A comprehensive evaluation method for herbal baijiu based on in-vitro gastrointestinal digestion

Abstract

The present application belongs to the technical field of wine analysis and in vitro digestion evaluation, and discloses a comprehensive evaluation method for herbal liquor based on in vitro gastrointestinal digestion. The herbal liquor to be tested and the reference liquor of the same base liquor flavor, the same alcohol content and without the introduction of homologous plant materials are subjected to oral, gastric and small intestinal simulation digestion under the same conditions, and the volatile and non-volatile metabolites in the gastric and small intestinal stages are detected; a semi-continuous anaerobic fermentation system is constructed by using the small intestinal digestion products and human mixed flora, part of the fermentation broth is replaced every 24 h, and fresh culture medium and small intestinal digestion products are supplemented, and 16S rRNA sequencing is performed at early and long time points; combined evaluation is carried out by combining the metabolites and flora indexes at each stage, so as to improve the stability and discrimination ability of the evaluation of herbal liquor.

Description

Technical Field

[0001] This invention belongs to the field of alcohol analysis and in vitro digestion evaluation technology, specifically involving a comprehensive evaluation method for herbal liquor based on in vitro gastrointestinal digestion. In particular, it involves a method that integrates the simulated digestion process of herbal liquor in the oral cavity, stomach, and small intestine with the detection of digestive product metabolites, in vitro semi-continuous fermentation, and intestinal flora detection, and comprehensively evaluates herbal liquor based on the detection data obtained from different digestion and fermentation stages. Background Technology

[0002] Herbal baijiu typically incorporates one or more medicinal and edible plant materials during the baijiu production process. This results in a baijiu containing not only conventional baijiu components such as ethanol, esters, higher alcohols, and organic acids, but also polyphenols, flavonoids, saponins, terpenes, and their derivatives derived from these plant materials. Compared to regular baijiu, herbal baijiu has a more complex composition, containing both volatile and non-volatile components, both polar and non-polar. These different types of components exhibit variations in stability, release state, and transformation processes within the gastrointestinal environment.

[0003] Currently, the evaluation of herbal spirits or other alcoholic beverages containing plant-derived ingredients typically employs analytical techniques such as gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) to detect the chemical composition of the sample. Alternatively, in vitro simulated digestion methods can be used to study changes in the sample within the oral cavity, stomach, and small intestine. Existing in vitro gastrointestinal simulated digestion techniques can study the compositional changes of food or beverages after they pass through the upper digestive tract by controlling conditions such as digestive fluid composition, digestive enzymes, bile, temperature, pH, and digestion time.

[0004] Chinese invention patent application CN111100787A discloses a fully automated device and method for simulating human gastrointestinal digestion and fermentation. By sequentially setting up simulation units for the oral cavity, stomach, small intestine, and different colonic segments, it achieves continuous digestion, fermentation, transport, and sampling of the analyte in an in vitro gastrointestinal environment. The focus of this disclosure is on the automated simulation of the entire human gastrointestinal digestion and fermentation environment and the continuous transport between different digestive segments. However, it does not establish a cross-stage comprehensive evaluation rule for herbal liquor that connects information on metabolites from the stomach and small intestine stages with subsequent long-term semi-continuous microbial succession information.

[0005] However, for herbal baijiu, directly detecting the composition of the raw liquor is not entirely equivalent to evaluating its state after passing through the gastrointestinal environment. After entering the gastrointestinal tract, the volatile and non-volatile components of herbal baijiu are affected by dilution, pH changes, enzymatic action, bile action, and interactions between different components. Therefore, the components and their relative content detected in the raw liquor cannot directly represent the material state after digestion and subsequent entry into the intestinal environment. Evaluation based solely on the results of raw liquor testing or the results of a single digestion endpoint can easily miss information about continuous changes between different digestive stages.

[0006] On the other hand, existing in vitro gut microbiota fermentation studies typically involve adding the analyte or its extract to a fecal microbiota culture system and evaluating the analyte's impact on the gut microbiota based on changes in microbiota composition or metabolites after short-term fermentation. However, when raw, undigested alcoholic samples are directly added to the microbiota culture system, the actual substrate state encountered by the microbiota differs from the state of the substances reaching the subsequent gut environment after digestion in the mouth, stomach, and small intestine. Furthermore, in static culture systems, as the culture time increases, nutrients are gradually consumed, accompanied by the accumulation of metabolites. Early microbiota responses and microbiota succession after longer cultures are easily influenced by changes in the culture system itself. Relying solely on microbiota changes at a single time point or within a short period is insufficient to fully reflect the continuous changes in the analyte after digestion.

[0007] Furthermore, the detection of volatile metabolites, non-volatile metabolites, and gut microbiota analysis typically yield independent data results. For the same herbal baijiu, if different sample pretreatment bases are used in each testing stage, or if there is a lack of continuous sample source relationships between different test results, it is difficult to form a unified evaluation basis among various types of test information. At the same time, individual indicators are easily affected by batch-to-batch differences in samples, which may lead to fluctuations in individual indicators for the same type of sample.

[0008] Therefore, it is necessary to establish an in vitro comprehensive evaluation method suitable for herbal baijiu, to obtain chemical composition information of different digestion stages based on the same gastrointestinal digestion, and to continue in vitro microbial fermentation with the actual obtained small intestinal digestion products, to maintain a long-term culture by periodic drainage and continuous replenishment of small intestinal digestion products, and to conduct cross-stage evaluation by combining early and long-term microbial information, so as to improve the stability and distinguishability of different herbal baijiu samples. Summary of the Invention

[0009] In view of this, the present invention proposes a comprehensive evaluation method for herbal liquor based on in vitro gastrointestinal digestion, in order to solve the problems of lack of correspondence between the direct detection results of raw liquor and the actual material state after digestion, separation between the chemical changes in the upper digestive tract and the subsequent microbial community evaluation, and the susceptibility of short-term or single-index evaluation to the influence of culture state and sample batch fluctuations in the existing evaluation process.

[0010] The technical solution of this invention is implemented as follows: This invention provides a comprehensive evaluation method for herbal liquor based on in vitro gastrointestinal digestion. The method involves setting up a test herbal liquor and a reference liquor with the same aroma and alcohol content as its base liquor, but without the introduction of medicinal and edible plant materials. Both are subjected to simulated oral, gastric, and small intestinal digestion under the same conditions. Digestion samples are obtained at the gastric and small intestinal stages for the detection of volatile and non-volatile metabolites. The obtained small intestinal digestion products are then subjected to anaerobic fermentation with a mixed bacterial suspension prepared from the feces of at least three healthy adults and an anaerobic liquid culture medium. From the start of fermentation, 10%–40% of the total volume of the fermentation liquid is replaced every 24 hours, and a feed solution equal in total volume to the excreted liquid, consisting of fresh anaerobic liquid culture medium and the corresponding small intestinal digestion products at a 5:1 volume ratio, is added. Samples are taken at two time ranges: 24–72 h and 7–28 d, and subjected to 16S analysis. High-throughput rRNA sequencing was performed. The herbal liquor to be tested was compared with the reference liquor at the same treatment stage. Evaluation indicators were selected from four types of data: gastric digestion, small intestinal digestion, early fermentation, and long fermentation. Cross-stage digestion-fermentation response characteristics were formed according to the preset judgment rules.

