Evaluation method for volatile peculiar smell components of dairy products

Abstract

The invention discloses a method for evaluating volatile peculiar smell components of dairy products. The invention provides a dairy product volatile peculiar smell component evaluation method which comprises the following steps: enriching volatile compounds; analyzing a volatile compound; analyzing aroma active substances; a step of sensory evaluation; and a step of correlation analysis. According to the evaluation method for the volatile peculiar smell components of the dairy product, the sensory guidance key peculiar smell substances can be screened out through comprehensive analysis of multiple detection means.

Description

Technical Field

[0001] This invention belongs to the field of off-odor component analysis of dairy products, and relates to a method for evaluating volatile off-odor components of dairy products. Specifically, it relates to a sensory-oriented method for screening, qualitative and quantitative analysis of volatile off-odor components in milk powder. Background Technology

[0002] Currently, complaints about off-odors in dairy products, especially milk powder, account for 37% of dairy product quality issues (according to a 2024 report by the China Dairy Industry Association). The current national standards rely on human sensory evaluation, which has an error rate as high as 20%-30%, and the accuracy and stability of the evaluation depend on the evaluator's condition.

[0003] Existing technologies have attempted to study and separate the sources of off-odors in milk powder. For example, Reference 1 discloses a method for analyzing off-odors in pine pollen milk powder products using GC-IMS technology based on flavor characteristics. This method applies gas chromatography-ion mobility spectrometry to the quality analysis of pine pollen milk powder products, identifying changes in volatile flavor compounds and achieving product flavor quality evaluation and similarity assessment. Another example is Reference 2, which monitors changes in volatile flavor compounds in infant formula (IF) during long-term storage (11 months) at room temperature (25°C) to identify key compounds affecting flavor changes. This method uses headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) to detect volatile compounds, uses odor activity value (OAV) to assess key odor contributors, orthogonal partial least squares discriminant analysis (OPLS-DA) to screen for markers affecting flavor changes, and uses cluster heatmaps and multivariate statistical methods to assist in the analysis.

[0004] However, current technologies lack an integrated approach that combines volatile substance content, aroma active substances, and sensory evaluation to determine off-odor components in milk powder. New methods for evaluating volatile off-odor components in dairy products, as cited in previous literature, still need to be developed.

[0005] Reference 1: CN116500182A

[0006] Reference 2: Yu, MG et al. "Comparison of aroma properties of infantformulas:Differences in key aroma compounds and their possible origins inprocessing." Journal of dairy science vol.106,9(2023):5970-5987.doi:10.3168 / jds.2022-22873 Summary of the Invention

[0007] The problem the invention aims to solve

[0008] While existing technologies have attempted to analyze the sources of off-odors in milk powder, they are not perfect. For example, reference 1 only identifies off-odor substances in milk powder based on changes in volatile compounds, without establishing a quantitative relationship with the sensory attributes of the off-odor. Reference 2 uses HS-SPME combined with GC-O to detect volatile substances in milk powder, but it does not solve the problems of stable detection and accurate detection of even trace amounts of volatile substances. Furthermore, SPME has low and unstable extraction and recovery rates for volatile compounds. In addition, existing technologies typically determine off-odor compounds by monitoring the trend of volatile compound content changes during storage, lacking a sensory-oriented method for identifying off-odor substances.

[0009] Based on the aforementioned deficiencies in existing technologies, this invention develops a method to identify key off-odor substances during the production, transportation, and storage of milk powder based on sensory evaluation, aroma-active compounds, and correlation analysis.

[0010] Solution for solving the problem

[0011] [1]. A method for evaluating volatile off-odor components in dairy products, wherein the evaluation method includes the following steps:

[0012] The enrichment step of volatile compounds involves using stir bar adsorption extraction technology to enrich the volatile compounds in the sample to be tested and then collecting the enriched sample.

[0013] The analytical steps for volatile compounds involve using gas chromatography-mass spectrometry to perform qualitative and quantitative analysis on the volatile compounds in the enriched sample, thereby identifying the volatile compounds in the sample to be tested.

[0014] The analysis steps for aroma active substances employ aroma activity values ​​and gas chromatography-olfactometry-mass spectrometry to screen the aroma active substances in the volatile compounds identified in the analysis steps for the volatile compounds.

[0015] The sensory evaluation process involves using quantitative descriptive analysis to evaluate the typical odor sensory characteristics of the sample to be tested, and obtaining an odor sensory characteristic evaluation score.

[0016] The correlation analysis step involves performing a correlation analysis between the aroma active substances obtained in the aroma active substance analysis step and the typical off-odor sensory characteristics obtained in the sensory evaluation step to identify key off-odor substances.

[0017] [2]. According to the evaluation method described in [1], in the stir bar adsorption extraction, a stir bar is used, and the extraction is performed at a speed of 300 to 700 rpm for 30 to 90 min;

[0018] Optionally, the stirring rod is aged at 200–300°C for 10–50 min before extraction.

[0019] [3]. According to the evaluation method described in [1] or [2], wherein, in the analytical step of the aroma active substance, the aroma activity value (OAV) is calculated by the following formula:

[0020]

[0021] In the formula: C i volatile compound concentration; OT i volatile compound threshold; and / or,

[0022] The aroma intensity of volatile compounds was determined by gas chromatography-olfactometry-mass spectrometry.

[0023] [4]. According to the evaluation method described in [3], in the analysis step of the aroma active substances, volatile compounds with an aroma activity value (OAV) greater than or equal to 1 or an aroma intensity greater than or equal to 1 are identified as aroma active substances.

[0024] [5]. According to any one of [1]-[4], the typical odor sensory characteristics include: fishy smell and oxidized smell.

[0025] [6]. The evaluation method according to any one of [1]-[5], wherein Spearman correlation analysis is used in the correlation analysis step.

