Simultaneous test method for organic acids and phenolic acids in lactic acid bacteria fermented fruit and vegetable juices

Abstract

This invention discloses a method for simultaneous testing of organic acids and phenolic acids in lactic acid bacteria fermented fruit and vegetable juices. The method comprises the following steps: Step (1): Mix the sample to be tested with a 50% methanol solution, collect the supernatant, and filter; Step (2): Inject the filtrate obtained in Step (1) into a high-performance liquid chromatograph (HPLC) for analysis, obtaining a HPLC chromatogram; Step (3): Obtain the peak areas corresponding to different organic acids and phenolic acids from the HPLC chromatogram, and then calculate the content of organic acids and phenolic acids involved in the sample to be tested based on the standard curve; wherein, the chromatographic column is a phenyl column, mobile phase A is methanol, mobile phase B is a 0.1%~0.4% phosphoric acid aqueous solution, the flow rate is 0.8~1.0 mL / min, and the temperature is 30~35℃. This invention can simultaneously determine 14 target substances and can regulate the production conditions of lactic acid bacteria fermented fruit and vegetable juices in real time, which is beneficial for monitoring product quality.

Description

Technical Field

[0001] This invention relates to the field of chemical analysis technology, specifically to a method for the simultaneous testing of organic acids and phenolic acids in lactic acid bacteria fermented fruit and vegetable juices. Background Technology

[0002] Lactic acid bacteria fermented fruit and vegetable juices include fermented fruit juice, fermented vegetable juice, and fermented compound fruit and vegetable juice. The organic acids and phenolic acids they contain are the core components that determine their flavor characteristics, functional activities, and quality stability.

[0003] The preparation method of lactic acid bacteria fermented fruit and vegetable juice is generally as follows: first, the raw materials are pretreated by cleaning, pulping, saccharification, sterilization and other steps, then suitable lactic acid bacteria strains are inoculated for temperature-controlled fermentation, and finally filtered and sterilized to prepare the finished product.

[0004] During the fermentation process described above, the organic acids such as lactic acid, malic acid, acetic acid, and citric acid produced by lactic acid bacteria metabolism are the material basis for the sweet and sour flavor of the product, and their content and proportion directly affect the balance of taste. Meanwhile, the free phenolic acids such as gallic acid, protocatechuic acid, ferulic acid, and cinnamic acid, which are generated by the enzymatic hydrolysis of bound polyphenols and other phenolic precursors from fruits and vegetables, are the main contributing factors to the product's antioxidant, anti-inflammatory, and other physiological activities.

[0005] During fermentation, the contents of organic acids and free phenolic acids change dynamically. Changes in organic acid content can, to some extent, reflect the metabolic activity level of lactic acid bacteria. The formation of free phenolic acids is also influenced by the metabolism of lactic acid bacteria, primarily originating from the following two sources:

[0006] First, the bound phenolic acids contained in fruits and vegetables are metabolized and transformed into monomers such as hydroxybenzoic acid, which can be metabolized into gallic acid, protocatechuic acid, etc.

[0007] Secondly, lactic acid bacteria can metabolize other phenolic precursors, such as hydroxycinnamic acid, to produce chlorogenic acid, ferulic acid, cinnamic acid, etc.

[0008] For the aforementioned organic acids and phenolic acids, existing methods only detect their single component category, for example:

[0009] 1. The national standard GB / T 5009.157-2016 uses high performance liquid chromatography to detect organic acids in fruit juice, beverages, chewing candies, pastries, jellies, canned fruits, etc. It only covers the determination of 7 organic acids, including tartaric acid, lactic acid, and malic acid, and does not cover phenolic acid components. The chromatographic column is a reverse-phase CAPECELL PAK MGS5-C18 column.

[0010] 2. Although the agricultural standard NY / T 3290-2018 can determine 14 phenolic acids, such as gallic acid, chlorogenic acid, and cinnamic acid, in fruits, vegetables, and their products, it does not cover organic acids, which are key metabolites in the fermentation process of fruit and vegetable juices fermented by lactic acid bacteria. Furthermore, this method requires quantitative analysis using liquid chromatography-mass spectrometry (LC-MS), meaning that the high-performance liquid chromatograph (HPLC) must be connected in series with a triple quadrupole mass spectrometer (MMS). The HPLC column used is a Waters HSS T3 column, and the price of a mid-range or higher triple quadrupole MMS can range from 1 million to 2 million yuan. Therefore, its detection cost is high and its universality is poor.

[0011] 3. Agricultural standard NY / T 2012-2011 uses high performance liquid chromatography to detect only four free phenolic acids in fruit products: caffeic acid, coumaric acid, ferulic acid, and sinapic acid. The chromatographic column used is a C18 column, which not only fails to detect a sufficient number of phenolic acids but also cannot take into account the analysis of organic acids.

[0012] 4. In the paper "Optimization of Roselle Infusion and Fermentation Process and Comparative Study of Organic Acids and Phenolic Acids Before and After Fermentation" published in Volume 39, Issue 4 of Food Science in 2018, the research team used three independent high-performance liquid chromatography (HPLC) methods to systematically detect seven organic acids, including lactic acid, acetic acid, and tartaric acid, and eight phenolic acid components, including protocatechuic acid and ferulic acid.

[0013] 5. The comparative study of organic acids and phenolic acids in root exudates of different soybean varieties published in the 23rd issue of Anhui Agricultural Sciences in 2007 designed two sets of high performance liquid chromatography detection schemes to determine seven common organic acids and five phenolic acids in batches.

[0014] 6. The article "Differential Analysis of Non-volatile Organic Acids and Phenolic Acids in Soy Sauce-flavored Baijiu with Different Processes" published in the 7th issue of Brewing Technology in 2024 also requires the use of two different detection methods to achieve comprehensive coverage of common organic acids and phenolic acids.