[0011] In some embodiments, the in vitro simulated oral, gastric, and small intestinal digestion are performed sequentially, with the digestive products obtained in the previous digestion stage serving as the processing target for the subsequent digestion stage. Specifically, the oral simulated digestion stage involves treatment with α-amylase at 37°C and pH 7.0 for 2 minutes; the gastric simulated digestion stage involves treatment with pepsin and gastric lipase at 37°C and pH 3.0 for 2 hours; and the small intestinal simulated digestion stage involves treatment with trypsin and bile at 37°C and pH 7.0 for 2 hours.

[0012] In some embodiments, the simulated in vitro digestion process utilizes simulated oral digestive fluid, simulated gastric digestive fluid, and simulated small intestinal digestive fluid, respectively, and calcium ions, digestive enzymes, and bile are added according to the corresponding digestion stage. The simulated digestive fluid contains one or more of KCl, KH₂PO₄, NaHCO₃, NaCl, MgCl₂, (NH₄)₂CO₃, and CaCl₂.

[0013] In some implementations, a gastric digestion sample is obtained after gastric digestion is completed, and a small intestinal digestion sample is obtained after small intestinal digestion is completed. Volatile metabolite detection and non-volatile metabolite detection are performed on the gastric digestion sample and the small intestinal digestion sample, respectively, to obtain metabolite data at different stages in the same continuous digestive chain.

[0014] In some embodiments, the volatile metabolites are detected using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HSP-GC-MS); the non-volatile metabolites are detected using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QFS-MS). With a reference sample included, the metabolite detection data of the tested herbal liquor and the reference sample are compared at the same digestion stage to screen for differentially expressed metabolites, and KEGG pathway enrichment analysis is performed. The screening criteria for differentially expressed metabolites are |log2FC| > 1 and p < 0.05, and pathway enrichment analysis is defined as p < 0.05 as significant enrichment.

[0015] In some embodiments, after the small intestine simulated digestion is completed, the small intestine digestion products are directly used as substrate components of the in vitro microbial fermentation system and mixed with human fecal bacterial suspension and anaerobic liquid culture medium. Every 24 hours after the start of fermentation, 10% to 40% of the total volume of the fermentation liquid is discharged and a feed liquid equal to the discharged liquid is added. The feed liquid consists of fresh anaerobic liquid culture medium and the corresponding small intestine digestion products in a volume ratio of 5:1, so that the total volume of the fermentation system remains unchanged and the substrate formed by the corresponding liquor digested in the gastrointestinal tract is continuously obtained.

[0016] In some embodiments, the initial composition of the semi-continuous in vitro fermentation system is 5 parts by volume of small intestinal digestion products, 10 parts by volume of human fecal microbial suspension, and 85 parts by volume of anaerobic liquid culture medium. The fermentation temperature is 37°C, and the total fermentation time is 28 days. After the fermentation system is started, 30% of the fermentation liquid is discharged every 24 hours, and 25% of the fresh anaerobic liquid culture medium and 5% of the small intestinal digestion products are added to replenish the system.

[0017] In some implementations, fermentation samples are collected at the start of fermentation and at 24 h, 48 h, 72 h, 7 d, 14 d and 28 d of fermentation. The samples used for detection at each time point from 24 h onwards are taken from the fermentation broth discharged in the corresponding cycle, and no additional samples are taken outside the cycle discharge volume. The sample at the start of fermentation is taken before the start of cultivation.

[0018] In some embodiments, the fermentation sample is subjected to 16S rRNA high-throughput sequencing, and the V4 region of the microbial 16S rRNA gene is amplified using 515F and 806R primers to obtain one or more data among the following: community taxonomic composition, community relative abundance, α diversity, β diversity, and differential microbial markers; for comprehensive evaluation, community data that can be stably obtained at the genus level are preferred, and species-level annotation results are used as auxiliary information.

[0019] In some implementations, a reference liquor with the same aroma and alcohol content as the base liquor of the herbal liquor to be tested, but without the introduction of medicinal and edible plant materials, is set up. Under the same conditions, in vitro simulated digestion, metabolite detection, semi-continuous fermentation, and microbial community detection are carried out. When it comes to screening for differential metabolites, comparing microbial communities, and LEfSe analysis, the herbal liquor group to be tested and the reference liquor group are compared with the same treatment stage or the same fermentation time point, and each group is set up with no less than 3 independent parallels.

[0020] In some embodiments, the comprehensive evaluation employs four cross-stage indicators: amyl acetate index during gastric digestion, metabolic pathway enrichment index during gastric and small intestinal stages, relative abundance change of Bifidobacterium spp. after 48 h of fermentation, and LEfSe difference index of Fusobacterium spp. between the reference baijiu group and the tested herbal baijiu group after 28 days of fermentation. When the tested herbal baijiu meets at least three of these indicators, it is determined to possess the aforementioned cross-stage digestion-fermentation response characteristics. This determination is used to evaluate the in vitro digestion-fermentation response characteristics of the tested herbal baijiu and is not equivalent to judging human disease states, treatment effects, or the activation status of human metabolic pathways.

[0021] In some embodiments, the volatile metabolite index during the gastric digestion stage is defined as the characteristic peak S / N ratio of amyl acetate in the gastric digestion sample of the herbal liquor being tested being ≥3, and both the forward and reverse matching degrees in the mass spectrometry database being greater than 700; the metabolic pathway index is defined as the enrichment analysis of the unsaturated fatty acid biosynthesis pathway of the herbal liquor being tested relative to the reference liquor during the gastric digestion stage (p < 0.05), the enrichment analysis of the primary bile acid biosynthesis pathway of the herbal liquor being tested relative to the reference liquor during the small intestinal digestion stage (p < 0.05), and the enrichment analysis of the glutathione metabolic pathway of the reference liquor relative to the herbal liquor being tested during the small intestinal digestion stage (p < 0.05); the microbial community index after 48 h of fermentation is defined as the relative abundance of Bifidobacterium spp. in the herbal liquor being tested group being at least 2 times higher than at the start of fermentation; the microbial community index after 28 days of fermentation is defined as the Fusobacterium spp. in the reference liquor group relative to the herbal liquor being tested group satisfying the Kruskal-Wallis test in LEfSe analysis (p < 0.05) and having an LDA score > 4.

