Composite beverage probiotic detection method and system

Abstract

The invention discloses a compound beverage probiotic detection method and system, and relates to the technical field of beverage ingredient detection.The method comprises the steps that stable isotope labeled D-lactic acid is added into a sample, and the stable isotope labeled D-lactic acid and natural D-lactic acid in the sample are mixed to be uniform; carrying out pretreatment on the uniformly mixed sample; the method comprises the following steps: performing chromatographic separation on a pretreated sample, and performing mass spectrometric detection to obtain a first signal of natural D-lactic acid and a second signal of stable isotope labeled D-lactic acid; the natural D-lactic acid in the sample is quantitatively corrected based on the ratio of the first signal to the second signal. The method provided by the invention aims to solve the problems of inaccurate chromatographic detection result and poor reproducibility caused by interference in a complex compound probiotic beverage matrix, and can accurately measure and correct loss and signal fluctuation in all processes so as to ensure the reliability of a final quantitative result.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of beverage ingredient detection, and particularly relates to a composite beverage probiotic detection method and system. BACKGROUND

[0002] In the modern food industry, the research and development of new composite probiotic beverages is showing a trend of vigorous development. Such products usually contain multiple active ingredients, among which probiotics are one of the core elements. In order to ensure product quality and claimed efficacy, manufacturers need to accurately and reliably quantify the probiotics in the beverage. However, traditional detection methods often fall short when faced with these complex substrates, making it difficult to provide stable and reliable results. For example, traditional plate colony counting method often results in poor reproducibility of the detection results when evaluating the number of live bacteria, because the high content of plant protein and fruit fiber in the beverage causes bacterial aggregation or difficulty in dispersion. More advanced detection methods at the genetic level, such as polymerase chain reaction (PCR) technology, although solve the problem of counting reproducibility, but cannot reflect the true metabolic activity of probiotics, because even if the bacterial DNA exists, its functionality may be impaired due to the inhibition of certain components in the substrate (such as phenolic compounds in plant extracts) on key metabolic enzymes, leading to deterioration of product flavor. This makes it impossible to accurately reflect the true "activity" level and functional state of the product by simply detecting the total DNA amount. Therefore, the quality control standard has been redefined, and the bacterial "metabolic activity" must be the core evaluation criterion, and the trace organic acid produced by active probiotics in a specific metabolic pathway, such as D-lactic acid, is determined as the most direct and reliable marker to judge its functional state. However, when detecting this trace marker using high-performance liquid chromatography, new challenges are encountered: complex beverage substrates, especially residual small molecule proteins or peptides, can bind with D-lactic acid, resulting in poor chromatographic peak shape and poor reproducibility, making the quantitative results unreliable.

[0003] In the modern food industry, in order to meet the dual needs of consumers for health and taste, the research and development of new composite probiotic beverages is showing a trend of vigorous development. Such beverages are usually not just simple fermented dairy products, in order to enhance their nutritional value and flavor level, high content of plant protein, such as pea protein, and natural fruit fiber, such as mango puree, are additionally introduced into the formula. These new ingredients, although enriching the taste and nutritional composition of the product, have significantly increased the complexity of the beverage substrate.

[0004] During routine factory quality inspections of these novel compound probiotic beverages, technicians discovered a common problem: the reproducibility of the test results was extremely poor when using the traditional plate count method to assess the number of live bacteria in the product. Specifically, even for the same batch of products, the number of live bacteria obtained after multiple samplings and tests showed irregular fluctuations. This phenomenon is mainly due to the high content of plant protein and fruit fiber in the beverage. During sample preparation, these components may cause probiotic cells to aggregate or form a microenvironment in the culture medium that is difficult to disperse, thus preventing the cells from spreading or being counted evenly, thereby affecting the final counting accuracy. For example, pea protein may form a gel-like structure under certain conditions, encapsulating some probiotics and preventing them from being effectively released and counted during dilution and coating; while fruit fiber may form a physical barrier in the petri dish, hindering the normal growth or observation of colonies.

[0005] To overcome the challenge of poor reproducibility of traditional counting methods in complex matrices, the quality control department decided to shift to a more advanced genetic-level detection method: polymerase chain reaction (PCR) technology to detect the DNA information of specific probiotics and infer their total amount. Initially, this method seemed to solve the counting reproducibility problem because DNA detection was less affected by physical interference from the matrix. However, during shelf-life tracking tests, a new contradiction emerged: data showed that even in the later stages of storage, the total DNA of probiotics in the beverage remained at a relatively high level, with almost no significant decline. But at the same time, the product's flavor deteriorated significantly; the characteristic tangy aroma representing probiotic vitality weakened considerably, and even unpleasant off-flavors began to appear. This phenomenon indicated that although the genetic material of the microorganisms remained, their functionality may have been impaired.

[0006] The research team immediately intervened and launched an in-depth investigation into the flavor degradation issue. Through meticulous analysis of the beverage's ingredients, they made a crucial discovery: certain plant extracts added to the formula to provide antioxidant function, such as green tea polyphenols, contain natural phenolic compounds that do not directly kill the probiotic cells and therefore do not lead to a decrease in total DNA. However, over time, during beverage storage, these phenolic compounds gradually interact with key metabolic enzymes within the probiotic cells, inhibiting their activity. The mechanism of this inhibition may include the binding of phenolic substances to the active sites of enzyme proteins or causing conformational changes in enzyme proteins, thereby reducing or even eliminating their catalytic function. For example, some polyphenols can bind to bacterial dehydrogenases or lactate dehydrogenases, preventing their normal catalytic conversion of substrates into metabolites.

[0007] This discovery perfectly explains the previously observed contradiction: probiotic cells are physically intact, and their DNA information is still present, but due to the inhibition of key metabolic enzyme activity, they have lost their normal metabolic functions and become functionally inactive cells. This means that these cells can no longer effectively produce specific metabolites that maintain the product's flavor and exert its health benefits, such as lactic acid and acetic acid. Therefore, simply measuring the total DNA amount can no longer accurately reflect the product's true "activity" level and functional state.

[0008] Based on these findings, the company's quality control standards were redefined: no longer solely relying on bacterial "quantity" as the evaluation indicator, but instead using bacterial "metabolic activity" as the core criterion. Further research by the R&D team identified a trace organic acid, such as D-lactic acid, produced by active probiotics in specific metabolic pathways, as the most direct and reliable marker for assessing their functional status. D-lactic acid is a unique metabolite of certain probiotics, and its content directly reflects the metabolic activity of the bacteria.

