Baijiu in-vitro simulated digestion method and application thereof

Abstract

The invention belongs to the technical field of wine brewing, and relates to an in-vitro simulated digestion method of baijiu and application thereof, the method comprises the following steps: mixing a baijiu sample, simulated saliva, an enzyme activator and salivary amylase with activity of 45-70U / mL to simulate oral digestion for 1.5-4.5 seconds to obtain oral digestion endpoint liquid; mixing the oral digestion endpoint liquid with simulated gastric juice, an enzyme activator, pepsase with the activity of 2000-2800U / mL and gastric lipase with the activity of 45-55U / mL, and digesting for 1.5-2.5 hours to obtain gastric digestion endpoint liquid; the gastric peristalsis frequency is 165-235 times / h, and the gastric emptying rate is 80-90 mL / h; mixing the gastric digestion endpoint liquid with simulated intestinal juice and an enzyme activator, and adding pancreatin with the activity of 110-190U / mL and bile acid with the concentration of 11-19mM to obtain small intestine digestion endpoint liquid; the intestinal peristalsis frequency is 560-670 times / h, the intestinal digestion time is 0.6-2.9 h, and the intestinal emptying rate is 51-59 mL / h. The dynamic process of digestion of Baijiu in a human body is simulated, simulated digestion parameters of digestion stages of an oral cavity, a stomach and a small intestine are optimized and simulated, and the digestion, metabolism and absorption conditions of the human body after Baijiu drinking are systematically reflected.

Description

Technical Field

[0001] The present invention belongs to the technical field of brewing, and relates to an in vitro simulated digestion method for liquor and its application. Background Art

[0002] How food exerts its health effects in the human body depends to a large extent on the absorption and utilization of food during gastrointestinal digestion. Animal and human clinical trials can more truly reflect the digestion and influence of food components in the human body. However, these methods have problems such as high cost, long cycle, large individual differences, and ethical and moral issues. In contrast, in vitro simulated digestion has many advantages such as low cost, easy evaluation, and good reproducibility.

[0003] According to different digestion forms, in vitro digestion is divided into static in vitro digestion and dynamic in vitro digestion. The static in vitro digestion model can effectively predict the digestion results in vivo, but it cannot study the digestion kinetics of complex food components and the relationship between the body and nutrient absorption. Therefore, static digestion is often used for screening research for mechanical purposes.

[0004] With the continuous progress of science and technology, more and more dynamic in vitro digestion models have emerged, such as the DGM (Dynamic Gastric Model) dynamic gastric model developed by the Norwich Institute in the UK, the HGS (Human Gastric Simulator) human gastric simulator developed by Kong and Singh, the DIDGI (The Dynamic Gastrointestinal Digester) developed by the French National Institute for Agricultural Research, the TIM (The TNO gastro-intestinal model) developed by the TNO Nutrition and Food Research Institute in the Netherlands, the SIMGI (The simulator of the gastrointestinal tract) developed by the Institute of Food Science in Madrid, Spain, etc. The Chinese Journal of Food Science and Technology, Vol. 23, No. 7, discloses a fully automatic device for simulating human gastrointestinal digestion and fermentation, and establishes the basis for constructing the simulation device according to the physiological environment characteristics of the human gastrointestinal tract from the oral cavity to the stomach, small intestine, and large intestine.

[0005] Liquor is one of the most widely consumed foods in China and is also an important element of traditional Chinese culture. There is a modern academic view that "moderate drinking is beneficial to physical and mental health", but what the healthy components are has always been an unsolved mystery. With the continuous development of modern detection technologies, more and more trace components in liquor have been successively detected, but the research on the in vitro digestion of liquor is not yet perfect, and the digestion, metabolism, and absorption mechanism of its trace components in the body after drinking are still unclear. Summary of the Invention

[0006] The object of the present invention is to provide an in vitro simulated digestion method for baijiu and its application. By using baijiu as a sample to simulate the kinetic process of baijiu digestion in the human body, and simultaneously optimizing the simulated digestion parameters such as enzyme activities and digestion times in the simulated oral, gastric, and small intestine digestion stages, it can systematically reflect the human digestion, metabolism, and absorption conditions after drinking baijiu, deepen the understanding of the digestion properties of baijiu, and provide support for the scientific development of the baijiu industry.

[0007] In the first aspect, the present invention provides an in vitro simulated digestion method for baijiu, comprising the following steps:

[0008] Step 1, Simulating oral digestion: Mix the baijiu sample, simulated saliva, and enzyme activator, adjust the pH to 6.5 - 7.5, add salivary amylase, and perform simulated oral digestion to obtain the end solution of oral digestion; the activity of the salivary amylase is 45 - 70 U / mL, and the time for simulated oral digestion is 1.5 - 4.5 seconds;

[0009] Step 2, Simulating gastric digestion: Mix the end solution of oral digestion with simulated gastric juice and enzyme activator, adjust the pH to 2.6 - 3.5, add pepsin and gastric lipase, and perform simulated gastric digestion to obtain the end solution of gastric digestion; the gastric peristalsis frequency for the simulated gastric digestion is 165 - 235 times / hour, the gastric digestion time is 1.5 - 2.5 hours, the gastric emptying rate is 80 - 90 mL / hour, the activity of the pepsin is 2000 - 2800 U / mL, and the activity of the gastric lipase is 45 - 55 U / mL;

[0010] Step 3, Simulating small intestine digestion: Mix the end solution of gastric digestion with simulated intestinal fluid and enzyme activator, adjust the pH to 6.5 - 7.5, add pancreatic enzymes and bile acids, and perform simulated small intestine digestion to obtain the end solution of small intestine digestion; the activity of the pancreatic enzymes is 110 - 190 U / mL, the concentration of bile acids is 11 - 19 mM, the peristalsis frequency of the small intestine is 560 - 670 times / hour, the digestion duration of the small intestine is 0.6 - 2.9 h, and the emptying rate of the small intestine is 51 - 59 mL / hour.