[0022] Compared with the prior art, the present invention has the following advantages: The present invention uses the actual digestion products after continuous simulated digestion in the mouth, stomach and small intestine as the basis for subsequent analysis and microbial fermentation, so that the chemical transformation information in the upper digestive tract stage and the time series information obtained in the subsequent microbial culture process have a continuous sample source relationship; by periodically draining the liquid and simultaneously replenishing fresh culture medium and small intestinal digestion products, the culture environment can be continuously updated and the substrate to be tested can be replenished during the long fermentation process, reducing the information decay caused by nutrient depletion and metabolite accumulation in the later stage of static culture; furthermore, by jointly evaluating the metabolite indicators in the digestion stage with the early and long-term microbial indicators, the inconsistency caused by the fluctuation of sample batches of a single indicator can be reduced, and the stability and discrimination ability of the evaluation can be improved. Attached Figure Description

[0023] Figure 1 Thermographs of the differential volatile metabolites of HB and TB after in vitro digestion in the oral cavity, stomach, and small intestine; Figure 2 The results of KEGG pathway enrichment analysis of differential nonvolatile metabolites during in vitro digestion are shown. A, B, and C are the KEGG pathway enrichment analysis results of differential metabolites between HB and TB at sample processing volumes of 100 mL, 200 mL, and 300 mL, respectively, during the gastric digestion stage. D and E are the KEGG pathway enrichment analysis results of differential metabolites between HB and TB at sample processing volumes of 100 mL and 300 mL, respectively, during the small intestinal digestion stage. Figure 3 The results of the analysis of gut microbiota structure and differences during semi-continuous fermentation are shown. A represents the Chao1 index and Shannon index, B represents the PCoA analysis of microbial communities among different fermentation groups, and C and D represent the relative abundance distribution at the phylum and genus levels, respectively. Figure 4 The results of the exploratory LEfSe analysis of the microbial community over the entire time sequence of semi-continuous fermentation are used to demonstrate the differential microbial distribution at different time stages. The comparison results of the two preset groups of LEfSe between the tested herbal liquor group and the reference liquor group at 28 days of fermentation are presented as statistical results in the specific implementation method. Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are used to illustrate the technical solutions of the present invention, and not to limit the scope of protection of the present invention. Adaptive adjustments made by those skilled in the art to the sample amount, digestion system scale, detection instrument model, and corresponding operating parameters without departing from the technical concept of the present invention can all be used to implement the present invention.

[0025] Unless otherwise specified, all reagents, culture media, chromatographic columns, DNA extraction kits, and analytical instruments used in the examples are commercially available products. In this embodiment, the herbal baijiu is designated as HB, with an alcohol content of 42% vol. Its preparation process uses medicinal and edible plant materials such as kudzu root, wolfberry, and mulberry leaves. A common light-aroma baijiu with the same aroma as HB base liquor, an alcohol content of 42% vol, and without the introduction of medicinal and edible plant materials was selected as a reference baijiu and designated as TB.

[0026] When parallel experiments are involved, each experimental group is set up with 3 independent replicates. Quantitative results are expressed as mean ± standard deviation. Independent samples t-tests are used for comparisons between two groups; one-way ANOVA with multiple comparisons is used when more than three treatment groups are involved; appropriate nonparametric tests are used for data that do not conform to a normal distribution, and p < 0.05 is considered statistically significant.

[0027] In Example 3, the human fecal samples used were voluntarily provided by healthy volunteers. All donors signed an "Informed Consent Form for Fecal Sample Collection Research," agreeing to participate in this study and provide fecal samples. The human samples used were solely for in vitro microbial fermentation and microbial composition analysis, and were not used to diagnose any disease status in the donors.

[0028] Example 1: In vitro gastrointestinal digestion and volatile metabolite detection of herbal baijiu (Chinese liquor) 1. Preparation of simulated digestive fluid Stock solutions of KCl, KH₂PO₄, NaHCO₃, NaCl, MgCl₂·6H₂O, (NH₄)₂CO₃, and CaCl₂·2H₂O were prepared separately. Simulated oral digestive fluid (SSF), simulated gastric digestive fluid (SGF), and simulated small intestinal digestive fluid (SIF) were prepared according to the dosages shown in Table 1. All simulated digestive fluids were sealed after preparation and stored at 4°C for later use.

[0029] Table 1 Electrolyte stock solutions used in simulated digestion solutions

[0030] 2. In vitro gastrointestinal tract simulation digestion 200 mL of the liquor to be tested was used as the baseline treatment volume. HB and TB were treated as follows, with three independent replicates for each group.

[0031] Oral digestion stage: 200 mL of the liquor to be tested was pumped into the oral digestion container, and 80 mL of SSF, 0.5 mL of CaCl2·2H2O solution with a concentration of 45 g / L and 10 mL of α-amylase solution with an enzyme activity of 1500 U / mL were added. The pH of the system was adjusted to 7.0, and the mixture was stirred at 37℃ in the dark for 2 min to obtain the oral digestion product.

[0032] Gastric digestion stage: The obtained oral digestion products were transferred into a gastric digestion container, and 160 mL of SGF, 0.5 mL of CaCl2·2H2O solution, 20 mL of pepsin solution with an enzyme activity of 40000 U / mL and 1.2 mL of gastric lipase solution with an enzyme activity of 20000 U / mL were added. The pH of the system was adjusted to 3.0, and the mixture was stirred at 37℃ in the dark for 2 h. After digestion was completed, a sample was taken to obtain the gastric digestion sample.

[0033] Small intestinal digestion stage: The gastric digestion products were transferred into the small intestinal digestion container, and 170 mL of SIF, 0.8 mL of CaCl2·2H2O solution, 50 mL of trypsin solution with an enzyme activity of 1600 U / mL and 50 mL of bile solution with a concentration of 38.4 g / L were added. The pH of the system was adjusted to 7.0, and the mixture was stirred at 37°C in the dark for 2 h to obtain the small intestinal digestion products.

[0034] When the amount of the liquor sample to be tested is changed, the amounts of each simulated digestive fluid, enzyme solution, bile, and calcium salt added are adjusted proportionally according to the above-mentioned 200mL baseline treatment volume to ensure that the sample and digestion system ratios of different sample treatment groups remain consistent. The above-mentioned simulated digestion process is carried out using an in vitro gastrointestinal biomimetic digestion system capable of quantitative delivery, mixing, temperature control, pH adjustment, and sample transfer. The in vitro gastrointestinal biomimetic digestion system used can be any existing device capable of achieving the above digestion conditions. This invention does not require the structure of a specific gastrointestinal biomimetic digestion device as a necessary condition for implementing this evaluation method.

