JP2013210217A - Method for evaluating quality of cocoa bean - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for easily and accurately evaluating the quality of cocoa beans.SOLUTION: The quality evaluation method of cocoa beans includes the steps of: obtaining an analytic sample by performing the preprocessing of cacao beans; obtaining an analytic result by subjecting the analytic sample to instrumental analysis (combined with liquid chromatography or mass analysis); performing multivariable analysis (especially, principal component analysis) by converting the analytic result into numerical data; and evaluating quality from the obtained analytic result. Thus, it is possible to achieve the evaluation of the total quality of cocoa beams by a simple method which used to be difficult in a conventional manner. That is, it is possible to quickly and beneficially evaluate the quality of cocoa beams by screening cocoa beams by instrumental analysis such as chromatography.

Description

The present invention relates to a method for evaluating the quality of cacao beans. More specifically, the present invention relates to a quality evaluation method by instrumental analysis of metabolites or degradation products in cocoa beans.

Conventionally, cacao has been used as a main raw material for chocolate and cocoa.
Cocoa is a tropical plant grown mainly in hot and humid areas near the equator, and the seeds (cocoa beans and cocoa beans) in the fruit of the cocoa tree are used as raw materials for cocoa mass used in chocolate and cocoa. It is used.

Cacao for making chocolate is made from dried and fermented cocoa tree seeds. Open the harvested cocoa pod, remove the pulp and cocoa beans, and discard the shell. The pulp and beans are then piled up, boxed or spread into a lattice, usually for 6-7 days. The fermented pulp is liquefied and spilled, the remaining cocoa beans are collected, dried, and further processed to make cocoa butter and cocoa powder. In some cases, the product is treated with alkali to reduce the acidity of the powder. Fermentation is important for the quality and flavor of beans that originally have a strong bitter taste. Unfermented or poorly fermented cocoa beans have a flavor similar to raw potatoes and are very sensitive to the growth of white mold and fungi, so they are used to produce chocolate for consumption as food. It is not done.

Currently, the final quality of cocoa beans is determined by sensory inspection based on the appearance, aroma, and color of beans by experts. It takes years of experience to acquire these skills. In addition, there is a drawback that objectivity and reproducibility are low. Therefore, a simple mechanized quality evaluation method is necessary for manufacturing process management and product quality management.

Chemical analysis appears to be the most reliable method for assessing the quality of cocoa beans. The combination of instrumental analysis methods and powerful computer-driven pattern recognition technology offers new possibilities for quality control and characterization of complex materials. Therefore, an object of the present invention is to provide a method for simply evaluating the quality of cacao beans.

The flavor and taste of cocoa beans are attributed to volatile components, sugars, and flavonoids. Its astringent taste is due to catechin (tannin) or the like, and its bitter taste is due to theobromine or the like. Due to cultivar differences, environmental effects, processing methods, etc., the apparent steady state amount of intermediate pathway metabolites and / or terminal accumulation of final metabolites also varies. In addition, degradation products from fermentation also affect the quality of cocoa beans. Therefore, monitoring metabolites and degradation products in cocoa beans both spatially and temporally is important for quality assessment.

In chromatography, fingerprinting based on pattern recognition methods is the process monitoring and control (raw material grading, routine online quality check, or product manufacturing process) in various analytical areas such as food and nutrition. ), And geographic origin (measurement of the source by the chemical composition of the ingredients, tracking of the origin of the final product by flavor and aroma components). (Patent Documents 1 to 3)

Japanese Patent Publication No. 2009-14700 Japanese Patent Publication No. 2009-244015 Japanese Patent Publication No. 2011-80812

Thus, the present invention provides a limited number of individual groups of specific groups that are considered important for specific functions, such as antioxidant compounds for medical purposes, and catechins and other phenolic compounds for taste and aroma. Instead of characterizing these compounds, the focus is on metabolites and degradation products, combined with chemical fingerprinting and multivariate analysis of these to complete a comprehensive and rapid analysis of cocoa beans. It was.

That is, the present invention provides a method for evaluating the quality of cocoa beans,
Pre-treating cocoa beans to obtain an analytical sample;
Subjecting the analysis sample to instrumental analysis to obtain an analysis result;
Converting the analysis result into numerical data and performing multivariate analysis; and evaluating the quality from the obtained analysis result.