[0011] In some embodiments, the activity of the salivary amylase is 45 - 55 U / mL, and the time for simulated oral digestion is 2.8 - 3.3 seconds; the gastric peristalsis frequency for the simulated gastric digestion is 190 - 210 times / hour, the gastric digestion time is 1.8 - 2.2 hours, the gastric emptying rate is 82 - 88 mL / hour, the activity of the pepsin is 2400 - 2600 U / mL, and the activity of the gastric lipase is 48 - 52 U / mL; the activity of the pancreatic enzymes is 140 - 160 U / mL, the concentration of bile acids is 13 - 17 mM, the peristalsis frequency of the small intestine is 600 - 620 times / hour, the digestion duration of the small intestine is 0.8 - 1.2 h, and the emptying rate of the small intestine is 53 - 58 mL / hour.

[0012] In some embodiments, the enzyme activator is a CaCl2·2H2O solution, and the concentration of the CaCl2·2H2O solution is 40-50 g / L.

[0013] In some embodiments, the concentration of the CaCl2·2H2O solution is 42-47 g / L.

[0014] In some embodiments, the volume ratio of the liquor sample, simulated saliva, and salivary amylase activator is 80-120:70-90:0.3-1.

[0015] In some embodiments, the simulated saliva includes a KCl solution, a KH2PO4 solution, a NaHCO3 solution, a MgCl2(H2O)6 solution, and an (NH4)2CO3 solution.

[0016] In some embodiments, the volume of the KCl solution is 15-30 mL, the concentration is 35-40 g / L, the volume of the KH2PO4 solution is 3-10 mL, the concentration is 65-70 g / L, the volume of the NaHCO3 solution is 5-10 mL, the concentration is 80-90 g / L, the volume of the MgCl2(H2O)6 solution is 0.5-1 mL, the concentration is 30-35 g / L, and the volume of the (NH4)2CO3 solution is 0.05-0.1 mL, the concentration is 45-50 g / L.

[0017] In some embodiments, the simulated gastric juice includes a KCl solution, a KH2PO4 solution, a NaHCO3 solution, a NaCl solution, and an (NH4)2CO3 solution.

[0018] In some embodiments, the volume of the KCl solution is 5-10 mL, the concentration is 35-40 g / L, the volume of the KH2PO4 solution is 1-2 mL, the concentration is 65-70 g / L, the volume of the NaHCO3 solution is 10-20 mL, the concentration is 80-90 g / L, the volume of the NaCl solution is 10-20 mL, the concentration is 110-120 g / L, and the volume of the (NH4)2CO3 solution is 0.5-1.0 mL, the concentration is 45-50 g / L.

[0019] In some embodiments, the simulated intestinal fluid includes a KCl solution, a KH2PO4 solution, a NaHCO3 solution, a NaCl solution, and a MgCl2(H2O)6 solution.

[0020] In some embodiments, the volume of the KCl solution is 5 - 10 mL, the concentration is 35 - 40 g / L, the volume of the KH2PO4 solution is 0.5 - 1.5 mL, the concentration is 65 - 70 g / L, the volume of the NaHCO3 solution is 50 - 100 mL, the concentration is 80 - 90 g / L, the volume of the NaCl solution is 20 - 30 mL, and the concentration is 110 - 120 g / L. The volume of the MgCl2(H2O)6 solution is 2.5 - 3.0 mL, and the concentration is 25 - 35 g / L.

[0021] In some embodiments, the alcohol content of the baijiu sample is 50 - 55%.

[0022] In some embodiments, the pancreatin includes trypsin and / or lipase.

[0023] In a second aspect, the present invention provides an application of the in vitro simulated digestion method described in any one of the above in reflecting the in vivo digestion, metabolism, and absorption of baijiu components.

[0024] In summary, the present application includes at least one of the following beneficial technical effects:

[0025] By using baijiu as a sample to simulate the kinetic process of baijiu digestion in the human body and simultaneously optimizing simulated digestion parameters such as enzyme activity and digestion time in the simulated oral, gastric, and small intestine digestion stages, it is possible to systematically reflect the in vivo digestion, metabolism, and absorption of baijiu after drinking, deepen the understanding of the digestive properties of baijiu, and provide support for the scientific development of the baijiu industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a comparative diagram of the effects of different salivary amylase activities in the simulated oral digestion stage in Example 1 of the present invention;

[0027] Figure 2 It is a comparative diagram of the effects of different digestion times in the simulated oral digestion stage in Example 1 of the present invention;

[0028] Figure 3 It is a comparative diagram of the effects of different gastric peristalsis frequencies in the simulated gastric digestion stage in Example 1 of the present invention;

