CN111850067A - A kind of degradation process of clover pectin - Google Patents

Abstract

The invention discloses a process for degrading cloverleaf pectin, belonging to the field of biotechnology, comprising the following steps: (1) hydrolysis treatment, (2) ventilation treatment, (3) ultrasonic treatment, (4) centrifugal treatment, ( 5) Enzymatic hydrolysis treatment. The overall process of the method of the invention is simple, and by popularization and application, the oligogalacturonic acid can be prepared efficiently and in a large amount, which lays a foundation for the subsequent separation of the pure oligogalacturonic acid and the identification of its activity and its structure.

Description

A kind of degradation process of clover pectin

technical field

The invention belongs to the field of biotechnology, and in particular relates to a degradation process of clover pectin.

Background technique

Akebia trifoliata (Scientific name: Akebia trifoliata (Thunb.) Koidz.) is Akebia trifoliata (Thunb.) Koidz., which belongs to the deciduous woody vine. Stem bark gray-brown, palmately compound leaves alternate or clustered on short branches; petiole straight, leaf blade papery or thinly leathery, ovate to broadly ovate, apex usually obtuse or slightly concave, base truncated or rounded , margin with wavy teeth or lobes, dark green above, light green below; racemes drawn from clustered leaves on short branches, pedicels slender, male flowers: pedicels filiform, stalks lavender, broadly elliptic or elliptic , filaments extremely short, drug chambers incurved at flowering; degenerate carpels oblong-conical. Female flower: pedicel slightly thicker than male flower, stigma capitate, papillae, orange-yellow. Fruit oblong, straight or slightly curved, seeds very numerous, flat ovate, testa reddish brown or dark brown, slightly shiny. Blooms in April-May and bears fruit in July-August.

The peel of Sanyemutong is rich in pectin. Pectin is a kind of acidic heteropolysaccharide with complex structure, mainly composed of galacturonan (HGA), rhamnogalacturonan-I (RG- I) and rhamnogalacturonan-II (RG-II) three structural regions, wherein HGA is a smooth region, RG-I and RG-II are whisker-like regions. Each pectin varies considerably with the plant source, tissue and developmental stage, the number, type and connection mode of residues in its side chain and the presence of other substituents, reflecting the structure of pectin polysaccharides. of high complexity.

At present, most studies on the structure and activity of pectin-oligosaccharide mixtures are obtained by degrading pectin polysaccharides. The main components of pectin-oligosaccharide mixtures are oligogalacturonic acid, which is a kind of acidic Sugar, composed of 2-20 monogalacturonic acid molecules connected by a-1,4 glycosidic bonds, oligogalacturonic acid has a variety of biological activities, can be used as an inducing factor of plant defense response, inducing plant resistance It can be used as a proliferation factor of bifidobacteria, which can efficiently and exclusively promote the growth of bifidobacteria and improve the structure of the flora; in addition, it has remarkable effects in controlling cotton verticillium wilt, apple mosaic disease, pepper virus disease, etc.

The existing degradation methods of pectin polysaccharides mainly include chemical degradation and enzymatic degradation. Chemical degradation is usually acid hydrolyzed pectin, the biggest feature is that the degradation rate is fast (especially under heating conditions), the degradation is relatively complete, and a large amount of monosaccharide is obtained, and the yield of oligosaccharide is low. In addition, due to the introduction of various reaction reagents in the degradation process, the control of the degradation reaction process is more difficult, and the separation and purification of the degradation products is also difficult to carry out. Therefore, the method of simple acid hydrolysis is rarely used to prepare oligogalacturonic acid. Enzymatic degradation of pectin polysaccharides, that is, using a specific one or several enzymes to degrade pectin molecules, allowing them to selectively cut off the a-1, 4 glycosidic bonds in the pectin molecules, so as to obtain specific pectin Oligosaccharides. The method for degrading the pectin polysaccharide does not cause side reactions, the reaction conditions are mild, the process is easy to control, and the molecular structure of pectin is hardly damaged, so it is an ideal degradation method. However, because the structure of pectin is very complex, the molecular weight is large, and the microenvironment in the molecule affects the contact between pectinase and the action site, the use of pectinase to directly degrade pectin has certain limitations, the degradation efficiency is low, and the yield of oligosaccharides is low. It is difficult to select suitable enzymes to efficiently prepare pectic oligosaccharides.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a degradation process of clover pectin.