[0035] 3. Detection of volatile metabolites To obtain Figure 1 The complete phased changes in volatile components are shown. Oral digestion samples were collected at the end of the oral digestion process. Eight mL samples from the oral, gastric, and small intestinal digestions were placed in 20 mL headspace vials, 1.6 g of NaCl was added, and the vials were sealed. Extraction was performed using a 65 μm CAR / PDMS extractor at 80 °C with shaking for 8 min, followed by desorption at 250 °C for 5 min, and then GC-MS analysis was performed. Data from the gastric and small intestinal digestion phases were used for combined evaluation, while the oral digestion samples were used for phased full-spectrum comparison.

[0036] The gas chromatography conditions were as follows: HP-5 MS column, 30 m × 0.25 mm × 0.25 μm; high-purity helium as carrier gas, flow rate 1.0 mL / min; injection port temperature 250℃, splitless injection. The initial column temperature was 40℃, held for 1 min, increased to 70℃ at 2℃ / min, then increased to 160℃ at 4℃ / min, and subsequently increased to 250℃ at 10℃ / min and held for 5 min.

[0037] Mass spectrometry was performed using an EI ion source with an ionization energy of 70 eV, an ion source temperature of 230℃, an interface temperature of 280℃, and a scan range of m / z 40–550. Compound identification was performed using the NIST 20 mass spectrometry database, with both forward and reverse matching scores greater than 700 as the qualitative standard. A signal-to-noise ratio (S / N) ≥ 3 for characteristic peaks was considered detectable, and an S / N ≥ 10 was considered to have reached the limit of quantitation. The relative peak area was expressed as the percentage of the peak area of ​​each characteristic peak relative to the total peak area of ​​all identified volatile components in the corresponding sample.

[0038] 4. Results of volatile metabolite detection Table 2. Changes in representative volatile metabolites of HB and TB after digestion in the stomach and small intestine.

[0039] Note: ND indicates not detected. Amyl acetate remained consistently detectable in HB after gastric digestion, while the compound was not detected in TB under the same conditions. After entering the small intestine digestion stage, the amyl acetate signal in HB decreased but remained detectable, indicating a continuous change in its concentration at different digestive stages.

[0040] In addition to amyl acetate, which is used as a joint evaluation index, Figure 1 The changes in other volatile components at different digestion stages are also shown to illustrate the differences in the overall volatile composition of HB and TB during continuous digestion. Exploratory volatile components not included in the preset joint judgment rules are not considered necessary conditions for the validity of the evaluation results of this invention.

[0041] Example 2: Detection of non-volatile metabolites at different digestion stages of herbal baijiu (Chinese white liquor) 1. Sample processing HB and TB were subjected to simulated gastrointestinal digestion according to Example 1, and gastric digestion samples and small intestinal digestion samples were collected.

[0042] Take 2 mL of each digested sample and add 6 mL of pre-cooled methanol / acetonitrile mixture (1:1, v / v). Vortex for 2 min, then sonicate in an ice-water bath for 10 min. Let stand at -20℃ for 30 min to precipitate macromolecular components, then centrifuge at 4℃ and 12000×g for 10 min. Take the supernatant, dry it under nitrogen, redissolve it in 30% acetonitrile aqueous solution, vortex for 5 min, filter through a 0.22 μm filter membrane, and perform UPLC-Q-TOF / MS analysis. This pretreatment is used to extract both polar and moderately nonpolar nonvolatile metabolites; pathway analysis is only based on differentially detected metabolites that meet the identification criteria.

[0043] For each sample, an equal volume of extract was mixed to prepare a QC sample. Before the formal sample analysis, the QC sample was continuously injected to stabilize the detection system. Thereafter, one QC sample was inserted every six samples analyzed to evaluate the instrument's stability during the detection process.

[0044] 2. UPLC-Q-TOF / MS detection An ACQUITY UPLC HSS T3 column was used at a column temperature of 40℃, a flow rate of 0.3 mL / min, and an injection volume of 2 μL. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was acetonitrile. The gradient elution conditions were: 0–8 min, 5%–30% B; 8–16 min, 30%–65% B; 16–20 min, 65% B; 20–24 min, 65%–90% B; 24–26 min, 90% B; 26–30 min, 90%–5% B.

[0045] Mass spectrometry was performed using an electrospray ionization source, acquiring data in both positive and negative ion modes. The scanning range was m / z 50–1200; the ion source temperature was 120℃, the desolvation temperature was 450℃, and the desolvation gas flow rate was 800 L / h; the capillary voltage was 3.0 kV in positive ion mode and 2.5 kV in negative ion mode; the low collision energy was 6 eV, and the high collision energy was acquired using a gradient collision energy of 20–40 eV.

[0046] Raw data were processed using MS-DIAL software for peak extraction, retention time correction, peak alignment, and normalization. Peaks with a relative standard deviation of no more than 20% for repeated detection in QC samples were used for subsequent statistical analysis. Metabolites were matched with HMDB, METLIN, and MS-DIAL built-in databases using precise mass numbers, isotope distributions, and MS / MS fragment information, with the precursor ion mass error controlled within 10 ppm. When standards were available, further confirmation was performed using retention time and MS / MS fragment consistency. At the same digestion stage, metabolite detection data from HB and TB were compared to screen for differentially metabolites. The screening criteria were |log2FC|>1 and p<0.05. The screened differentially metabolites were subjected to KEGG metabolic pathway enrichment analysis using MetaboAnalyst, with p<0.05 considered as significant enrichment.

[0047] 3. Changes in metabolites at different stages of digestion Table 3. Enrichment results of representative metabolic pathways at different digestion stages of HB and TB.

[0048] Taking the HB-300 sample as an example, 94.1% of differentially expressed metabolites were upregulated during the gastric digestion stage, while 98.5% were downregulated during the small intestinal digestion stage. Further comparison of the gastric and small intestinal digestion data of various HB samples showed that their main differentially expressed metabolites exhibited different trends in the two digestion stages, while TB did not show the same stage-specific change pattern.

[0049] The above results are used to characterize the enrichment features of differential metabolites associated with the corresponding metabolic pathways, and do not interpret the pathway enrichment results in the in vitro digestive system as the activation of the corresponding metabolic pathways in the human body.

[0050] To ensure the traceability of metabolite basis for pathway enrichment results, further analysis was conducted on representative differentially expressed metabolites associated with the corresponding enriched pathways. During gastric digestion, HB relative to TB detected linoleic acid (FC=2.45, p=0.012) and oleic acid (FC=2.12, p=0.018), corresponding to enrichment related to unsaturated fatty acid biosynthesis and linoleic acid metabolism. During small intestinal digestion, HB relative to TB detected bile acids (FC=1.88, p=0.009) and chenodeoxycholic acid (FC=1.74, p=0.015), corresponding to enrichment related to primary bile acid biosynthesis. During small intestinal digestion, TB relative to HB detected reduced glutathione (FC=2.21, p=0.011), corresponding to enrichment related to glutathione metabolism.