[0015] In summary, current technologies require separate detection methods for organic acids and phenolic acids, making simultaneous determination of both impossible. In other words, under current technological conditions, simultaneous detection of organic acids and phenolic acids is not feasible. This is because achieving simultaneous determination of organic acids and phenolic acids presents two major challenges:

[0016] Firstly, organic acids and phenolic acids have different structures, and current technologies have not found chromatographic columns that can simultaneously separate them. Organic acids can be well separated on strong cation exchange columns through ion exchange with the stationary phase. For example, existing technologies using CAPECELL PAK MGS5-C18 columns with phosphate methanol as the mobile phase, or Aminex HPX-87H columns (300 × 7.8 mm) with 0.045N H2SO4 as the mobile phase, can successfully separate compounds such as acetic acid, citric acid, malic acid, and lactic acid simultaneously. However, these types of columns cannot separate phenolic acids, and Aminex... The HPX-87H column cannot use an organic phase as the mobile phase; while phenolic acids show better separation on the T3 column. For example, gallic acid has a short retention time on the T3 column, while ferulic acid has a longer retention time due to the presence of hydrophobic substituents such as methoxy groups, and the two can be separated. However, in the non-polar environment of the T3 column, the hydrophobic interaction between organic acids and the stationary phase is weak, and most of them will elute rapidly with the mobile phase, making it difficult to retain and separate them effectively. Common monobasic organic acids such as acetic acid and propionic acid elute almost simultaneously on the T3 column, resulting in poor separation.

[0017] Secondly, organic acids and phenolic acids have different ultraviolet absorption wavelengths. Both have carboxyl groups (-COOH), and some also contain auxiliary functional groups such as hydroxyl groups (-OH), carbonyl groups (C=O), and phenolic hydroxyl groups (-Ar-OH, where Ar is a benzene ring). These functional groups all have certain ultraviolet absorption, but due to structural differences, their ultraviolet absorption wavelengths are different. The wavelengths used in existing technologies are only suitable for the detection of a single component category, making it difficult to simultaneously determine organic acids and phenolic acids.

[0018] In the industrial-scale production of lactic acid bacteria fermented fruit and vegetable juices, because organic acids and phenolic acids cannot be measured simultaneously, companies need to use multiple methods to detect the two types of components separately. The drawbacks of this approach are:

[0019] 1. Data discrepancies may occur due to testing time differences, making it difficult to accurately control product quality: For example, organic acids may be tested in the morning, but phenolic acids can only be tested in the afternoon. During this period, the fermentation state may have changed, making it impossible to reflect the quality status at the same time point in real time. This will lead to delays in process adjustments and make it impossible to control production conditions in real time to ensure product quality. That is, when the organic acid content meets the requirements, the fermentation maturity may be misjudged because the free phenolic acid status is not known at the same time, which may easily cause product flavor imbalance or insufficient functional activity.

[0020] 2. High testing costs and long testing time: Existing technologies require at least 2-3 testing methods to detect organic acids and phenolic acids in the production process of lactic acid bacteria fermented fruit and vegetable juices. Each method requires separate sample processing, instrument debugging, and repeated experiments. Calculated based on the cost per test (including reagents, labor, and instrument wear and tear), the cost of testing with a single method is approximately 500-800 yuan. Completing the testing of both increases the cost by at least 1-2 times. At the same time, multiple tests lead to a longer time cycle, with each test requiring 5-6 hours and a cumulative time exceeding 12 hours, resulting in reduced production batch turnover efficiency.

[0021] 3. There are significant limitations in the detection of organic acids and phenolic acids during the production of lactic acid bacteria fermented fruit and vegetable juices: Because there is no method for simultaneously measuring the content of organic acids and phenolic acids, research on the fermentation mechanism of lactic acid bacteria fermented fruit and vegetable juices and plant-derived fermented products remains at the level of single components. It is difficult to simultaneously reveal the dynamic relationship between organic acids and phenolic acids, and cannot reflect the metabolic pathways of lactic acid bacteria and the laws governing polyphenol conversion. Therefore, it is also impossible to further clarify the relationship between the regulation of organic acid content and the increase in free phenolic acid release. Consequently, the detection results cannot provide an accurate basis for real-time control of production conditions, thus affecting product quality monitoring, which is particularly significant for the industry of live bacteria fermented fruit and vegetable juices with short shelf lives. Summary of the Invention

[0022] The purpose of this invention is to provide a method for the simultaneous testing of organic acids and phenolic acids in lactic acid bacteria fermented fruit and vegetable juices, enabling simultaneous and efficient quantitative determination of organic acids and phenolic acids, and allowing for real-time control of the production conditions of lactic acid bacteria fermented fruit and vegetable juices, which is beneficial for monitoring product quality.

[0023] To achieve the above objectives, the present invention adopts the following technical solution.

[0024] A method for simultaneous testing of organic acids and phenolic acids in lactic acid bacteria fermented fruit and vegetable juices, characterized by comprising the following steps:

[0025] Step (1): Mix the sample to be tested with 50% methanol solution, take the supernatant and filter it;

[0026] Step (2): Inject the filtrate obtained in step (1) into a high performance liquid chromatograph for analysis and obtain a liquid chromatogram;

[0027] Step (3): Obtain the peak areas corresponding to different organic acids and phenolic acids from the liquid chromatogram, and then calculate the content of organic acids and phenolic acids involved in the sample to be tested based on the standard curve;

[0028] The conditions for the high-performance liquid chromatography are as follows:

[0029] The chromatographic column used is a phenyl column, i.e., a Phenyl column, and its packing material is a phenyl (-C6H5) bonded phase;

[0030] Mobile phase A was methanol, mobile phase B was 0.1%~0.4% (volume percentage) phosphoric acid aqueous solution, the flow rate was 0.8~1.0 mL / min; the temperature was 30~35℃, and the injection volume was 10 μL;

[0031] The gradient elution conditions for the mobile phase are:

[0032] From 0 to 8.5 min, mobile phase A changed from 2.5% to 5%, and mobile phase B changed from 97.5% to 95%.