[0009] Analysts immediately set out to establish a high-performance liquid chromatography (HPLC) method to detect the newly identified biomarker, D-lactic acid. They attempted industry-standard sample pretreatment: first, adjusting the sample pH to precipitate most of the proteins, then centrifuging, and finally filtering the supernatant for direct injection. However, the results were perplexing and unpredictable. The peak shape of the target analyte, D-lactic acid, was extremely poor, often exhibiting tailing, broadening, or even splitting. Furthermore, multiple injections of the same sample showed very poor reproducibility of peak area, rendering the quantitative results completely unreliable. After repeated troubleshooting and diagnostics, they finally realized that the problem was not with the chromatographic instrument itself, but rather stemmed from the complex interference of the beverage sample matrix. Although most proteins were removed through precipitation, some small protein or peptide molecules remained dissolved in the supernatant. These residual components had a strong binding interaction with D-lactic acid. This binding affected the interaction between D-lactic acid and the stationary phase of the column during chromatographic separation, leading to unstable retention behavior and resulting in poor peak shape and reproducibility issues. Meanwhile, other components in the matrix that were not completely removed, such as residual sugars, pigments, or plant extracts, also significantly inhibited or enhanced the detection signal, further interfering with the accurate quantification of D-lactic acid. For example, in mass spectrometry, these matrix components may compete with the target analyte for ionization, resulting in a suppressed signal intensity of D-lactic acid, leading to detection results lower than the actual value.

[0010] The resulting technical challenge is that a completely new testing process must be established to accurately assess the true activity of compound probiotic beverages. The core of this process lies in the need for an efficient sample pretreatment technique. This technique must not only effectively break the binding of the marker D-lactic acid with residual proteins and peptides in the beverage matrix, but also thoroughly purify and enrich it from a system containing complex interfering substances such as fruit fiber, sugar, and pigments, thus eliminating matrix interference with the chromatographic detection signal. Subsequently, chromatographic techniques are used to perform precise and reproducible quantitative analysis of trace amounts of D-lactic acid in the purified sample. This combined method is the only way to solve the problem of unreliable current test results and the inability to accurately assess product activity. (Invention Content) The purpose of this invention is to provide a method and system for detecting probiotics in complex probiotic beverages. This invention aims to solve the problems of inaccurate chromatographic detection results and poor reproducibility caused by the binding of D-lactic acid with residual proteins and peptides, as well as interference from other matrices, in complex probiotic beverage matrices. It can accurately measure and correct for losses and signal fluctuations throughout the process, thereby ensuring the reliability of the final quantitative results. To achieve the above objective, this invention employs the following technical effects: According to one aspect of the present invention, a method for detecting probiotics in a compound beverage is provided, comprising the following steps: S1. Add a known amount of stable isotope-labeled D-lactic acid to the compound probiotic beverage sample, and mix the stable isotope-labeled D-lactic acid with the natural D-lactic acid in the compound probiotic beverage sample evenly. S2. Perform pretreatment on the well-mixed sample; S3. By performing chromatographic separation on the pretreated sample, and by performing mass spectrometry detection on the chromatographically separated natural D-lactic acid and stable isotope-labeled D-lactic acid, the first signal of natural D-lactic acid and the second signal of stable isotope-labeled D-lactic acid are obtained. S4. Based on the ratio of the first signal to the second signal, the natural D-lactic acid in the compound probiotic beverage sample is quantitatively corrected.

[0011] In a further preferred embodiment of the above scheme, in step S1, the stable isotope-labeled D-lactic acid is mixed evenly with the natural D-lactic acid in the compound probiotic beverage sample through the following steps: Prepare an organic solvent mixture containing stable isotope-labeled D-lactic acid; the organic solvent mixture contains acidic substances; By rapidly injecting a compound probiotic beverage sample into an organic solvent mixture, the acidic substances in the organic solvent mixture break down the microscopic gel network in the compound probiotic beverage sample, resulting in a uniformly mixed sample.

[0012] In a further preferred embodiment of the above scheme, the acidic substance is an organic acid, including formic acid, acetic acid, or trifluoroacetic acid.

[0013] In a further preferred embodiment of the above scheme, the acidic substance is an inorganic acid, including hydrochloric acid, sulfuric acid, or phosphoric acid.

[0014] In a further preferred embodiment of the above scheme, step S2, the specific steps for pretreatment of the uniformly mixed sample include: By extracting a homogeneously mixed sample, specific trace active ingredients in the matrix are separated from natural D-lactic acid and stable isotope-labeled D-lactic acid. During the extraction process, the conformational changes or aggregation of specific trace active ingredients are inhibited by controlling the solvent composition or pH of the extraction environment. The extracted sample was subjected to solid-liquid separation to obtain a solution containing natural D-lactic acid and stable isotope-labeled D-lactic acid.

[0015] In a further preferred embodiment of the above scheme, step S3, the specific steps of chromatographic separation of the pretreated sample include: When performing chromatographic separation on the pretreated sample, a pre-separation unit is set up before the main chromatographic column; the pre-separation unit is used to selectively adsorb residual trace substances in the compound probiotic beverage sample; After a preset number of sample injections, the pre-separation unit is cleaned.

[0016] In a further preferred embodiment of the above scheme, the cleaning process includes switching the flow path or changing the solvent composition.

[0017] In a further preferred embodiment of the above scheme, step S3, which involves mass spectrometry detection of the chromatographically separated natural D-lactic acid and the stable isotope-labeled D-lactic acid to obtain the first signal of natural D-lactic acid and the second signal of stable isotope-labeled D-lactic acid, includes the following specific steps: Between the chromatographic column outlet and the mass spectrometer ion source inlet, a processing unit is set up and controlled to add auxiliary solvent to the chromatographic effluent; The effect of residual matrix components on the ionization of natural D-lactic acid and stable isotope-labeled D-lactic acid was homogenized by mixing auxiliary solvents with residual matrix components in chromatographic effluents. Mass spectrometry was used to detect the homogenized chromatographic effluent to obtain the first signal of natural D-lactic acid and the second signal of stable isotope-labeled D-lactic acid.

[0018] In a further preferred embodiment of the above scheme, the auxiliary solvent is a volatile organic solvent, including acetonitrile or methanol.

[0019] According to another aspect of the present invention, the present invention also provides a probiotic detection system for compound beverages, comprising: The control mixing module is used to add a known amount of stable isotope-labeled D-lactic acid to the compound probiotic beverage sample and to mix the stable isotope-labeled D-lactic acid with the natural D-lactic acid in the compound probiotic beverage sample evenly. The pretreatment module is used to pretreat the uniformly mixed sample. The separation and detection module is used to perform chromatographic separation on the pretreated sample and to perform mass spectrometry detection on the chromatographically separated natural D-lactic acid and stable isotope-labeled D-lactic acid to obtain the first signal of natural D-lactic acid and the second signal of stable isotope-labeled D-lactic acid. The calibration module is used to quantitatively calibrate the natural D-lactic acid in the compound probiotic beverage sample based on the ratio of the first signal to the second signal.