[0051] Example 3: 28-day semi-continuous in vitro fermentation of small intestinal digestion products 1. Preparation of human fecal microbial suspension Six healthy adults were selected as fecal donors (3 males and 3 females), aged 22–26 years, with a BMI of 18.5–23.9 kg / m². All donors had not taken antibiotics or had a history of gastrointestinal disease within the six months prior to sampling. All donors signed an informed consent form for fecal sample collection, agreeing to participate in this study and provide fecal samples.

[0052] Fresh fecal samples were immediately transferred to an anaerobic environment after collection. The samples were diluted 1:5 (w:v) with PBS containing 0.1% (w / v) L-cysteine ​​hydrochloride, thoroughly homogenized, and filtered through four layers of sterile gauze. Equal volumes of the filtrates from the six donors were mixed, and glycerol was added to a final concentration of 10% (v / v). The mixtures were then aliquoted and stored at -80°C. The samples were rapidly resuscitated in an anaerobic environment before use, and inoculation was completed within 1 hour of resuscitation.

[0053] 2. Anaerobic liquid culture medium Each 1 L of anaerobic liquid culture medium contains 10 g peptone, 5 g yeast extract, 2 g glucose, 0.4 g NaHCO3, 0.5 g NaCl, 0.2 g KH2PO4, 0.2 g K2HPO4, 0.01 g MgSO4·7H2O, 0.01 g CaCl2, 0.5 g L-cysteine ​​hydrochloride, and 0.001 g resazurin. Adjust the pH of the culture medium to 6.0–6.8, dispense into 250 mL sealed culture flasks, replace the gas phase with a N2 / CO2 / H2 mixture with a volume ratio of 90:5:5, and sterilize at 121 °C for 15 min.

[0054] 3. Semi-continuous fermentation system Small intestinal digestion products obtained by HB and TB according to Example 1 were used as fermentation substrates, and a blank digestion group (NC) was set up. Each 100 mL initial fermentation system contained 5 mL of small intestinal digestion products, 10 mL of human fecal bacterial suspension, and 85 mL of anaerobic liquid culture medium. The NC group used an equal volume of blank digestion products instead of the small intestinal digestion products of baijiu.

[0055] Each experimental group was cultured at 37℃ and 180 rpm for a total fermentation time of 28 days. The small intestinal digestate used for continuous feeding during the 28 days was prepared once from the same batch of digestion system and aseptically aliquoted into 5 mL portions, stored at -80℃. One portion was thawed at 4℃ before each day's use, without repeated freeze-thaw cycles. Every 24 hours after fermentation, 30 mL of fermentation broth was drawn, followed by the addition of 25 mL of fresh anaerobic liquid culture medium and 5 mL of the small intestinal digestate corresponding to the experimental group, restoring the total volume to 100 mL. Daily broth draining and replenishment were performed in an anaerobic glove box. Fresh culture medium and small intestinal digestate were pre-equilibrated with a N2 / CO2 / H2 mixture of 90:5:5 before replenishment to minimize oxygen exposure during the feeding process.

[0056] Samples were taken at 0 h, 24 h, 48 h, 72 h, 7 d, 14 d, and 28 d. For each time point from 24 h onwards, 1.5 mL of the sample was aliquoted from the 30 mL fermentation broth discharged that day; no additional samples were taken beyond the 30 mL discharge volume. The 0 h sample was aliquoted after system establishment but before the start of formal culture. All samples were immediately stored at -80℃.

[0057] 4.16S rRNA high-throughput sequencing Total DNA was extracted from each fermentation sample, and the V4 region of the 16S rRNA gene was amplified using primers 515F and 806R. PCR products were separated, purified, and quantified by 2% agarose gel electrophoresis to construct sequencing libraries, and paired-end sequencing was performed using the Illumina MiSeq platform.

[0058] The obtained sequences underwent quality control using the QIIME 2 workflow, and DADA2 was used for paired-end denoising, splicing, and chimera removal to obtain the ASV table. Taxonomic annotation was performed using the SILVA 138.1 database, with a classification confidence threshold set to 0.70. Based on the ASV table, the Chao1 index, Shannon index, and Bray-Curtis distance were calculated, and PCoA analysis was performed. LEfSe analysis was used to compare the HB and TB groups at the same fermentation time points using a pre-defined intergroup comparison. The Kruskal-Wallis test was used to screen taxa with intergroup differences (p < 0.05), and LDA was used to evaluate the effect size. An LDA score > 4 was considered a significantly different microbial biomarker. Subgroups were not set in this embodiment. Considering the limited species-level resolution of the V4 region for some closely related species, genus-level microbial community data was used for comprehensive evaluation, with species-level annotation serving only as supplementary information.

[0059] 5. Time-series changes in gut microbiota Table 4. Relative abundance of Bifidobacterium during the semi-continuous fermentation of HB and TB.

[0060] At 48 h of fermentation, the Bifidobacterium spp. in group HB increased by approximately 2.68 times compared to 0 h, falling within the range of 2.3–3.1 times; the spp. in group TB increased by approximately 1.10 times compared to 0 h. The Chao1 and Shannon indices in group HB showed an overall trend of initial decrease followed by recovery. In PCoA analysis, PC1 and PC2 explained 52.1% and 24.0% of the data, respectively, with a cumulative explained rate of 76.1%. Samples from different time points in group HB showed continuous migration along the culture time.

[0061] During the period from 72 h to 14 d, *Veillonella* showed a sustained enrichment in the HB group, while no similar enrichment was observed in the TB group. At 28 d of fermentation, the relative abundance of Proteobacteria was 3.2% in the HB group and 7.8% in the TB group. When comparing the HB and TB groups using the pre-specified LEfSe test at 28 d of fermentation, the relative abundance of *Fusobacterium* in the TB group was significantly higher than that in the HB group, and it was identified as a significantly dominant microbial marker in the TB group compared to the HB group (Kruskal-Wallis test p=0.018, LDA score=4.8). No dominant *Fusobacterium* marker satisfying p<0.05 and LDA score>4 was detected in the HB group compared to the TB group. Figure 4 The results presented are exploratory LEfSe results over the entire time series, used to help illustrate the differences in the microbial community at different time stages, and are not intended to replace the two pre-set comparisons for the above 28 days.

[0062] Comparative Example 1 was evaluated using a short-term static fermentation method. To examine the differences between the information obtained from the semi-continuous long-term fermentation of this invention and conventional short-term static culture, a short-term static fermentation comparative example was set up. The initial systems of the HB-S and TB-S groups were the same as in Example 3, containing 5 mL of the corresponding small intestinal digestion products, 10 mL of human fecal bacterial suspension, and 85 mL of anaerobic liquid culture medium, respectively. Each group was cultured at 37°C and 180 rpm without draining or replenishing the culture medium during the culture period, and samples were taken at 24 h, 48 h, and 72 h. The HB-C and TB-C semi-continuous fermentation groups in Example 3 were also used as comparisons.