The above-mentioned technical purpose of the present invention is achieved through the following technical solutions:

A degradation process of clover pectin pectin, comprising the following steps:

(1) Hydrolysis treatment:

The peel pectin is put into the acid solution for acid hydrolysis treatment, the hydrolysis time is controlled to be 5-6h, and the acid hydrolysis solution is obtained after completion;

(2) Ventilation treatment:

The compound gas is introduced into the acid hydrolyzate obtained in step (1), and the duration of the controlled introduction is 6～8h;

(3) Ultrasonic treatment:

The probe of the ultrasonic instrument is immersed in the acid hydrolyzate after the treatment in step (2), and then ultrasonic treatment is performed for 50 to 55 minutes for standby;

(4) Centrifugal treatment:

The acid hydrolyzate treated in step (3) is centrifuged, and the centrifugal precipitate is obtained after filtering the filtrate for subsequent use;

(5) Enzymatic hydrolysis treatment:

First, the centrifugal precipitate obtained in step (4) is mixed with sodium acetate buffer, and then pectinase is added for enzymolysis treatment, followed by saponification, neutralization and demineralization, and finally freeze-drying to obtain pectin oligosaccharides. sugar mixture.

Further, the acid solution described in the step (1) is a phosphoric acid solution with a concentration of 1 to 1.5 mol/L, and the corresponding weight ratio of the peel pectin to the acid solution is 1:95 to 100; During the acid hydrolysis treatment, the temperature of the acid solution is always maintained at 70-75°C.

Further, the composite gas described in step (2) is formed by mixing oxygen and argon according to a volume ratio of 2-3:1.

Further, in the ultrasonic treatment described in step (3), the frequency of ultrasonic waves is controlled to be 300-340 kHz, the pulse time is controlled to be 2-3 s, the intermittent time is controlled to be 2 s, and the intensity is 260-280 W/cm ² .

Further, during the centrifugation described in step (4), the rotational speed of the centrifugation is controlled to be 2200-2400 rpm.

Further, the pH value of the sodium acetate buffer described in step (5) is 4-5.

Further, the corresponding weight ratio when the centrifugal precipitate described in step (5) is blended with the sodium acetate buffer is 1:80-85.

Further, the conditions during the saponification treatment described in step (5) are controlled as follows: 1-1.2 mol/L sodium hydroxide solution saponification treatment for 30-40 min.

Further, during the saponification treatment, the temperature of the control solution is 30-33°C.

Further, during the freeze-drying treatment described in step (5), the temperature of the controlled freezing is -25～-30°C.

Compared with the prior art, the present invention has the following advantages:

The invention provides a degradation process of pectin, which effectively realizes efficient and precise degradation of pectin through reasonable matching of treatment operations, wherein the acid hydrolysis treatment is performed first, which can degrade most of the neutrality in the pectin polysaccharide. sugar, followed by aeration treatment, and the saturated absorption of the solution was carried out by using the unique compound gas, which changed the bubble content and thermal conductivity of the solution, which could significantly enhance the cavitation and heating effects during ultrasonic treatment, and then accelerated the pectin Finally, enzymatic hydrolysis treatment was carried out to further ensure the purity of the degradation and the yield of the product. The overall process of the method of the invention is simple, and by popularization and application, the oligogalacturonic acid can be prepared efficiently and in a large amount, which lays a foundation for the subsequent separation of the pure oligogalacturonic acid and the identification of its activity and its structure.

Detailed ways

Example 1

A degradation process of clover pectin pectin, comprising the following steps:

(1) Hydrolysis treatment:

The peel pectin is put into the acid solution for acid hydrolysis, and the hydrolysis time is controlled to be 5h, and the acid hydrolyzed solution is obtained after completion; the acid solution is a phosphoric acid solution with a concentration of 1 mol/L, and the peel pectin is put into the solution. When the acid solution is used, the corresponding weight ratio of the two is 1:95; during the acid hydrolysis treatment, the temperature of the acid solution is always kept at 70°C;

(2) Ventilation treatment:

In the acid hydrolyzate of step (1) gained, pass into composite gas, and control the duration of feeding to be 6h; Described composite gas is to be mixed by oxygen and argon correspondingly according to volume ratio 2:1;

(3) Ultrasonic treatment:

The probe of the ultrasonic instrument is immersed in the acid hydrolyzate after the step (2) treatment, then ultrasonic treatment is carried out, and standby after 50min; during the described ultrasonic treatment, the frequency of the ultrasonic wave is controlled to be 300 kHz, the pulse time is controlled to be 2s, and the intermittent time is controlled. is 2s and the intensity is 260W/cm ² ;

(4) Centrifugal treatment:

The acid hydrolyzate treated in step (3) is centrifuged, and the centrifugal sediment is obtained after filtering the filtrate for subsequent use; during the described centrifugation, the rotating speed of the centrifugation is controlled to be 2200rpm;

(5) Enzymatic hydrolysis treatment:

First, the centrifugal precipitate obtained in step (4) is mixed with sodium acetate buffer, and then pectinase is added for enzymolysis treatment, followed by saponification, neutralization and demineralization, and finally freeze-drying to obtain pectin oligosaccharides. Sugar mixture; the pH value of the sodium acetate buffer is 4; the corresponding weight ratio when the centrifugal precipitate is blended with the sodium acetate buffer is 1:80; the condition control during the saponification treatment is: 1mol /L sodium hydroxide solution for saponification treatment for 30min; control the temperature of the solution to be 30°C during the saponification treatment; control the freezing temperature to be -25°C during the freeze-drying treatment.

