bath time is 6-30 hours.

[0019] The detoxified cooked ginkgo kernel obtained by the present application is used for preparing ginkgo food, medicine, cosmetics and as raw materials or ingredients for daily use.

[0020] The MPN and MPNG obtained by the method have potential application in preparation of drugs, biological pesticides and biological insecticides.

[0021] The prepared attenuated cooked Chinese white chestnut whole kernel is obtained by the method for reducing toxic components of Chinese white chestnut kernel without damage.

[0022] The method uses MPN and MPNG as detoxification objects, and uses the water-soluble characteristics of MPN and MPNG to cook and cool the whole Chinese white chestnut kernel, and then add high-temperature-resistant alpha-amylase enzyme solution to perform constant-temperature water bath treatment. The method effectively reduces the content of MPN and MPNG in Chinese white chestnut, and ensures the integrity of the Chinese white chestnut kernel. The whole detoxification process directly uses deionized water as a solvent without using toxic organic solvents. The process is simple, convenient, safe and efficient.

[0023] The method directly realizes the heating and soaking detoxification of the whole Chinese white chestnut kernel by a specific method. The method not only removes ginkgo poison, but also does not damage the epidermal structure of Chinese white chestnut.

[0024] Advantages: Compared with the prior art, the method has the following advantages:

[0025] (1) The method is based on the water-soluble characteristics of MPN and MPNG, the constant-temperature water bath method is used to realize efficient removal of MPN and MPNG in the whole Chinese white chestnut kernel. The process is simple, and the removal rate of MPN and MPNG is high. The removal rates of MPN and MPNG are 83.34±4.69% and 89.87±1.10%, respectively.

[0026] (2) The method is a method for reducing toxic components of Chinese white chestnut kernel without damage. The final product is a complete Chinese white chestnut kernel with low MPN and MPNG.

[0027] (3) The method uses water bath detoxification. In the detoxification process, no toxic organic solvents are used. There is no multi-step repeated extraction of toxic organic solvents. The process is safe, and the product has no toxic organic solvent residue.

[0028] (4) The product obtained by the method is a Chinese white chestnut kernel with reduced toxic components. The method provides a safe Chinese white chestnut raw material or ingredient for food processing scenes that require whole Chinese white chestnut kernel.

[0029] (5) The purpose of the method is to obtain low-toxic and complete Chinese white chestnut kernel. Current research mainly focuses on detoxification treatment of Chinese white chestnut powder or homogenate. In the method, high-temperature-resistant alpha-amylase is added in the high-temperature water bath process of the whole Chinese white chestnut kernel. The method protects the epidermis of Chinese white chestnut from being damaged, and ensures that more than 80% of MPN and MPNG are removed.

[0030] (6) The present application is simple in operation, and mainly involves a constant temperature water bath pot, and compared with the resin adsorption method and the membrane filtration method, the present application does not produce a large amount of acidic and alkaline waste liquid in the cleaning process, and is more convenient and low in cleaning cost. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 The process flow chart of the present application is shown in the following figure:

[0032] Figure 2 The influence relationship diagram of water bath at different temperatures for a certain time on the removal of MPN, MPNG and TMPN in cooked white ginkgo fruit kernels is shown in the following figure (different lowercase letters represent that the removal rates of MPN, MPNG and TMPN under different treatment conditions are significantly different (P<0.05));

[0033] Figure 3 The appearance diagram of cooked white ginkgo fruit kernels treated by water bath at different temperatures for a certain time is shown in the following figure;

[0034] Figure 4 The influence relationship diagram of the ratio of material to liquid on the removal of MPN, MPNG and TMPN in cooked white ginkgo fruit kernels is shown in the following figure (different lowercase letters represent that the removal rates of MPN, MPNG and TMPN under different ratios of material to liquid are significantly different (P<0.05));

[0035] Figure 5 The appearance diagram of cooked white ginkgo fruit kernels treated by different ratios of material to liquid is shown in the following figure;

[0036] Figure 6 The influence relationship diagram of different concentrations of enzyme liquid on the removal of MPN, MPNG and TMPN in cooked white ginkgo fruit kernels is shown in the following figure (different lowercase letters represent that the removal rates of MPN, MPNG and TMPN after treatment by different concentrations of enzyme liquid are significantly different (P<0.05));