[0033] 8.5~10 min, mobile phase A 5%→10%, mobile phase B 95%→90%;

[0034] 25-30 min, mobile phase A 20%, mobile phase B 80%;

[0035] 30-40 min, mobile phase A 20%→40%, mobile phase B 80%→60%;

[0036] 40-55 min, mobile phase A 40%→50%, mobile phase B 60%→50%;

[0037] 55-60 min, mobile phase A 50%, mobile phase B 50%;

[0038] Over 60-61 min, mobile phase A decreased from 50% to 2.5%, and mobile phase B decreased from 50% to 97.5%.

[0039] 61~65 min, mobile phase A 2.5%, mobile phase B 97.5%;

[0040] The detection wavelength is set to 210nm for 0~8min and 254nm for 8~65min.

[0041] The lactic acid bacteria fermented fruit and vegetable juice of the present invention is a lactic acid bacteria fermented fruit and vegetable juice made from one or more of the following raw materials, including but not limited to apples, pears, cantaloupes, watermelons, pomegranates, prickly pears, citrus fruits, tomatoes, and cucumbers.

[0042] This invention can simultaneously determine 7 organic acids, 6 phenolic acids, and 1 phenolic acid precursor. The organic acids are: tartaric acid, malic acid, lactic acid, acetic acid, citric acid, succinic acid, and fumaric acid; the phenolic acids are: gallic acid, protocatechuic acid, chlorogenic acid, ferulic acid, cinnamic acid, and ellagic acid; and the phenolic acid precursor is: punicin. The above 14 substances cover the types of organic acids and phenolic acids and their precursors produced by the above fruits and vegetables during fermentation. In the following invention, phenolic acids and their precursors are collectively referred to as polyphenols.

[0043] The inventors, through practical experience in detecting organic acids and phenolic acids during the production of lactic acid bacteria-fermented fruit and vegetable juices, deeply felt the shortcomings of existing single-component detection methods, and thus initiated a research project to simultaneously detect both. The inventors conducted extensive experiments on various conditions suitable for simultaneous detection of both, particularly addressing the two major difficulties of simultaneous detection in existing technologies. Specifically, they tested various chromatographic columns, wavelengths, mobile phases, flow rates, and mobile phase gradient elution conditions suitable for simultaneous detection of organic acids and phenolic acids. Based on a large amount of data, they summarized and achieved simultaneous determination of organic acids and phenolic acids through synergistic optimization of "stationary phase interaction matching - dynamic adaptation of mobile phase elution control."

[0044] Through experimental research, the inventors discovered that by utilizing the unique structure of the phenyl-bonded phase of the Phenyl chromatographic column, it is possible to achieve differentiated retention of 7 organic acids and 7 polyphenols through multiple interactions, including van der Waals forces (i.e., the π-π interaction between phenyl and aromatic compounds), hydrogen bonds (i.e., the interaction between hydrogen on the phenyl and the polar groups of the target compound), and hydrophobic interactions. This solves the problem of insufficient separation of polybasic acids with small polarity differences and similar functional groups by traditional chromatographic columns.

[0045] The Phenyl column can be either an Agilent ZORBAX SB-Phenyl column or an XBridge BEHPhenyl column.

[0046] Meanwhile, this invention precisely controls the partition coefficient (K value) of the target analytes between the stationary and mobile phases by adjusting the ratio gradient and flow rate of the mobile phase, namely the methanol and 0.1%~0.4% phosphoric acid aqueous solution system. For example, by gradually increasing the proportion of the organic phase (methanol) to enhance elution capacity, phenolic acids with strong retention can be eluted rapidly. At the same time, the pH value is adjusted by phosphoric acid to inhibit the dissociation of organic acids, enhance their hydrophobic interaction with the stationary phase, avoid peak broadening, and ultimately achieve baseline separation of 14 target analytes, overcoming the co-eluting problem caused by the similar polarity of polybasic acids.

[0047] Preferably, in step (1), the sample to be tested is mixed with 50% methanol and subjected to ultrasonication for 15 minutes.

[0048] Compared with the prior art, the present invention has the following advantages:

[0049] 1. This invention proposes a method for simultaneous testing of organic acids and phenolic acids in lactic acid bacteria fermented fruit and vegetable juices, which enables simultaneous and efficient quantitative detection of seven common organic acids and seven polyphenols in the lactic acid bacteria fermented fruit and vegetable juices.

[0050] In this invention, a Phenyl chromatographic column is used, along with adjustments to the mobile phase, its flow rate, and its ratio, to achieve good separation of 14 target substances and to accurately detect the 14 target substances simultaneously using appropriate detection wavelengths.

[0051] 2. This invention is applicable to various types of lactic acid bacteria fermented fruit and vegetable juice samples, and the detection results are highly accurate. At the same time, the testing method of this invention is short, generally taking about 60 minutes, and can be used for real-time or periodic monitoring of organic acids and phenolic compounds in lactic acid bacteria fermented fruit and vegetable juice.

[0052] 3. This invention can be applied to the research and development of a series of beverage products made from single or compound fruits and vegetables through fermentation, which helps to control product quality.

[0053] Taking the 14 organic acids and polyphenols described in this invention as examples,

[0054] (1) The present invention greatly simplifies the detection method. Existing technologies require at least 2 to 3 detection methods, such as GB / T5009.157-2003 for the determination of organic acids and NY / T 3290-2018 for the determination of phenolic acids. Each method requires separate sample processing, instrument debugging, and repeated experiments. However, the present invention only requires one detection operation to complete the detection of the 14 organic acids and polyphenols in lactic acid bacteria fermented fruit and vegetable juice.

