CN102898485A - Method for preparing functional alpha-galactooligosaccharides and chickpea sugar alcohol from chickpeas - Google Patents

Abstract

The invention relates to a method for preparing functional α-galactooligosaccharides and chickpea sugar alcohols by using chickpeas as raw materials. The method comprises the following steps: selecting chickpea seeds, pulverizing them with a pulverizer, sieving, adding ethanol water solution according to a certain material-liquid ratio for extraction, centrifuging the extract, collecting supernatant, and repeating the above steps three times to obtain the extract. A medium-pressure chromatographic column was used for elution with a series of linear concentration gradients of aqueous ethanol. The sugar content of the eluate was measured, and according to the measurement results, the sugar-containing components were collected for HPLC analysis to obtain chickpea α-galacto-oligosaccharides with different purities. Chickpea α-galacto-oligosaccharides were passed through Biogel P-2 gel filtration chromatography column to obtain chickpea sugar alcohol. The results of in vitro anaerobic feces mixed culture experiment showed that chickpea α-galacto-oligosaccharides and chickpea sugar alcohol have good intestinal beneficial function, so they have broad application prospects.

Description

Method for preparing functional α-galactooligosaccharides and chickpea sugar alcohols from chickpeas

technical field

The invention relates to a method for preparing functional α-galactooligosaccharides and chickpea sugar alcohols by using medium-pressure column chromatography and Biogel P-2 gel filtration chromatography column and using chickpeas as raw materials. the

Background technique

α-Galactooligosaccharides (α-Galactooligosaccharides, α-GOS) is a kind of low molecular weight soluble oligosaccharides containing α-galactosidic bonds, widely distributed in legumes. In the past, it has been considered that α-galacto-oligosaccharides are an anti-nutritional factor and the main cause of flatulence, so they are strongly removed during the processing of soy foods. As Japan began to study functional oligosaccharides in the 1980s, people also had a new understanding of α-galacto-oligosaccharides, and found that it can promote the proliferation of beneficial bacteria Bifidobacteria in the intestinal tract, while inhibiting the growth of spoilage bacteria. grow. Many scholars have re-researched α-galacto-oligosaccharides and found that it is not the cause of flatulence, and α-galacto-oligosaccharides have only begun to be re-researched and utilized. the

At present, α-galactooligosaccharides are mainly extracted from legumes at home and abroad. The reported extraction materials mainly include soybeans, lupines, cowpeas, peas, etc. Among them, the research on extracting α-galactooligosaccharides from soybeans is relatively extensive. Has been used in beverages, yogurt, health food and other fields. However, soybean-derived α-galacto-oligosaccharides contain high content of sucrose, which limits its application in obese people. Chickpeas are rich in carbohydrates, and the functional oligosaccharides include raffinose, chickpea sugar alcohol, stachyose and verbascose. It is reported that the content of total oligosaccharides in chickpeas is 5.89%. -10.1%, is one of the important sources of α-galacto-oligosaccharides. Compared with soybeans, the content of α-galacto-oligosaccharides in chickpea is higher, while the content of sucrose is lower, which can well solve the problem of limiting the application range of soybean oligosaccharides due to the high content of sucrose. Therefore, chickpea is an ideal raw material for the production of α-galacto-oligosaccharides. The oligosaccharides in chickpea include sucrose, raffinose, chickpea sugar, stachyose, and a small amount of verbascose, among which chickpea sugar is a characteristic trisaccharide in chickpea. the

Chickpea is currently one of the most widely cultivated edible bean species in the world. It is the second largest consumer bean in the world, and its output ranks third in the world. Because chickpeas are very resistant to drought and barrenness, they are very suitable for planting in western my country. They have been grown in Xinjiang for 2,500 years. Chickpea is rich in nutrients and is a rare ideal natural green health food with broad market development prospects. The research, development and utilization of chickpea is also of great significance to the economic development of western my country. the

Contents of the invention

The purpose of the present invention is to use chickpea as raw material, utilize medium-pressure column chromatography, Biogel P-2 gel filtration chromatography column, separate and purify to prepare alpha-galacto-oligosaccharides and pure chickpea sugar alcohols with different purities. the

Description of drawings

Fig. 1 is the HPLC graph of chickpea α-galacto-oligosaccharides

Figure 2 is the ESI-TOF-MS spectrum of chickpeal alcohol

Figure 3 is the ¹ H-NMR spectrum of chickpea sugar alcohol

Figure 4 Effects of different extraction times on the extraction rate of chickpea oligosaccharides

Figure 5 Effects of different extraction material-liquid ratios on the extraction rate of chickpea oligosaccharides

Figure 6 Effects of different extraction temperatures on the extraction rate of chickpea oligosaccharides

The influence of different concentrations of ethanol aqueous solution on the extraction rate of chickpea oligosaccharides in Fig.