[0020] In summary, the present invention adopts the above technical solution, and the present invention has the following technical effects: This invention can significantly improve the accuracy and reproducibility of D-lactic acid detection results in compound probiotic beverages, and effectively overcome the influence of various interference factors such as protein binding, physical loss and matrix effect in complex matrices on quantitative analysis, thus providing reliable technical support for accurately assessing the metabolic activity of probiotics and product quality. Attached Figure Description

[0021] Figure 1 This is a flowchart of a method for detecting probiotics in compound beverages provided in an embodiment of the present invention; Figure 2 This is a schematic diagram of a compound beverage probiotic detection system provided in an embodiment of the present invention; In the attached diagram, 100 is the control and mixing module; 200 is the preprocessing module; 300 is the separation and detection module; and 400 is the calibration module. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and preferred embodiments. However, it should be noted that many details listed in the specification are merely to provide the reader with a thorough understanding of one or more aspects of the present invention, and these aspects of the invention can be implemented even without these specific details.

[0023] Combination Figure 1 As shown, the method for detecting probiotics in compound beverages according to the present invention includes the following steps: S1. Add a known amount of stable isotope-labeled D-lactic acid to the compound probiotic beverage sample, and mix the stable isotope-labeled D-lactic acid with the natural D-lactic acid in the compound probiotic beverage sample evenly. S2. Pretreatment is performed on the homogeneously mixed sample to ensure that the stable isotope-labeled D-lactic acid undergoes matrix interference and loss simultaneously with the natural D-lactic acid; S3. By performing chromatographic separation on the pretreated sample, and by performing mass spectrometry detection on the chromatographically separated natural D-lactic acid and stable isotope-labeled D-lactic acid, the first signal of natural D-lactic acid and the second signal of stable isotope-labeled D-lactic acid are obtained. S4. Based on the ratio of the first signal to the second signal, the natural D-lactic acid in the compound probiotic beverage sample is quantitatively corrected.

[0024] Stable isotope-labeled D-lactic acid refers to D-lactic acid molecules in which some atoms are replaced by stable isotopes, resulting in a slight change in molecular weight, but maintaining a high degree of consistency with natural D-lactic acid in chemical properties and physical behavior. It can be obtained through chemical or biological synthesis, such as by using deuterated reagents. Its main purpose is to distinguish it from natural D-lactic acid in mass spectrometry detection and to serve as an internal standard for correcting matrix effects and losses during sample pretreatment and detection. Known quantification refers to the pre-determined and recorded amount of stable isotope-labeled D-lactic acid added, for example, by weighing or preparing a standard solution. This primarily provides a reference standard for subsequent quantitative correction using signal ratios. Uniform mixing refers to the complete dispersion of stable isotope-labeled D-lactic acid and natural D-lactic acid in the compound probiotic beverage sample at the molecular level, achieving a homogeneous state. This can be achieved through mechanical stirring, ultrasonic treatment, or rapid injection of an acidic organic solvent mixture. It primarily ensures that the internal standard and the target analyte experience the same matrix effects and losses in all subsequent processing steps, thus guaranteeing the effectiveness of quantitative correction. Pretreatment refers to the pre-treatment of the sample to remove or reduce interfering substances and concentrate the target analyte. This can be achieved through extraction, solid-liquid separation, precipitation, filtration, or centrifugation. It mainly serves to purify the sample matrix, reduce the interference of complex matrices on subsequent chromatographic separation and mass spectrometry detection, and ensure that the internal standard and natural D-lactic acid undergo matrix interference and loss simultaneously. Simultaneous matrix interference and loss refers to the fact that the degree and manner of influence and potential physical or chemical loss of stable isotope-labeled D-lactic acid and natural D-lactic acid during sample pretreatment are basically the same as those of other components in the matrix. This is mainly used to ensure that the internal standard can reflect the behavior of natural D-lactic acid throughout the detection process, thus providing a basis for subsequent quantitative correction. Chromatographic separation refers to the process of separating different components in a sample based on their differences in physicochemical properties using chromatographic techniques. This can be achieved using methods such as liquid chromatography, gas chromatography, or ion chromatography. For example, high-performance liquid chromatography (HPLC) is mainly used to separate D-lactic acid from complex sample matrices, reducing interference from co-elutions on mass spectrometry detection and improving detection specificity. Mass spectrometry refers to the mass analysis of chromatographically separated components using a mass spectrometer to obtain their mass-charge ratio information and signal intensity. It can be achieved using plasma sources such as electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), or matrix-assisted laser desorption / ionization (MALDI), combined with mass analyzers such as quadrupole, time-of-flight, or orbital trap. It is mainly used for the identification and signal intensity measurement of natural D-lactic acid and stable isotope-labeled D-lactic acid.The ratio of the first signal to the second signal refers to the mathematical proportional relationship between the signal intensity of natural D-lactic acid obtained by mass spectrometry detection and the signal intensity of stable isotope-labeled D-lactic acid obtained by mass spectrometry detection. It is mainly used to eliminate the influence of factors such as sample matrix, pretreatment process, instrument fluctuation and mass spectrometry ionization efficiency on the absolute signal intensity of D-lactic acid, thereby realizing the quantitative correction of natural D-lactic acid.

[0025] The core innovation of this application lies in introducing a known quantitative stable isotope-labeled D-lactic acid as an internal standard into the compound probiotic beverage sample, and ensuring that it undergoes matrix interference and loss synchronously with natural D-lactic acid throughout the entire detection process. This effectively overcomes the interference of complex beverage matrix on the quantitative detection of D-lactic acid, thereby improving the accuracy, reliability and reproducibility of the detection results.

[0026] Specifically, this detection method systematically solves the challenge of quantitative detection of D-lactic acid in complex matrices through a series of synergistic steps. First, in the sample preparation stage, a known quantification of stable isotope-labeled D-lactic acid is precisely added to the compound probiotic beverage sample, ensuring thorough and homogeneous mixing with the naturally occurring D-lactic acid in the sample. This initial step is crucial, providing a reliable internal standard reference for subsequent quantitative calibration and ensuring a high degree of consistency between the internal standard and the target analyte in terms of chemical properties and behavior. Subsequently, the homogeneously mixed sample undergoes pretreatment. This pretreatment process is designed to allow the stable isotope-labeled D-lactic acid and the naturally occurring D-lactic acid to simultaneously experience matrix interference effects and any losses that may occur during the pretreatment process. This synchronicity is the core of the effectiveness of the internal standard method; it ensures that regardless of the complexity of the matrix or the losses caused by the pretreatment process, the relative ratio between the internal standard and the naturally occurring D-lactic acid remains constant, effectively offsetting the impact of matrix effects and sample losses on the accuracy of the detection results. Next, the pretreated sample underwent chromatographic separation to further purify D-lactic acid, separating it from the residual complex matrix, reducing interference from co-effluents in subsequent detection, and improving detection specificity. Following this, mass spectrometry was used to detect the chromatographically separated natural D-lactic acid and stable isotope-labeled D-lactic acid, obtaining their specific signals. Because the influences on both during chromatographic behavior and mass spectrometric ionization are highly consistent, even with residual matrix interference, their effects on the two signals are synchronous. Finally, based on the ratio of the first signal of natural D-lactic acid to the second signal of stable isotope-labeled D-lactic acid, the natural D-lactic acid in the compound probiotic beverage sample was quantitatively corrected. This ratio correction mechanism effectively eliminates the influence of matrix complexity, pretreatment losses, instrument fluctuations, and changes in mass spectrometric ionization efficiency on the absolute signal intensity of D-lactic acid throughout the detection process, thereby achieving accurate, reliable, and reproducible quantification of natural D-lactic acid in the compound probiotic beverage sample.