[0029] Figure 4 It is a comparative diagram of the effects of different pepsin activities in the simulated gastric digestion stage in Example 1 of the present invention;

[0030] Figure 5 It is a comparative diagram of the effects of different gastric lipase activities in the simulated gastric digestion stage in Example 1 of the present invention;

[0031] Figure 6 It is a comparative diagram of the effects of different gastric pH values in the simulated gastric digestion stage in Example 1 of the present invention;

[0032] Figure 7 This is the comparison chart of the effects of different gastric digestion times in the simulated gastric digestion stage of Example 1 of the present invention;

[0033] Figure 8 This is the comparison chart of the effects of different gastric emptying rates in the simulated gastric digestion stage of Example 1 of the present invention;

[0034] Figure 9 This is the comparison chart of the effects of different small intestine emptying rates in the simulated small intestine digestion stage of Example 1 of the present invention;

[0035] Figure 10 This is the comparison chart of the effects of different bile acid concentrations in the simulated small intestine digestion stage of Example 1 of the present invention;

[0036] Figure 11 This is the comparison chart of the effects of different pancreatic enzyme activity rates in the simulated small intestine digestion stage of Example 1 of the present invention;

[0037] Figure 12 This is the comparison chart of the effects of different small intestine pH values in the simulated small intestine digestion stage of Example 1 of the present invention;

[0038] Figure 13 This is the comparison chart of the effects of different small intestine digestion durations in the simulated small intestine digestion stage of Example 1 of the present invention;

[0039] Figure 14 This is the comparison chart of the effects of different small intestine peristalsis frequencies in the simulated small intestine digestion stage of Example 1 of the present invention;

[0040] Figure 15 This is the chart of the number of digestion products at different stages of Baijiu sample 1 in Example 2 of the present invention;

[0041] Figure 16 This is the chart of the number of digestion products at different stages of Baijiu sample 2 in Example 3 of the present invention. Detailed implementation manners

[0042] The technical solution of the present invention is further described below through specific embodiments. The specific embodiments do not represent a limitation on the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0043] Example 1 explores the optimal parameters for in vitro simulated digestion of simulated Baijiu

[0044] Based on the physiological processes of the human gastrointestinal tract, an in vitro model is used to simulate the digestive fluid environments of the human oral cavity, stomach, and intestine. Among them, the electrolyte stock solution concentrations of the initial simulated salivary fluid (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) are all 1.25 times. Mix them with the necessary amounts of enzyme solution, CaCl2·2H2O solution, HCl / NaOH, and water to obtain a working solution diluted to 1-fold ion concentration, which is consistent with the electrolyte concentration recommended based on in vivo human data. After preparation, it is stored at 4°C. Before use, it is preheated to 37°C. The components and dosages of the 1.25-fold electrolyte stock solution are shown in Table 1 below.

[0045] Table 1 Components and Dosages for Preparing 1 L of 1.25-Fold Electrolyte Stock Solution

[0046]

[0047] As shown in Table 2 below, control groups numbered 1 - 14 are set, and the variable for each group is a different digestion parameter. For example, the variable for Group 1 is the activity of salivary amylase, including four digestion parameters: 50 U / mL, 75 U / mL, 100 U / mL, and 125 U / mL.

[0048] Table 2 Digestion Parameters for Control Groups 1 - 14

[0049]

[0050]

[0051]

[0052]

[0053] 1. Simulate the oral digestion phase

[0054] Pump 50 mL of liquor sample 1 (alcohol content: 53%) into the oral digestion tank for control groups 1 - 14. At the same time, pump in 40 mL of SSF and 250 μL of a 44.1 g / L calcium chloride dihydrate (CaCl₂·2H₂O) solution. Use 1 M HCl or NaOH to adjust the pH of the mixed system to 7. For group 1, add 3.3 mL, 5 mL, 6.7 mL, and 8.3 mL of α - amylase solution (1500 U / mL, dissolved in SSF) respectively. For groups 2 - 14, add 5 mL of α - amylase solution (1500 U / mL, dissolved in SSF). Finally, add distilled water to make the volume up to 100 mL, so that the α - amylase activity in the final system of group 1 is 50 U / mL, 75 U / mL, 100 U / mL, 125 U / mL, and the α - amylase activity in the final system of groups 2 - 14 is 75 U / mL. Collect partial samples from groups 1 and 2 and store them temporarily in a 4°C refrigerator. This part of the samples is the starting point of oral - stage digestion. Set the temperature of the oral digestion tank to 37°C, stir in the dark. For groups 2, oscillate for 1 s, 3 s, and 5 s respectively. For group 1 and groups 3 - 14, oscillate for 2 min. After the simulated oral digestion period ends, collect all samples from groups 1 and 2 and store them in a 4°C refrigerator to obtain the oral digestion end - point fluid of groups 1 - 2.

[0055] 2. Simulate the gastric digestion period

[0056] Immediately drain the digestive fluids of groups 3 - 14 into the gastric digestion tank, and at the same time, pump in 80 mL of SGF and 50 μL of a 44.1 g / L CaCl₂·2H₂O solution.