[0037] Figure 7 The appearance diagram of cooked white ginkgo fruit kernels treated by different concentrations of enzyme liquid is shown in the following figure;

[0038] Figure 8 The influence relationship diagram of water bath time on the removal of MPN, MPNG and TMPN in cooked white ginkgo fruit kernels is shown in the following figure (different lowercase letters represent that the removal rates of MPN, MPNG and TMPN under different water bath times are significantly different (P<0.05));

[0039] Figure 9 The appearance diagram of cooked white ginkgo fruit kernels under the optimal conditions after attenuation is shown in the following figure;

[0040] Figure 10Figure of the effect of different temperature and time on detoxification of MPN and, MPNG and TMPN in boiled whole kernel of Chinese white nut without adding α-amylase; (different lower case letters indicate that there is significant difference in removal rate of MPN and, MPNG and TMPN under different treatments (P<0.05));

[0041] Figure 11 Figure of appearance of boiled whole kernel of Chinese white nut after treatment at different temperature and time without adding α-amylase;

[0042] Figure 12 Figure of the effect of different temperature and time on detoxification of MPN and, MPNG and TMPN in boiled whole kernel of Chinese white nut without adding α-amylase; (different lower case letters indicate that there is significant difference in removal rate of MPN and, MPNG and TMPN under different treatments (P<0.05));

[0043] Figure 13 Figure of appearance of boiled whole kernel of Chinese white nut after treatment at different temperature and time without adding α-amylase; DETAILED DESCRIPTION

[0044] The present application is further illustrated below in conjunction with examples.

[0045] The materials, reagents and the like used in the examples can be obtained from commercial channels unless otherwise specified.

[0046] Main reagents and drugs:

[0047] Chinese white nut (large Buddha finger) was purchased from Taixing, Jiangsu.

[0048] The α-amylase used in the examples is commercially available high-temperature resistant α-amylase (food grade), 4000 U / g.

[0049] 1. Determination of MPN and, MPNG and TMPN content

[0050] (1) Test the content of MPN and MPNG in Chinese white nut and extract liquid in examples 1-8 of the present application.

[0051] After the static water bath ended, the ginkgo kernels were removed, 3 mol / L guanidine hydrochloride was added for homogenization, the homogenate was diluted to 100 mL, 10 mL of the homogenate was taken into a 50 mL centrifuge tube, diluted twice, and then the homogenate was placed in a 25°C, 200 rpm shaking table for extraction for 120 min, 2 mL of the homogenate was taken and centrifuged at 12000 rpm for 10 min, the supernatant was filtered through a 0.22 μm filter membrane, and then the MPNG and MPN contents were determined by liquid phase. After the solution in the conical flask was diluted, 2 mL was taken, centrifuged at 12000 rpm for 10 min, filtered through a 0.22 μm filter membrane, and then the MPNG and MPN contents were determined by liquid phase. Finally, the removal rates of MPN and MPNG in the ginkgo kernels and the solution were calculated by the mass of MPN and MPNG.

[0052] (2) Chromatographic conditions: the chromatographic column type was Elite SinoChrom ODS-BP C18 (4.6 x 150 mm, 5 μm); the column temperature was set to 30°C; the mobile phase A was 5 mM sodium pentanesulfonate-potassium phosphate solution (pH adjusted to 2.5 with phosphoric acid), and the mobile phase B was acetonitrile; the flow rate was 1 mL / min; the injection volume was 20 μL; the ultraviolet detection wavelength was 291 nm; and the liquid phase elution conditions are shown in Table 1.

[0053] Table 1 Liquid phase gradient elution conditions

[0054]

[0055] (3) Preparation of a standard curve: 10.0 mg of each of MPN, MPNG, PNP and PN standard substances was accurately weighed, diluted to 100 mL to prepare a 100 μg / mL stock solution, and serially diluted to 10, 8, 6, 4, 2, 1, 0.5, 0.25 and 0.1 μg / mL standard solution, filtered through a 0.22 μm filter membrane, and then detected by liquid phase according to the above liquid phase determination method. The injection amount (μg) was taken as the abscissa (x), and the peak area was taken as the ordinate (y) for linear regression.

[0056] 2, Removal rates of MPN and MPNG and TMPN were calculated, and the results of Examples 1-8 are shown in Table 2.

