CN102944622B - Extraction and detection method of Jerusalem artichoke fructan - Google Patents

Abstract

The invention belongs to the field of analysis and detection, and relates to a method for extracting and detecting frisee polyphenols (levan). An inventor adopts a GracePure<TM> SPE C18-Max column produced by GRACE Company to filter a sample in the extraction of frisee plant samples. The column can remove the polar matters from a water solution, nearly cannot absorb sugar, and can effectively reduce the detection background. A common HPLC (high performance liquid chromatography) instrument is adopted, but a PREVAIL Carbohydrate ES special sugar column produced by the GRACE Company is adopted and has good sugar separation function. The sugar column is an amino sugar column bonded by polystyrene and divinylbenzene, so the problem of disturbing other amino columns is avoided, the chemical degradation is avoided, and the stability is realized at room temperature and under the gradient elution condition; an ELSD (evaporative light-scattering detector) is used for the sugar detection, and one complete set of extraction and detection flow of the polyphenols (levan) of the frisee plant samples is firstly established.

Description

Extraction and detection method of Jerusalem artichoke fructan

technical field

The invention belongs to the field of analysis and detection, and relates to a method for extracting and detecting Jerusalem artichoke fructan.

Background technique

Plant fructan is a general term for a class of water-soluble carbohydrates that are mainly composed of multiple fructosyl groups connected by glycosidic bonds. Although fructans can be found in 15% of angiosperms, the total amount of fructans in plants is generally low, only in the tubers of Jerusalem artichoke (Helianthus tuberosus, also known as devil ginger, Jerusalem artichoke); chicory (Cichoriumintybus ), burdock (Arctium lappal), and chicken feet (Dactylis glomeratus); bulbs of lilies (Liliumbrownii) and onions (Allium cepa); ryegrass (Lolium perenne), barley (Hordeumvulgare), and wheat (Triticum aestivum) stems and leaves The content is relatively high in the tissues of a few species of plants (VanLaere and Van Den Ende, 2002; Ritsema and Smeekens, 2003a).

Inulin (Inulin) extracted from Jerusalem artichoke tubers is a kind of fructan, which is basically connected by fructosyl groups through β (2-1) bonds, and there is a glucose group at the end. The degree of polymerization of inulin in Jerusalem artichoke is between 3 and 50, and the degree of polymerization is the number of fructosyl groups (Van Der Meer et al., 1998). The inulin of Jerusalem artichoke is a mixture of fructans with different degrees of polymerization. Inulin can constitute up to 20% fresh weight or 90% dry weight of Jerusalem artichoke tubers (Van den Ende et al., 2004).

Fructans are a class of soluble carbohydrates that are very beneficial to human health. Since the human body does not contain enzymes that decompose fructan, humans cannot digest and absorb fructan, but enter the colon through fructan, where it becomes a nutrient for intestinal flora, selectively promoting bifidobacteria and Digestion and absorption by the growth of lactic acid bacteria. At the same time, the growth of these probiotic bacteria can reduce the production of substances related to the occurrence of human tumors. In addition, short-chain fructans, as a low-calorie sweetening ingredient, can be used as sugar or lipid substitutes in yogurt and ice cream, etc., and can also be used in special foods for diabetics (Li Xingjun, 2010) .

Because the content of fructan in Jerusalem artichoke, especially tubers, is high and has high economic value, but its components are complex, fructans with different degrees of polymerization are mixed together, and fructans with different components have different functions, so these fructans are separated and detected Sugar content is particularly important.

Because the traditional spectrophotometer or thin layer chromatography method can only detect the total water-soluble sugar of plants or a few sugars, it is not used much in the detection of various sugars in plants. The current method for detecting various sugars in plants is basically high performance liquid chromatography (HPLC). The specific methods of HPLC can be divided into two categories: one is the ordinary HPLC method, which is detected by using a differential detector (RID) or an evaporative light detector (ELSD), for example, using a RID detector to detect fructose in oligosaccharides and three sugars and four sugars (Jiang Shiqiong, 1996), detection of high degree of polymerization fructans (polymerization degree higher than 6) in Jerusalem artichoke (Sun Xuemei et al., 2011), etc., using ELSD detector to detect monosaccharides or disaccharides in tobacco (Sun Yuan, 2004 ; Cheng Yong, 2010); one is relatively special chromatography-ion chromatography (HPAEC), which mainly uses pulsed amperometric detector (PAD), etc., such as the detection of small molecule monosaccharides such as glucose in tobacco (Xu Yiran et al., 2010), detect monosaccharides and disaccharides or trisaccharides such as sucrose and melibiose in soybeans (Li Renyong et al., 2009), PAD detectors can theoretically detect picogram-level sugars.