[0055] (2) The present invention greatly saves detection costs. Based on the cost of a single detection (including reagents, labor, and instrument wear and tear), a single method costs about 500 to 800 yuan. Existing technologies require at least 2 to 3 detection methods to complete the detection of organic acids and phenolic acids in lactic acid bacteria fermented fruit and vegetable juices. The detection cost is at least 1 to 2 times higher than the cost of a single detection, i.e., 1,000 to 2,400 yuan. However, the detection cost of the present invention is generally only 400 to 500 yuan.

[0056] (3) The present invention greatly saves time and costs. Existing technologies require multiple tests, which leads to a longer time cycle. A single test takes 5 to 6 hours, and 2 to 3 tests take a total of 10 to 18 hours, usually exceeding 12 hours. However, the present invention can complete the detection of the 14 organic acids and polyphenols in about 1 hour. This not only improves the efficiency of production batch turnover, but also allows for real-time adjustment of production conditions based on the test results, which is beneficial for controlling product quality.

[0057] (4) In their research on the simultaneous detection of organic acids and phenolic acids during the production process of lactic acid bacteria fermented fruit and vegetable juice, the inventors found that the core characteristic is the "synchronous generation and mutual influence" of organic acids and phenolic acids. The dynamic correlation between the two directly reflects the metabolic pathway of lactic acid bacteria and the polyphenol conversion law. In other words, the simultaneous determination of the content and changes of organic acids and phenolic acids helps researchers establish a quantitative correlation model of the two types of components, analyze key mechanisms such as "regulating organic acids to assist phenolic acid conversion", and play a positive role and important significance in clarifying the relationship between the regulation of organic acid content and the increase in the release of free phenolic acids, revealing the essential law of the formation of product quality of lactic acid bacteria fermented fruit and vegetable juice and the regulation of product quality.

[0058] In particular, existing products often use multi-strain or multi-raw material compound fermentation, resulting in relatively complex product compositions. Therefore, this invention provides a method applicable to lactic acid bacteria fermented fruit and vegetable juice substrates, capable of simultaneously and quantitatively detecting multiple organic acids and free phenolic acids. This method efficiently and synchronously monitors both, and by timely replenishment of materials and real-time adjustment of fermentation condition parameters, it regulates the rate of organic acid synthesis and metabolism, significantly improving the control and enhancement of the production quality of lactic acid bacteria fermented fruit and vegetable juices. This method has immense research value for the improvement and new product development of lactic acid bacteria fermented fruit and vegetable juices. Attached Figure Description

[0059] Figure 1 The scanned images are of the 14 reference standards in Example 1;

[0060] Figure 2 The liquid chromatogram of the compound fruit and vegetable fermentation juice;

[0061] Figure 3 The liquid chromatogram of pomegranate fermentation juice;

[0062] Figure 4 The liquid chromatogram of prickly pear fermented juice;

[0063] Figure 5 Liquid chromatogram for spiked determination of compound fruit and vegetable fermented juice;

[0064] Figure 6 Liquid chromatogram for spiked determination of pomegranate fermentation juice;

[0065] Figure 7 Liquid chromatogram for spiked determination of prickly pear fermented juice;

[0066] Figure 8 The liquid chromatograms of the compound fruit and vegetable fermentation juice before and after fermentation are shown.

[0067] Figure 9 The liquid chromatograms of pomegranate fermentation juice before and after fermentation are shown.

[0068] Figure 10 The images show the liquid chromatograms of prickly pear fermented juice before and after fermentation.

[0069] Figure 11 Comparison of liquid chromatograms measured under three flow rate conditions;

[0070] Figure 12 Comparison of liquid chromatograms measured with different mobile phases B;

[0071] Figure 13 For comparison of liquid chromatograms for determination of different mobile phase gradients in national and industry standards;

[0072] Figure 14 Comparison of liquid chromatograms for different methanol ratios during the 0–8.5 min period;

[0073] Figure 15 Comparison of liquid chromatograms measured at different column temperatures;

[0074] Figure 16 Comparison of liquid chromatograms measured using different liquid chromatography columns.

[0075] Figure 17 The liquid chromatogram for Example 9 is shown. Detailed Implementation

[0076] The present invention will be further described in detail below through specific implementation examples, so that those skilled in the art can better understand and implement the technical solution of the present invention.

[0077] The lactic acid bacteria fermented fruit and vegetable juice products tested in the following embodiments of the present invention include three types: compound fruit and vegetable fermented juice, pomegranate fermented juice, and prickly pear fermented juice.

[0078] The method for preparing lactic acid bacteria fermented fruit and vegetable juice is as follows:

[0079] (1) Take the raw material and pulp it. Add water at 0.1 to 0.3 times its mass to prepare a solution. Add calcium carbonate to adjust the pH value to 4.0 to 5.0. Add white sugar, which accounts for 4.0 to 6.0% of the total mass of the raw material, and stir until dissolved to obtain the original pulp before fermentation.

[0080] Fruit and vegetable pulp, pomegranate pulp, and prickly pear pulp were prepared according to the above method. Among them, the ratio of apple:pear:cantaloupe:watermelon:tomato:cucumber in the fruit and vegetable pulp was 7~10:7~10:1~2:1~2:1~2:1~2 (mass ratio); the pomegranate pulp was whole pomegranate pulp; and the prickly pear pulp was prickly pear pulp.

[0081] (2) Inoculate the plant pulp with Leuconostoc mesenteroides subsp. mesenteroides: Lactobacillus delbrueckii subsp. bulgaricus: Streptococcus thermophilus: Lactobacillus plantarum in a mass ratio of 1:1:1:1, so that the inoculum of lactic acid bacteria in the pulp is controlled at 10. 6 ~10 7CFU / mL; Fruit and vegetable pulp was fermented at 37℃ for 25 days to produce compound fruit and vegetable fermented juice, while pomegranate pulp and prickly pear pulp were fermented at 37℃ for 72 hours to produce pomegranate fermented juice and prickly pear fermented juice, respectively.