[0026] [7]. According to the evaluation method described in [6], the formula for calculating the correlation coefficient in the Spearman correlation analysis is as follows:

[0027]

[0028] In the formula: d i The rank difference represents the difference in ranking between two variables to be analyzed; n is the total number of features.

[0029] The variables to be analyzed include the content of aroma active compounds and the evaluation score of odor sensory characteristics; the total number of characteristics is the number of samples to be tested.

[0030] [8]. The evaluation method according to any one of [1]-[7], wherein, prior to the analysis step of the volatile compound, a step of desorption and enrichment of the volatile compound enriched by stir bar adsorption extraction technology is further included, wherein the desorption and enrichment is performed using a thermal desorption system and a cold sample introduction system-programmed temperature evaporator.

[0031] [9]. The evaluation method according to any one of [1]-[8], wherein, in the analytical step of the volatile compound, the quantitative analysis is performed by external standard method, internal standard method or a combination of two methods.

[0032]

[10] . The evaluation method according to any one of [1]-[9], wherein, in the analytical step of the volatile compound, the qualitative analysis includes at least one of retention index (RI), NIST14.0 database search (MS), standard compound comparison (STD) and olfaction (O).

[0033]

[11] . The evaluation method according to any one of [1]-

[10] , wherein the sample to be tested is milk powder, preferably infant milk powder.

[0034]

[12] . According to any one of [1]-

[11] , the evaluation method wherein the key odor substance comprises:

[0035] Key odor substances that cause fishy smell include (E)-2-octen-1-ol, nonanal, (E,E)-2,4-nonadienal, cis-4,5-epoxytrans-2-decenal, heptanal, (E)-2-octenal, and 2-n-pentylfuran.

[0036] Key off-odor substances that cause oxidative odor include (E)-2-octen-1-ol, cis-4,5-epoxytrans-2-decenal, nonanal, (E,E)-2,4-nonadienal, and heptanal.

[0037] The effects of the invention

[0038] The method for evaluating volatile off-odor components in dairy products provided by this invention can screen out key off-odor substances that guide sensory perception through comprehensive analysis using multiple detection methods, and has at least the following advantages:

[0039] (1) SBSE-TD was used instead of HS-SPME to improve stability and detection limit;

[0040] (2) The addition of sensory evaluation and two methods for identifying aroma-active compounds (GC-O and OAV values) helps to identify key off-odor compounds, making the analysis more comprehensive;

[0041] (3) Determine the odor compounds by comprehensively considering sensory characteristics and volatile substance characteristics through correlation analysis;

[0042] (4) Construct a replicable standard detection and analysis process to achieve accurate identification of key compounds. Attached Figure Description

[0043] Figure 1 : Schematic diagram of the evaluation method for volatile odor components in milk powder provided by the present invention.

[0044] Figure 2 : Results of the comparison of the number of compounds.

[0045] Figure 3 : Schematic diagram of correlation analysis results, where ** indicates a highly significant positive correlation between the compound and the relevant sensory attributes (P > 0.01); * indicates a significant positive correlation between the compound and the relevant sensory attributes (P > 0.05). Detailed Implementation

[0046] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0047] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0048] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0049] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0050] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0051] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0052] The following will provide a detailed description of the method for evaluating volatile off-odor components in dairy products according to the present invention.

[0053] This invention provides a method for evaluating volatile off-odor components in dairy products, wherein the evaluation method includes the following steps:

[0054] The enrichment step of volatile compounds involves using stir bar adsorption extraction technology to enrich the volatile compounds in the sample to be tested and then collecting the enriched sample.

[0055] The analytical steps for volatile compounds involve using gas chromatography-mass spectrometry to perform qualitative and quantitative analysis on the volatile compounds in the enriched sample, thereby obtaining the volatile compounds in the sample to be tested.

[0056] The analysis steps for aroma active substances employ aroma activity values ​​and gas chromatography-olfactometry-mass spectrometry to screen the aroma active substances in the volatile compounds obtained in the analysis steps for the volatile compounds.

[0057] The sensory evaluation process involves using quantitative descriptive analysis to evaluate the typical odor sensory characteristics of the sample to be tested, and obtaining an odor sensory characteristic evaluation score.

[0058] The correlation analysis step involves performing a correlation analysis between the aroma active substances obtained in the aroma active substance analysis step and the typical off-odor sensory characteristics obtained in the sensory evaluation step to identify key off-odor substances.

[0059] The method for evaluating volatile odor components in dairy products provided by this invention can screen out key odor substances that provide sensory guidance through comprehensive analysis using multiple detection methods.

[0060] <Steps for enriching volatile compounds>

[0061] In the volatile compound enrichment step of the evaluation method of the present invention, the volatile compounds of the test sample are enriched by stir bar adsorption extraction technology, and the enriched sample is collected.

[0062] Test sample

[0063] In the evaluation method of this invention, the sample to be tested is a dairy product.

[0064] In this invention, there are no particular restrictions on the source of "dairy products" or test samples, which can refer to food products produced by animals such as cattle, goats, sheep, yaks, horses, camels and other mammals.

[0065] Examples of dairy products include low-fat milk (e.g., 0.1%, 0.5%, or 1.5% fat), nonfat milk, milk powder, whole milk, whole milk products, butter, buttermilk, buttermilk products, skim milk, skim milk products, high-fat products, condensed milk, fresh cream, cheese, ice cream and confectionery products, probiotic beverages, or probiotic yogurt-type beverages. "Milk powder" refers to artificial dairy products made by evaporating milk until it is dry.

[0066] In some preferred embodiments of the present invention, the samples in the evaluation method of the present invention are derived from cow or sheep milk powder products.

[0067] In some preferred embodiments of the present invention, the samples used in the evaluation method of the present invention are various infant formula milk products (milk powder), including stage 1, stage 2, stage 3, etc. In some specific embodiments, these samples may be in liquid, block, or powder form.