Although the detection limit of plant sugars detected by ion chromatography is theoretically lower than that of ordinary HPLC, but because the mobile phase in the ion chromatography instrument system is strong acid and strong alkali, the price of the instrument is generally more expensive, which is basically 2 to 3 times the price of traditional HPLC. In normal use, the requirements for mobile phase water are extremely high, and the maintenance cost of daily use is also high. In addition, the versatility of the PAD detector is not strong, and the detection of sugars will cause oxidation or reduction of certain molecules on the electrode surface. Since traditional HPLC has a much larger application range and objects than ion chromatography, organic non-polar, polar and ionic compounds can be used, while ion chromatography is currently mainly used for the analysis of inorganic ions and a small amount of organic ionic compounds. Therefore, as the most widely used HPLC in the use of analytical instruments, how to use ordinary HPLC to establish an efficient method for separating and detecting plant fructans is particularly important.

In traditional HPLC, although the RID detector can also be used to detect carbohydrates, the RID detector is based on the continuous measurement of the change in the refractive index of the effluent from the chromatographic column, and is only suitable for the determination of carbohydrates with weak ultraviolet (UV) absorption ; At the same time, RID is extremely sensitive to temperature, making the baseline very unstable, incompatible with gradient elution, and the detection sensitivity is very low. Therefore, there are considerable limitations in the use of RID for the detection of carbohydrates.

Although the previous studies also used the HPLC-ELSD method to determine sugars in plants, there are certain defects, which are specifically reflected in (1): some studies use ordinary silica gel-based amino columns, which are not durable (due to moisture can cause self-hydrolysis of the amino stationary phase), and the life of the column is not long. At the same time, during sample extraction, heating and centrifugation are simply performed, and non-sugar substances interfere with the detection; (2): Previous studies have mostly analyzed and detected small molecules such as monosaccharides, disaccharides, or sugar alcohols, etc. For oligosaccharides and There are fewer studies on sugars with higher degrees of polymerization. The content of glucose, fructose, sucrose, and oligofructans in Jerusalem artichoke tissue is relatively high, and other sugars are little or almost non-existent; (3) For plants with high fructan content, such as Jerusalem artichoke, there are few detections, and for plants such as tobacco and soybean More detection.

Contents of the invention

The purpose of the present invention is to provide a kind of extraction and detection method of Jerusalem artichoke fructan aiming at the above-mentioned defect of prior art.

The purpose of the present invention can be achieved through the following technical solutions:

The extraction and detection method of Jerusalem artichoke fructan comprises the following steps:

(1) Extraction and purification of total soluble sugars from Jerusalem artichoke tissue:

a Fresh Jerusalem artichoke tissue samples were dried in an oven at a constant temperature of 70-80°C for 40-55 hours,

b After the tissue sample is ground, mix it with deionized water at a mass volume ratio of 1:2~5, put it in a water bath at 100°C for 15~25 minutes, repeat the extraction 3~5 times, and collect all the solutions extracted three times to remove protein,

c Filtrate the solution obtained in b, centrifuge the filtrate at 8000-12000 g for 20-30 minutes, collect the supernatant,

The filtrate obtained in the last step is passed through the solid phase extraction column GracePureTM SPE C18-Max,

e The filtrate is filtered through a 0.45 μm aqueous phase filter membrane, and used as a sample for the next step of HPLC;

(2) Detection of Jerusalem artichoke fructan: the sample prepared in the previous step was detected by HPLC, and the detection conditions were as follows:

Chromatographic column: Prevail ^TM Carbohydrate ES Coloumn-W250*46mm 5um;

Detector: evaporative light detector ELSD, Alltech 3300;

The injection volume is 10ul;

The flow rate is 1ml/min, and the running time of each sample is 55min,

Mobile phase: B phase: water, C phase: acetonitrile;

Elution method: gradient elution, the elution program is shown in Table 1:

Table 1

time (min) %B %C 1 0 25 75 2 15 35 65 3 30 50 50 4 40 50 50 5 42 25 75 6 55 25 75

"%" in the table means volume percentage.

The extraction and purification of the Jerusalem artichoke tissue soluble total sugar preferably include:

a Fresh Jerusalem artichoke tissue samples were dried in an oven at a constant temperature of 80°C for 48 hours,

b After the tissue sample is ground, mix it with deionized water at a mass volume ratio of 1:3, put it in a water bath at 100°C for 20 minutes, and repeat the extraction 3 times. Collect all the solutions extracted three times to remove the protein.

c Filtrate the solution obtained in b, centrifuge the filtrate at 10000 g for 20 minutes, collect the supernatant,

d The filtrate obtained in the previous step is passed through the solid phase extraction column GracePure ^TM SPE C18-Max,

e The filtrate was filtered through a 0.45 μm aqueous phase filter, and used as a sample for the next step of HPLC.