In a preferred embodiment, the pretreatment comprises extracting the material in the cocoa beans with an extraction solution, and the instrumental analysis comprises liquid chromatography, high performance liquid chromatography, or ultra high performance liquid chromatography and mass. It is a combination with analysis. In a more preferred embodiment, the analysis result is quality evaluated based on the holding time.

In another preferred embodiment, the pretreatment includes a step of extracting a hydrophilic compound from the cocoa beans to obtain an extract.

In a more preferred embodiment, data of a compound having a composition formula of C15H14O6, C30H28O12, C30H26O12, C45H38O18, C12H22O11, C13H13NO7 is analyzed in the analysis results.

In another preferred embodiment, the instrumental analysis is a combination of liquid chromatography and mass spectrometry, a combination of high performance liquid chromatography and mass spectrometry, or a combination of ultra high performance liquid chromatography and mass spectrometry.

In another preferred embodiment, the pretreatment includes a step of pulverizing the cocoa beans to obtain a powder.

In another preferred embodiment, the analysis result is an analysis result by a combination of high performance liquid chromatography and mass spectrometry, or a combination of ultra high performance liquid chromatography and mass spectrometry.

According to the present invention, it is possible to evaluate the quality of cacao beans, which has been difficult in the past, with a more accurate and simple method.

In the present invention, the cocoa beans to be subjected to quality evaluation prediction may be before fermentation and after fermentation, drying and roasting, but preferably cocoa beans after fermentation and drying.

In the present invention, cocoa beans are appropriately pretreated according to the instrumental analysis described below, and then subjected to various instrumental analyzes.

In the present invention, instrumental analysis refers to analysis / measurement means using an analytical instrument, such as liquid chromatography (LC) (for example, high performance liquid chromatography (HPLC), ultrahigh performance liquid chromatography (UPLC)), mass spectrometry ( MS). These instrumental analyzes may be combined, and examples include LC / MS, HPLC / MS, UPLC / MS, and the like. The apparatus used for these instrumental analysis is not specifically limited, If the metabolite (for example, an amino acid, an organic acid, sugar) contained in cocoa beans can be measured, the apparatus normally used is used. Can be used. Measurement conditions can be appropriately set by those skilled in the art so as to be appropriate for the measurement of these substances.

The pretreatment is performed according to the instrumental analysis as described above in order to make the substance to be analyzed in the cocoa beans suitable for the instrumental analysis. Examples of the pretreatment include treatments such as drying, cutting, pulverization, and extraction. For example, the pulverization can be performed using an appropriate instrument such as a blender or a ball mill. The extraction can be performed using water, an organic solvent, or a mixture of these solvents. Examples of the organic solvent that can be used for extraction include methanol, ethanol, n-propanol, isopropanol, acetone, chloroform, and the like. As an extraction operation, cocoa beans may be heated in water and / or an organic solvent. The heating temperature is appropriately determined according to the solvent used. The heating time is also set appropriately. These unit operations are singly or combined to set appropriate preprocessing conditions.

For LC, in particular HPLC, as instrumental analysis, preferably the pretreatment comprises a step of extracting the cocoa beans with water and / or a hydrophilic solvent to obtain an extract. Examples of the column to be used include an affinity column, a reverse phase column, and an ion exchange column. HPLC conditions (for example, flow rate, detector, mobile phase, etc.) are appropriately selected according to the sample.

In the case of performing ultra high performance liquid chromatography (UPLC) as the instrumental analysis, the pretreatment preferably includes a step of extracting the cocoa beans with water and / or a hydrophilic solvent to obtain an extract. UPLC passes a high linear velocity mobile phase through small column particles to achieve fast analysis speed, high resolution, and high sensitivity. Column particles used in UPLC are column particles having a smaller diameter (eg, 1.7 μm) than column particles (eg, 5 μm or 3.5 μm) used in HPLC, such as affinity columns, reverse phase columns, and An ion exchange column may be mentioned. The UPLC conditions (for example, flow rate, detector, mobile phase, etc.) are appropriately selected depending on the sample.

The cocoa bean sample subjected to the above pretreatment is subjected to arbitrary instrumental analysis to obtain an analysis result. The resulting analytical result can be a fingerprint of the cocoa bean sample. Multi-variate analysis is performed by converting this fingerprint into numerical data. Results obtained by analysis include spectral data such as retention time and signal intensity (or ionic strength).