[0063] The relative abundance of Bifidobacterium and the Shannon index were measured, and the differences in the bacterial community structure between HB and TB were evaluated based on the Bray-Curtis distance. Table 5 shows the HB / TB Bray-Curtis distance as the average pairwise Bray-Curtis distance between samples from the HB and TB groups at the same time point; the parallel sample index (CV) is the coefficient of variation of the relative abundance of Bifidobacterium in three independent parallel samples from each group at the corresponding time point.

[0064] Table 5 Comparison of static fermentation and semi-continuous fermentation

[0065] Under static culture conditions, HB and TB showed some differences between 24 and 48 hours, but the difference in Bifidobacterium genus between the two groups decreased at 72 hours, while the dispersion of parallel samples increased. In contrast, the semi-continuous system, by periodically removing part of the fermentation broth and adding fresh culture medium and corresponding small intestinal digestion products, was able to maintain the directional differences in bacterial communities between HB and TB during a longer culture period. This result indicates that the role of semi-continuous culture in this invention is not simply to prolong the culture time, but to obtain long-term bacterial succession information that is difficult to stably retain in short-term static culture by continuously updating part of the culture environment and continuously supplementing the test substrate after small intestinal digestion.

[0066] Comparative Example 2: Direct fermentation of raw wine that has not undergone gastrointestinal digestion. To investigate the impact of continuous gastrointestinal digestion on subsequent microbiota evaluation, a direct fermentation group of raw wine without gastrointestinal digestion was established. Five groups were set up: HB-D, TB-D, HB-R, TB-R, and E. HB-D and TB-D used small intestinal digestion products of HB and TB, respectively; HB-R and TB-R used HB and TB without simulated gastrointestinal digestion, diluted with sterile water as supplementary substrates; and group E used an ethanol-water solution as a control. The residual ethanol concentration in the small intestinal digestion products was determined using headspace gas chromatography-flame ionization detection, and a calibration curve was established using an ethanol standard series. Groups HB-R, TB-R, and E were adjusted to equal amounts of ethanol based on the measured residual ethanol concentrations of the small intestinal digestion products of HB-D and TB-D to maintain comparable ethanol levels among different groups.

[0067] Each group was cultured for 28 days according to the semi-continuous culture method in Example 3.

[0068] Table 6. Changes in microbial community under different substrate treatments

[0069] When undigested raw wine was used directly as a substrate, although some differences in microbiota still existed between HB-R and TB-R, the magnitude of these differences was smaller than when the corresponding small intestinal digestion products were used as substrates. Particularly regarding the changes in Bifidobacteria at 48 h, the relative abundance of Proteobacteria at 28 d, and the relative abundance of Fusobacterium at 28 d, HB-D and TB-D, which were digested in the gastrointestinal tract before entering the fermentation system, showed a clearer trend of separation. This indicates that using small intestinal digestion products as substrates for subsequent microbiota culture is beneficial for maintaining the material connection between upper gastrointestinal treatment and subsequent microbiota evaluation.

[0070] Example 4: Screening of different semi-continuous replacement ratios To investigate the effect of the daily replacement ratio on long-term culture results during semi-continuous fermentation, the daily system replacement ratio was set to 10%, 20%, 30%, and 40%, respectively, while keeping other conditions constant. After the corresponding proportion of fermentation broth was removed, equal volumes of fresh anaerobic liquid culture medium and small intestinal digestion products were added, with the volume ratio of fresh culture medium to small intestinal digestion products maintained at 5:1. Each group was cultured for 28 days, and the Bray-Curtis distance between HB and TB at 28 days, the coefficient of variation (CV) of key microbial community indicators in parallel samples, and the pH fluctuation of the system were used as comparative indicators. Table 7 lists the HB / TB Bray-Curtis distance as the average pairwise Bray-Curtis distance between samples from the HB and TB groups at 28 days; the CV of key microbial community indicators is the average coefficient of variation of the relative abundance of Bifidobacterium at 48 h and the relative abundance of Proteobacteria at 28 days in their respective three independent parallel samples.

[0071] Table 7 Comparison of different daily replacement ratios

[0072] When the daily replacement ratio is 10%, the accumulation of culture metabolites in the system is relatively high, leading to increased sample dispersion in the later stages. When the replacement ratio is increased to 20% and 30%, the system stability improves. Further increasing to 40%, although the culture environment is sufficiently refreshed, the cumulative difference between HB and TB decreases. In the culture system of this embodiment, a daily replacement of 30% can balance culture environment refresh, continuous input of analyte substrates, and maintenance of long-term microbial community differences.

[0073] Example 5: Repeatability verification of multiple production batches of samples and ethanol controls of equal alcohol content Five independent production batches of HB and five independent production batches of TB were selected, and each batch was subjected to in vitro gastrointestinal digestion, metabolite detection and 28-day semi-continuous fermentation according to Examples 1 to 3. An ethanol-water solution E with the same alcohol content as HB and TB was set up as an ethanol control.

[0074] Table 8 Representative evaluation indicators for different batches of HB and TB

[0075] Table 9 Evaluation results of ethanol controls with equal alcohol content

[0076] The five HB production batches showed largely consistent trends in gastric digestion, small intestinal digestion, and semi-continuous fermentation stages. The five TB production batches, as ordinary baijiu batches, exhibited different cross-stage response characteristics. The ethanol group at the same alcohol concentration did not show the increased Bifidobacteria and long-term bacterial separation trend observed in HB, indicating that the differences observed between HB and TB cannot be simply attributed to the ethanol concentration itself.

[0077] Example 6: Comparison of Single Evaluation Dimension and Cross-Stage Joint Evaluation Five HB production batches and five TB production batches from Example 5 were used as 10 batches to be evaluated. Each batch was divided into three independent parallel treatments, and a fixed reference liquor was used to form three independent parallel treatments, creating an evaluation unit of "batch to be evaluated - reference group" under the same digestion, fermentation, and testing conditions. Each batch was randomly coded by personnel not involved in the analysis, and the analysts completed the index calculations and final determinations for each evaluation unit without knowing the true category of the batch. Therefore, the B and D indices related to differential metabolite enrichment and LEfSe were based on inter-group statistics between the three independent parallel treatments of each batch and the corresponding three independent parallel treatments of the reference group, rather than calculating inter-group p-values ​​or LDA scores for a single sample.