Detailed ways

Below by embodiment, the present invention will be further described. the

Example 1:

(1) Extraction, separation and purification of chickpea α-galacto-oligosaccharides

Select chickpeas with full grains, crush them with a grinder, and pass through a 60-mesh sieve. Weigh an appropriate amount of chickpea powder, add 50% ethanol aqueous solution according to the ratio of material to liquid 1:10, shake and extract in a constant temperature oscillator at 50°C, then centrifuge at 5000 rpm, collect the supernatant, and extract the lower layer of soybean powder according to the above conditions , and the supernatant was collected by centrifugation. The two supernatants were combined, concentrated and freeze-dried to obtain chickpea crude extract. the

The chickpea crude extract was dissolved in an appropriate amount of deionized water, filtered through a filter membrane, and the filtrate was applied to an activated carbon-diatomaceous earth (1:1) medium-pressure chromatography column. The chromatographic column is eluted with a series of linear gradient ethanol solutions (0% to 50%), the eluate is collected, the sugar content and composition of the eluate are analyzed by phenol-sulfuric acid method and HPLC method, and suitable components are combined , and through vacuum concentration and freeze-drying, respectively prepare chickpea α-galacto-oligosaccharide samples with α-galacto-oligosaccharide content of 70%-80%, 85%-90% and more than 90%. the

(2) Determination method of α-galacto-oligosaccharide content

The content of oligosaccharides in the samples was determined by HPLC external standard method. Chromatographic conditions: chromatographic column Sugar-D, detector RID, column temperature 40°C, mobile phase acetonitrile-water volume ratio 75:25, flow rate 1.0mL/min, injection volume 20μL. Preparation of sugar standard curve: Accurately weigh standard samples of fructose, sucrose, raffinose, garbanose, stachyose, and verbascose, dissolve them in water, add an equal amount of acetonitrile, and prepare a series of standard samples with different concentration gradients. The solution was filtered through a filter membrane and analyzed by HPLC. the

(3) Preparation and structural analysis of chickpea sugar alcohol

Chickpea extract (freeze-dried extract) was dissolved in an appropriate amount of deionized water, filtered through a filter membrane, and the filtrate was passed through an activated carbon-diatomaceous earth (1:1, w/w) medium-pressure chromatography column. The chromatographic column is eluted with a series of ethanol with a linear concentration gradient (0% to 50%). After the eluate is initially detected by the phenol-sulfuric acid method, the eluate containing sugar is collected, and the elution peaks are further detected by HPLC. sugar components in the solution. The eluate with high content of unknown trisaccharides was combined and concentrated, and the concentrated solution was applied to Biogel P-2 chromatography column, and eluted with water. The eluate was detected by phenol sulfuric acid method and HPLC, and the eluate containing only unknown trisaccharides The eluates were combined, concentrated, and freeze-dried to obtain a white powder. the

The purified unknown trisaccharide sample was analyzed by ¹ H-NMR and ESI-TOF-MS. During ¹ H-NMR analysis, D ₂ O was used as the solvent and tetramethylsilane was used as the internal standard.

(4) Evaluation of intestinal beneficial function of chickpea α-galacto-oligosaccharides

Using chickpea α-galacto-oligosaccharides of different purity as carbon source, the in vitro anaerobic feces mixed culture method was adopted. After the culture, the bacteria were counted by fluorescence in situ hybridization technology. Finally, the prebiotic index (PI) was used as the evaluation index for the intestinal beneficial function of chickpea α-galacto-oligosaccharides. the

PI＝(Bif/Total)+(Lac/Total)—(Bac/Total)—(His/Total)

In the formula: Bif represents the number of Bifidobacterium spp. at different time points divided by the number of inoculation; Lac represents the number of Lactobacillus-Enterococcus spp. at different time points divided by the number of inoculation; Bac represents the number of Bacteroides-Prevotella group at different time points Divided by the number of inoculation; His represents the number of Clostridiu mhistolyticum group divided by the number of inoculation at different time points; Total represents the number of total flora at different time points divided by the number of inoculation. the

Example 2:

Extraction method and condition optimization of chickpea α-oligomeric half-emulsion

(1) Extraction method of chickpea α-oligomeric half-emulsion

① Whole grain extraction method: Soybean grains are soaked in water at low temperature without crushing, and then extracted with ethanol aqueous solution at high temperature. the

② Grinding and extraction method: After crushing and degreasing, the grains are extracted with aqueous solution, methanol or ethanol aqueous solution at high temperature. It can be seen from Table 1 that the extraction rates of the two extraction methods are different. In contrast, the contents of sucrose and α-galacto-oligosaccharides obtained by crushing extraction method were significantly higher than those obtained by whole grain extraction method. the

Table 1 Extraction rate of oligosaccharides by two extraction methods

(2) Optimization of the extraction conditions of chickpea α-oligomeric half-emulsion

Based on the pulverization extraction method, the extraction condition optimization experiment of chickpea oligosaccharides was carried out. the