[0057] MPN removal rate % = c1 x v1 x N1 / (c1 x v1 x N1 + c2 x v2 x N2);

[0058] MPNG removal rate % = c3 x v1 x N1 / (c3 x v1 x N1 + c4 x v2 x N2);

[0059] TMPN removal rate % = c5 x v1 x N1 / (c5 x v1 x N1 + c6 x v2 x N2);

[0060] Wherein, c1 is the concentration of MPN in the removal liquid (μg / mL); v1 is the volume of the removal liquid; N1 is the dilution multiple; c2 is the concentration of MPN in the ginkgo pulp liquid (μg / mL); v2 is the volume of the ginkgo pulp; N2 is the dilution multiple; c3 is the concentration of MPNG in the removal liquid (μg / mL); c4 is the concentration of MPNG in the ginkgo pulp liquid (μg / mL); c5 is the concentration of TMPN in the removal liquid (μg / mL); and c6 is the concentration of TMPN in the ginkgo pulp liquid (μg / mL).

[0061] Both MPN and MPNG exist in ginkgo, and MPN is mainly in raw ginkgo, while MPNG is mainly in boiled ginkgo. In order to consider the removal of MPN and MPNG, the removal rate of TMPN (the total of MPN and MPNG after conversion) is used as the index in the experiment. In addition, the specific indexes of different batches of experiments may have certain differences, but the direction of each index is clear.

[0062] 3. Calculation of the ginkgo kernel epidermis damage rate

[0063] Ginkgo kernel epidermis damage rate = the number of damaged ginkgo kernels / the total number of ginkgo kernels in the experiment * 100%

[0064] Note: None: the damage rate is 0; very little: 0 < the damage rate ≤ 10%; little: 10 < the damage rate ≤ 20%; much: 20% < the damage rate ≤ 40%; very much: the damage rate > 40%.

[0065] Example 1

[0066] (1) After removing the seed coat and inner seed coat of raw ginkgo, raw ginkgo whole kernels are obtained;

[0067] (2) 10 g of raw ginkgo whole kernels (dry weight 5 g or so) are placed in a conical flask, 50 mL of ultrapure water is added, and then boiled for 10 min to obtain boiled ginkgo whole kernels;

[0068] (3) The boiled ginkgo whole kernels and water in the conical flask are cooled to room temperature;

[0069] (4) 200 U / g of ginkgo dry weight of α-amylase is added to the conical flask, and the volume is made up to 50 mL;

[0070] (5) The conical flask is placed in a 45℃ constant temperature water bath, and the enzyme hydrolysis is carried out for 24 hours;

[0071] (6) After the water bath is completed, the boiled ginkgo whole kernels are filtered out to obtain the water bath extraction liquid and attenuated boiled ginkgo whole kernels;

[0072] (7) Determination of the contents of MPN and MPNG in the water bath extraction solution and the whole cooked Chinese white fruit kernel after water bath;

[0073] (8) Recovery of MPN and MPNG in the water bath extraction solution.

[0074] The process flow of the present example is shown in Figure 1 .

[0075] Example 2

[0076] Example 2 is the same as the method of Example 1, except that step (5) is placed in a constant temperature water bath at 55°C and water bath enzymolysis is carried out for 24 hours.

[0077] Example 3

[0078] Example 3 is the same as the method of Example 1, except that step (5) is placed in a constant temperature water bath at 65°C and water bath enzymolysis is carried out for 12 hours.

[0079] Example 4

[0080] Example 4 is the same as the method of Example 1, except that step (5) is placed in a constant temperature water bath at 75°C and water bath enzymolysis is carried out for 6 hours.

[0081] Example 5

[0082] Example 5 is the same as the method of Example 1, except that step (5) is placed in a constant temperature water bath at 85°C and water bath enzymolysis is carried out for 4 hours.

[0083] Example 6

[0084] Example 6 is the same as the method of Example 1, except that step (1) takes 4 conical flasks, respectively adds 10 g of raw Chinese white fruit whole kernel (dry weight of about 5 g) and then respectively adds 50, 100, 150 and 200 mL of ultrapure water, then boils for 10 min to obtain cooked Chinese white fruit kernel; step (4) adds high temperature α-amylase (200 U / g of white fruit dry weight) and then respectively supplements the solution volume to 50, 100, 150 and 200 mL; and step (5) places the conical flasks in a constant temperature water bath at a temperature of 55°C and water bath for 24 hours.