[0082] Example 1

[0083] 1. Preparation of the test solution:

[0084] Accurately pipette 1-5 mL of the sample to be tested into a 25 mL volumetric flask, then add about 15 mL of 50% methanol and sonicate for 15 min. Then, dilute to 25 mL with 50% methanol and mix well. Take the supernatant, filter it through a 0.22 μm filter membrane, and then perform the test.

[0085] 2. Preparation of mixed reference solution:

[0086] Accurately weigh specific amounts of different reference standards and place them in brown volumetric flasks. Dissolve the solutions in methanol, shake well, and dilute to the mark. Pipette specific volumes of the mother liquor into 20 mL volumetric flasks and dilute to the mark with 50% methanol to obtain mixed reference standard solutions. The sample amounts of different reference standards and the volumes of mother liquor taken are detailed in Table 1. Pipette 0.1 mL, 0.3 mL, 0.5 mL, 1.0 mL, 2.0 mL, and 3 mL of the mixed reference standard solution into 5 mL volumetric flasks and dilute to the mark with 50% methanol to obtain a series of mixed reference standard solutions for the preparation of standard curves.

[0087] Table 1: Preparation of Mixed Standard Solutions

[0088]

[0089] 3. Chromatographic conditions

[0090] The mobile phase ratio is detailed in Table 2. The concentration of mobile phase B, an aqueous solution of phosphoric acid, is 0.1%. The suitable flow rate is 1.0 mL / min. Column: Agilent ZORBAX SB-Phenyl column, 5 μm × 4.6 mm × 250 mm. Temperature: 35℃. Injection volume: 10 μL.

[0091] Table 2: Volume Ratio of Mobile Phase

[0092]

[0093] Referring to the above chromatographic conditions, and with the diode array detector enabled for full-wavelength scanning (190 nm–400 nm), the mixed reference solution was tested; the scan chromatogram is shown below. Figure 1 .

[0094] Tartaric acid, malic acid, lactic acid, acetic acid, citric acid, succinic acid, and fumaric acid exhibit strong absorption at wavelengths between 206 nm and 210 nm. Specifically, the 210 nm wavelength was selected for the detection of these seven organic acids. Among the seven polyphenols, gallic acid shows strong absorption between 254 nm and 290 nm, with the strongest absorption at 272 nm; protocatechuic acid shows strong absorption between 254 nm and 265 nm, with the strongest absorption at 260 nm; punicalin A and punicalin B show strong absorption between 250 nm and 266 nm, with the strongest absorption at 258 nm; chlorogenic acid shows strong absorption between 230 nm and 254 nm and between 306 nm and 345 nm, with two strongest absorption peaks at 242 nm and 326 nm; ferulic acid shows strong absorption between 236 nm and 254 nm and between 310 nm and 334 nm. Under the specified nm conditions, cinnamic acid exhibits strong absorption with two strongest absorption peaks at 245 nm and 324 nm. Cinnamic acid also shows strong absorption in the 254 nm–300 nm range, with two strongest absorption peaks at 278 nm. Ellagic acid shows strong absorption in the 250 nm–258 nm range, with a maximum absorption wavelength of 254 nm. Considering the strong absorption wavelength range of all seven polyphenols, 254 nm was selected as the absorption wavelength for all seven polyphenols. The seven organic acids completed their peak elution before 8 minutes, while the seven polyphenols began eluting after 8 minutes. Therefore, the detection wavelength was set at 210 nm for 0–8 minutes and at 254 nm for 8–65 minutes.

[0095] Example 2:

[0096] 1. Establishment of the standard curve

[0097] The mixed reference solution was diluted to six concentration levels as shown in Example 1, analyzed, and standard curves were plotted (see Table 3) to determine the linear range of each component. The data in Table 3 show that the correlation coefficients of the standard curves for the 14 components met the condition R > 0.999, indicating good linearity for all 14 components within their respective ranges. The linear ranges for each component were: tartaric acid 15.93 μg / mL–477.80 μg / mL, malic acid 48.12 μg / mL–1443.54 μg / mL, lactic acid 68.13 μg / mL–2043.82 μg / mL, acetic acid 39.14 μg / mL–1174.09 μg / mL, citric acid 41.90 μg / mL–1257.00 μg / mL, succinic acid 89.80 μg / mL–2694.00 μg / mL, and fumaric acid 0.82 μg / mL–24 μg / mL. 58 μg / mL, gallic acid 2.85 μg / mL~85.48 μg / mL, protocatechuic acid 1.99 μg / mL~59.85 μg / mL, punicin (A+B) 8.34 μg / mL~250.14 μg / mL, chlorogenic acid 1.99 μg / mL~59.81 μg / mL, ferulic acid 2.01 μg / mL~60.30 μg / mL, cinnamic acid 0.75 μg / mL~22.54 μg / mL, ellagic acid 3.39 μg / mL~101.75 μg / mL.

[0098] Table 3: Standard curves for 14 components

[0099]

[0100] 2. Precision and accuracy tests of blank spiked samples

[0101] The accuracy of the method of this invention was examined by spike recovery tests on blank samples. Distilled water was used instead of the sample, and an appropriate amount of reference standard was added and mixed thoroughly. Fourteen samples were prepared repeatedly according to the preparation method of the test solution in Example 1, and then tested under the chromatographic conditions in Example 1. The spike recovery rate of each reference standard was calculated. The results are shown in Table 4. The spike recovery rate of each reference standard was between 90% and 110%, and the RSD was less than 10%, which meets the requirements of GB / T27417-2017 "Guideline for Conformity Assessment and Validation of Chemical Analysis Methods", indicating that the method has high accuracy and accurate detection results.

[0102] Table 4 Results of Spiked Recovery Test

[0103]

[0104] 3. Results of the applicability and precision determination of compound fruit and vegetable fermented juice, pomegranate fermented juice, and prickly pear fermented juice.