[0057] For group 3, use 1 M HCl to adjust the pH to 3, add 5 mL of pepsin solution (80000 U / mL, dissolved in SGF), and 1200 μL of gastric lipase solution (10000 U / mL, dissolved in SGF). Finally, add distilled water to make the final volume 200 mL, so that the pepsin activity in the final system of group 3 is 2000 U / mL and the gastric lipase activity is 60 U / mL. Collect partial samples from group 3 and store them temporarily in a 4°C refrigerator. This part of the samples is the starting point of variable digestion for group 3. Set the temperature of the gastric digestion tank to 37°C, stir in the dark for 2 h. The stirring rates of group 3 are 120 times / h, 160 times / h, 200 times / h, and 240 times / h respectively. Use a digestive pump to drain the digestive fluid of group 3 at a discharge rate of 85 mL / h and collect it immediately to obtain the gastric digestion end - point fluid of group 3.

[0058] For group 4, add 3.8 mL, 5 mL, 6.25 mL, and 7.5 mL of pepsin solution (80000 U / mL, dissolved in SGF) respectively, so that the pepsin activity in the final system of group 4 is 1500 U / mL, 2000 U / mL, 2500 U / mL, and 3000 U / mL respectively. Set the stirring rate to 240 times / h, and keep other conditions the same as those of group 3 to obtain the gastric digestion end - point fluid of group 4.

[0059] Five groups were respectively added with 1000 μL, 1200 μL, 1400 μL, 1600 μL of gastric lipase solution (10000 U / mL, dissolved in SGF) to make the gastric lipase activities in the final systems of the four groups be 50 U / mL, 60 U / mL, 70 U / mL, 80 U / mL respectively. Then 5 mL of pepsin solution (80000 U / mL, dissolved in SGF) was added to make the pepsin activities in the final systems of the three groups be 2000 U / mL. Other conditions were the same as those of the four groups, and the gastric digestion end solutions of the five groups were obtained.

[0060] Six groups were respectively adjusted to pH values of 1, 1.5, 2, 2.5, 3 with 1 M HCl, and 1200 μL of gastric lipase solution (10000 U / mL, dissolved in SGF) was added to make the gastric lipase activity in the final system be 60 U / mL. Other conditions were the same as those of the five groups, and the gastric digestion end solutions of the six groups were obtained.

[0061] Seven groups were respectively stirred in the dark for 0.5 h, 1 h, 2 h, and adjusted to pH 3 with 1 M HCl. Other conditions were the same as those of the six groups, and the gastric digestion end solutions of the seven groups were obtained.

[0062] Eight groups were respectively drained the digestive fluids at the emptying rates of 55 mL / h, 65 mL / h, 75 mL / h, 85 mL / h with a digestive pump and stirred in the dark for 2 h. Other conditions were the same as those of the seven groups, and the gastric digestion end solutions of the eight groups were obtained.

[0063] Groups 9 - 14 were added with 5 mL of pepsin solution (80000 U / mL, dissolved in SGF) and 1200 μL of gastric lipase solution (10000 U / mL, dissolved in SGF), and finally distilled water was added to make the final volume be 200 mL to make the pepsin activity in the final system be 2000 U / mL and the gastric lipase activity be 60 U / mL. The temperature of the gastric digestion tank was set at 37 °C, stirred in the dark for 2 h, the stirring rate was 240 times / h, and the digestive fluid was drained at the emptying rate of 85 mL / h with a digestive pump and immediately collected, and the gastric digestion end solutions of groups 9 - 14 were obtained.

[0064] 3. Simulating the small intestine digestion period

[0065] The gastric digestion end solutions of groups 9 - 14 were pumped into the small intestine digestion tank. After gastric digestion, immediately 85 mL of SIF and 400 μL of 44.1 g / L CaCl₂·2H₂O solution were pumped into the digestive fluids of groups 9 - 14.

[0066] For 9 groups, adjust the pH of the mixed sample to 7 with 1 M NaOH, add 50 mL of trypsin solution (based on protease: 800 U / mL, dissolved in SIF), 25 mL of bile (38.4 mg / mL), and finally add distilled water to make the final volume 400 mL, so that the final system has a trypsin activity of 100 U / mL and a bile concentration of 2.4 mg / mL. Collect partial samples from 9 groups and store them temporarily in a refrigerator at 4°C. This part of the sample is the starting point of digestion for the 9-group variable. Set the temperature of the digestion tank to 37°C, stir in the dark for 2 h, with a stirring rate of 610 times / h, and use a digestion pump to discharge the digestive juice at emptying rates of 45 mL / h, 50 mL / h, 55 mL / h, and 60 mL / h respectively, and immediately collect it at low temperature to obtain the small intestine digestion end-point liquid of 9 groups.

[0067] For 10 groups, add bile respectively to make the final system bile concentration 5 mM, 10 mM, 15 mM, 20 mM, use a digestion pump to discharge the digestive juice at an emptying rate of 60 mL / h, and keep other conditions the same as those of 9 groups to obtain the small intestine digestion end-point liquid of 10 groups.

[0068] For 11 groups, add trypsin solution respectively to make the final system trypsin solution activity 50 U / mL, 100 U / mL, 150 U / mL, 200 U / mL, add bile to make the final system bile concentration 2.4 mg / mL, and keep other conditions the same as those of 10 groups to obtain the small intestine digestion end-point liquid of 11 groups.