[0078] The following four evaluation indicators were set: A. At least two of the three independent parallel gastric digestion samples of the batch to be evaluated had an amyl acetate characteristic peak with an S / N ≥ 3 and a forward and reverse match score in the mass spectrometry database greater than 700; B. During the gastric digestion stage, the enrichment analysis of the unsaturated fatty acid biosynthesis pathway of the batch to be evaluated relative to the reference group (p < 0.05); during the small intestinal digestion stage, the enrichment analysis of the primary bile acid biosynthesis pathway of the batch to be evaluated relative to the reference group (p < 0.05); and the enrichment analysis of the glutathione metabolism pathway of the reference group relative to the batch to be evaluated (p < 0.05); C. The relative abundance of Bifidobacterium spp. in the three independent parallel samples of the batch to be evaluated increased by at least 2-fold from the average at 0 h after 48 h of fermentation; D. In the LEfSe intergroup analysis of the reference group relative to the batch to be evaluated after 28 d of fermentation, Fusobacterium spp. met the Kruskal-Wallis test (p < 0.05) and had an LDA score > 4.

[0079] Batch-level evaluation is performed using individual indicators from A, B, C, and D respectively; in addition, a combined evaluation using four indicators from A to D is performed. When the same batch to be evaluated meets at least three of these indicators, it is determined that it has the cross-stage digestion-fermentation response characteristics described in this invention.

[0080] Table 10 Batch-level blind method differentiation results for different evaluation methods

[0081] When using volatile and non-volatile metabolic indicators, 48-hour microbial community indicators, or 28-day microbial community indicators alone for batch-level evaluation, all can distinguish between hepatic oxalate (HB) and tuberculous thromboembolism (TB) to some extent, but inconsistencies still exist in the identification of individual batches. After using a combined evaluation of four cross-stage indicators, 9 out of 10 blinded batches were correctly identified, achieving a batch-to-batch consistency rate of 90.0%, higher than any single evaluation dimension. This result indicates that information from different digestion and fermentation stages can complement each other, reducing inconsistencies caused by batch-to-batch variations in individual indicators.

[0082] Example 7: The impact of different sampling time combinations on evaluation results The 10 batches to be evaluated obtained in Example 5 were evaluated using different bacterial community sampling combinations. Scheme I used only 48-hour bacterial community data; Scheme II used 24-hour, 48-hour, and 72-hour bacterial community data; Scheme III used 48-hour and 28-day bacterial community data; and Scheme IV used complete time-series data for 0-hour, 24-hour, 48-hour, 72-hour, 7-day, 14-day, and 28-day periods. The discriminant consistency rate of the 10 batches to be evaluated and the consistency of the change directions of HB and TB in each batch were used as evaluation indicators.

[0083] Table 11 Evaluation results of different sampling time combinations

[0084] When using only an early single time point, inconsistencies in judgment can arise in some batches due to short-term microbial community fluctuations; however, by utilizing information from the early 48 hours and the long-term 28 days, the consistency of evaluation improves. A complete time series can further reflect the changes in the microbial community from the early response and transition phases to the longer-term culture phases.

[0085] Example 8: Evaluation of the applicability of the method under different sample processing volumes Treatment groups of baijiu (Chinese white liquor) were set up with volumes of 100 mL, 200 mL, and 300 mL. Using the 200 mL treatment group as a baseline, the corresponding simulated digestion system was added at 0.5 times the volume of the 100 mL treatment group and at 1.5 times the volume of the 300 mL treatment group, ensuring a consistent ratio between the sample, the simulated digestion system, and the corresponding digestion components. Digestion and fermentation were evaluated according to Examples 1-3.

[0086] Table 12 Representative evaluation results under different sample processing volumes

[0087] While maintaining the same proportion of sample to each digestion component, the evaluation results obtained from different treatment scales showed basically the same direction of change, indicating that this method does not depend on a single absolute sample processing volume and the system can be adjusted proportionally according to the volume of the digestion equipment and the detection requirements.

[0088] Comprehensive analysis The above embodiments and comparative examples show that the present invention first simulates the digestion of the herbal liquor to be tested through the oral cavity, stomach, and small intestine, and retains the detection information of the gastric digestion and small intestinal digestion stages respectively; then the obtained small intestinal digestion products are used as the continuous input substrate of the semi-continuous fermentation system, and long-term culture is carried out under the conditions of periodic drainage and replenishment of fresh culture medium and corresponding digestion products, thereby making the chemical transformation information of the upper digestive tract stage and the subsequent microbial community time series information have a continuous sample source relationship.

[0089] Compared with direct microbial culture of undigested raw wine, using small intestinal digestion products as fermentation substrates yields a clearer HB / TB separation trend. Compared with short-term static culture, semi-continuous culture can continuously replenish small intestinal digestion products and update part of the culture environment over a longer period of time, thereby preserving information on early response and subsequent microbial succession stages. The comparison of different replacement ratios further shows that the replacement ratio and evaluation effect are not simply a monotonic relationship. In this embodiment, 30% replacement per day takes into account both discrimination and repeatability.

[0090] Further, by jointly evaluating volatile metabolites from the gastric digestion stage, non-volatile metabolites from the small intestinal digestion stage, and early and long-term microbial community indicators, inconsistencies caused by batch-to-batch fluctuations in individual indicators can be reduced. Based on the above continuous processing relationship and joint evaluation method, this invention forms a comprehensive in vitro evaluation process for herbal baijiu with a sequential material connection.

[0091] The above description is merely a specific embodiment of the present invention, used to illustrate the technical concept of the present invention. Those skilled in the art can make adjustments to specific testing instruments, system scale, or equivalent operating parameters based on the disclosure of the present invention without changing the technical essence of the present invention, and these adjustments can all be used to implement the present invention.

Claims

1. A comprehensive evaluation method for herbal baijiu based on in vitro gastrointestinal digestion, characterized in that, Includes the following steps: (1) Set up the herbal liquor to be tested and the reference liquor. The reference liquor is a liquor with the same aroma and alcohol content as the base liquor of the herbal liquor to be tested and without the introduction of medicinal and edible plant raw materials. The herbal liquor to be tested and the reference liquor are subjected to in vitro simulated oral digestion, gastric digestion and small intestinal digestion under the same conditions. Samples are taken at the end of gastric digestion and the end of small intestinal digestion to obtain gastric digestion sample and small intestinal digestion sample respectively. (2) Volatile metabolites and non-volatile metabolites were detected in the gastric digestion samples and small intestinal digestion samples, respectively; (3) The small intestinal digestion products, human fecal microbial suspension prepared from the feces of no less than 3 healthy adults and mixed in equal volumes, and anaerobic liquid culture medium are mixed for anaerobic fermentation; every 24 hours after the start of fermentation, 10% to 40% of the total volume of the fermentation system is discharged, and a feed liquid equal to the volume of the discharged fermentation liquid is added. The feed liquid is composed of fresh anaerobic liquid culture medium and small intestinal digestion products corresponding to the corresponding liquor at a volume ratio of 5:1; samples are taken at at least one time point within the fermentation range of 24 to 72 hours and at least one time point within the fermentation range of 7 to 28 days to obtain fermentation samples. (4) Perform 16S rRNA high-throughput sequencing on the fermentation sample; (5) At the same digestion stage or the same fermentation time point, the test data of the herbal liquor to be tested and the reference liquor are compared. At least one evaluation index is selected from the metabolite data of the gastric digestion stage, the metabolite data of the small intestine digestion stage, the microbial community data of 24-72 h of fermentation and the microbial community data of 7-28 d of fermentation. The joint evaluation is carried out according to the preset judgment rules to obtain the cross-stage digestion-fermentation response characteristics of the herbal liquor to be tested.