[0085] Example 7

[0086] Example 7 is the same as the method of Example 1, except that step (4) adds high temperature α-amylase to the conical flasks in an amount of 40, 80, 120, 160 and 200 U / g of white fruit dry weight, respectively, and supplements the volume to 100 mL; and step (5) is carried out in a constant temperature water bath at a temperature of 55°C for 24 hours.

[0087] Example 8

[0088] Example 8 is the same as Example 1, except that: in step (4), 120 U / g of high-temperature α-amylase is added to the conical flask and the volume is made up to 100 mL; in step (5), the temperature of the constant temperature water bath is 55°C and the water bath time is 6, 12, 21, 24, 27 and 30 hours respectively.

[0089] The removal of MPN and MPNG from whole roasted ginkgo kernels by different embodiments described above was investigated, and the results are shown in Table 2 below.

[0090] Table 2. Removal of MPN and MPNG from whole ripe ginkgo kernels.

[0091]

[0092]

[0093]

[0094] Note: Different lowercase letters indicate that there are significant differences in the removal rates of MPN, MPNG and TMPN under different treatment conditions (P < 0.05), and the same applies below.

[0095] During a prolonged high-temperature water bath, the outer skin of whole roasted ginkgo kernels may crack. This invention, while ensuring the removal of MPN and MPNG, preserves the integrity of the ginkgo kernel outer skin through enzymatic hydrolysis by α-amylase at high temperature. (See Table 2 and...) Figures 2-8 It can be seen that under different water bath temperatures, different material-to-liquid ratios, the addition of high-temperature α-amylase with a certain enzyme activity, and different water bath times, the removal rates of MPN, MPNG, and TMPN in whole roasted ginkgo kernels are different, and the degree of damage to the kernel skin also varies. The ginkgo kernels crack earlier with increasing temperature. For example, at 45℃, the ginkgo skin did not crack; at 50℃, the cracking time was 24 hours; when the temperature increased to 65, 75, and 85℃, the cracking time was 12, 6, and 4 hours, respectively. Figure 3 (As shown). Furthermore, from Figure 2 It can be seen that the MPNG removal rate was highest under the treatment conditions of 55℃-24h; and the MPN removal rate was highest under the treatment conditions of 65℃-12h. High-temperature treatment can improve the dissolution of MPN and MPNG, but excessively high temperatures can easily cause the outer skin of the ginkgo kernel to crack. Figure 3 (As shown). The effect of the material-to-liquid ratio (dry weight of ginkgo:water) on the removal of MPN, MPNG, and TMPN is as follows. Figure 4 As shown. From Figure 4 It can be seen that when the material-to-liquid ratio is 1:30 (g / mL), the removal of MPN, MPNG, and TMPN is at its maximum, but from...Figure 5 It can be found that a large number of ginkgo skin has obvious rupture when the ratio of material to liquid is 1:30 (g / mL). Considering the removal rates of MPN, MPNG and TMPN and the integrity of ginkgo skin, the ratio of material to liquid is 1:20 (g / mL) when the effect is the best. From the above, Figure 6 and Figure 7 It can be seen that the addition of a certain enzyme activity of high-temperature alpha-amylase can protect the ginkgo kernel skin to a certain extent, ensuring the integrity of the kernel skin. From the above, Figure 6 It can be seen that the addition of enzyme has no significant effect on the removal rate of MPNG when the addition amount of enzyme is 40-160 (U / g ginkgo dry weight). The toxic substance in cooked ginkgo is mainly MPNG, so the addition amount of enzyme has no significant effect on the removal rate of total toxic substance. But from the above, Figure 7 It can be seen that the addition amount of enzyme has a significant effect on the integrity of ginkgo skin. With the increase of the addition amount of enzyme, the integrity of ginkgo kernel skin is better maintained. From the above, the addition amount of enzyme is 120 U / g when the ginkgo skin is basically intact and the cost is saved. Figure 8 is the effect of water bath time on the removal rates of MPN, MPNG and TMPN under the optimal ratio of material to liquid, temperature and enzyme addition amount. From the above, Figure 8 It can be seen that with the extension of water bath time, the removal rate of MPNG shows an increasing trend within the first 21 h, while there is no significant difference in the removal rate of MPNG from 21 h to 30 h; the removal rate of MPN shows a significant increasing trend from 6 h to 27 h, while there is no significant difference at 27 h and 30 h. The removal rate of TMPN increases first and then shows a stable trend, and there is no significant difference in the removal rate of TMPN at 24 h, 27 h and 30 h. Considering the removal rate of TMPN, the saving of time and energy cost, the present application selects 24 h as the optimal water bath time. Figure 9 The ginkgo kernel skin is smooth and intact without obvious rupture after the whole ginkgo kernel is treated under the optimal water bath condition. In summary, the removal rates of MPN, MPNG and TMPN of the whole ginkgo kernel under the optimal conditions (55℃, the ratio of material to liquid is 1:20 (g / mL), the addition amount of enzyme is 120 U / g ginkgo dry weight, and the water bath time is 24 h) are 83.34±2.35%, 89.87±1.10% and 88.03±0.52%, respectively.

