A kit and method for rapidly detecting the content of fatty acids in malanin

Abstract

This invention discloses a rapid detection kit and method for fatty acid content in garlic berries. By optimizing the sample pretreatment process and employing liquid nitrogen grinding combined with freeze-drying, homogenization and stabilization of samples can be completed in a short time, effectively preventing fatty acid oxidation or degradation and reducing the interference of moisture on subsequent reactions. The pre-prepared standards and reaction systems (such as internal standard solution, saponification reagent, and methyl esterification reagent) in the kit standardize the operating procedures, significantly reducing human error and ensuring consistency of test results across different batches. Furthermore, the integrated saponification and methyl esterification reaction conditions are precisely controlled, greatly shortening the total detection time while ensuring complete reaction, making it suitable for rapid screening of high-throughput samples.

Description

Technical Field

[0001] This invention belongs to the field of garlic fruit detection technology, specifically a reagent kit and method for rapidly detecting the fatty acid content in garlic fruit. Background Technology

[0002] Garlic kernels are rich in fatty acids, and their compositional analysis is of great significance in the food and pharmaceutical fields. Fatty acid detection in garlic kernels is an important method for analyzing the composition and content of fatty acids in garlic kernels, primarily using gas chromatography-mass spectrometry (GC-MS). This method can accurately identify saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids in garlic kernels, such as oleic acid, linoleic acid, and palmitic acid, providing a scientific basis for assessing the nutritional value of garlic kernels and developing their potential applications. During the detection process, the sample undergoes pretreatment and methylation before being introduced into the GC-MS system for analysis. By comparing the retention times and mass spectra of standards, qualitative and quantitative analysis of fatty acid composition is achieved, thereby revealing the compositional characteristics of fatty acids in garlic kernels.

[0003] However, the existing technology lacks an integrated detection tool for garlic kernels, resulting in time-consuming operation and poor repeatability. Summary of the Invention

[0004] The purpose of this invention is to provide a rapid reagent kit and method for detecting the fatty acid content in garlic cloves in order to solve the problems mentioned above.

[0005] The technical solution adopted in this invention is as follows: a kit and method for rapid detection of fatty acid content in garlic fruit, the kit comprising: a sealed bottle pre-packaged with heptadecanoic acid methanol solution (24-30 mg / mL), a sealed bottle with 7.5% KOH methanol solution, a sealed bottle with 14% BF3 methanol solution, a sealed bottle with 0.9% NaCl solution, a sealed bottle with n-hexane, and graduated centrifuge tubes.

[0006] In a preferred embodiment, the centrifuge tube is made of double-layered high-temperature resistant glass, and the tube wall is marked with "70℃ water bath zone" and "room temperature operation zone".

[0007] In a preferred embodiment, the amount of heptadecanoic acid methanol solution used is 40-60 μL / 0.05 g sample.

[0008] In a preferred embodiment, a kit and method for rapidly detecting the fatty acid content in garlic cloves include the following steps: S1. Take garlic kernels, remove the green outer skin and the hard shell in the middle, grind the kernels with liquid nitrogen, freeze-dry them, seal and store them for later use; S2. Prepare heptadecanic acid with a concentration of 24-30 mg / ml using methanol as a solvent; S3. Take approximately 0.05 g of the garlic fruit sample obtained in S1 and 40-60 μL of heptadecanic acid from S2 into a 15 ml centrifuge tube, add 3-5 mL of 7.5% (W / V) KOH (solvent is CH3OH) and seal. S4. Mix the centrifuge tube sealed in S3, place it in a 70℃ water bath for 4-6 hours (mix it every 30 minutes during the water bath), then remove it and let it cool naturally. S5. Take the cooled centrifuge tube from S4, add 2 mL of concentrated HCl acid, add 2 mL of 14% (V / V) BF3 (boron trifluoride) (solvent CH3OH), seal, place in a 70℃ water bath, and incubate for 1.5~2 hours. Then remove and allow to cool naturally. S6. Take the centrifuge tube that has cooled naturally in S5, add 1 mL of 0.9% (W / V) NaCl solution, add 2 mL of n-hexane, mix well for 1-2 minutes, centrifuge (4000 rpm, 3 min) and take the supernatant. S7. Repeat step S6; S8. Combine the supernatants from S6 and S7; S9 takes the extract from S8 and performs gas phase analysis.

[0009] In a preferred embodiment, during step S1, the garlic kernels are pretreated by removing the green outer skin and the hard inner shell, then rapidly ground into powder using liquid nitrogen to ensure uniform particle size and increase the contact area for subsequent reactions. The ground sample is immediately transferred to a freeze dryer and dried at -50°C to -80°C for 24 to 48 hours until all moisture is removed. The dried sample should be sealed in a light-proof and moisture-proof container and stored at -20°C to prevent fatty acid oxidation or degradation and ensure the accuracy of the test results.