[0027] As a preferred embodiment, the specific implementation of this application is as follows: First, an organic solvent mixture containing a known quantitative amount of stable isotope-labeled D-lactic acid is prepared. This mixture may contain acetonitrile and a small amount of formic acid. Then, the compound probiotic beverage sample is rapidly injected into the organic solvent mixture. Vigorous stirring or ultrasonic treatment ensures that the natural D-lactic acid and the stable isotope-labeled D-lactic acid in the sample are thoroughly and uniformly mixed. Simultaneously, the organic solvent and acidic substances help to break down the microscopic gel network in the sample, promoting the release and uniform dispersion of D-lactic acid. Next, the uniformly mixed sample is pretreated, for example, using solid-phase extraction (SPE) or liquid-liquid extraction (LLE) methods to remove most interfering substances such as proteins, sugars, and pigments, while ensuring that the natural D-lactic acid and the stable isotope-labeled D-lactic acid undergo matrix effects and recovery losses simultaneously during the extraction process. For example, a C18 solid-phase extraction column can be used, and by optimizing the elution solvent composition and pH value, effective separation and enrichment of the target analyte can be achieved. The pretreated sample was then separated using a high-performance liquid chromatography (HPLC) system, such as a hydrophilic interaction chromatography (HILIC) column or a chiral column, to ensure effective separation of D-lactic acid from residual matrix components. The effluent from the column exit was directly fed into the ion source of a mass spectrometer, such as an electrospray ionization (ESI) source, for tandem mass spectrometry (MS / MS) detection. During mass spectrometry detection, characteristic ion pairs of natural D-lactic acid and stable isotope-labeled D-lactic acid were monitored separately to obtain their respective signal intensities. Finally, using mass spectrometry software or a data processing system, the ratio between the signal intensity of natural D-lactic acid and that of stable isotope-labeled D-lactic acid was calculated, and combined with a pre-established standard curve, the content of natural D-lactic acid in the compound probiotic beverage sample was quantitatively corrected.

[0028] Through the above-described scheme, this application addresses the issues of accuracy, reliability, and reproducibility in the detection of D-lactic acid in the complex matrix of compound probiotic beverages. By introducing a stable isotope-labeled internal standard and ensuring that it undergoes matrix interference and loss synchronously with natural D-lactic acid throughout the detection process, this application eliminates the influence of matrix effects, target binding, and unstable detection signals on the quantitative results, thereby achieving the quantitative detection of D-lactic acid in compound probiotic beverages.

[0029] In some embodiments, in step S1, the stable isotope-labeled D-lactic acid is mixed evenly with the natural D-lactic acid in the compound probiotic beverage sample through the following steps: Prepare an organic solvent mixture containing stable isotope-labeled D-lactic acid; the organic solvent mixture contains an acidic substance; the acidic substance is an organic acid, including formic acid, acetic acid or trifluoroacetic acid, or an inorganic acid, including hydrochloric acid, sulfuric acid or phosphoric acid; By rapidly injecting a compound probiotic beverage sample into an organic solvent mixture, the acidic substances in the organic solvent mixture break down the microscopic gel network in the compound probiotic beverage sample, thereby forcibly releasing the natural D-lactic acid in the compound probiotic beverage sample and causing it to be uniformly mixed at the molecular level with stable isotope-labeled D-lactic acid, resulting in a uniformly mixed sample.

[0030] Organic solvent mixtures refer to solvent systems containing stable isotopically labeled D-lactic acid. Their main component is an organic solvent, which may include, but is not limited to, acetonitrile, methanol, isopropanol, or acetone. These solvents can dissolve D-lactic acid and are miscible with aqueous samples or form stable dispersions. Acidic substances are chemical substances that can provide hydrogen ions and lower the pH of a solution. These can be organic acids, such as formic acid, acetic acid, and trifluoroacetic acid, or inorganic acids, such as hydrochloric acid, sulfuric acid, phosphoric acid, or nitric acid. Microgel networks refer to colloidal systems with a three-dimensional network structure formed under specific conditions in compound probiotic beverage samples by plant proteins, fruit pulp fibers, or other macromolecules. These networks can physically bind or encapsulate small molecules. Rapid injection refers to the operation of adding compound probiotic beverage samples to organic solvent mixtures at a high flow rate or rapidly within a short time. This can be achieved using syringe pumps, rapid titration devices, or high-pressure jetting devices. Disintegration refers to the destruction of the structural integrity of microgel networks through chemical or physical processes, causing them to transform from a solid or semi-solid gel state to a liquid or dispersed state. Forced release refers to the active, non-natural diffusion release of naturally occurring D-lactic acid, which was originally physically bound or chemically combined within the microscopic gel network, into the solution system after the network is disrupted. Homogeneous mixing at the molecular level refers to the sufficient dispersion and interpenetration of stable isotope-labeled D-lactic acid and naturally occurring D-lactic acid at the molecular scale in solution. This ensures that the relative concentration ratio of the two remains consistent within any small volume, thereby guaranteeing the representativeness and accuracy of subsequent analyses.

[0031] This method ensures homogeneous molecular-level mixing of stable isotope-labeled D-lactic acid and natural D-lactic acid in the compound probiotic beverage sample through a series of synergistic operations. First, an organic solvent mixture containing stable isotope-labeled D-lactic acid is prepared, pre-dissolving the internal standard in a chemically active environment. The acidic substances in this mixture, whether organic acids such as formic acid, acetic acid, and trifluoroacetic acid, or inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid, provide the chemical basis for subsequent destruction of the sample matrix. Then, by rapidly injecting the compound probiotic beverage sample into the pre-prepared organic solvent mixture, active and rapid contact between the internal standard and the sample is achieved. This rapid injection, combined with the chemical action of the acidic substances in the organic solvent mixture, efficiently breaks down the microscopic gel network formed by plant proteins and fruit fibers in the compound probiotic sample. Once these gel networks are broken down, the natural D-lactic acid, which was previously physically encapsulated or bound within them, is forcibly released into the solution system. At this point, because the stable isotope-labeled D-lactic acid has been pre-dissolved in the organic solvent mixture, and natural D-lactic acid is forcibly released, the two can be thoroughly and uniformly mixed at the molecular level. This method of actively disrupting the matrix and forcibly releasing the analyte overcomes the limitation of traditional simple mixing in penetrating complex matrices, ensuring a high degree of homogeneity between the internal standard and the analyte in the initial stage. This uniform mixing is crucial for subsequent detection methods because it ensures that the stable isotope-labeled D-lactic acid and natural D-lactic acid undergo matrix interference and loss simultaneously in all subsequent steps such as pretreatment, chromatographic separation, and mass spectrometry detection. This allows quantitative correction based on the signal ratio of the two to accurately reflect the true content of natural D-lactic acid in the sample. It is precisely because of this unique mixing method that accurate D-lactic acid quantification in complex matrices becomes possible, providing a reliable starting point for the quality control of compound probiotic beverages.