[0105] For the compound fruit and vegetable fermented juice, pomegranate fermented juice, and prickly pear fermented juice, seven test solutions were prepared repeatedly according to the preparation method of the test solution in Example 1, and then tested according to the chromatographic conditions in Example 1. The applicability chromatograms for the determination of compound fruit and vegetable fermented juice, pomegranate fermented juice, and prickly pear fermented juice are shown below. Figures 2-4 As shown, no chromatographic peaks with the same retention times as those in the mixed reference solution were observed in the chromatogram of the blank control solution, proving that there was no interference. Meanwhile, seven organic acids and one polyphenol were detected in the compound fruit and vegetable fermentation juice; four organic acids and seven polyphenols were detected in pomegranate; and five organic acids and six polyphenols were detected in prickly pear. The RSD% (n=7) of each component was calculated using the standard curve, and all RSD values ​​were less than 10%, indicating good repeatability and high precision of the method. Detailed data are shown in Tables 5-7.

[0106] Table 5: Parallel Test Results of Compound Fruit and Vegetable Fermented Juice Samples

[0107]

[0108] Table 6: Results of Parallel Tests on Pomegranate Fermented Juice Samples

[0109]

[0110] Table 7: Parallel Test Results of Prickly Pear Fermented Juice Samples

[0111]

[0112] 4. Accuracy results of the determination of samples of compound fruit and vegetable fermented juice, pomegranate fermented juice, and prickly pear fermented juice

[0113] The accuracy of this method was assessed through spiked recovery tests on samples. 1–5 mL of sample was precisely pipetted into a 25 mL volumetric flask, and the corresponding mixed reference solution was added. Then, approximately 15 mL of 50% methanol was added, and the mixture was sonicated for 30 min. The volume was then brought to 25 mL with 50% methanol and thoroughly mixed. The supernatant was filtered through a 0.22 μm filter before analysis. Seven test solutions were prepared repeatedly, and the samples were analyzed under optimal chromatographic conditions. The spiked recoveries of each reference standard were calculated. Spiking tests were conducted on compound fruit and vegetable fermented juice, pomegranate fermented juice, and prickly pear fermented juice to assess the accuracy of the detection method. The amount of reference standard added and the test results are detailed in Tables 8–10. Tables 8–10 show that the spiked recoveries of each reference standard ranged from 90% to 110%, meeting the requirements of GB / T27417-2017 "Guideline for Conformity Assessment and Validation of Chemical Analysis Methods," indicating that the method has high accuracy and provides accurate detection results. Chromatograms of the spiked test results for compound fruit and vegetable fermented juice, pomegranate fermented juice, and prickly pear fermented juice are shown below. Figures 5-7 As shown.

[0114] Table 8: Spike Test Results of Compound Fruit and Vegetable Fermented Juice

[0115]

[0116] Table 9: Spike Test Results of Pomegranate Fermented Juice

[0117]

[0118] Table 10: Spike Test Results of Prickly Pear Fermented Juice

[0119]

[0120] Example 3:

[0121] Changes in organic acid and phenolic acid content before and after fermentation of compound fruit and vegetable fermented juice, pomegranate fermented juice, and prickly pear fermented juice

[0122] Following the preparation method of the test solution in Example 1, three samples were prepared repeatedly for the fermented fruit and vegetable juice, pomegranate, and prickly pear samples before and after fermentation. The samples were then tested under the chromatographic conditions described in Example 1, and the results are as follows: Figures 8-9 As shown in Tables 11-13.

[0123] The results showed that after fermentation, the levels of four organic acids—malic acid, succinic acid, citric acid, and fumaric acid—in the compound fruit and vegetable fermentation juice decreased, while the levels of two other organic acids—lactic acid and acetic acid—significantly increased. This demonstrates that the fermentation process has a certain capacity for organic acid metabolism. While lactic acid bacteria produce lactic acid and acetic acid during fermentation, malic acid, succinic acid, citric acid, and fumaric acid are also consumed. Furthermore, protocatechuic acid and chlorogenic acid, two phenolic acids, were produced in the fermented fruit and vegetable juice. The method of this invention can provide good support for the research of fermentation processes for fruit and vegetable fermentation juice samples.

[0124] After fermentation, pomegranate juice showed a significant increase in tartaric acid, succinic acid, lactic acid, and acetic acid, a significant decrease in pungent glycosides and ellagic acid, and a significant increase in gallic acid and chlorogenic acid. During pomegranate fermentation, the large-molecule phenolic acids pungent glycosides and ellagic acid were decomposed to produce gallic acid. Although the microbial strains used in pomegranate fermentation were the same as those used in fruit and vegetable fermentation juices, the patterns of change in organic acids and polyphenols differed significantly.

[0125] After fermentation, the levels of tartaric acid, lactic acid, acetic acid, and succinic acid in the fermented prickly pear juice all increased significantly, while the levels of gallic acid, chlorogenic acid, and gallic acid also increased significantly.

[0126] The method of this invention for monitoring the fermentation process of compound fruit and vegetable juice, pomegranate, and prickly pear not only facilitates process monitoring, but also significantly improves the phenolic acid content in the samples, providing a material basis for the development of product health benefits.

[0127] Table 11: Test Results of Compound Fruit and Vegetable Fermented Juice Before and After Fermentation

[0128]

[0129] Table 12: Test results of pomegranate fermented juice before and after fermentation

[0130]

[0131] Table 13: Test results of prickly pear fermented juice before and after fermentation

[0132]

[0133] Example 4:

[0134] To further investigate the applicability of the chromatographic conditions of this invention, comparative experiments were conducted on the mobile phase flow rate and phosphoric acid concentration.

[0135] 1) Mobile phase flow rates of 1.0 mL / min, 0.8 mL / min, and 0.5 mL / min were set respectively, and gradient elution was performed on the mixed reference solution according to the conditions in Example 1. The results are as follows: Figure 11 As shown in Table 14, the resolutions were as follows. The results showed that ellagic acid did not elute at a mobile phase flow rate of 0.5 mL / min; at mobile phase flow rates of 0.8 mL / min and 1.0 mL / min, the resolutions of all standards were greater than 1.5. Therefore, a flow rate of 0.8 mL / min to 1.0 mL / min is preferred for separation.