[0069] For 12 groups, adjust the pH to 7, 7.5, 8, 8.5 respectively with 1 M NaOH, add trypsin solution to make the final system trypsin solution activity 100 U / mL, and keep other conditions the same as those of 11 groups to obtain the small intestine digestion end-point liquid of 12 groups.

[0070] For 13 groups, stir in the dark for 0.5 h, 1 h, 3 h, 6 h respectively, adjust the pH to 7, and keep other conditions the same as those of 12 groups to obtain the small intestine digestion end-point liquid of 13 groups.

[0071] For 14 groups, set the stirring rate to 480 times / h, 550 times / h, 610 times / h, 680 times / h respectively, stir in the dark for 2 h, and keep other conditions the same as those of 13 groups to obtain the small intestine digestion end-point liquid of 14 groups.

[0072] 4. Component detection

[0073] Conduct component detection of volatile substances and non-volatile substances on the digestion end-point liquids obtained from the above groups 1 - 14 respectively. Among them, for the detection of volatile substances, headspace-solid phase microextraction combined with gas chromatography-mass spectrometry (HS-SPME-GC-MS) coupling technology is used, and for the detection of non-volatile substances, ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC-Triple-TOF / MS) is used. The specific operation and pretreatment methods are as follows:

[0074] Detection of volatile substances: Take 8 mL of the digestion end-point solution collected from groups 1 - 14 and place it in a 20 mL headspace vial. Add 1.6 g of sodium chloride (to increase the system saturation and promote the volatilization of volatile substances). The SPME extraction head is aged for 30 - 60 min before use. Set the parameters of the autosampler as follows: extraction temperature 50 °C, extraction time 30 min, and desorption time 7 min.

[0075] Set the chromatographic conditions as follows: injection port temperature 250 °C, column oven temperature program: hold at 40 °C for 1 min, increase to 70 °C at 2 °C / min, then increase to 160 °C at 4 °C / min, and then increase to 250 °C at 10 °C / min and hold for 5 min; carrier gas: 99.999% high-purity helium, carrier gas flow rate: 1 mL / min, splitless injection.

[0076] Set the mass spectrometry conditions as follows: EI ion source, ion source temperature 230 °C, ionization voltage 70 eV, interface temperature 280 °C, quadrupole temperature 150 °C, solvent delay 1 min, ionization mode EI, mass scan range m / z 40 - 550.

[0077] Detection of non-volatile substances: Centrifuge an appropriate amount of the digestion end-point solution collected from groups 1 - 14. This step is to remove macromolecular protease substances in the digestion solution. Take 2 mL of the supernatant and dry it at room temperature under a nitrogen purging instrument. At this time, the volatile substances have volatilized, and the non-volatile substances precipitate at the bottom of the tube. Add 1 mL of chloroform to redissolve and vortex for 5 min. At this time, the organic substances in the liquor can dissolve in chloroform, while the inorganic salts and a small amount of protease incompletely introduced during centrifugation deposit at the bottom of the tube. Take 800 μL of the chloroform layer (note not to take the precipitate or particulate matter) and inject it into a new centrifuge tube. Purge it to dryness with nitrogen at room temperature, add 30% acetonitrile water, and vortex for 5 min. This step is to avoid directly injecting the sample using chloroform as the solvent onto the instrument, as chloroform will damage the liquid chromatography-mass spectrometry column. Finally, filter through a 0.22 μm organic filter membrane and inject it into an injection vial for instrument analysis.

[0078] 5. Data processing

[0079] (1) Headspace-solid phase microextraction-gas phase data analysis

[0080] As shown in Table 3 are the analysis results of n-alkane (C4 - 26) standards.

[0081] Table 3 Qualitative analysis of n-alkane standards

[0082] Component RI Number of carbon chains Name Molecular formula CAS number 3.284 C7 Heptane C7H16 142-82-5 5.272 C8 Octane C8H18 111-65-9 9.029 C9 Nonane C9H20 111-84-2 14.479 C10 Decane C10H22 124-18-5 20.234 C11 Undecane C11H24 1120-21-4 24.757 C12 Dodecane C12H26 112-40-3 28.608 C13 Tridecane C16H34 629-50-5 32.051 C14 Tetradecane C14H30 629-59-4 35.222 C15 Pentadecane C15H32 629-62-9 38.183 C16 Hexadecane C16H34 544-76-3 40.569 C17 Heptadecane C17H36 629-78-7 42.275 C18 Octadecane C18H38 593-45-3 43.654 C19 Nonadecane C19H40 629-92-5 44.838 C20 Eicosane C20H42 112-95-8 45.899 C21 Heneicosane C21H44 629-94-7 46.871 C22 Docosane C22H46 629-97-0 47.786 C23 Tricosane C23H48 638-67-5 48.793 C24 Tetracosane C24H50 646-31-1 49.987 C25 Pentacosane C25H52 629-99-2 51.465 C26 Hexacosane C26H54 630-01-3

[0083] Import the off-line data of the samples into Masshunter, delete all the substance peaks with signal-to-noise ratio < 3, integrate and select the target substance peaks, bring them into the NIST 2014 database for preliminary qualitative screening to obtain the most likely substance information, and the RI value of the spectral library can be obtained by uploading the CAS number of the substance through the online NIST website. Combine with n-alkanes, calculate the actual RI value through the formula, and define that when the difference between the RI value of the spectral library and the actual RI is ≤ 10, the substance is determined to be the required target substance.