[0032] In one embodiment, to ensure uniform mixing of stable isotope-labeled D-lactic acid with natural D-lactic acid in a compound probiotic beverage sample, an organic solvent mixture containing stable isotope-labeled D-lactic acid can be prepared first. This mixture can use acetonitrile as the organic solvent and add an appropriate amount of formic acid as the acidic substance. Specifically, a known quantitative amount of stable isotope-labeled D-lactic acid can be dissolved in a certain volume of acetonitrile, and then a small amount of formic acid can be added, for example, to achieve a concentration of 0.1%, forming an acidic organic solvent mixture. Subsequently, a compound probiotic beverage sample, such as a probiotic beverage containing pea protein and mango puree, is rapidly injected into the prepared acidic organic solvent mixture using a syringe pump at a flow rate of 5 ml per minute. During the rapid injection of the sample, the formic acid in the organic solvent mixture can quickly contact the beverage sample and disrupt the microscopic gel network formed by plant proteins and fruit fibers in the sample. This disruption forces the natural D-lactic acid, which was originally bound by the gel network, to be released into the acetonitrile-formic acid mixture. Because the stable isotope-labeled D-lactic acid is pre-dissolved in the mixture and native D-lactic acid is released, the two can rapidly diffuse at the molecular level and achieve homogeneous mixing. In this way, a homogeneous sample can be obtained, providing a reliable starting material for subsequent detection steps.

[0033] This method utilizes the chemical action of the acidic substances in an organic solvent mixture containing stable isotope-labeled D-lactic acid, combined with the physical action of rapidly injecting the compound probiotic beverage sample, to effectively dismantle the microscopic gel network induced by environmental factors within the sample. This dismantling effect forces the release of the previously physically encapsulated or bound natural D-lactic acid, ensuring that the newly added stable isotope-labeled D-lactic acid rapidly penetrates and achieves uniform mixing at the molecular level with the released natural D-lactic acid. This overcomes the problem of uneven initial distribution caused by limited diffusion, thereby improving the accuracy of subsequent quantitative correction.

[0034] In some embodiments, step S2, the specific steps of pretreatment of the uniformly mixed sample, include: By extracting a homogeneously mixed sample, specific trace active ingredients in the matrix are separated from natural D-lactic acid and stable isotope-labeled D-lactic acid. During the extraction process, by controlling the solvent composition or pH of the extraction environment, conformational changes or aggregation of specific trace active ingredients are inhibited, thereby avoiding asymmetric interactions between specific trace active ingredients and natural D-lactic acid and stable isotope-labeled D-lactic acid. The extracted sample was subjected to solid-liquid separation to obtain a solution containing natural D-lactic acid and stable isotope-labeled D-lactic acid, thereby allowing the stable isotope-labeled D-lactic acid to undergo matrix interference and loss simultaneously with the natural D-lactic acid.

[0035] Extraction refers to the process of separating target analytes from complex sample matrices using the selectivity of solvents or adsorbents, while simultaneously removing or reducing interfering components. This can be achieved using techniques such as liquid-liquid extraction, solid-phase extraction, or supercritical fluid extraction. Specific trace active ingredients refer to substances present in minute quantities in compound probiotic beverage samples that possess biological or chemical activity. These substances may non-specifically bind to D-lactic acid, form complexes, or interfere with detection; examples include residual small-molecule proteins, peptides, phenolic compounds, pigments, or certain sugar derivatives. Controlling the solvent composition or pH of the extraction environment refers to strategies for optimizing the recovery rate of target analytes and suppressing the behavior of interfering components by precisely adjusting the type, ratio, or pH of the solvents used in the extraction process. This can be achieved by selecting organic solvents of different polarities, adjusting the ratio of aqueous to organic phases, or adding acids or bases to adjust the pH of the solution. Conformational changes or aggregation refer to the phenomenon where the molecular structure of a specific trace active ingredient changes or multiple molecules combine to form larger particles under specific solvent environments or pH conditions. These changes may lead to alterations in its interaction mode with D-lactic acid, which can manifest as protein denaturation, polyphenol polymerization, or colloid formation. Asymmetric interactions refer to the phenomenon where, due to differences in microdynamics or binding sites, the degree of influence on natural D-lactic acid and stable isotope-labeled D-lactic acid interacting with specific trace active ingredients in the matrix is ​​inconsistent. This inconsistency undermines the assumption in isotope dilution methods that the internal standard and analyte simultaneously undergo matrix effects, thus introducing quantitative errors. Solid-liquid separation refers to the process of separating solid particles or insoluble substances from a liquid sample to obtain a clear solution, which can be achieved using physical methods such as centrifugation, filtration, or sedimentation.