[0068] In this specification, volatile flavor compounds (VOCs) refer to a class of organic chemical substances that readily evaporate or sublimate spontaneously from a liquid or solid state at room temperature and pressure, exist in the air in gaseous form, and can be detected by the sense of smell. In this specification, volatile flavor compounds are also referred to simply as volatile compounds.

[0069] Stirring rod adsorption extraction technology

[0070] Stirred rod adsorption-extraction (SPE) is a sample pretreatment technique based on adsorption principles, integrating sampling, extraction, and concentration. It uses a glass magnetic stir bar coated with an adsorbent material (most commonly polydimethylsiloxane, PDMS). By placing this stir bar into the liquid sample and stirring, the target analytes in the sample are selectively adsorbed or absorbed into the coating. Afterward, the stir bar is removed, and the enriched analytes are released through thermal desorption or solvent desorption, and then introduced into an analytical instrument (such as gas chromatography-mass spectrometry, GC-MS) for detection.

[0071] In this invention, there are no special restrictions on the adsorption material, thickness, or length of the stirring rod, as long as it can effectively adsorb the volatile compounds of the sample to be tested. Those skilled in the art can obtain the stirring rod commercially.

[0072] In some preferred embodiments, the stirring rod is coated with a divinylbenzene / carbon molecular sieve / polydimethylsiloxane (DVB / CAR / PDMS) coating, with a coating length of about 20 mm and a thickness of about 1.0 mm.

[0073] In some embodiments, during the stir bar adsorption extraction, a stir bar is used to extract for 30 to 90 minutes at a rotation speed of 300 to 700 rpm.

[0074] In some preferred embodiments, in the stir bar adsorption extraction, a stir bar is used to extract at a speed of 400 to 700 rpm, for example, at 400 rpm, 500 rpm, 600 rpm or 700 rpm.

[0075] In some preferred embodiments, extraction is performed for 40 to 80 minutes, more preferably 40 to 70 minutes, for example 40 minutes, 50 minutes, 60 minutes or 70 minutes.

[0076] In some embodiments, the stirring rod is aged at 200–300°C for 10–50 min prior to extraction.

[0077] In some embodiments, the stirring rod is aged at 220–280°C for 20–50 min prior to extraction. For example, the stirring rod is aged at 250°C for 30 min prior to extraction.

[0078] Desorption and enrichment steps

[0079] In some embodiments of the present invention, prior to the analysis of the volatile compounds, a step of desorption and enrichment of the volatile compounds enriched using stir bar adsorption extraction technology is included, wherein the desorption and enrichment is performed using a thermal desorption system and a cold sample introduction system-programmed temperature evaporator.

[0080] In some specific implementations, in the thermal desorption system, the desorption temperature is increased from 30°C to 200°C at a rate of 100°C / min and held at 200°C for 10 min; the carrier gas (helium) flow rate is 80 mL / min for desorption.

[0081] In some specific implementations, in a cold sample introduction system-programmed temperature evaporator (CIS-PTV), the sample is first cooled to 10°C, then increased to 240°C at a rate of 10°C / s and held for 5 minutes for enrichment.

[0082] The SPME used in the prior art has low extraction recovery rate and is unstable for volatile compounds. The present invention creatively uses SBSE-TD to replace HS-SPME in the prior art, thereby improving stability and detection limit.

[0083] <Analytical Procedures for Volatile Compounds>

[0084] In the volatile compound analysis step of the evaluation method of the present invention, gas chromatography-mass spectrometry is used to perform qualitative and quantitative analysis on the volatile compounds of the enriched sample to obtain the volatile compounds in the sample to be tested.

[0085] In some implementations, the quantitative analysis of the volatile compounds is performed using an external standard method, an internal standard method, or a combination of both.

[0086] In some specific implementation schemes, external standard method is used for quantification of substances with standard compounds, while internal standard method is used for quantification of substances without relevant standard compounds.

[0087] In some embodiments, the qualitative analysis of the volatile compounds includes at least one of retention index (RI), NIST 14.0 database search (MS), standard compound comparison (STD), and olfaction (O).

[0088] In some implementation schemes, the column temperature conditions for gas chromatography are as follows:

[0089] The initial temperature is 30-50℃, held for 1-3 min, then increased to 100-120℃ at 2-6℃ / min, then increased to 140-180℃ at 1-3℃ / min, and finally increased to 210-250℃ at 8-12℃ / min, held for 1-5 min; the carrier gas flow rate is 1-2 mL / min.

[0090] In some preferred embodiments, the column temperature conditions for gas chromatography are as follows:

[0091] The initial temperature was 40℃, held for 2 min, increased to 110℃ at 4℃ / min, then increased to 160℃ at 2℃ / min, and finally increased to 230℃ at 10℃ / min, held for 3 min; the carrier gas flow rate was 1.5 mL / min.

[0092] In some implementations, the gas chromatography column can be a non-polar gas chromatography column, such as a capillary column.

[0093] In some embodiments, the stationary phase used in the gas chromatography column can be (5% phenyl)-methylpolysiloxane (equivalent to USP stationary phase G27) or a stationary phase with similar properties. In some specific embodiments, the gas chromatography column can be an HP-5MS capillary column, or gas chromatography columns with similar stationary phases such as Rtx-5ms, Rxi-5ms, Rxi-5Sil MS, PTE-5, BPX-5, AT-5ms, ZB-5ms, ZB-5ms Plus, SLB-5ms, and Equity-5.

[0094] In some embodiments, the gas chromatography column has a length of 10–70 m (e.g., commercially available 15 m, 30 m, 50 m, and 60 m gas chromatography columns), an inner diameter of 0.10–0.53 mm, and a film thickness of 0.1–5 μm (e.g., thin liquid films with a thickness of 0.1–0.2 μm; standard liquid films with a thickness of 0.25–0.33 μm; and thick liquid films with a thickness of 0.5–5.0 μm). In some preferred embodiments, the film thickness is 0.25 μm.