As multivariate analysis, various analysis tools are employed for analysis of instrumental analysis data. For example, PCA (principal component analysis), HCA (hierarchical cluster analysis), PLS regression analysis (projection to latent structure: projection to latent structure), discriminant analysis (discriminant analysis, etc.) Multivariate tools. Many of these analysis tools are commercially available as software, and arbitrary ones are available. Such a commercially available tool is generally provided with an operation manual so that multivariate analysis can be performed without knowledge of difficult mathematics and statistics.

For example, when a metabolite is extracted from cocoa beans and analyzed by LC / MS, the obtained analysis results include retention time, mass spectrum, ionic strength, and the like of various metabolites. Using the present invention, six kinds of cocoa beans having different flavor and taste were analyzed. As a result, it has been clarified that compounds having a composition formula of C15H14O6, C30H28O12, C30H26O12, C45H38O18, C12H22O11, and C13H13NO7 play an important role in quality evaluation of cocoa beans.

In order to use the six index components of C15H14O6, C30H28O12, C30H26O12, C45H38O18, C12H22O11, and C13H13NO7 that have been found this time for the evaluation of cocoa beans, for example, the target cocoa beans are analyzed by LC or LC / MS. The quality of the cocoa beans can be evaluated by detecting the peak of the component and comparing it with a predetermined reference value.

In addition, the accuracy of the evaluation model obtained by the above method can be increased by accumulating data. Therefore, for example, if the tendency of the relative amount of metabolites in cocoa beans becomes clearer, it is possible to obtain a fingerprint from the analysis result of cocoa beans of unknown quality and to evaluate the quality based on this fingerprint Become.

(Example 1: Preparation of analytical sample for LC / MS)
The cacao beans were used as samples after fermentation and fermentation. Six kinds of cocoa beans having different taste and aroma when made into a chocolate product were used as samples.

First, 2 mL of methanol was added to dry cocoa beans (200 mg) in a 15 mL Falcon tube, sonicated in a warm bath at 45 ° C. for 15 minutes, and centrifuged at 3,000 × g for 10 minutes. 1 mL of the supernatant was then transferred to an 8 mL glass tube. 1 mL of methanol was added to the residue, sonicated again in a 45 ° C. warm bath for 15 minutes, and centrifuged at 3,000 × g for 10 minutes. 1 mL of the supernatant was transferred to the glass tube, and the collected supernatant was dried with nitrogen, and 200 ml of methanol was added and redissolved. After adding 1.8 mL of water, the sample was purified using a 0.45 mm filter to obtain an analytical sample.

(Example 2: Analysis of cocoa bean sample by LC / MS)
5 μL of the analytical sample obtained in Example 1 was injected into the LC / MS. The LC / MS apparatus used in this example was a MicroCROFTO TOF mass spectrometer (trade name, manufactured by BRUKER Daltonics) and 100 mm × 2.1 mm i. d. Ultimate 3000 (trade name, manufactured by Dionex) equipped with an AQUITY UPLC HSS T-3 column having a particle diameter of 1.8 mm. In the mobile phase, water and acetonitrile were mixed at 95: 5 as a high polarity solvent, and 0.05% formic acid was added as an additive, and acetonitrile and water were mixed at 95: 5 and added as a low polarity solvent. A solution containing 0.05% formic acid was used as an agent. The column oven temperature was 40 ° C. The solvent flow rate through the column was 0.3 mL / min. The proportion of low polarity solvent was kept at 2% for 3 minutes, then increased to 30% in 17 minutes, then increased to 100% in the next 20 minutes and finally kept at 100% for 25 minutes. MS used an electrospray ionization (ESI) source in negative mode.

FIG. 1 shows a total ion chromatogram of the cocoa bean (2) among the cocoa bean samples. All samples showed similar chromatogram patterns. However, as shown in FIG. 2, a difference in peak intensity was observed due to differences in cocoa beans.

(Example 3: Analysis of data by LC / MS and fingerprinting of cocoa bean metabolites).
Multivariate analysis was then performed to reveal important compounds and information from this complex data. Principal component analysis (PCA) (Profile Analysis 2.0 (trade name of software manufactured by Bruker Daltonics)) was selected in order to grasp the relationship expressed by similarity or dissimilarity among groups of multivariate data. Six types of cocoa bean samples with different aroma and taste could be separated on the score plot (FIG. 3). A loading plot corresponding to the score plot is shown in FIG. Differences in quality indicated differences in metabolic profiling of cocoa bean samples.