[0010] In a preferred embodiment, in step S3, 0.05 g of garlic fruit sample is mixed with 40 to 60 μL of heptadecanic acid internal standard solution with a concentration of 24 to 30 mg / mL in a 15 mL centrifuge tube, followed by the addition of 3 to 5 mL of a methanol solution containing 7.5% potassium hydroxide. It is essential to ensure that the potassium hydroxide is completely dissolved in the methanol to create a homogeneous alkaline environment that promotes fatty acid saponification. The centrifuge tube must be tightly sealed to prevent methanol evaporation, and gentle shaking should be used to ensure sufficient contact between the sample and the reagent, providing stable reaction conditions for subsequent high-temperature hydrolysis.

[0011] In a preferred embodiment, in step S4, the sealed centrifuge tubes are placed in a 70°C constant-temperature water bath and heated continuously for 4 to 6 hours, during which time they are manually shaken and mixed every 30 minutes to ensure uniform heating of the reaction system and accelerate the saponification process. The water bath temperature must be maintained within a fluctuation range of no more than ±1°C using a precision temperature control device to avoid excessively high temperatures that could lead to fatty acid decomposition or excessively low temperatures that could affect reaction efficiency. After the reaction is complete, the solution must be allowed to cool naturally to room temperature to prevent sudden cooling from causing solution stratification or component loss.

[0012] In a preferred embodiment, in step S5, 2 ml of concentrated hydrochloric acid and 2 ml of a methanol solution containing 14% boron trifluoride are added sequentially to the saponified cooling solution, slowly dripping and gently shaking to avoid violent exothermic reactions. The centrifuge tube after mixing needs to be resealed and heated in a 70°C water bath for 1.5 to 2 hours to promote the conversion of fatty acids into more volatile methyl ester derivatives. This step requires strict control of the reaction time to ensure complete methyl esterification, while avoiding prolonged high temperatures that could lead to side reactions or degradation of the target product.

[0013] In a preferred embodiment, in step S6, 1 mL of 0.9% sodium chloride solution and 2 mL of n-hexane are added to the reaction solution, and the mixture is manually shaken for 1 to 2 minutes to ensure thorough mixing of the two phases. Then, it is centrifuged at 4000 rpm for 3 minutes. After centrifugation, the supernatant should be quickly transferred to a clean container to avoid interface disturbance leading to impurity contamination. The extraction is repeated once to improve the recovery rate of fatty acid methyl esters. The combined supernatants from both extractions should be stored away from light to minimize the impact of photolysis or volatilization on the detection results.

[0014] In a preferred embodiment, in step S9, the extract is analyzed using an Agilent 8860 gas chromatograph system with an HP-5 capillary column. The initial column temperature is set at 50°C, increased to 280°C at a rate of 10°C per minute, and held for 5 minutes. High-purity nitrogen is used as the carrier gas, with a flow rate of 1.0 mL per minute. The injection port and flame ionization detector temperatures are 250°C and 300°C, respectively. The injection volume is controlled at 1 μL, and the split ratio is 10:1 to ensure sharp peaks and resolution meeting quantitative requirements. Finally, the fatty acid content is accurately determined by comparing retention time and peak area.

[0015] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are: 1. In this invention, by optimizing the sample pretreatment process and employing liquid nitrogen grinding combined with freeze-drying technology, homogenization and stabilization of samples can be completed in a short time, effectively avoiding fatty acid oxidation or degradation, and reducing the interference of moisture on subsequent reactions. The pre-prepared standards and reaction systems (such as internal standard solutions, saponification reagents, and methyl esterification reagents) in the kit standardize the operating procedures, significantly reducing human error and ensuring the consistency of test results across different batches. Furthermore, the integrated saponification and methyl esterification reaction conditions are precisely controlled, greatly shortening the total detection time while ensuring complete reaction, making it suitable for rapid screening of high-throughput samples.