[0095] In some embodiments of the present invention, the mass spectrometer employs an electron impact (EI) ion source with an electron energy of 70 eV and a temperature set to 230°C. The interface temperature is 250°C, and the mass scan range (m / z) is set to 40–350.

[0096] <Analytical Procedures for Aroma Active Substances>

[0097] In the aroma active substance analysis step of the evaluation method of the present invention, aroma activity values ​​and gas chromatography-olfactometry-mass spectrometry are used to screen the aroma active substances in the volatile compounds obtained in the volatile compound analysis step.

[0098] In this specification, aroma-active substances or aroma-active compounds refer to volatile compounds that can be perceived by the human olfactory system and contribute to specific odor characteristics.

[0099] In some implementations, the aroma activity value (OAV) is calculated using the following formula:

[0100]

[0101] In the formula: C i volatile compound concentration; OT i , volatile compound threshold.

[0102] In some embodiments, the concentration of the volatile compound is obtained by quantitative analysis in the aforementioned analytical steps of the volatile compound analysis.

[0103] In some implementations, the volatile compound threshold is obtained from existing technologies, such as the papers listed in subsequent embodiments.

[0104] In some implementations, the aroma intensity of volatile compounds is determined by gas chromatography-olfactometry-mass spectrometry.

[0105] In this specification, aroma intensity refers to a sensory measure of the strength of stimulation produced by an odor or aroma active substance on the human olfactory system.

[0106] In some implementations, the olfaction is detected using a commercially available olfactometer (e.g., ODP-3, Gerstel, Germany), and the aroma intensity of the aroma active substance is determined by the olfactometer.

[0107] In some specific implementations, in the analysis step of the aroma active substances, volatile compounds with an aroma activity value (OAV) greater than or equal to 1 or an aroma intensity greater than or equal to 1 are identified as aroma active substances.

[0108] This invention innovatively employs both GC-O and OAV values ​​to screen important compounds (i.e., aroma active substances) that contribute to the overall aroma of a sample's volatile compounds. An unexpected discovery was made: some substances can only be detected by GC-O analysis, while others can only be calculated using OAV values. Therefore, this invention combines two methods to identify aroma active substances, resulting in a more comprehensive list of compounds.

[0109] <Steps of Sensory Evaluation>

[0110] In the sensory evaluation step of the evaluation method of the present invention, quantitative descriptive analysis is used to evaluate the typical odor sensory characteristics of the sample to be tested, and an odor sensory characteristic evaluation score is obtained.

[0111] In this specification, quantitative descriptive analysis is a systematic and objective sensory evaluation method in which a trained evaluation team identifies, defines, quantifies, and assesses the sensory characteristics of a product.

[0112] In this specification, typical odor sensory characteristics refer to a systematic method for qualitatively describing, quantitatively assessing, and classifying abnormal or unpleasant odors through human olfaction (and sometimes taste, i.e., "olfaction").

[0113] In some specific implementations, the typical odor sensory characteristics include: fishy smell and oxidized smell.

[0114] In some specific implementation schemes, the definition and reference for fishy smell are as follows: a smell similar to that of live fish, 20mL of purified water + 0.4g DHA + 0.2g ARA (n=7).

[0115] In some specific implementation schemes, the definition and reference for oxidative odor are as follows: an odor similar to rancid oil, expressed as 30 mL of purified water + 0.2 μL of 2,4-nonadialdehyde + 0.7 μL of 3-methylbutyraldehyde (n = 6).

[0116] In some specific implementation plans, sensory evaluation evaluators can be screened according to ISO 3972:1991, then trained and assessed according to ISO 4212:2003, and finally formed an evaluation team of 10 evaluators (3 men and 7 women).

[0117] In some specific implementations, the sensory evaluation can use a 9-point scale to evaluate the samples, with a minimum scoring interval of 1, where 1-2 indicates very weak, 3-4 indicates very weak, 5-6 indicates moderate intensity, 7-8 indicates strong, and 9 indicates very strong; the sensory evaluation is conducted using a randomized block design according to ISO 8589,2007; during the evaluation, evaluators are required to take sufficient rest while evaluating different samples to reduce sensory fatigue.

[0118] <Steps of Correlation Analysis>

[0119] In the correlation analysis step of the evaluation method of the present invention, a correlation analysis is performed on the aroma active substances obtained in the analysis step of the aroma active substances and the typical off-odor sensory characteristics obtained in the sensory evaluation step to determine the key off-odor substances.

[0120] In some specific implementations, a correlation analysis is performed between the content of aroma active substances obtained in the analysis step and the odor sensory characteristic score obtained in the sensory evaluation step to identify key odor substances.

[0121] In some implementations, Spearman correlation analysis is used in the correlation analysis step.

[0122] In some implementations, the correlation coefficient (ρ) in the Spearman correlation analysis is calculated using the following formula:

[0123]

[0124] In the formula: d i The rank difference represents the difference in ranking between two variables to be analyzed; n is the total number of features.

[0125] The variables to be analyzed include the content of aroma active compounds and the evaluation score of odor sensory characteristics; the total number of characteristics is the number of samples to be tested.

[0126] In some embodiments, the content of the aroma-active compounds is determined by quantitative analysis in the analytical steps of the volatile compounds.

[0127] In some implementations, the odor sensory characteristic evaluation score of the sample to be tested is determined by the sensory evaluation step.

[0128] In some implementation schemes, the correlation coefficient (ρ) is 0-1, which indicates that the two variables are positively correlated, and the larger the value, the stronger the correlation; -1-0 indicates that the two variables are negatively correlated, and the smaller the value, the stronger the correlation; 0 indicates that there is no correlation between the two variables.

[0129] In some implementations, the sample to be tested is infant formula.

[0130] In some implementations, the key odor substance includes:

[0131] Key odor substances that cause fishy smell include (E)-2-octen-1-ol, nonanal, (E,E)-2,4-nonadienal, cis-4,5-epoxytrans-2-decenal, heptanal, (E)-2-octenal, and 2-n-pentylfuran.