Six compounds of C15H14O6, C30H28O12, C30H26O12, C45H38O18, C12H22O11, C13H13NO7 were deduced by comparing the prominent compounds with their isotopic patterns in the mass spectrum and the theoretical spectrum. For example, in cocoa beans (2), the amount of compounds that are presumed to be flavonoids and compounds that are presumed to be flavonoid polymers is relatively large. The amount of the compound presumed to be a flavonoid polymer was relatively small. This indicated that metabolomics analysis could be useful for quality assessment of cocoa beans.

In order to use the above 6 index components for the evaluation of cocoa beans, for example, the target cocoa beans are analyzed by LC or LC / MS, and the peak of 6 index components is detected and compared with a predetermined reference value. By doing so, the quality of the cocoa beans can be evaluated.

(Example 4: Quality evaluation of unknown sample using 6 index components by LC / MS).
The 6 index components obtained in Example 3 in the samples of unknown quality (sample) and cocoa beans (1) to (6) of known quality are analyzed, and the peak area areas of the obtained chromatograms are compared. did. In FIG. 5, the graph of the peak area area of 6 index components in each cocoa bean is shown. As a result of the analysis, the cacao bean (6) was the closest to the sample of unknown quality (sample) in the content pattern of the 6 index components. From the above, it was predicted that the unknown quality sample analyzed this time was cocoa beans with similar quality to cocoa beans (6).

According to the present invention, it is possible to evaluate the overall quality of cacao beans, which has been difficult in the past, by a simple method. That is, cocoa beans can be quickly and beneficially evaluated for quality by screening by instrumental analysis such as chromatography.

Traditional instrumental analysis of cocoa beans focuses on a specific group of compounds that may be important for a specific function, such as antioxidant compounds for medical purposes, and catechins and other phenolic compounds for flavor and aroma. Has been applied. However, the characterization of cocoa beans can come from a combination rather than from a single metabolite. Therefore, if the method of this invention by analyzing a several component at once is used, more accurate quality evaluation is possible and the further improvement of a chocolate manufacturing process or a cacao bean distribution storage process can be anticipated.

It is a total ion chromatogram of LC of cacao bean (2). It is a chromatogram which shows the ionic strength of LC. Figure 2 is a PCA score plot by LC / MS analysis of all cocoa bean samples. It is a figure which shows the PCA analysis result of LC / MS analysis of a cocoa bean sample, and is a loading plot of PC1 and PC2. It is an analysis result of 6 index components in a quality unknown sample (sample) and a quality known sample (cocoa beans (1)-(6)).

Claims (9)

A method for evaluating the quality of cocoa beans,
Pre-treating cocoa beans to obtain an analytical sample;
Subjecting the analysis sample to instrumental analysis to obtain an analysis result;
Converting the analysis result into numerical data and performing multivariate analysis; and evaluating the quality from the obtained analysis result;
Including a method. The method according to claim 1, wherein the multivariate analysis is a principal component analysis. The method according to claim 1 or 2, wherein the instrumental analysis is liquid chromatography, high performance liquid chromatography, or a combination of ultra high performance liquid chromatography and mass spectrometry. The method according to claim 3, wherein the analysis result is quality evaluated based on a holding time. The method according to claim 1 or 2, wherein the pretreatment includes a step of extracting a hydrophilic compound from the cocoa beans to obtain an extract. 6. The method of claim 5, wherein the instrumental analysis is liquid chromatography, high performance liquid chromatography, or a combination of ultra high performance liquid chromatography and mass spectrometry. The method according to claim 6, wherein data of C15H14O6 C30H28O12 C30H26O12 C45H38O18 C12H22O11 C13H13NO7 is analyzed as a quality index among the analysis results. Pre-treating a plurality of cocoa beans of known quality to obtain individual analytical samples;
Subjecting the individual analysis samples to instrumental analysis to obtain individual analysis results; and converting the relationship between the individual analysis results and the quality into numerical data for multivariate analysis;
Is a quality evaluation model for cocoa beans. A method for evaluating the quality of cocoa beans,
Pre-treating cocoa beans to obtain an analytical sample;
Subjecting the analysis sample to instrumental analysis to obtain an analysis result;
A step of converting the analysis result into numerical data and performing multivariate analysis; and a step of collating the obtained analysis result with the quality evaluation model according to claim 12;
Including a method.

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