[0016] 2. This invention utilizes a dual liquid-liquid extraction technique to improve the recovery rate of target analytes. Combined with the high sensitivity and specificity of a gas chromatography system, it enables simultaneous qualitative and quantitative analysis of multiple fatty acids in complex samples. The modular design of the kit simplifies the experimental process, eliminating the need for complex equipment or specialized operational experience, making it particularly suitable for routine laboratory testing or the practical needs of small and medium-sized research institutions. The overall solution balances detection speed and result accuracy, providing an efficient and low-cost solution for the development of garlic fruit resources, quality monitoring, and research on functional components. Attached Figure Description

[0017] Figure 1 This is the gas chromatography chromatogram of the methyl alkyl ester standard of the present invention; Figure 2 These are the gas chromatography spectra of 37 fatty acid methyl ester standards in this invention; Figure 3 This is the gas chromatography spectrum of the garlic fruit sample in this invention; Figure 4 This is the gas chromatography spectrum of the garlic fruit sample in this invention; Figure 5 This is the overall flowchart of the present invention. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Example

[0019] Reference Figure 1-4 , A rapid reagent kit and method for detecting fatty acid content in garlic fruit. The reagent kit includes: a sealed bottle containing pre-packaged heptadecanoic acid methanol solution (24-30 mg / mL), a sealed bottle containing 7.5% KOH methanol solution, a sealed bottle containing 14% BF3 methanol solution, a sealed bottle containing 0.9% NaCl solution, a sealed bottle containing n-hexane, and graduated centrifuge tubes.

[0020] The centrifuge tubes are made of double-layered high-temperature resistant glass, and the tube walls are marked with "70℃ water bath zone" and "normal temperature operation zone".

[0021] The amount of heptadecanoic acid methanol solution used is 40-60 μL / 0.05 g sample.

[0022] A method for rapidly detecting the fatty acid content in garlic cloves includes the following steps: S1. Take garlic kernels, remove the green outer skin and the hard shell in the middle, grind the kernels with liquid nitrogen, freeze-dry them, seal and store them for later use; S2. Prepare heptadecanic acid with a concentration of 24-30 mg / ml using methanol as a solvent; S3. Take approximately 0.05 g of the garlic fruit sample obtained in S1 and 40-60 μL of heptadecanic acid from S2 into a 15 ml centrifuge tube, add 3-5 mL of 7.5% (W / V) KOH (solvent is CH3OH) and seal. S4. Mix the centrifuge tube sealed in S3, place it in a 70℃ water bath for 4-6 hours (mix it every 30 minutes during the water bath), then remove it and let it cool naturally. S5. Take the cooled centrifuge tube from S4, add 2 mL of concentrated HCl acid, add 2 mL of 14% (V / V) BF3 (boron trifluoride) (solvent CH3OH), seal, place in a 70℃ water bath, and incubate for 1.5~2 hours. Then remove and allow to cool naturally. S6. Take the centrifuge tube that has cooled naturally in S5, add 1 mL of 0.9% (W / V) NaCl solution, add 2 mL of n-hexane, mix well for 1-2 minutes, centrifuge (4000 rpm, 3 min) and take the supernatant. S7. Repeat step S6; S8. Combine the supernatants from S6 and S7; S9 takes the extract from S8 and performs gas phase analysis.

[0023] In step S1, during sample pretreatment, the green outer skin and inner hard shell of the garlic cloves need to be removed. Then, the cloves are rapidly ground into powder using liquid nitrogen to ensure uniform particle size and increase the contact area for subsequent reactions. The ground sample must be immediately transferred to a freeze dryer and dried at -50°C to -80°C for 24 to 48 hours until all moisture is removed. The dried sample should be sealed in a light-proof and moisture-proof container and stored at -20°C to prevent fatty acid oxidation or degradation and ensure the accuracy of the test results.

[0024] In step S3, 0.05 g of garlic fruit sample is mixed with 40 to 60 μL of heptadecanoic acid internal standard solution at a concentration of 24 to 30 mg / mL in a 15 mL centrifuge tube. Then, 3 to 5 mL of a methanol solution containing 7.5% potassium hydroxide is added. It is essential to ensure that the potassium hydroxide is completely dissolved in the methanol to create a homogeneous alkaline environment that promotes fatty acid saponification. The centrifuge tube must be tightly sealed to prevent methanol evaporation, and gentle shaking should be used to ensure sufficient contact between the sample and reagents, providing stable reaction conditions for subsequent high-temperature hydrolysis.

[0025] In step S4, the sealed centrifuge tubes need to be placed in a 70°C constant temperature water bath and heated continuously for 4 to 6 hours. During this period, the mixture should be manually shaken every 30 minutes to ensure that the reaction system is heated evenly and to accelerate the saponification process. The water bath temperature needs to be maintained within ±1°C using a precision temperature control device to avoid excessively high temperatures that could lead to fatty acid decomposition or excessively low temperatures that could affect reaction efficiency. After the reaction is complete, the mixture needs to be allowed to cool naturally to room temperature to prevent sudden cooling that could cause solution stratification or component loss.