[0084] Actual RI = 100*n + 100[(t x -t n ) / (t n+1 -t n )]

[0085] Among them, RI is the retention index of the analyte;

[0086] tx is the retention time of volatile substance x, in min;

[0087] tn, tn+1 are the retention times of n-alkanes Cn, Cn+1, in min;

[0088] n is the number of carbon atoms of the volatile substance.

[0089] (2) Ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry data analysis

[0090] The liquid chromatography-mass spectrometry uses Prognosis QI (Waters Corporation) to process the off-line results. The QC samples are used for the correction of instrument signals and data processing, and the data matrix is further processed by the 80% rule. Match the MS and MS / MS information with the HMDB database and the METLIN database for metabolite annotation. The mass errors of MS1 and MS2 are set to 5 ppm.

[0091] Bring the list of qualitative substances (including peak areas) into the metaboanalyst 6.0 metabolomics online website for screening of differential compounds. By comparing with the blank digestion group and before and after digestion, extract the metabolites with FC value > 2 or FC value < 0.5, and define them as the differential metabolites generated by digestion.

[0092] (3) Experimental results

[0093] According to the above-mentioned component detection method and data analysis method, the number of digestion products in the digestion end solution of groups 1-14 is obtained, and the corresponding results are as Figure 1 - 14 shown:

[0094] Simulated oral digestion stage: When the activity of salivary amylase in the simulated oral digestion system is 50 U / mL ( Figure 1) and the simulated oral digestion time is 3 seconds ( Figure 2 ) when the production amount of the digestion products reaches the peak, indicating that the optimal simulated digestion parameters in the oral digestion stage are: the activity of salivary amylase is 50 U / mL, and the simulated oral digestion time is 3 seconds.

[0095] Simulated gastric digestion stage: In the simulated gastric digestion system, when the gastric peristalsis frequency is 200 times per hour ( Figure 3 ), the activity of pepsin is 2500 U / mL ( Figure 4 ), the activity of gastric lipase is 50 U / mL ( Figure 5 ), the gastric pH value is 3 ( Figure 6 ), the gastric digestion time is 2 hours ( Figure 7 ), and the gastric emptying rate is 85 mL / hour ( Figure 8 ), more digestion products can be produced.

[0096] Simulated small intestine digestion stage: By finely regulating key parameters such as intestinal emptying rate, bile concentration, pancreatic enzyme activity, pH value, intestinal digestion duration, intestinal peristalsis frequency, etc., it is found that when the small intestine emptying rate is 55 mL / hour ( Figure 9 ), the bile acid concentration is 15 mM ( Figure 10 ), the pancreatic enzyme activity is 150 U / mL ( Figure 11 ), the small intestine pH value is maintained at 7 ( Figure 12 ), the small intestine digestion duration is 1 hour ( Figure 13 ), and the small intestine peristalsis frequency is 610 times per hour ( Figure 14 ), the simulation system can achieve the highest efficiency and produce the largest amount of digestion products.

[0097] All the above simulated digestion parameters are within the normal human physiological range.

[0098] Among them, the above CaCl2·2H2O solution is an enzyme activator. Calcium ion is an enzyme activator. Enzymes need a specific ionic environment to play a catalytic role. Calcium ions can bind to the enzyme protein, and by changing the spatial structure of the enzyme, the active center of the enzyme can be better exposed, thereby enhancing the enzyme activity and improving its hydrolysis efficiency for starch, which helps to simulate the real digestion process in the oral cavity. Secondly, the calcium ions and chloride ions produced after the CaCl2·2H2O solution is dissolved can regulate the ionic strength of the solution, making the ionic environment in the solution closer to the physiological conditions in the human body, providing a stable environment for biomolecules such as salivary amylase, and ensuring that various chemical reactions and enzymatic catalytic reactions in the simulated digestion process can proceed smoothly.

[0099] α - amylase is a general term for a class of enzymes that can act on starch. Salivary amylase is one of the α - amylases secreted by the salivary glands. Salivary amylase is a hydrolase secreted by the salivary glands, belonging to glycoside - bond hydrolases, and can catalyze the decomposition of starch into maltose, thus playing an important role in the process of food digestion.

[0100] Pepsin is a digestive protease that has the function of decomposing proteins. Pepsinogen is secreted by the chief cells of the gastric mucosa and is activated into pepsin by gastric acid, which can decompose the proteins in food into small peptide fragments. Lipase is a general term for a class of enzymes that can hydrolyze long - chain fatty acid triglycerides, mainly in the pancreas, and a small amount of lipase is also contained in the stomach.

[0101] Gastric peristalsis refers to the process in which after food enters the stomach, the middle part of the stomach transports the food towards the pylorus through rhythmic contraction movements.

[0102] Gastric emptying is the process by which food is discharged from the stomach into the duodenum.

[0103] Bile is composed of bile salts, bilirubin, cholesterol, lecithin, potassium, sodium, calcium, etc., and can be directly discharged from the liver and gallbladder into the duodenum in large amounts, which can promote the digestion and decomposition of fat and the absorption of fat - soluble vitamins.