[0096] The present application first uses different temperatures for water bath detoxification, effectively reduces the MPN and MPNG in cooked ginkgo whole kernel, and the process operation is simple, only uses water and heat treatment, has high safety, and the obtained detoxified raw or cooked ginkgo whole kernel can be used as raw material of various ginkgo foods, and is particularly suitable for ginkgo foods which need to use whole kernel for cooking. At the same time, the ginkgo detoxification uses water as the extraction agent, and there is no organic solvent residue in the detoxified ginkgo, which is convenient for subsequent processing and use.

[0097] Test Example 1

[0098] Test Example 1 was carried out according to the following experimental procedures, and the results are shown in Table 3:

[0099] (1) After removing the middle and inner seed coats of raw ginkgo, raw ginkgo whole kernels were obtained;

[0100] (2) 10 g of raw ginkgo whole kernels (dry weight about 5 g) were placed in a conical flask, 50 mL of ultrapure water was added, and then boiled for 10 min to obtain boiled ginkgo whole kernels;

[0101] (3) The boiled ginkgo whole kernels and water in the conical flask were cooled to room temperature;

[0102] (4) The volume of the conical flask was made up to 50 mL;

[0103] (5) The conical flask was placed in a water bath at different temperatures for static water bath;

[0104] (6) After different time of static water bath, the boiled ginkgo whole kernels were filtered out to obtain water bath extraction liquid and attenuated boiled ginkgo whole kernels;

[0105] (7) The contents of MPN and MPNG in the water bath extraction liquid and the boiled ginkgo whole kernels after water bath were determined;

[0106] (8) The MPN and MPNG in the water bath extraction liquid were recovered.

[0107] Table 3 Effect of temperature on removal of MPN and MPNG from boiled ginkgo whole kernels (without adding α-amylase)

[0108]

[0109] In combination with the results of Test Example 1 in Table 3 and Figures 10-11 It can be found that, compared with Examples 1-5, from the number and degree of damage to the kernel skin, it can be seen that the boiled ginkgo kernels are more prone to skin damage in a short time during the constant temperature water bath without adding enzymes. For example, in Example 3 with the addition of α-amylase, the skin damage is very small after 12 h of water bath at 65°C, while in Test Example 1 without the addition of α-amylase, the number of skin damage is already large after 3 h of water bath. Since the water bath time in Test Example 1 without the addition of α-amylase is shorter than that in Examples 1-5, the removal rates of MPN, MPNG and TMPN are also lower than those in the experimental group with the addition of α-amylase at the same temperature. The results show that in Test Example without the addition of α-amylase, the ginkgo skin is more prone to damage during the constant temperature water bath, and a large amount of ginkgo skin will be damaged after 8 h of water bath at 55°C without the addition of amylase.

[0110] Test Example 2

[0111] Test Example 2 was carried out according to the following experimental procedures, and the results are shown in Table 4:

[0112] (1) After removing the middle and inner seed coats of raw ginkgo, raw ginkgo whole kernels were obtained;

[0113] (2) 10 g of raw ginkgo whole kernels (dry weight about 5 g) were placed in a conical flask, and 50, 100, 150 and 200 mL of ultrapure water were added respectively, and then boiled for 10 min to obtain boiled ginkgo whole kernels;

[0114] (3) The boiled ginkgo whole kernels and water in the conical flask were cooled to room temperature;

[0115] (4) The volume of the conical flask was supplemented to 50, 100, 150 and 200 mL respectively;

[0116] (5) The conical flask was placed in a 55°C water bath for static water bath;

[0117] (6) After 24 h of static water bath, the boiled ginkgo whole kernels were filtered out to obtain water bath extraction liquid and attenuated boiled ginkgo whole kernels;

[0118] (7) The contents of MPN and MPNG in the water bath extraction liquid and the boiled ginkgo whole kernels after water bath were determined;

[0119] (8) The MPN and MPNG in the water bath extraction liquid were recovered.