Beneficial effect:

The present invention firstly establishes a whole set of extraction and detection process of fructan from Jerusalem artichoke plant samples. In the extraction of Jerusalem artichoke plant samples, the GracePure ^TM SPE C18-Max reverse solid phase extraction column from GRACE was used for sample filtration, which is suitable for complex sample detection. Since sugar is non-polar or moderately polar in aqueous solution, the column can effectively remove polar substances in aqueous solution, has almost no adsorption on sugar, can effectively reduce the background of detection, and can concentrate samples. Utilizing the method established by the present invention, by using conventional HPLC instruments, HPAEC instruments, which are expensive and rarely used in conventional laboratories, are avoided. Through the GracePure ^TM SPE C18-Max column used in the sample extraction process, the interference of non-sugar substances on the sugar content can be effectively removed; the special sugar column of PREVAIL Carbohydrate ES is used, and the sugar column is a polystyrene-divinylbenzene bonded amino group The sugar column does not have the problems that plague other amino columns, there is no chemical degradation phenomenon, and it is also very stable at room temperature and gradient elution, and can separate various sugar compounds for a long time and efficiently. Through the established detection method, in a routine analysis laboratory, using ordinary HPLC instruments, the effect of separating and detecting various sugars from HPAEC can be achieved, and the cost of use and maintenance is low.

Description of drawings

Figure 1: Fructose standard curve (fructose concentration 0.01~6mg/ml, 5-fold serial dilution)

The abscissa is the logarithm of the respective sugar concentration with the base 10, and the ordinate is the logarithm of the peak area with the base 10.

Figure 2: Standard curve of glucose (glucose concentration range is 6.7~4×103μg/ml, 5-fold serial dilution)

The abscissa is the logarithm of the respective sugar concentration with the base 10, and the ordinate is the logarithm of the peak area with the base 10.

Figure 3: Standard curve of sucrose (concentration range 0.01~6mg/ml, 5-fold serial dilution)

The abscissa is the logarithm of the respective sugar concentration with the base 10, and the ordinate is the logarithm of the peak area with the base 10.

Figure 4: Standard curve of kestose (concentration range: 8.3~5×103μg/ml, 5-fold serial dilution)

The abscissa is the logarithm of the respective sugar concentration with the base 10, and the ordinate is the logarithm of the peak area with the base 10.

Figure 5: Standard curve of kestose (concentration range: 8.3~5×103μg/ml, 5-fold serial dilution)

The abscissa is the logarithm of the respective sugar concentration with the base 10, and the ordinate is the logarithm of the peak area with the base 10.

Figure 6: Standard curve of kestopentaose (concentration range: 8.3~5×103μg/ml, 5-fold serial dilution)

The abscissa is the logarithm of the respective sugar concentration with the base 10, and the ordinate is the logarithm of the peak area with the base 10.

Figure 7: HPLC charts of 6 sugars

Fructose concentration, 0.1mg/ml; Glucose concentration, 0.067mg/ml; Sucrose concentration, 0.1mg/ml; Kestose triose concentration, 0.083mg/ml; Kestose tetraose concentration, 0.083mg/ml; Concentration, 0.083mg/ml.

Figure 8: HPLC analysis of Jerusalem artichoke leaves

Figure 9: HPLC analysis of Jerusalem artichoke stems

Figure 10: HPLC analysis of Jerusalem artichoke tubers

Detailed ways

Example 1

sample:

Jerusalem artichoke varieties: Regular Jerusalem artichoke

Sugar: fructose (SIGMA), sucrose (SIGMA), glucose (SIGMA), kestose (Wako), kestose (Wako), sucrose pentaose (Wako) standard solution

experiment method:

1. Extraction and purification of total soluble sugars from Jerusalem artichoke tissue:

a. Fresh samples such as Jerusalem artichoke stems, leaves or tubers are baked in an oven at a constant temperature of 80°C for 48 hours;

b After the tissue sample is ground, mix it with deionized water at a ratio of 1:3 (g/ml), put it in a water bath at 100°C for 20 minutes, repeat the extraction 3 times, and collect all the solutions extracted three times to remove the protein;

c The solution obtained in the second step is filtered, the filtrate is centrifuged at 10000g for 20 minutes, and the supernatant is collected;

d The filtrate obtained in the previous step is passed through the solid phase extraction column GracePure ^TM SPE C18-Max (GRACE company);

e The filtrate was filtered through a 0.45 μm aqueous membrane filter and injected into HPLC.

2. HPLC sugar determination

Required equipment: high performance liquid chromatography (Agilent1200), sugar column (Prevail ^TM Carbohydrate ESColoumn-W250*46mm 5um), evaporative light detector (ELSD, Alltech 3300)

Required reagents: HPLC grade acetonitrile (analytical grade), HPLC grade water HPLC determination method:

The sample injection volume is 10ul.