[0104] Pancreatic enzymes mainly include trypsin, pancreatic amylase, and pancreatic lipase, etc. Trypsin can convert proteins into peptones, pancreatic amylase converts starch into dextrin and sugar, and pancreatic lipase decomposes fat into glycerol and fatty acids.

[0105] Small intestine peristalsis usually overlaps with rhythmic segmental movements, and squeezes food into the large intestine through peristalsis and rhythmic contractions.

[0106] Example 2

[0107] An in vitro simulated digestion method for white liquor, comprising the following steps:

[0108] Step 1, Simulate oral digestion

[0109] Pump 100 mL of white liquor sample 1, 80 mL of SSF, and 500 μL of a calcium chloride (CaCl2·2H2O) solution with a concentration of 44.1 g / L into the oral digestion tank, adjust the pH to 7 using 1 M hydrochloric acid or 1 M sodium hydroxide, pump in a salivary amylase solution (1500 U / mL, dissolved in SSF), and make up with pure water to a volume of 200 mL. The final activity of salivary amylase in the oral digestion tank is 50 U / mL. Stir in the dark at 37 °C for 3 seconds (i.e., the simulation time of oral digestion is 3 seconds) to perform simulated oral digestion and obtain the oral digestion end - point liquid.

[0110] Among them, Baijiu sample 1 is a certain commercially available Baijiu with an alcohol content of 53%, which is the same as Baijiu sample 1 in Example 1.

[0111] Step 2: Simulate gastric digestion

[0112] Pump all the oral digestion end - point liquid into the gastric simulation digestion tank. At the same time, pump in 180 mL of SGF, 100 μL of a CaCl₂·2H₂O solution with a concentration of 44.1 g / L, adjust the pH to 3 with 1 M HCl or 1 M NaOH, then pump in 10 mL of pepsin solution (80000 U / mL, dissolved in SGF), 1200 μL of gastric lipase solution (10000 U / mL, dissolved in SGF), and make up with pure water to a final volume of 400 mL for the whole system. The final activity of pepsin in the gastric simulation digestion tank is 2500 U / mL, and the final activity of gastric lipase is 50 U / mL. Set the temperature in the gastric simulation digestion tank to 37 °C, the stirring rate to 200 times per hour (equivalent to a gastric peristalsis frequency of 200 times per hour), stir in the dark for 2 hours (i.e., the gastric digestion time is 2 hours), and the gastric emptying rate is 85 mL / hour to carry out simulated gastric digestion to obtain the gastric digestion end - point liquid.

[0113] Step 3: Simulate small intestine digestion

[0114] Pump all the gastric digestion end - point liquid into the small intestine simulation digestion tank, pump in 280 mL of SIF, 800 μL of a CaCl₂·2H₂O solution with a concentration of 44.1 g / L, adjust the pH to 7 with 1 M HCl or 1 M NaOH, then add 50 mL of pancreatic enzyme (1600 U / mL, dissolved in SIF) and 50 mL of bile (614.4 mg / mL, dissolved in SIF), and make up with pure water to a final volume of 800 mL for the small intestine digestive juice. The final concentration of bile acids in the small intestine simulation digestion tank is 15 mM, and the final activity of pancreatic enzyme is 150 U / mL. Set the temperature in the small intestine simulation digestion tank to 37 °C, the stirring rate to 610 times per hour (i.e., the intestinal peristalsis frequency is 610 times per hour), stir in the dark for 1 h (i.e., the small intestine simulation digestion duration is 1 h), and the emptying rate of the small intestine simulation digestion tank is 55 mL / hour (i.e., the intestinal emptying rate is 55 mL / hour) to carry out simulated small intestine digestion to obtain the small intestine digestion end - point liquid.

[0115] Example 3

[0116] Based on Example 2, another Maotai - flavored Baijiu sample 2 is subjected to in vitro simulated digestion in the same manner as in Example 2.

[0117] Using the same component detection method and data analysis method as in Example 1, detect and analyze the digestion end - point liquid obtained in Examples 2 and 3. The results are as Figure 15 and 16As shown above: By performing in vitro digestion simulation with the optimized simulated digestion parameters such as enzyme activity and digestion time in the oral cavity, stomach, and small intestine digestion stages, more thorough decomposition of baijiu can be achieved, and a larger number of digestion products are obtained in each stage.

[0118] It can be understood that the present invention is described through some embodiments. Those skilled in the art know that without departing from the spirit and scope of the present invention, various changes or equivalent replacements can be made to these features and embodiments. Additionally, under the teaching of the present invention, these features and embodiments can be modified to adapt to specific situations and materials without departing from the spirit and scope of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of this application belong to the scope protected by the present invention.