[0036] This approach employs a refined pretreatment strategy to proactively separate and suppress the harmful behaviors of specific trace active ingredients, thereby fundamentally eliminating or avoiding asymmetric interactions and ensuring the accuracy of the internal standard method throughout the entire process. Specifically, during the pretreatment of the homogeneously mixed sample, extraction is first used to effectively separate the specific trace active ingredients in the matrix that interfere with the detection of D-lactic acid from the target analytes, natural D-lactic acid and stable isotope-labeled D-lactic acid. This separation directly solves the problem of interfering substances binding or interacting with D-lactic acid in complex matrices, providing a purer sample for subsequent chromatographic and mass spectrometric detection. During this extraction process, by controlling the solvent composition or pH of the extraction environment, conformational changes or aggregation of these specific trace active ingredients can be suppressed. This is because some active ingredients may change their structure or form aggregates under specific conditions, thereby enhancing their non-specific binding or interference with D-lactic acid, leading to separation difficulties or unstable detection signals. By precisely controlling the extraction conditions, the stability of these interfering components can be maintained, making them easier to separate. More importantly, this avoids asymmetric interactions between these specific trace active components and natural D-lactic acid and stable isotope-labeled D-lactic acid. Such asymmetric interactions would undermine the assumption in isotope dilution methods that the internal standard and analyte undergo matrix effects simultaneously, thus introducing quantitative errors. By suppressing this asymmetric interaction, the accuracy of the internal standard's correction is ensured. Subsequently, solid-liquid separation is performed on the extracted sample to remove solid impurities formed during extraction or insoluble substances in the separated liquid phase, resulting in a clear solution containing both natural and stable isotope-labeled D-lactic acid. This step further purifies the sample, reducing matrix effects in subsequent chromatographic and mass spectrometric analyses. Finally, through these refined pretreatment steps, it is ensured that stable isotope-labeled D-lactic acid and natural D-lactic acid undergo matrix interference and loss simultaneously throughout the entire pretreatment process. This means that regardless of the recovery rate of D-lactic acid during extraction and separation, or the presence of trace interferences in the matrix, both the internal standard and the analyte will be affected to the same extent. Therefore, the signal of natural D-lactic acid can be accurately corrected using the signal from the internal standard, significantly improving the accuracy and reproducibility of D-lactic acid quantification in complex matrices. This method, combined with the previous step of uniformly mixing stable isotope-labeled D-lactic acid with natural D-lactic acid at the molecular level, forms a complete and high-precision quantitative detection system. Thorough mixing ensures that the internal standard and analyte are highly homogeneous before entering the pretreatment stage. Based on this, this method actively eliminates or suppresses factors in the matrix that may lead to asymmetric interactions through refined extraction and separation, further enhancing the reliability of the internal standard correction method.This combination enables the quantitative analysis of D-lactic acid to not only overcome the problems of recovery fluctuations and signal suppression or enhancement caused by matrix effects, but also to solve the problem of differences in behavior between internal standards and analytes caused by trace amounts of active ingredients that are difficult to detect in traditional methods. This allows for accurate and reliable quantification of D-lactic acid in complex probiotic beverage samples.

[0037] To further illustrate the embodiments of this application, a specific example can be provided. When pretreating a homogeneously mixed sample, a liquid-liquid extraction method can be used. For example, the homogeneously mixed sample is mixed with a certain proportion of organic solvents such as acetonitrile or methanol, and then subjected to vortexing or ultrasonic treatment to transfer D-lactic acid from the aqueous phase to the organic phase, while most large molecular interferences such as proteins and polysaccharides precipitate or remain in the aqueous phase. During this process, specific trace active ingredients such as phenolic compounds or residual small molecule peptides that may be present in the matrix may form weak bindings with D-lactic acid. To suppress conformational changes or aggregation of these components, the proportion of the organic solvent can be precisely controlled. For example, the proportion of acetonitrile can be controlled between 60% and 80%, or the pH of the extraction system can be adjusted to the range of 3.0 to 5.0 by adding a small amount of acid (such as formic acid or acetic acid) to maintain the stability of these active ingredients and avoid asymmetric interactions with D-lactic acid. After extraction, the mixture can be centrifuged at high speed, for example at 10,000 rpm for 10 minutes, to fully separate the precipitated protein, aggregated active ingredients and other insoluble substances to the bottom. Then, carefully aspirate the supernatant to obtain a clear solution containing natural D-lactic acid and stable isotope-labeled D-lactic acid. This solution can be directly used for subsequent chromatographic and mass spectrometric analysis.

[0038] By performing refined extraction on a homogeneously mixed sample, specific trace active components that interfere with D-lactic acid detection in the matrix can be effectively separated. During this process, by precisely controlling the solvent composition or pH of the extraction environment, conformational changes or aggregation of these active components are suppressed, thereby fundamentally avoiding microscopic kinetic asymmetric interactions between them and both natural and stable isotope-labeled D-lactic acid. Subsequently, a solid-liquid separation step further purifies the sample, ensuring that both stable isotope-labeled and natural D-lactic acid undergo matrix interference and loss simultaneously throughout the pretreatment process. This significantly improves the accuracy and reproducibility of D-lactic acid quantification in complex matrices, ensuring the precision of isotope tracer-based quantitative correction throughout the entire process.

[0039] In some embodiments, step S3, specifically the chromatographic separation of the pretreated sample, includes: When performing chromatographic separation on the pretreated sample, a pre-separation unit is set up before the main chromatographic column; the pre-separation unit is used to selectively adsorb residual trace substances in the compound probiotic beverage sample; After a preset number of sample injections, the pre-separation unit is cleaned. The cleaning process includes switching the flow path or changing the solvent composition to remove trace substances adsorbed on the pre-separation unit. By removing trace substances from the pre-separation unit, trace substances are prevented from entering the main chromatographic column, thereby maintaining the efficiency and lifespan of the main chromatographic column.

[0040] The pre-separation unit is a component installed before the main chromatographic column for preliminary sample purification and selective adsorption of interfering substances. It can be implemented using a guard column, an online solid-phase extraction column, or a pretreatment column with specific adsorption materials. The scavenging process refers to the removal of trace substances adsorbed on the pre-separation unit. This can be achieved through backwashing, high-concentration solvent elution, or regeneration via heating, electrochemistry, or other methods.

[0041] This protocol incorporates a pre-separation unit before the main column in the chromatographic separation of pretreated samples. This allows the sample to undergo a preliminary purification process before entering the core separation component (main column). The pre-separation unit selectively adsorbs residual trace substances in the compound probiotic beverage sample, meaning it possesses specific adsorption capabilities to target residual matrix components that may negatively impact chromatographic separation and mass spectrometry detection. Through this selective adsorption, the target analyte D-lactic acid and its stable isotope label can pass through smoothly, while interfering substances are retained, ensuring higher sample purity entering the main column and laying the foundation for subsequent accurate separation and detection. Since the pre-separation unit continuously adsorbs trace substances from the sample, its adsorption capacity gradually becomes saturated. Therefore, after a predetermined number of sample injections, the pre-separation unit is purged. Periodic regeneration of the pre-separation unit restores its adsorption capacity, ensuring its continued effective protective function and preventing interfering substances from penetrating into the main column due to saturation. Purge procedures include switching the flow path or changing the solvent composition, providing a flexible and efficient purge method. Switching the flow path can disconnect the pre-separation unit from the main column and introduce a cleaning solution for backwashing or high-intensity elution, thereby eluting the adsorbed trace substances from the unit. Changing the solvent composition can disrupt the binding between trace substances and the adsorbent by adjusting the polarity, ionic strength, or pH of the eluent, causing them to desorb from the pre-separation unit. These methods thoroughly remove contaminants accumulated on the pre-separation unit, ensuring it maintains high adsorption performance during subsequent sample injections. It is through the interception and periodic cleaning of the pre-separation unit that the main column is kept in a relatively clean working environment. This not only effectively prevents the main column from becoming contaminated, avoiding problems such as decreased column efficiency, peak shape deterioration, and reduced reproducibility, thus ensuring the accuracy and reliability of D-lactic acid detection results, but also significantly extends the lifespan of the expensive main column, reducing operating costs and maintenance frequency. Based on this, combined with the overall method described in this application, which involves adding stable isotope-labeled D-lactic acid and performing simultaneous pretreatment and mass spectrometry detection, the introduction of the pre-separation unit further enhances the stability and accuracy of the entire detection process. Even after pretreatment, a small amount of residual interfering substances may still exist in complex matrices. The pre-separation unit ensures the purity of the sample entering the main chromatographic column, thereby enabling more stable and accurate signals for natural D-lactic acid and stable isotope-labeled D-lactic acid during chromatographic separation and mass spectrometry detection. This ensures the reliability of quantitative correction based on signal ratios and ultimately achieves accurate quantification of natural D-lactic acid in compound probiotic beverage samples.