[0132] Key off-odor substances that cause oxidative odor include (E)-2-octen-1-ol, cis-4,5-epoxytrans-2-decenal, nonanal, (E,E)-2,4-nonadienal, and heptanal.

[0133] As can be seen, this invention uses correlation analysis to comprehensively consider sensory characteristics and volatile substance characteristics to determine odor compounds, and constructs a reproducible standard detection and analysis process to achieve accurate identification of key compounds.

[0134] Example

[0135] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0136] The method for evaluating volatile odor components provided by this invention employs Stir Bar Sorptive Extraction-Thermal Desorption (SBSE-TD) technology to replace Headspace-Solid Phase Micro-Extraction (HS-SPME), achieving high enrichment of volatile components and effectively and stably capturing volatile odor components. Simultaneously, Gas Chromatography-Olfactometry-Mass Spectrometry (GC-O-MS) and Odor Activity Value (OAV) are used to analyze aroma-active compounds. These two methods complement each other, enabling accurate identification of aroma-active compounds. Sensory evaluation combined with correlation analysis further assists in identifying key odor compounds, achieving sensory-guided odor substance analysis. The specific steps of the method for evaluating volatile odor components are as follows;

[0137] (1) Instruments and reagents:

[0138] Stirring rod, coated with divinylbenzene / carbon molecular sieve / polydimethylsiloxane (DVB / CAR / PDMS), coating thickness 1.0 mm, length 20 mm (Gerstel, Germany);

[0139] Thermal Desorption System (TDS, Gerstel, Germany);

[0140] Gas chromatography-mass spectrometry (GC-MS) instrument (8890GC-5977BMS, Agilent, USA);

[0141] Chromatographic column: HP-5MS (30m × 0.25mm × 0.25μm, J&W, USA);

[0142] Smell tester (ODP-3, Gerstel, Germany);

[0143] Docosahexaenoic acid powder (DHA≥7%, Jiabiyou Biotechnology (Wuhan) Co., Ltd., Wuhan);

[0144] Arachidonic acid powder (ARA≥10%, Jiabiyou Biotechnology (Wuhan) Co., Ltd., Wuhan); 2,4-Nonadialdehyde (purity≥89%, Sigma-Aldrich, USA);

[0145] 3-Methylbutyraldehyde (purity ≥87%, Sigma-Aldrich, USA);

[0146] Pentanal (purity ≥97%, Aladdin, Shanghai);

[0147] Hexanal (purity ≥99%, Macklin, Shanghai);

[0148] Heptanal (purity ≥98%, Aladdin, Shanghai);

[0149] (E,E)-2,4-hexadienal (purity ≥95%, Aladdin, Shanghai);

[0150] (E,E)-2,4-Heptadienal (purity ≥90%, Macklin, Shanghai);

[0151] Octaldehyde (purity ≥99%, Macklin, Shanghai);

[0152] (E)-2-Octenal (purity ≥95%, Macklin, Shanghai);

[0153] Octaldehyde (purity ≥99%, Macklin, Shanghai);

[0154] Nonanal (purity ≥96%, Macklin, Shanghai);

[0155] (E,Z)-2,6-nonadienal (purity ≥96%, Macklin, Shanghai);

[0156] (E)-2-Nonanal (purity ≥95%, Aladdin, Shanghai);

[0157] (E)-2-Decanal (purity ≥95%, Aladdin, Shanghai);

[0158] (E)-2-Heptaneal (purity ≥95%, Aladdin, Shanghai);

[0159] (E)-2-hexaneal (purity ≥98%, Aladdin, Shanghai);

[0160] (E,E)-2,4-decadienal (purity ≥90%, Aladdin, Shanghai);

[0161] 1-Octen-3-ol (purity ≥98%, Aladdin, Shanghai);

[0162] 2-Ethylhexanol (purity ≥99.5%, CNW, Shanghai);

[0163] furfuryl alcohol (purity ≥98%, Macklin, Shanghai);

[0164] Benzaldehyde (purity ≥99.5%, Aladdin, Shanghai);

[0165] 2-Hepanotone (purity ≥99.8%, Aladdin, Shanghai);

[0166] δ-decanolide (purity ≥98%, Aladdin, Shanghai);

[0167] 1-Octen-3-one (purity ≥95%, Macklin, Shanghai);

[0168] 2-Pentylfuran (purity ≥98%, Macklin, Shanghai);

[0169] Furfural (purity ≥99%, Aladdin, Shanghai);

[0170] 4-Methyl-2-heptanone (purity ≥98%, CNW, Shanghai);

[0171] Series of alkanes (C7-C 40 (Sigma-Aldrich, USA).

[0172] sample:

[0173] Taking Feihe Xingfeifan infant formula (stages 1 and 3) as examples, the sample information is as follows:

[0174]

[0175]

[0176] (2) Methods for analyzing volatile substances:

[0177] Qualitative and quantitative analysis of volatile compounds in the sample was performed. Details are as follows:

[0178] Sample preparation: Weigh 2.58g of sample into a 20mL headspace vial, add 20μL of internal standard (4-methyl-2-heptanone, 0.2μg / μL), then add 18mL of purified water, mix well, and seal with a polytetrafluoroethylene septum vial for later use.

[0179] Extraction (stirred rod adsorption extraction) conditions: The stirring rod was aged at 250℃ for 30 min before extraction, and then extracted at 500 rpm at room temperature (25℃±2℃) for 60 min.

[0180] Desorption (thermal desorption) conditions: After extraction, the stirred rod was desorbed in a TDS (Cooled Injection System-Programmed Temperature Vaporizer, CIS-PTV) with the desorption temperature increased from 30℃ to 200℃ at 100℃ / min and held at 200℃ for 10 min; the carrier gas (helium) flow rate was 80 mL / min; enrichment conditions: the CIS-PTV was first cooled to 10℃, then increased to 240℃ at 10℃ / s and held for 5 min.