①Effect of different extraction time on the extraction rate of chickpea oligosaccharides

The extraction time was 15min, 30min, 60min, 90min, and the extraction temperature, solid-liquid ratio, and extraction times were 70°C, 1:2, and 2 times, respectively. The main soluble oligosaccharides in chickpea, sucrose and Extraction rate of galacto-oligosaccharides. Repeat each level 3 times. The results showed that when the extraction time was increased to more than 30min, the extraction time had no significant effect on the extraction rate of oligosaccharides. When the extraction time is 30min, the oligosaccharides can be completely extracted. the

②Effect of different extraction material-liquid ratios on the extraction rate of chickpea oligosaccharides

The ratio of solid to liquid for extraction is 1:5, 1:8, 1:10, 1:15, 1:20, and the extraction temperature, time and extraction times are 70°C, 30min and 2 times respectively. Calculate the extraction rate of the main soluble oligosaccharides sucrose and α-galacto-oligosaccharides in chickpea by using different extraction solid-liquid ratios. Repeat each level 3 times. The results showed that when the solid-liquid ratio was 1:10, the oligosaccharides could be completely leached out. the

③Effect of different extraction temperatures on the extraction rate of chickpea oligosaccharides

The extraction temperatures were room temperature (20°C), 50°C, 70°C, and 90°C, and the extraction time, solid-liquid ratio, and times were 30 min, 1:10, and 2 times, respectively. The extraction rates of the main soluble oligosaccharides sucrose and α-galacto-oligosaccharides in chickpea were calculated at different extraction temperatures. Repeat each level 3 times. The results showed that 50℃ was an optimal extraction temperature. the

④Effect of different concentrations of ethanol aqueous solution on the extraction rate of chickpea oligosaccharides

The concentration of the ethanol aqueous solution of the extract is 0% (pure water), 30%, 50%, 70%, 90%, and the extraction time, solid-liquid ratio and times are respectively 30min, 1:10 and 2 times. Calculate the extraction rate of the main soluble oligosaccharides sucrose and α-galacto-oligosaccharides in chickpea when using different concentrations of extracts. Repeat each level 3 times. The results show that 50% ethanol solution is an ideal extraction solvent. the

Claims (2)

1. a method for preparing functional α-galactooligosaccharides and chickpea sugar alcohols with chickpeas as raw material, characterized in that it comprises the following steps: (1) Using chickpea produced in the western region of my country as a raw material, select chickpea seeds with plump grains, no insect damage, and no loss, pulverize with a grinder, and sieve to obtain chickpea powder for grinding and extraction. (2) Weigh an appropriate amount of soybean powder in step (1) into a container, add ethanol aqueous solution according to a certain amount of feed liquid, and vibrate and extract under constant temperature conditions. Centrifuge the extract, collect the supernatant, repeat the above steps twice for the bean flour in the lower layer, collect the supernatant, and combine the supernatant. (3) The extract obtained in the freeze-drying step (2) is reconstituted with an appropriate amount of water, filtered through a filter membrane, and the filtrate is passed through an activated carbon-diatomaceous earth medium-pressure chromatography column. (4) Use the phenol sulfuric acid method to measure the sugar content of the eluent obtained in step (3). According to the measurement results, collect the sugar-containing components for HPLC analysis, collect suitable components, and obtain α-galactooligosaccharides with different contents. chickpea α-galacto-oligosaccharide samples. (5) The chickpea α-galacto-oligosaccharide obtained in step (4) is passed through a Biogel P-2 gel filtration chromatography column, and the fraction containing only chickpea sugar alcohol is collected, concentrated in vacuo, and freeze-dried to obtain chickpea Pure soybean sugar alcohol. (6) The method for identifying the structure of the chickpeat alcohol obtained in step (5) is as follows: separating and purifying the obtained chickpeg sugar alcohol sample for ¹ H-NMR and ESI-TOF-MS analysis. (7) In vitro anaerobic feces mixed culture method was used to evaluate the intestinal beneficial function of chickpea α-galacto-oligosaccharides with different purity prepared in step (4). 2. In vitro anaerobic feces mixed culture experiment results show that chickpea α-galacto-oligosaccharides have a good proliferation effect on bifidobacteria and lactic acid bacteria, and have a certain inhibitory effect on the growth of Bacteroides and Clostridium ; Chickpea α-galacto-oligosaccharides only changed the composition of the bacteria in the intestine, and basically had no effect on the number of overall flora; The lactose sample has the strongest effect on the proliferation of beneficial bacteria and the strongest inhibitory effect on harmful bacteria, showing the highest PI value (2.00), and the α-galacto-oligosaccharide content is 70-80%, 85-90% The PI values of chickpea α-galacto-oligosaccharide sample and chickpea crude extract were 1.39, 1.73 and 0.89, while the control group was negative. The experimental results show that chickpea α-galacto-oligosaccharides have a good intestinal beneficial function.

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