[0136] Table 14: Separation degree under three flow rate conditions

[0137]

[0138] 2) Simultaneously, 0.4% phosphoric acid, 0.1% phosphoric acid, and pure water were set as mobile phase B. The separation effect of the mixed reference solution was studied with reference to the gradient ratio conditions of the mobile phase in Example 1. The results are as follows: Figure 12 As shown in Table 15, the standards cannot be effectively separated under pure water conditions, failing to meet the requirement of a resolution greater than 1.5. When 0.1% and 0.4% phosphoric acid are used as mobile phase B, the resolution of each component is within the recommended range. However, under pure water conditions, the resolution of each component exceeds the recommended range. Therefore, 0.1%~0.4% phosphoric acid is preferred as mobile phase B.

[0139] Table 15: Separation degree under conditions B for the two mobile phases

[0140]

[0141] Example 5:

[0142] This embodiment includes one experimental group and three control groups. The testing conditions for the experimental group are the same as in Example 1. The differences between the control groups and Example 1 are as follows: For control group 1, the mobile phase was set according to the existing national standard method GB 5009.157-2016, "Determination of Organic Acids in Food," as shown in Table 16; for control group 2, the mobile phase was set according to NY / T 3290-2018, "Determination of Phenolic Acid Content in Fruits, Vegetables and Their Products," using liquid chromatography-mass spectrometry (LC-MS), with a flow rate of 1.0 mL / min, and the mobile phase gradient is shown in Table 17; for control group 3, the mobile phase was set according to NY / T 2012-2011, "Determination of Free Phenolic Acid Content in Fruits and Their Products," with a flow rate of 0.9 mL / min, and the mobile phase gradient is shown in Table 18. Other chromatographic parameters for the control groups remained consistent with those in Example 1. Test results are shown in Table 18. Figure 13 .

[0143] Table 16: Mobile phase setting ratio of control group 1

[0144]

[0145] Table 17: Mobile phase setting ratio of control group 2

[0146]

[0147] Table 18: Mobile phase setting ratio of control group 3

[0148]

[0149] Depend on Figure 13 It can be seen that, under the elution conditions of GB5009.157-2016 (reference standard 157-2016), the seven organic acids and gallic acid were well separated, but the remaining six polyphenols could not be separated, eluting mainly between 60 and 62.5 min. Under the elution conditions of NY / T3290-2018 (rapid elution), the overall separation effect of organic acids and polyphenols was poor. Under the elution conditions of NY / T 2012-2011 (reference standard 257-2011), tartaric acid could not be separated between 3.0 and 3.5 min (resolution less than 0.85 and less than 1.5), and at 40.05 min, ferulic acid, cinnamic acid, and ellagic acid (three phenolic acids) eluted simultaneously, failing to be effectively separated. The above reference standards cannot meet the requirements for the simultaneous determination of 14 organic acids and polyphenols. The mobile phase elution conditions of this invention are significantly superior to the above three elution methods, enabling the simultaneous determination of 14 organic acids and polyphenols.

[0150] Because the elution peaks of organic acids are relatively concentrated during the 0-8.5 min period in this invention, to further investigate the potential impact of mobile phase fluctuations on the detection results, the proportion of the mobile phase during the 0-8.5 min period was mainly adjusted, with the methanol ratio adjusted to four elution conditions: 2.5%→5%, 5%, 2.5%→10%, and 10%. (See Table 19 and...) Figure 14 It can be seen that during the 0-8.5 min period, when the methanol ratio was adjusted to 10%, the separation degree of malic acid, tartaric acid, lactic acid, and acetic acid was less than 1.5, and the target peak could not be separated. When the methanol ratio was adjusted to 2.5%→5%, 5%, and 2.5%→10%, all seven organic acids could be separated well. The separation degree of the seven polyphenols was also good, so they are not compared here. From the perspective of cost saving and environmental protection, the methanol ratio of 2.5%→5% during the 0-8.5 min period is preferred.

[0151] Table 19: Resolution under four mobile phase conditions

[0152]

[0153] Example 6: Comparison of Column Temperatures

[0154] This embodiment includes three experimental groups for comparing chromatographic column temperatures. The column temperatures were adjusted to 30℃, 35℃, and 40℃, respectively, with other conditions identical to those in Example 1. Figure 15 Table 20 shows that the separation of the seven organic acids was good at 30℃ and 35℃. At 40℃, the separation degree of tartaric acid was 1.0, which is less than the standard requirement of 1.5. The separation degrees of the seven phenolic acids were all good, so they will not be compared here. Therefore, a column temperature of 30℃~35℃ is more suitable.

[0155] Table 20: Organic acid separation degree at different column temperatures

[0156]

[0157] Example 7: Comparison of different chromatographic column models

[0158] This embodiment sets up three experimental groups to compare different types of chromatographic columns. The column brands and models are Agilent Extend C18 (a common and widely used column), XBridge BEH Phenyl, and Agilent ZORBAX SB-Phenyl, with column parameters of 250mm*5um*1.6mm. Figure 16Table 21 shows that the Agilent ExtendC18 column had poor separation performance. The separation efficiency of the Agilent ZORBAX SB-Phenyl and XBridge BEH Phenyl was basically the same, and both were able to obtain the target peaks. Since both Agilent ZORBAX SB-Phenyl and XBridge BEH Phenyl use a phenyl (-C6H5) bonded phase, the phenyl (-C6H5) bonded phase Phenyl column has a better separation effect for organic acids and phenolic acids.