[0042] In one embodiment, when performing chromatographic separation on a pretreated compound probiotic beverage sample, a guard column can be connected in series as a pre-separation unit before the main chromatographic column (e.g., a C18 reversed-phase column). This guard column can be filled with packing material similar to the main column but with larger particle size or wider pore size, or with a polymer matrix packing material having specific adsorption capabilities to selectively adsorb residual trace substances such as proteins, polysaccharides, or pigments in the sample. For example, an online solid-phase extraction column with an inner diameter of 2.1 mm and a length of 10 mm can be selected, with a polymer reversed-phase adsorbent as packing material. After, for example, 100 sample injections, the pre-separation unit can be purged. The purge process may include switching the flow path via a six-way valve to separate the pre-separation unit from the main column flow path and introducing a high-concentration organic solvent (e.g., a 90% acetonitrile aqueous solution) for backwashing to elute trace substances adsorbed on the pre-separation unit. Alternatively, the elution process may involve altering the solvent composition, for example, increasing the organic phase content of the mobile phase from the conventional 5% to 80% without switching the flow path, and maintaining this level for a period of time to strongly elute contaminants on the pre-separation unit. These operations ensure that the pre-separation unit continues to perform its elution function in subsequent sample analyses, thereby protecting the main column.

[0043] This method effectively intercepts and removes residual trace substances from compound probiotic beverage samples by setting up a pre-separation unit before the main chromatographic column and periodically cleaning it. This prevents these trace substances from entering the main chromatographic column and causing contamination or damage, thus solving the problems of decreased column efficiency, retention time drift, and increased column pressure. At the same time, this method significantly extends the lifespan of the main chromatographic column, reduces analytical costs and maintenance frequency, and ensures the long-term stability and accuracy of D-lactic acid detection results.

[0044] In some embodiments, step S3, which involves mass spectrometry detection of the chromatographically separated natural D-lactic acid and the stable isotope-labeled D-lactic acid to obtain a first signal of natural D-lactic acid and a second signal of stable isotope-labeled D-lactic acid, includes the following specific steps: Between the chromatographic column outlet and the mass spectrometer ion source inlet, a processing unit is set up and controlled to add an auxiliary solvent to the chromatographic effluent; the auxiliary solvent is a volatile organic solvent, including acetonitrile or methanol; The effect of residual matrix components on the ionization of natural D-lactic acid and stable isotope-labeled D-lactic acid was homogenized by mixing auxiliary solvents with residual matrix components in chromatographic effluents. Mass spectrometry was used to detect the homogenized chromatographic effluent to obtain the first signal of natural D-lactic acid and the second signal of stable isotope-labeled D-lactic acid.

[0045] A processing unit is a device used to precisely control and introduce auxiliary solvents into the chromatographic effluent between the column outlet and the mass spectrometer ion source inlet. It can be implemented using, for example, one or more micro pumps, syringe pumps, or a liquid addition module integrated into a flow path control valve.

[0046] This solution aims to address the issue of residual matrix components affecting the differential ionization of natural D-lactic acid and stable isotope-labeled D-lactic acid in a mass spectrometry ion source after chromatographic separation of compound probiotic beverage samples, thereby ensuring the accuracy of signal acquisition ratios. Its overall operational logic is as follows: First, an auxiliary solvent is added to the chromatographic effluent between the column outlet and the mass spectrometry ion source inlet by a processing unit precisely controlled. This is a crucial pretreatment step, actively introducing a controllable medium before the target analyte enters the mass spectrometry ion source. The auxiliary solvent is selected as a volatile organic solvent, such as acetonitrile or methanol. These solvents have good compatibility with the chromatographic mobile phase and are easily volatilized in the mass spectrometry ion source, without introducing new interferences. Simultaneously, they effectively alter the solvent composition of the chromatographic effluent, creating conditions for subsequent homogenization. Second, the added auxiliary solvent is thoroughly mixed with any trace amounts of residual matrix components that may be present in the chromatographic effluent. This mixing effect allows the auxiliary solvent to dilute, dissolve, or otherwise alter the physicochemical properties of these matrix components, thereby making their influence on the ionization process of natural D-lactic acid and stable isotope-labeled D-lactic acid more consistent or significantly reduced. This "homogenization" ensures that the ionization efficiency of natural D-lactic acid and its stable isotope-labeled internal standard in the mass spectrometry ion source is no longer affected by the random or asymmetric interference of residual matrix components, making their behavior during ionization more synchronized and predictable. Finally, the chromatographic effluent after being homogenized by the auxiliary solvent is analyzed by mass spectrometry to obtain the first signal of natural D-lactic acid and the second signal of stable isotope-labeled D-lactic acid. Because the ionization environment is optimized and stabilized, the obtained first and second signals will more accurately reflect their relative content in the sample, and the interference from matrix effects will be significantly reduced. This method combines the steps of adding a known amount of stable isotope-labeled D-lactic acid to a compound probiotic beverage sample and ensuring homogeneous mixing of the stable isotope-labeled D-lactic acid with the natural D-lactic acid in the sample, and the pretreatment of the homogeneous sample to ensure that the stable isotope-labeled D-lactic acid and the natural D-lactic acid undergo matrix interference and loss simultaneously. Together, these steps constitute a complete system for accurate quantitative analysis of D-lactic acid in complex matrices. By actively homogenizing the matrix effect before mass spectrometry detection, the behavior of natural D-lactic acid and its stable isotope-labeled internal standard is ensured to be highly consistent during ionization. This significantly improves the accuracy and reliability of quantitative correction based on the signal ratio of the two, effectively overcoming the interference of complex matrices on the mass spectrometry detection signal, resulting in more accurate and reliable final quantitative results.