[0120] Table 4 Effect of solid-liquid ratio on removal of MPN and MPNG in boiled ginkgo whole kernels (without adding α-amylase)

[0121]

[0122]

[0123] By comparing Test Example 2 with Example 6 (as shown in Figure 4 , Figure 5 , Figure 12 and Figure 13 ), for the case of damage to the kernel epidermis, after 24 h of water bath at the same temperature of 55°C, the ginkgo epidermis was severely damaged (as shown in Figure 13 ) in Test Example 2 without adding α-amylase, while in Example 6 with the addition of a certain amount of α-amylase, the ginkgo epidermis remained basically intact (as shown in Figure 5 ) within a solid-liquid ratio of 1:30 (g / mL). Under the optimal condition of adding enzyme, when the solid-liquid ratio was 1:20 g / mL, there was no damage to the ginkgo epidermis, while in Test Example 2 without adding α-amylase, there were a large number of damaged ginkgo. This indicates the important role of adding α-amylase, i.e. to ensure that the ginkgo epidermis does not appear damaged during the process of high-temperature long-time water bath.

[0124] From the removal of MPN, MPNG and TMPN, it can be seen that both Test Example 2 and Example 6 achieved removal rates of 75.73% or more. At the optimal material to liquid ratio of 1:20 (g / mL), the removal rate of MPNG in Test Example 2 without the addition of alpha-amylase was slightly higher than that of Example 6, which can be because the damage to the white ginkgo fruit skin facilitated the dissolution of MPNG, and the content of MPN was not detected in Test 2, so the removal rate of MPN was 100%. The removal rate of TMPN in Test 2 compared with Example 6 was not significantly different. The purpose of the present application is to obtain intact low-toxicity white ginkgo kernels. Therefore, one important role of adding alpha-amylase is to protect the integrity of the white ginkgo kernel skin, while also not substantially affecting the removal efficiency of MPN and MPNG.

Claims

1. A method for lossless reduction of toxic components in ginkgo nut kernel, characterized in that, It comprises the following steps: (1) removing the middle seed coat and inner seed coat of raw ginkgo, and obtaining raw ginkgo whole kernel; (2) boiling the raw ginkgo whole kernel after adding water, and obtaining boiled ginkgo whole kernel; (3) cooling the boiled ginkgo whole kernel and water boiling liquid to room temperature; (4) adding α-amylase to the boiled ginkgo whole kernel and water boiling liquid; (5) adding α-amylase and performing constant temperature water bath; (6) after the water bath, filtering out the cooked ginkgo whole kernel to obtain water bath extraction liquid and attenuated cooked ginkgo whole kernel; (7) recovering MPN and MPNG in the water bath extraction liquid; In step (4), the addition amount of α-amylase is 40-200 U / g of ginkgo dry weight; in step (5), the temperature of constant temperature water bath is 45-85 ℃; and the constant temperature water bath time is 6-30 hours.

2. The method according to claim 1, wherein the white ginkgo toxin component is reduced without damage. In step (2), the water added in the water adding process is 10-40 times of the dry weight of ginkgo kernel.

3. The method of claim 1, wherein the white camphor toxin component is reduced without damage. In step (2), the boiling time is 5-15 minutes.

4. The method of claim 1, wherein the white camellia nut toxic principle is reduced without loss. In step (4), the α-amylase is high-temperature-resistant α-amylase.

5. An attenuated cooked ginkgo whole kernel prepared by the method for lossless reduction of ginkgo toxic components according to claim 1.

6. Application of the attenuated cooked ginkgo whole kernel obtained by the method for lossless reduction of ginkgo toxic components according to claim 1 in the preparation of ginkgo food, medicine, cosmetics and as raw materials or ingredients for daily use.

Images