[0159] Table 21: Resolution under different chromatographic columns

[0160]

[0161] Example 8: Comparison of Sample Pretreatment Conditions

[0162] This embodiment uses pomegranate fermentation juice as an example to compare the extraction results of pure water, 50% (volume percentage) methanol solution, and methanol used in the preparation of the test sample solution. The results are shown in Table 22. The extraction effects of methanol and 50% methanol solution are significantly better than those of punicalin A+B and ellagic acid under pure water conditions. This indicates that the extraction effect of pure methanol and 50% methanol solution is better, and large molecular weight polyphenols such as punicalin and ellagic acid are extracted more fully and in higher contents. This may be because large molecular weight polyphenols are more alcohol-soluble than water-soluble.

[0163] Table 22: Test results of extraction effects of pomegranate fermentation juice with different solvents

[0164]

[0165] Example 9: Experiment on optimizing the fermentation conditions of pomegranate juice using the method of the present invention

[0166] The non-intervention group (control group) prepared pomegranate fermented juice by short-cycle fermentation of pomegranate pulp at 37℃ for 72 h.

[0167] The intervention group involved the directed fermentation of ellagic acid and gallic acid. The test method described in Example 1 was used to monitor the changes in the content of seven organic acids and seven polyphenols in pomegranate fermentation juice during the fermentation process online. Samples were taken and analyzed every hour during the monitoring process. When the total amount of the seven organic acids exceeded 10,000 mg / L, the pH of the fermentation broth was adjusted to 5.0-6.0 using a buffer solution. At the same time, 1%-2% (m / V) of yeast powder was added to the fermentation broth to supplement the nitrogen source in the fermentation system, and the fermentation temperature was adjusted to 28-30℃. Fermentation continued for a total duration of 72 h.

[0168] After intervention in fermentation, the rate of organic acid formation in the system slowed down significantly, the rate of consumption of pungent glycosides increased, and the yields of ellagic acid and gallic acid significantly improved. Ultimately, the ellagic acid content achieved positive growth, and the gallic acid yield was also significantly higher than before the intervention. Specifically, the ellagic acid content increased from 214 mg / L before the intervention to 472 mg / L, and the gallic acid content increased from 168 mg / L to 420 mg / L.

[0169] Table 23: Comparison of organic acid and polyphenol content in pomegranate fermentation juice before and after intervention.

[0170]

[0171] Example 10: Further verification of the changes in the content of organic acids and polyphenols in lactic acid bacteria fermented compound fruit and vegetable juice using the method of the present invention.

[0172] This embodiment tested a compound fruit and vegetable fermented juice using carrots, apples, tomatoes, and pomegranates as the main raw materials, while adding the medicinal and edible ingredient, *Phyllanthus urinaria*, to prepare a medicinal and edible fermented fruit and vegetable pulp, thus creating a compound fruit and vegetable fermented juice product with potential health benefits. The ratio of carrots:apples:tomatoes:*Phyllanthus urinaria in the fruit and vegetable pulp used to prepare the compound fruit and vegetable fermented juice was 7~10:5~10:3~5:1~2 (mass ratio); other process parameters remained consistent with the compound fruit and vegetable fermented juice described above. The compound fruit and vegetable fermented juice was tested using the method of this invention, and the results are shown in Table 24 and... Figure 17 .

[0173] The results showed that the method of the present invention is also applicable to the determination of organic acids and polyphenols in fermented fruit and vegetable juices rich in glycosides. After fermentation, the free phenolic acids, such as gallic acid, increased significantly, while ellagic acid decreased significantly to the point of disappearing; among organic acids, malic acid disappeared and lactic acid increased significantly.

[0174] Table 24: Comparison of organic acid and polyphenol content before and after fermentation of compound fruit and vegetable fermented juice in Example 9

[0175]

Claims

1. A method for simultaneous testing of organic acids and phenolic acids in fruit and vegetable juice fermented by lactic acid bacteria, characterized by, Comprise the following steps: Step (1): take the sample to be tested and 50% methanol solution, take the supernatant and filter; Step (2): the filtrate obtained in step (1) is injected into a high performance liquid chromatograph for analysis, and a liquid chromatogram is obtained; Step (3): the peak area corresponding to different organic acids and phenolic acids is obtained from the liquid chromatogram, and then the content of the organic acids and phenolic acids involved in the sample to be tested is calculated according to the standard curve; The conditions of the high performance liquid chromatograph are as follows: The chromatographic column uses a phenyl chromatographic column, and the filler is a phenyl bonded phase; The mobile phase A is methanol, the mobile phase B is 0.1%-0.4% phosphoric acid aqueous solution, the flow rate is 0.8-1.0 mL / min; the temperature is 30-35℃, and the injection amount is 10uL; The gradient elution conditions of the mobile phase are as follows: 0-8.5 min, mobile phase A 2.5%→5%, mobile phase B 97.5%→95%; 8.5-10 min, mobile phase A 5%→10%, mobile phase B 95%→90%; 25-30 min, mobile phase A 20%, mobile phase B 80%; 30-40 min, mobile phase A 20%→40%, mobile phase B 80%→60%; 40-55 min, mobile phase A 40%→50%, mobile phase B 60%→50%; 55-60 min, mobile phase A 50%, mobile phase B 50%; 60-61 min, mobile phase A 50%→2.5%, mobile phase B 50%→97.5%; 61-65 min, mobile phase A 2.5%, mobile phase B 97.5%; The detection wavelength is: 0-8min, set the detection wavelength to 210nm, 8-65min, set the detection wavelength to 254nm.

2. The method for simultaneous testing of organic acids and phenolic acids in lactic acid bacteria-fermented fruit and vegetable juice according to claim 1, characterized in that, In step (1), the sample to be tested is mixed with 50% methanol and ultrasonic treated for 15min.

3. The method for simultaneous testing of organic acids and phenolic acids in lactic acid bacteria-fermented fruit and vegetable juice according to claim 1, characterized in that, The phenyl chromatographic column is one of Agilent ZORBAX SB-Phenyl column and XBridge BEH Phenyl column.

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