Gradient elution method: the flow rate is 1ml/min, the running time of each sample is 55min,

Mobile phase: B phase: water, C phase: acetonitrile. The volume ratio of water and acetonitrile in the gradient elution is shown in the table below.

Table 1 Levan HPLC elution program

time (min) %B %C 1 0 25 75 2 15 35 65 3 30 50 50 4 40 50 50 5 42 25 75 6 55 25 75

"%" in the table means volume percentage.

3. Establishment of fructan detection method

3.1 Establishment of standard curve

Through serial dilution, the sugars of a single component were measured separately, and the standard curves of various sugars were established, as shown in Figures 1-6. As can be seen from the standard curves of the above-mentioned 6 kinds of sugars, using the HPLC-ELSD method established by the inventor, several main sugars in Jerusalem artichoke tissue can be measured, and the lower limit of detection is basically a microgram level. Compared with the ion chromatography method, The lower limit of detection is basically the same (Wang Jianhua et al., 2007), which can meet the needs of most experiments. Since there is no standard product for fructans with a higher degree of polymerization than fructopentose, experimenters have no way to identify them.

3.2 Separation effect of various sugars

In order to detect multiple sugars, the inventors mixed the above six sugars together and separated them on a column. The results are shown in Figure 7. From Figure 7, it can be found that through the method established by the inventor, the above six sugars can be well separated, indicating that the experimental method established by the inventor can be used for the detection of complex samples of Jerusalem artichoke.

3.3 Jerusalem artichoke tissue samples

According to the separation and analysis method established by the inventor, the inventor analyzed and measured different tissues of Jerusalem artichoke (leaves, stems, tubers 5 months after planting).

From the test results in Figure 8, it is found that in the Jerusalem artichoke leaves that have grown for 5 months, the sugar content in 1g of dry Jerusalem artichoke leaves is as follows: fructose, 7.83mg; glucose, 10.08mg; sucrose, 19.44mg; kestose : 0.9mg; Kestetose or fructan with high degree of polymerization was almost detected. This also shows that in Jerusalem artichoke leaves, small molecular sugars such as monosaccharides and disaccharides are basically the main ones.

From the detection results in Figure 9, in the Jerusalem artichoke stalks that have grown for 5 months, the sugar contents in 1g of Jerusalem artichoke stem samples are: fructose, 77.04mg; glucose, 18.18mg; sucrose, 38.25mg; Sugar, 24.21mg; Kestose, 26.55mg; Kestose, 36.09mg. In the stalks of the same period, there is gradually already a high degree of polymerization of sugars and the stalk acts as a temporary sugar storage organ.

From the test results in Figure 10, it is found that during the tuber development period, the sugar content in 1g of Jerusalem artichoke tuber dry sample: fructose, 33.03mg; glucose, 7.56mg; sucrose, 30.78mg; kestose, 20.97mg; kestetose , 32.85mg; cane pentaose, 39.06mg, and other highly polymerized sugars appeared in large quantities. It shows that in tubers, fructans with different degrees of polymerization have taken the dominant position.

Claims (1)

1. the extraction of jerusalem artichoke levulan and detection method, is characterized in that comprising following steps:

(1) jerusalem artichoke is organized the extraction and purification of total Soluble Sugar:

A jerusalem artichoke organize fresh sample in baking oven at 80 ℃ constant temperature dry 48h,

After b tissue sample grinds, mix by the mass volume ratio of 1:3 with deionized water, put in the water-bath of 100 ℃ 20 minutes into, extracting is 3 times repeatedly, collects all solution of extracting for three times with removal albumen,

C is by the solution filter of gained in b, and centrifugal 20 minutes of filtrate 10000g, collects supernatant,

The filtrate that the upper step of d obtains is crossed solid phase extraction and is carried post GracePure ^tMsPE C ₁₈-Max,

E filtrate is crossed 0.45 μ m water membrane filtration, as the sample of next step HPLC;

(2) detection of jerusalem artichoke levulan: sample prepared by previous step detects by HPLC, and testing conditions is as follows:

Chromatographic column: Prevail ^tMcarbohydrate ES Coloumn-W250 * 46mm5 μ m;

Detecting device: evaporative light detecting device ELSD, Alltech3300;

Sample size: 10 μ l;

Flow velocity: 1ml/min, be 55min the working time of each sample,

Mobile phase: B phase: water, C phase: acetonitrile;

Type of elution: gradient elution, elution program is as shown in table 1:

Table 1

? Time (min) %B %C 1 0 25 75 2 15 35 65 3 30 50 50 4 40 50 50 5 42 25 75 6 55 25 75

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