[0047] In one specific embodiment, this method incorporates a T-type mixing connector as a processing unit between the chromatographic column outlet and the mass spectrometer ion source inlet. One end of this connector connects to the column outlet, and the other end connects to a micro-injection pump. The micro-injection pump precisely controls the flow rate and dosage of the auxiliary solvent. The auxiliary solvent can be chromatographically pure acetonitrile, injected into the chromatographic eluent at a constant flow rate via the micro-injection pump. The chromatographic eluent and acetonitrile are thoroughly mixed in the T-type mixing connector, ensuring that the acetonitrile can fully contact and interact with the residual matrix components in the eluent. The mixed eluent is then directly introduced into the electrospray ionization source of the mass spectrometer for detection. In this way, acetonitrile, as an auxiliary solvent, can effectively dilute or alter the properties of the residual matrix components, making their ionization effects on natural D-lactic acid and stable isotope-labeled D-lactic acid more consistent. This results in first and second signals less affected by matrix effects during mass spectrometry detection, providing a more reliable data foundation for subsequent quantitative analysis.

[0048] This method actively homogenizes the influence of residual matrix components on the ionization of natural D-lactic acid and stable isotope-labeled D-lactic acid by adding an auxiliary solvent and mixing it between the column outlet and the mass spectrometry ion source inlet. This ensures a high degree of consistency in the ionization efficiency of natural D-lactic acid and its stable isotope-labeled internal standard during mass spectrometry detection, thereby significantly reducing the interference of complex matrices on the accuracy of signal acquisition ratios. Ultimately, the obtained first and second signals more accurately reflect the relative content of the target analyte in the sample, greatly improving the accuracy and reliability of quantitative correction based on signal ratios, and effectively solving the technical problem of inaccurate D-lactic acid quantification in complex matrices.

[0049] Combination Figure 2 As shown, the compound beverage probiotic detection system according to the present invention includes: The control mixing module 100 is used to add a known amount of stable isotope-labeled D-lactic acid to the compound probiotic beverage sample and to mix the stable isotope-labeled D-lactic acid with the natural D-lactic acid in the compound probiotic beverage sample evenly. The pretreatment module 200 is used to pretreat the uniformly mixed sample. The separation and detection module 300 is used to perform chromatographic separation on the pretreated sample and to perform mass spectrometry detection on the chromatographically separated natural D-lactic acid and stable isotope-labeled D-lactic acid to obtain the first signal of natural D-lactic acid and the second signal of stable isotope-labeled D-lactic acid. The calibration module 400 is used to quantitatively calibrate the natural D-lactic acid in the compound probiotic beverage sample based on the ratio of the first signal to the second signal.

[0050] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting probiotics in compound beverages, characterized in that, Includes the following steps: S1. Add a known amount of stable isotope-labeled D-lactic acid to the compound probiotic beverage sample, and mix the stable isotope-labeled D-lactic acid with the natural D-lactic acid in the compound probiotic beverage sample evenly. S2. Perform pretreatment on the well-mixed sample; S3. By performing chromatographic separation on the pretreated sample, and by performing mass spectrometry detection on the chromatographically separated natural D-lactic acid and stable isotope-labeled D-lactic acid, the first signal of natural D-lactic acid and the second signal of stable isotope-labeled D-lactic acid are obtained. S4. Based on the ratio of the first signal to the second signal, the natural D-lactic acid in the compound probiotic beverage sample is quantitatively corrected.

2. The method for detecting probiotics in compound beverages according to claim 1, characterized in that, In step S1, the stable isotope-labeled D-lactic acid is mixed evenly with the natural D-lactic acid in the compound probiotic beverage sample through the following steps: Prepare an organic solvent mixture containing stable isotope-labeled D-lactic acid; the organic solvent mixture contains acidic substances; By rapidly injecting a compound probiotic beverage sample into an organic solvent mixture, the acidic substances in the organic solvent mixture break down the microscopic gel network in the compound probiotic beverage sample, resulting in a uniformly mixed sample.

3. The method for detecting probiotics in compound beverages according to claim 2, characterized in that, Acidic substances are organic acids, including formic acid, acetic acid, or trifluoroacetic acid.

4. The method for detecting probiotics in compound beverages according to claim 2, characterized in that, Acidic substances are inorganic acids, including hydrochloric acid, sulfuric acid, or phosphoric acid.

5. The method for detecting probiotics in compound beverages according to claim 1, characterized in that, In step S2, the specific steps for pretreatment of the uniformly mixed sample include: By extracting a homogeneously mixed sample, specific trace active ingredients in the matrix are separated from natural D-lactic acid and stable isotope-labeled D-lactic acid. During the extraction process, the conformational changes or aggregation of specific trace active ingredients are inhibited by controlling the solvent composition or pH of the extraction environment. The extracted sample was subjected to solid-liquid separation to obtain a solution containing natural D-lactic acid and stable isotope-labeled D-lactic acid.

6. The method for detecting probiotics in compound beverages according to claim 1, characterized in that, In step S3, the specific steps for chromatographic separation of the pretreated sample include: When performing chromatographic separation on the pretreated sample, a pre-separation unit is set up before the main chromatographic column; the pre-separation unit is used to selectively adsorb residual trace substances in the compound probiotic beverage sample; After a preset number of sample injections, the pre-separation unit is cleaned.

7. The method for detecting probiotics in compound beverages according to claim 6, characterized in that, Cleaning processes may include switching flow paths or changing solvent composition.

8. The method for detecting probiotics in compound beverages according to claim 1, characterized in that, In step S3, the specific steps for obtaining the first signal of natural D-lactic acid and the second signal of stable isotope-labeled D-lactic acid by mass spectrometry detection of the chromatographically separated natural D-lactic acid and stable isotope-labeled D-lactic acid include: Between the chromatographic column outlet and the mass spectrometer ion source inlet, a processing unit is set up and controlled to add auxiliary solvent to the chromatographic effluent; The effect of residual matrix components on the ionization of natural D-lactic acid and stable isotope-labeled D-lactic acid was homogenized by mixing auxiliary solvents with residual matrix components in chromatographic effluents. Mass spectrometry was used to detect the homogenized chromatographic effluent to obtain the first signal of natural D-lactic acid and the second signal of stable isotope-labeled D-lactic acid.

9. The method for detecting probiotics in compound beverages according to claim 8, characterized in that, The auxiliary solvent is a volatile organic solvent, including acetonitrile or methanol.

10. A probiotic detection system for compound beverages, characterized in that, include: The control mixing module is used to add a known amount of stable isotope-labeled D-lactic acid to the compound probiotic beverage sample and to mix the stable isotope-labeled D-lactic acid with the natural D-lactic acid in the compound probiotic beverage sample evenly. The pretreatment module is used to pretreat the uniformly mixed sample. The separation and detection module is used to perform chromatographic separation on the pretreated sample and to perform mass spectrometry detection on the chromatographically separated natural D-lactic acid and stable isotope-labeled D-lactic acid to obtain the first signal of natural D-lactic acid and the second signal of stable isotope-labeled D-lactic acid. The calibration module is used to quantitatively calibrate the natural D-lactic acid in the compound probiotic beverage sample based on the ratio of the first signal to the second signal.

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