2. The comprehensive evaluation method for herbal liquor according to claim 1, characterized in that, In step (3), the initial fermentation system of the anaerobic fermentation consists of 5 parts of small intestinal digestion products, 10 parts of human fecal bacterial suspension and 85 parts of anaerobic liquid culture medium. The fermentation temperature is 37°C and the total fermentation time is 28 days. After the fermentation starts, 30% of the total volume of the fermentation liquid is discharged every 24 hours, and 25% of the total volume of the fermentation system of fresh anaerobic liquid culture medium and 5% of the total volume of the corresponding small intestinal digestion products are added.

3. The comprehensive evaluation method for herbal liquor according to claim 1 or 2, characterized in that, In step (3), fermentation samples are collected at the start of fermentation and at 24 h, 48 h, 72 h, 7 d, 14 d and 28 d of fermentation. The samples taken at each sampling time point after 24 h of fermentation are taken from the fermentation liquid discharged in the corresponding cycle, and the samples taken at the start of fermentation are taken before the start of cultivation.

4. The comprehensive evaluation method for herbal liquor according to claim 1, characterized in that, In step (1), the in vitro simulated oral digestion was treated with α-amylase for 2 min at 37°C and pH 7.0; the in vitro simulated gastric digestion was treated with pepsin and gastric lipase for 2 h at 37°C and pH 3.0; and the in vitro simulated small intestinal digestion was treated with trypsin and bile for 2 h at 37°C and pH 7.

0.

5. The comprehensive evaluation method for herbal liquor according to claim 1, characterized in that, In step (2), the volatile metabolites are detected using headspace solid-phase microextraction-gas chromatography-mass spectrometry (GC-MS). 8 mL of the digested sample to be tested is taken, 1.6 g of sodium chloride is added, and the sample is extracted with a 65 μm CAR / PDMS extractor at 80 °C for 8 min with shaking. After desorption at 250 °C for 5 min, GC-MS detection is performed. The detection of characteristic volatile metabolites is determined by the characteristic ion peak signal-to-noise ratio (S / N) ≥ 3 and the forward and reverse matching degrees of the mass spectrometry database both being greater than 700.

6. The comprehensive evaluation method for herbal liquor according to claim 1, characterized in that, The human fecal microbial suspension was prepared from fresh feces of 3 to 8 healthy adults with a BMI of 18.5 to 23.9 kg / m² and who had not taken antibiotics within 6 months prior to sampling. The feces from each donor were diluted with PBS containing 0.1% L-cysteine ​​hydrochloride at a mass-to-volume ratio of 1:5, filtered, and the resulting filtrates were mixed in equal volumes.

7. The comprehensive evaluation method for herbal liquor according to claim 1, characterized in that, In step (2), the detection of non-volatile metabolites is performed using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry. During the same digestion stage, the metabolite detection data of at least three independent parallel samples of the herbal liquor to be tested and the reference liquor are compared to screen for differential metabolites. KEGG metabolic pathway enrichment analysis is then performed on the differential metabolites. The screening conditions for differential metabolites are |log2FC|>1 and p<0.

05. Metabolic pathway enrichment analysis is defined as p<0.05 as significant enrichment.

8. The comprehensive evaluation method for herbal liquor according to claim 7, characterized in that, In step (4), the V4 region of the 16S rRNA gene of microorganisms in the fermentation sample was amplified using primers 515F and 806R. The amplified products were subjected to high-throughput sequencing, and the taxonomic composition and relative abundance of the microbial community were obtained based on ASV. At the same fermentation time point, the microbial community data of at least three independent parallel samples of the herbal liquor to be tested and the reference liquor were compared between groups. The LEfSe analysis used the Kruskal-Wallis test p < 0.05 and LDA score > 4 as the criteria for determining significant differences in microbial markers.

9. The comprehensive evaluation method for herbal liquor according to claim 8, characterized in that, The cross-stage digestion-fermentation response characteristics were evaluated using the following four indicators: A, the detection results of the characteristic peak of amyl acetate in the gastric digestion sample of the herbal baijiu to be tested; B, the enrichment results of the unsaturated fatty acid biosynthesis pathway of the herbal baijiu to be tested relative to the reference baijiu during the gastric digestion stage, the enrichment results of the primary bile acid biosynthesis pathway of the herbal baijiu to be tested relative to the reference baijiu during the small intestinal digestion stage, and the enrichment results of the glutathione metabolism pathway of the reference baijiu relative to the herbal baijiu to be tested during the small intestinal digestion stage. C, The change in the relative abundance of Bifidobacterium spp. after 48 h of fermentation of the tested herbal baijiu relative to the beginning of fermentation; D, The intergroup analysis results of Fusobacterium LEfSe in the reference baijiu group and the tested herbal baijiu group after 28 days of fermentation.

10. The comprehensive evaluation method for herbal liquor according to claim 9, characterized in that, When the herbal liquor to be tested meets at least three of the above four evaluation indicators, it is determined that it has the cross-stage digestion-fermentation response characteristics. Among them, indicator A is that the signal-to-noise ratio (S / N) of the characteristic peak of amyl acetate in the gastric digestion sample is ≥3 and the forward and reverse matching degrees of the mass spectrometry database are both greater than 700; indicator B is that the enrichment analysis of the unsaturated fatty acid biosynthesis pathway in the gastric digestion stage is p<0.05, the enrichment analysis of the primary bile acid biosynthesis pathway in the small intestine digestion stage is p<0.05, and the enrichment analysis of the glutathione metabolism pathway of the reference liquor relative to the herbal liquor to be tested in the small intestine digestion stage is p<0.05; indicator C is that the relative abundance of Bifidobacterium spp. at 48 h of fermentation is at least 2 times higher than that at the beginning of fermentation; indicator D is that the Fusobacterium spp. in the reference liquor group relative to the herbal liquor group at 28 d of fermentation meets the Kruskal-Wallis test in LEfSe analysis, p<0.05 and the LDA score is >4.

Images