Claims

1. An in vitro simulated digestion method for Chinese liquor, characterized in that, It includes the following steps: Step 1, simulating oral digestion: Mix the liquor sample, simulated saliva and enzyme activator, adjust the pH to 6.5 - 7.5, add salivary amylase, and conduct simulated oral digestion to obtain the end - point solution of oral digestion; the activity of the salivary amylase is 45 - 70 U / mL, and the time of simulated oral digestion is 1.5 - 4.5 seconds; Step 2, simulating gastric digestion: Mix the end - point solution of oral digestion with simulated gastric juice and enzyme activator, adjust the pH to 2.6 - 3.5, add pepsin and gastric lipase, and conduct simulated gastric digestion to obtain the end - point solution of gastric digestion; the gastric peristalsis frequency of the simulated gastric digestion is 165 - 235 times per hour, the gastric digestion time is 1.5 - 2.5 hours, the gastric emptying rate is 80 - 90 mL per hour, the activity of the pepsin is 2000 - 2800 U / mL, and the activity of the gastric lipase is 45 - 55 U / mL; Step 3, simulating small intestine digestion: Mix the end - point solution of gastric digestion with simulated intestinal fluid and enzyme activator, adjust the pH to 6.5 - 7.5, add pancreatic enzyme and bile acid, and conduct simulated small intestine digestion to obtain the end - point solution of small intestine digestion; the activity of the pancreatic enzyme is 110 - 190 U / mL, the concentration of bile acid is 11 - 19 mM, the peristalsis frequency of the small intestine is 560 - 670 times per hour, the digestion duration of the small intestine is 0.6 - 2.9 h, and the emptying rate of the small intestine is 51 - 59 mL per hour.

2. The in vitro simulated digestion method according to claim 1, wherein The activity of the salivary amylase is 45 - 55 U / mL, and the time of simulated oral digestion is 2.8 - 3.3 seconds; the gastric peristalsis frequency of the simulated gastric digestion is 190 - 210 times per hour, the gastric digestion time is 1.8 - 2.2 hours, the gastric emptying rate is 82 - 88 mL per hour, the activity of the pepsin is 2400 - 2600 U / mL, and the activity of the gastric lipase is 48 - 52 U / mL; the activity of the pancreatic enzyme is 140 - 160 U / mL, the concentration of bile acid is 13 - 17 mM, the peristalsis frequency of the small intestine is 600 - 620 times per hour, the digestion duration of the small intestine is 0.8 - 1.2 h, and the emptying rate of the small intestine is 53 - 58 mL per hour.

3. The in vitro simulated digestion method according to claim 1, wherein The enzyme activator is a CaCl₂·2H₂O solution, and the concentration of the CaCl₂·2H₂O solution is 40 - 50 g / L; Preferably, the concentration of the CaCl₂·2H₂O solution is 42 - 47 g / L.

4. The in vitro simulated digestion method according to claim 1, wherein The volume ratio of the liquor sample, simulated saliva and salivary amylase activator is 80 - 120:70 - 90:0.3 - 1.

5. The in vitro simulated digestion method according to claim 1, characterized in that, The simulated saliva includes KCl solution, KH₂PO₄ solution, NaHCO₃ solution, MgCl₂(H₂O)₆ solution and (NH₄)₂CO₃ solution; Preferably, the volume of the KCl solution is 15 - 30 mL, and the concentration is 35 - 40 g / L; the volume of the KH₂PO₄ solution is 3 - 10 mL, and the concentration is 65 - 70 g / L; the volume of the NaHCO₃ solution is 5 - 10 mL, and the concentration is 80 - 90 g / L; the volume of the MgCl₂·6H₂O solution is 0.5 - 1 mL, and the concentration is 30 - 35 g / L; the volume of the (NH₄)₂CO₃ solution is 0.05 - 0.1 mL, and the concentration is 45 - 50 g / L.

6. The in vitro simulated digestion method according to claim 1, wherein, The simulated gastric juice includes a KCl solution, a KH₂PO₄ solution, a NaHCO₃ solution, a NaCl solution, and an (NH₄)₂CO₃ solution; Preferably, the volume of the KCl solution is 5 - 10 mL, and the concentration is 35 - 40 g / L; the volume of the KH₂PO₄ solution is 1 - 2 mL, and the concentration is 65 - 70 g / L; the volume of the NaHCO₃ solution is 10 - 20 mL, and the concentration is 80 - 90 g / L; the volume of the NaCl solution is 10 - 20 mL, and the concentration is 110 - 120 g / L; the volume of the (NH₄)₂CO₃ solution is 0.5 - 1.0 mL, and the concentration is 45 - 50 g / L.

7. The in vitro simulated digestion method according to claim 1, characterized in that, The simulated intestinal fluid includes a KCl solution, a KH₂PO₄ solution, a NaHCO₃ solution, a NaCl solution, and a MgCl₂·6H₂O solution; Preferably, the volume of the KCl solution is 5 - 10 mL, and the concentration is 35 - 40 g / L; the volume of the KH₂PO₄ solution is 0.5 - 1.5 mL, and the concentration is 65 - 70 g / L; the volume of the NaHCO₃ solution is 50 - 100 mL, and the concentration is 80 - 90 g / L; the volume of the NaCl solution is 20 - 30 mL, and the concentration is 110 - 120 g / L; the volume of the MgCl₂·6H₂O solution is 2.5 - 3.0 mL, and the concentration is 25 - 35 g / L.

8. The in vitro simulated digestion method according to claim 1, characterized in that, The alcohol content of the liquor sample is 50 - 55%.

9. The in vitro simulated digestion method according to any one of claims 1-8, characterized in that, The pancreatic enzyme includes trypsin and / or pancreatic lipase.

10. Use of an in vitro simulated digestion method according to any one of claims 1 - 9 in reflecting the in vivo digestion, metabolism, and absorption of liquor components.

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