[0181] GC temperature program: starting temperature 40℃, hold for 2 min, increase to 110℃ at 4℃ / min, then increase to 160℃ at 2℃ / min, then increase to 230℃ at 10℃ / min, hold for 3 min; carrier gas flow rate 1.5 mL / min.

[0182] Mass spectrometry conditions: interface temperature 250℃; ion source temperature 230℃; electron ionization source EI, electron energy 70eV; mass scan range m / z 40~350.

[0183] Qualitative analysis of volatile compounds:

[0184] The retention index (RI) is used to qualitatively identify compounds detected by GC-MS by comparing the calculated retention index with the retention index retrieved from the database. The calculation method for the retention index is as follows:

[0185]

[0186] In the formula: N is the number of carbon atoms in the adjacent n-alkane to the left of the target compound; and The values ​​represent the retention times of the unknown compound and two adjacent n-alkane compounds, respectively.

[0187] MS: The compounds were identified by searching the NIST 14.0 database.

[0188] STD: Qualitative compounds are identified by comparing their spectra with those of standard compounds.

[0189] O: Qualitative characterization is achieved by comparing the aroma of compounds.

[0190] Quantitative analysis of volatile compounds: For substances with available standard compounds, the external standard method is used for quantification; for other compounds, the internal standard method is used. The formula for the internal standard method is as follows:

[0191]

[0192] In the formula: C i Compound content (μg / kg); M isM0 represents the mass of the internal standard; M0 represents the mass of the milk powder; A i The peak area of ​​the target compound; A is Internal standard peak area; f i The correction factor is 1.00.

[0193] Experimental Results: Qualitative results of volatile compounds in the SBSE-TD-GC-MS method for evaluating volatile odor components of this invention.

[0194] A total of 76 substances were detected, including 26 aldehydes, 17 ketones, 15 alcohols, 6 acids, 5 esters, 3 lactones, and 4 other substances (see Table 1 below for details). Compared with the HS-SPME-GC-MS analysis results of the same type of milk powder (Feihe Xingfeifan infant formula) (Li, Yilin et al. “Monitoring volatile changes in infant formula during long-term storage at room temperature.” Current research in food science vol.7100645.19Nov.2023,doi:10.1016 / j.crfs.2023.100645), more volatile substances were detected. The comparison results are shown below. Figure 2 HS-SPME-GC-MS can only detect 45 volatile compounds, while the SBSE-TD-GC-MS of this invention performs better in detecting major aroma compounds in milk powder, such as aldehydes, ketones, alcohols, and esters. Its detection of lactones and other terpenes is slightly weaker than SBSE-TD-GC-MS. Compared to aldehydes, ketones, alcohols, and esters, lactones and terpenes have higher aroma thresholds and are present in low concentrations in milk powder, contributing very little to its aroma characteristics. This indicates that SBSE-TD can effectively enrich the major aroma compounds in milk powder, solving the problem of insufficient enrichment of aroma compounds in milk powder by HS-SPME.

[0195] Table 1 Qualitative results of volatile compounds in infant formula milk powder

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203] In Table 1, the aroma threshold refers to the compound threshold (OT) mentioned earlier. i Milk matrix and water matrix refer to the solvents used for the corresponding compounds in aroma threshold determination. For example, if benzaldehyde is noted as being in milk matrix, it means that the aroma threshold is determined by dissolving benzaldehyde in milk. In the sample source, "stage 1" and "stage 3" refer to the sample category. For example, "stage 1" for benzaldehyde means that the substance is only detected in stage 1 milk powder, (E)-2-hexenal means that the substance is only detected in stage 3 milk powder, and hexanal is detected in both stage 1 and stage 3 milk powder.

[0204] (3) Calculation of GC-O and OAV values:

[0205] GC-O analysis and OAV value calculation were performed on the volatile compounds in step (2) above to screen out the volatile substances that contribute to the aroma of the sample (e.g., milk powder) (i.e. aroma active substances).

[0206] GC-O analysis method: The temperature of the GC to the olfactory port transfer tube was 280℃, and the water vapor flow rate was 45mL / min; the sample was smelled by 6 expert evaluators, each evaluator smelled the sample 3 times; the retention time, aroma description, and aroma intensity (1-very weak, 2-medium, 3-strong, 4-very strong) were recorded; the average smell intensity of each compound was defined as aroma intensity.

[0207] OAV calculation method:

[0208] OAV is calculated using the following formula:

[0209]

[0210] In the formula: C i volatile compound concentration; OT i , volatile compound threshold.

[0211] The threshold values ​​for volatile compounds can be found in literature and books. For example:

[0212] Book: Odour Thresholds (by L.J. van Gemert, second edition);

[0213] Paper: Fenaille, et al.“Comparison of mass spectrometry-basedelectronic nose and solid phase microextraction gas chromatography-massspectrometry technique to assess infant formula oxidation.”Journal ofagricultural and food chemistry vol.51,9(2003):2790-6.doi:10.1021 / jf026131w;

[0214] Tian,Huaixiang et al.“Flavoromics approach to identifying the keyaroma compounds in traditional Chinese milk fan.”Journal of dairy sciencevol.102,11(2019):9639-9650.doi:10.3168 / jds.2019-16796;

[0215] Johanna Spitzer,Andrea Buettner,Monitoring aroma changes during humanmilk storage at-19℃ by quantification experiments,Food ResearchInternational,Volume 51,Issue 1,2013,Pages 250-256,ISSN 0963-9969,doi:10.1016 / j.foodres.2012.12.002.;

[0216] Muelbert,Mariana et al.“Olfactory Cues in Infant Feeds:VolatileProfiles of Different Milks Fed to Preterm Infants.”Frontiers in nutritionvol.7 603090.15Jan.2021,doi:10.3389 / fnut.2020.603090;