[0026] In step S5, 2 ml of concentrated hydrochloric acid and 2 ml of methanol solution containing 14% boron trifluoride are added sequentially to the cooled saponified solution, dropwise while gently shaking to avoid violent exothermic reactions. The centrifuge tube after mixing needs to be resealed and heated in a 70°C water bath for 1.5 to 2 hours to promote the conversion of fatty acids into more volatile methyl ester derivatives. This step requires strict control of the reaction time to ensure complete methyl esterification while avoiding prolonged high temperatures that could lead to side reactions or degradation of the target product.

[0027] In step S6, add 1 mL of 0.9% sodium chloride solution and 2 mL of n-hexane to the reaction solution, and manually shake for 1 to 2 minutes to ensure thorough mixing of the two phases. Then centrifuge at 4000 rpm for 3 minutes. After centrifugation, quickly transfer the supernatant to a clean container to avoid interfacial disturbance that could introduce impurities. Repeat the extraction once to improve the recovery rate of fatty acid methyl esters. Combine the two supernatants and store them in the dark to minimize the impact of photolysis or volatilization on the detection results.

[0028] In step S9, the extract was analyzed using an Agilent 8860 gas chromatograph system with an HP-5 capillary column. The initial column temperature was set at 50°C, increased to 280°C at a rate of 10°C per minute, and held for 5 minutes. High-purity nitrogen was used as the carrier gas at a flow rate of 1.0 mL per minute. The injection port and flame ionization detector temperatures were 250°C and 300°C, respectively. The injection volume was controlled at 1 μL, with a split ratio of 10:1 to ensure sharp peaks and resolution meeting quantitative requirements. Finally, the fatty acid content was accurately determined by comparing retention time and peak area.

[0029] From the above, we can conclude that: In this invention, by optimizing the sample pretreatment process and employing liquid nitrogen grinding combined with freeze-drying technology, homogenization and stabilization of samples can be completed in a short time, effectively avoiding fatty acid oxidation or degradation, and reducing the interference of moisture on subsequent reactions. The pre-prepared standards and reaction systems (such as internal standard solutions, saponification reagents, and methyl esterification reagents) in the kit standardize the operating procedures, significantly reducing human error and ensuring consistency of test results across different batches. Furthermore, the integrated saponification and methyl esterification reaction conditions are precisely controlled, greatly shortening the total detection time while ensuring complete reaction, making it suitable for rapid screening of high-throughput samples.

[0030] This invention utilizes a dual liquid-liquid extraction technique to improve the recovery rate of target analytes. Combined with the high sensitivity and specificity of a gas chromatography system, it enables simultaneous qualitative and quantitative analysis of multiple fatty acids in complex samples. The modular design of the kit simplifies the experimental procedure, eliminating the need for complex equipment or specialized operational experience, making it particularly suitable for routine laboratory testing or the practical needs of small and medium-sized research institutions. The overall solution balances detection speed and result accuracy, providing an efficient and low-cost solution for the development of garlic fruit resources, quality monitoring, and research on functional components.

[0031] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the term "comprising" or any other variations thereof is intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes the element.

[0032] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A rapid reagent kit and method for detecting fatty acid content in garlic cloves, characterized in that: The kit includes: a sealed bottle containing pre-packaged heptadecanoic acid methanol solution, a sealed bottle containing 7.5% KOH methanol solution, a sealed bottle containing 14% BF3 methanol solution, a sealed bottle containing 0.9% NaCl solution, a sealed bottle containing n-hexane, and graduated centrifuge tubes.

2. The kit and method for rapid detection of fatty acid content in garlic cloves as described in claim 1, characterized in that: The centrifuge tubes are made of double-layered high-temperature resistant glass, with the tube walls marked with "70℃ water bath zone" and "normal temperature operation zone".

3. The kit and method for rapid detection of fatty acid content in garlic fruit as described in claim 1, characterized in that: The amount of heptadecanoic acid methanol solution used is 40-60 μL / 0.05 g sample.