[0217] Clarke, Holly J et al. “The Influence of Pasture and Non-pasture-Based Feeding Systems on the Aroma of Raw Bovine Milk.” Frontiers in nutrition vol. 9841454. 10 Mar. 2022, doi: 10.3389 / fnut.2022.841454;

[0218] Jia, H.xin, Chen, W.L., Qi, X.Y., & Su, M.Y. (2019). The stability of milk-based infant formulas during accelerated storage. CyTA-Journal of Food, 17(1), 96–104. doi: 10.1080 / 19476337.2018.1561519;

[0219] Terry Braggins, Paul Jamieson, Maree Luckman, Elizabeth Nickless, Gavin Scott, Derek Yang, Paul Andrewes, Variability between farms of New Zealand raw bovine milk flavour: Identification and characterisation of odorous outliers, International Dairy Journal, Volume 111, 2020, 104835, ISSN 0958-6946, doi: 10.1016 / j.idairyj.2020.104835. The above books and papers are incorporated herein by reference.

[0220] Aroma-active substances with odor in volatile compounds were identified using GC-O and OAV values. Simultaneously, GC-O and OAV values ​​were used to screen important compounds (i.e., aroma-active substances) contributing to the overall aroma of infant formula milk powder. Specifically, in this embodiment, volatile compounds with an aroma activity value (OAV) greater than or equal to 1 or an aroma intensity greater than or equal to 1 were identified as aroma-active substances. Specific experimental results are shown in Tables 2-1 to 2-4. Some substances can only be detected by GC-O analysis, while others can only be calculated using OAV values. Therefore, this invention combines two methods to identify aroma-active substances, resulting in a more comprehensive list of substances.

[0221] Table 2-1 Aroma Active Substances in Infant Formula (Aldehydes 1)

[0222]

[0223] Table 2-2 Aroma Active Substances (Aldehydes 2) in Infant Formula

[0224]

[0225] Table 2-3 Flavor Active Substances (Ketones) in Infant Formula

[0226]

[0227] Table 2-4 Aroma Active Substances in Infant Formula (Alcohols, Acids, Esters, Others)

[0228] (4) Evaluation of typical off-odor sensory characteristics using quantitative descriptive analysis (QDA):

[0229] The typical odor sensory characteristics of the sample to be tested were evaluated. Specifically:

[0230] Sample preparation: The sample was prepared by mixing with 45℃ warm water according to the instructions. Then, 10mL of the reconstituted milk powder was placed in a 20mL food-grade PET bottle for later use. The sample was marked with a three-digit random code.

[0231] Evaluation personnel selection and training: Evaluation personnel are selected according to ISO 3972:1991, and then trained and assessed according to ISO 4212:2003, finally forming an evaluation team of 10 evaluation personnel (3 men and 7 women).

[0232] Sensory evaluation: Samples were evaluated using a 9-point scale with a minimum scoring interval of 1, where 1-2 indicates very weak, 3-4 indicates very weak, 5-6 indicates moderate intensity, 7-8 indicates strong, and 9 indicates very strong. Sensory evaluation was conducted using a randomized block design in accordance with ISO 8589,2007. Evaluators were required to take sufficient breaks while evaluating different samples to reduce sensory fatigue.

[0233] Odor sensory descriptions, definitions, and references: Fishy odor, similar to the smell of live fish, 20mL purified water + 0.4g DHA + 0.2g ARA (n=7); Oxidized odor, similar to the smell of rancid oil, 30mL purified water + 0.2μL 2,4-nonadialdehyde + 0.7μL 3-methylbutyraldehyde (n=6). Evaluation scores for different odor sensory characteristics (fishy odor / oxidized odor) of the test samples were obtained.

[0234] (5) Correlation analysis:

[0235] A correlation analysis was performed on the content of aroma compounds obtained in step (3) and the odor sensory evaluation scores obtained in step (4). Odor compounds were screened based on the correlation coefficient (odor compounds and related sensory attributes showed a positive correlation). The details are as follows:

[0236] Spearman correlation analysis was used to perform a rank correlation analysis on aroma-active compounds and off-odor sensory characteristics to identify key off-odor substances. The correlation coefficient (ρ) was calculated using the following formula:

[0237]

[0238] In the formula: d i The rank difference represents the difference in ranking between two variables to be analyzed; n represents the total number of features.

[0239] Among them, the two variables to be analyzed are:

[0240] Variable 1: Content of aroma active compounds, the content of aroma active substances obtained in step (3), which is determined by GC-MS in step (2).

[0241] Variable 2: Odor sensory evaluation score, obtained in step (4).

[0242] The total number of features refers to the total number of values ​​for each variable. For example, if 8 samples are used in the example, then each variable will have 8 values, and the total number of features is 8.

[0243] Results analysis: 0-1 indicates a positive correlation between the two variables, with larger values ​​indicating a stronger correlation; -1-0 indicates a negative correlation between the two variables, with smaller values ​​indicating a stronger correlation; 0 indicates no correlation between the two variables.

[0244] Example of a specific calculation process:

[0245] Benzaldehyde is an aroma-active compound, and fishy odor is a characteristic odor. The correlation coefficient between benzaldehyde and fishy odor is calculated as follows:

[0246] First, the GC-MS quantification results of benzoic acid in the eight samples were sorted, and the fishy smell scores of the eight samples were sorted.

[0247] Then, find the ranking of sample 1's benzaldehyde content among the 8 samples, and the ranking of sample 1's fishy smell among the 8 samples. Subtracting these two rankings gives the d of sample 1. i ;

[0248] Similarly, calculate d for samples 2-8 respectively. i ;

[0249] Then these 8 d i Substituting the values ​​into the formula yields the correlation coefficient between benzoic acid and fishy odor.

[0250] As shown above, the correlation coefficient between each aroma active substance and odor sensory characteristic is calculated, and substances with a correlation coefficient > 0 are selected as odor substances that cause odor.

[0251] See the experimental results. Figure 3 Table 3 below lists the key odor substances screened through correlation analysis. Specifically, correlation analysis was performed on the screened aroma-active substances and odor sensory evaluation results to identify the main compounds that determine the odor of milk powder. The correlation analysis results are shown in [Table 3]. Figure 3 The graph shows the correlation between two corresponding indicators. Red dots indicate a negative correlation between the related attributes, while blue dots indicate a positive correlation. The darker the color of the dot, the higher the correlation.

[0252] Further screening revealed compounds positively correlated with both fishy and oxidized odors, which were identified as the primary compounds determining the off-odor of milk powder (primary off-odor compounds). Correlation analysis was conducted, and significance tests were performed on the corresponding compounds (significance analysis involves testing the correlation coefficients obtained from the analysis; this can be done through an online analysis website: https: / / www.bioinformatics.com.cn / plot_basic_corrplot_corrlation_plot_082). Key compounds causing off-odors in milk powder were identified and marked with "*" in Table 3. "**" indicates a highly significant positive correlation between the compound and the relevant sensory attribute (P > 0.01); "*" indicates a significant positive correlation between the compound and the relevant sensory attribute (P > 0.05). In summary, seven key compounds contribute to the fishy odor: (E)-2-octen-1-ol, nonanal, (E,E)-2,4-nonadienal, cis-4,5-epoxytrans-2-decenal, heptanal, (E)-2-octenal, and 2-n-pentylfuran, with (E)-2-octen-1-ol playing a crucial role. Five key compounds contribute to the oxidative odor: (E)-2-octen-1-ol, cis-4,5-epoxytrans-2-decenal, nonanal, (E,E)-2,4-nonadienal, and heptanal, again with (E)-2-octen-1-ol playing a significant role. Therefore, monitoring these substances can help monitor the appearance of off-odors in milk powder during storage and predict odor intensity.

[0253] Table 3. Main Odor Compounds in Infant Formula Milk Powder

[0254]

[0255]

[0256] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0257] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for evaluating volatile off-odor components in dairy products, wherein, The evaluation method includes the following steps: The enrichment step of volatile compounds involves using stir bar adsorption extraction technology to enrich the volatile compounds in the sample to be tested and then collecting the enriched sample. The analytical steps for volatile compounds involve using gas chromatography-mass spectrometry to perform qualitative and quantitative analysis on the volatile compounds in the enriched sample, thereby identifying the volatile compounds in the sample to be tested. The analysis steps for aroma active substances employ aroma activity values ​​and gas chromatography-olfactometry-mass spectrometry to screen the aroma active substances in the volatile compounds identified in the analysis steps for the volatile compounds. The sensory evaluation process involves using quantitative descriptive analysis to evaluate the typical odor sensory characteristics of the sample to be tested, and obtaining an odor sensory characteristic evaluation score. The correlation analysis step involves performing a correlation analysis between the aroma active substances obtained in the aroma active substance analysis step and the typical off-odor sensory characteristics obtained in the sensory evaluation step to identify key off-odor substances.

2. The evaluation method according to claim 1, wherein, In the adsorption extraction using the stirring rod, a stirring rod is used, and extraction is performed at a speed of 300–700 rpm for 30–90 min. Optionally, the stirring rod is aged at 200–300°C for 10–50 min before extraction.

3. The evaluation method according to claim 1 or 2, wherein, In the analysis step of the aroma active substances, the aroma activity value (OAV) is calculated using the following formula: In the formula: C i volatile compound concentration; OT i volatile compound threshold; and / or, The aroma intensity of volatile compounds was determined by gas chromatography-olfactometry-mass spectrometry.

4. The evaluation method according to claim 3, wherein, In the analysis step of the aroma active substances, volatile compounds with an aroma activity value (OAV) greater than or equal to 1 or an aroma intensity greater than or equal to 1 are identified as aroma active substances.

5. The evaluation method according to any one of claims 1-4, wherein, The typical odor sensory characteristics include: fishy smell and oxidized smell.

6. The evaluation method according to any one of claims 1-5, wherein, In the correlation analysis step, Spearman correlation analysis is used.

7. The evaluation method according to claim 6, wherein, The formula for calculating the correlation coefficient in the Spearman correlation analysis is as follows: In the formula: d i The rank difference represents the difference in ranking between two variables to be analyzed; n is the total number of features. The variables to be analyzed include the content of aroma active compounds and the evaluation score of odor sensory characteristics; the total number of characteristics is the number of samples to be tested.

8. The evaluation method according to any one of claims 1-7, wherein, Prior to the analysis of the volatile compounds, the method further includes a step of desorption and enrichment of the volatile compounds enriched using stir bar adsorption extraction technology, wherein the desorption and enrichment are performed using a thermal desorption system and a cold sample introduction system-programmed temperature evaporator.

9. The evaluation method according to any one of claims 1-8, wherein, In the analytical steps of the volatile compounds, the quantitative analysis is performed using the external standard method, the internal standard method, or a combination of both methods.

10. The evaluation method according to any one of claims 1-9, wherein, In the analytical steps for the volatile compounds, the qualitative analysis includes at least one of retention index (RI), NIST 14.0 database search (MS), standard compound comparison (STD), and olfaction (O).

11. The evaluation method according to any one of claims 1-10, wherein, The sample to be tested is milk powder, preferably infant formula.

12. The evaluation method according to any one of claims 1-11, wherein, The key odor-causing substances include: Key odor substances that cause fishy smell include (E)-2-octen-1-ol, nonanal, (E,E)-2,4-nonadienal, cis-4,5-epoxytrans-2-decenal, heptanal, (E)-2-octenal, and 2-n-pentylfuran. Key off-odor substances that cause oxidative odor include (E)-2-octen-1-ol, cis-4,5-epoxytrans-2-decenal, nonanal, (E,E)-2,4-nonadienal, and heptanal.

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