4. The kit and method for rapid detection of fatty acid content in garlic fruit as described in claim 1, characterized in that: The method includes the following steps: S1. Take garlic kernels, remove the green outer skin and the hard shell in the middle, grind the kernels with liquid nitrogen, freeze-dry them, seal and store them for later use; S2. Prepare heptadecanic acid with a concentration of 24-30 mg / ml using methanol as a solvent; S3. Take approximately 0.05 g of the garlic fruit sample obtained in S1 and 40-60 μL of heptadecanic acid from S2 into a 15 ml centrifuge tube, add 3-5 mL of 7.5% (W / V) KOH (solvent is CH3OH) and seal. S4. Mix the centrifuge tube sealed in S3, place it in a 70℃ water bath for 4-6 hours (mix it every 30 minutes during the water bath), then remove it and let it cool naturally. S5. Take the cooled centrifuge tube from S4, add 2 mL of concentrated HCl acid, add 2 mL of 14% (V / V) BF3 (boron trifluoride) (solvent CH3OH), seal, place in a 70℃ water bath, and incubate for 1.5~2 hours. Then remove and allow to cool naturally. S6. Take the centrifuge tube that has cooled naturally in S5, add 1 mL of 0.9% (W / V) NaCl solution, add 2 mL of n-hexane, mix well for 1-2 minutes, centrifuge (4000 rpm, 3 min) and take the supernatant. S7. Repeat step S6; S8. Combine the supernatants from S6 and S7; S9 takes the extract from S8 and performs gas phase analysis.

5. The kit and method for rapid detection of fatty acid content in garlic fruit as described in claim 1, characterized in that: In step S1, during the sample pretreatment process, the green outer skin and the hard inner shell of the garlic kernels need to be removed, and then rapidly ground into powder using liquid nitrogen to ensure uniform particle size and increase the contact area for subsequent reactions. The ground sample should be immediately transferred to a freeze dryer and dried at -50°C to -80°C for 24 to 48 hours until all moisture is removed. The dried sample should be sealed in a light-proof and moisture-proof container and stored at -20°C to prevent fatty acid oxidation or degradation and ensure the accuracy of the test results.

6. The kit and method for rapid detection of fatty acid content in garlic fruit as described in claim 1, characterized in that: In step S3, 0.05 g of garlic fruit sample is mixed with 40 to 60 μL of heptadecanic acid internal standard solution with a concentration of 24 to 30 mg / mL in a 15 mL centrifuge tube, followed by the addition of 3 to 5 mL of methanol solution containing 7.5% potassium hydroxide. It is necessary to ensure that the potassium hydroxide is completely dissolved in the methanol to form a uniform alkaline environment to promote fatty acid saponification. The centrifuge tube should be tightly sealed to prevent methanol evaporation, and the sample and reagent should be fully contacted by gentle shaking to provide stable reaction conditions for subsequent high-temperature hydrolysis.

7. The kit and method for rapid detection of fatty acid content in garlic fruit as described in claim 1, characterized in that: In step S4, the sealed centrifuge tubes need to be placed in a 70°C constant temperature water bath and heated continuously for 4 to 6 hours. During this period, the mixture is manually shaken and stirred every 30 minutes to ensure that the reaction system is heated evenly and to accelerate the saponification process. The water bath temperature needs to be maintained by a precision temperature control device to ensure that the fluctuation range does not exceed ±1°C, so as to avoid the fatty acid decomposition caused by excessively high temperature or the reaction efficiency being affected by excessively low temperature.

8. The kit and method for rapid detection of fatty acid content in garlic fruit as described in claim 1, characterized in that: In step S5, 2 ml of concentrated hydrochloric acid and 2 ml of methanol solution containing 14% boron trifluoride are added sequentially to the saponified cooling solution. The addition is done slowly with gentle shaking to avoid violent heat release. The centrifuge tube after mixing needs to be sealed again and heated in a 70°C water bath for 1.5 to 2 hours to promote the conversion of fatty acids into more volatile methyl ester derivatives.

9. The kit and method for rapid detection of fatty acid content in garlic fruit as described in claim 1, characterized in that: In step S6, 1 mL of 0.9% sodium chloride solution and 2 mL of n-hexane are added to the reaction solution. The mixture is manually shaken for 1 to 2 minutes to ensure thorough mixing of the two phases. Then, it is centrifuged at 4000 rpm for 3 minutes. After centrifugation, the supernatant should be quickly transferred to a clean container to avoid interface disturbance that could lead to the introduction of impurities. The extraction is repeated once to improve the recovery rate of fatty acid methyl esters. The supernatants from both extractions are combined and stored away from light to reduce the impact of photolysis or volatilization on the detection results.

10. The kit and method for rapid detection of fatty acid content in garlic fruit as described in claim 1, characterized in that: In step S9, the extract is analyzed using an Agilent 8860 gas chromatograph system with an HP-5 capillary column. The initial column temperature is set to 50°C, and then increased to 280°C at a rate of 10°C per minute and held for 5 minutes. The carrier gas is high-purity nitrogen, and the flow rate is set to 1.0 mL per minute. The temperatures of the injection port and the flame ionization detector are 250°C and 300°C, respectively. The injection volume is controlled at 1 μL, and the split ratio is 10:1.

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