Example 2

A degradation process of clover pectin pectin, comprising the following steps:

(1) Hydrolysis treatment:

The peel pectin is put into the acid solution for acid hydrolysis treatment, the hydrolysis time is controlled to be 5.5h, and the acid hydrolyzed solution is obtained after completion; the acid solution is a phosphoric acid solution with a concentration of 1.3mol/L, and the peel pectin is When put into the acid solution, the corresponding weight ratio of the two is 1:98; during the acid hydrolysis treatment, the temperature of the acid solution is always kept at 74°C;

(2) Ventilation treatment:

In the acid hydrolyzed solution of step (1) gained, pass into compound gas, and control the duration of passing in to be 7h; Described compound gas is to be mixed by oxygen and argon correspondingly according to volume ratio 2.6:1;

(3) Ultrasonic treatment:

The probe of the ultrasonic instrument is immersed in the acid hydrolyzate after the step (2) treatment, and then ultrasonically processed, for subsequent use after 52min; the frequency of the ultrasonic wave is controlled to be 330kHz, the pulse time is controlled to be 2s, and the intermittent time is controlled during the described ultrasonication. is 2s and the intensity is 270W/cm ² ;

(4) Centrifugal treatment:

The acid hydrolyzate after the step (3) treatment is centrifuged, and the centrifugal sediment is obtained after filtering the filtrate; during the described centrifugation, the rotating speed of the centrifugation is controlled to be 2300rpm;

(5) Enzymatic hydrolysis treatment:

First, the centrifugal precipitate obtained in step (4) is mixed with sodium acetate buffer, and then pectinase is added for enzymolysis treatment, followed by saponification, neutralization and demineralization, and finally freeze-drying to obtain pectin oligosaccharides. Sugar mixture; the pH value of the sodium acetate buffer solution is 4.5; the corresponding weight ratio when the centrifugal precipitate is blended with the sodium acetate buffer solution is 1:82; the condition control during the saponification treatment is: 1.1 The mol/L sodium hydroxide solution was saponified for 35 minutes; the temperature of the solution was controlled to be 32°C during the saponification treatment; the temperature of the freeze-dried solution was controlled to be -28°C.

Example 3

A degradation process of clover pectin pectin, comprising the following steps:

(1) Hydrolysis treatment:

The peel pectin is put into the acid solution for acid hydrolysis treatment, the hydrolysis time is controlled to 6h, and the acid hydrolyzed solution is obtained after completion; the acid solution is a phosphoric acid solution with a concentration of 1.5mol/L, and the peel pectin is put into When the acid solution is reached, the corresponding weight ratio of the two is 1:100; during the acid hydrolysis treatment, the temperature of the acid solution is always kept at 75°C;

(2) Ventilation treatment:

In the acid hydrolyzate of step (1) gained, pass into compound gas, and control the time length of passing in to be 8h; Described compound gas is to be mixed according to volume ratio 3:1 correspondingly by oxygen and argon;

(3) Ultrasonic treatment:

The probe of the ultrasonic instrument is immersed in the acid hydrolyzate after the step (2) treatment, then ultrasonic treatment is carried out, and standby after 55min; during the described ultrasonic treatment, the frequency of the control ultrasonic wave is 340kHz, the pulse time is controlled to be 3s, and the intermittent time is controlled. is 2s and the intensity is 280W/cm ² ;

(4) Centrifugal treatment:

The acid hydrolyzate after the step (3) treatment is centrifuged, and the centrifugal sediment is obtained after filtering the filtrate; during the described centrifugation, the rotating speed of the centrifugal control is 2400rpm;

(5) Enzymatic hydrolysis treatment:

First, the centrifugal precipitate obtained in step (4) is mixed with sodium acetate buffer, and then pectinase is added for enzymolysis treatment, followed by saponification, neutralization and demineralization, and finally freeze-drying to obtain pectin oligosaccharides. Sugar mixture; the pH value of the sodium acetate buffer is 5; the corresponding weight ratio when the centrifugal precipitate is blended with the sodium acetate buffer is 1:85; the condition control during the saponification treatment is: 1.2 The mol/L sodium hydroxide solution was saponified for 40 min; the temperature of the solution was controlled to be 33° C. during the saponification treatment; the temperature of the freeze-dried treatment was controlled to be -30° C.

Comparative Example 1

Compared with Example 3, the only difference between the present Comparative Example 1 is that the ventilation treatment operation in step (2) is omitted, and the other method steps are the same.

Comparative Example 2

Compared with Example 3, this Comparative Example 2 only differs in that step (3) ultrasonic treatment operation is omitted, and other method steps are the same.

Comparative Example 3

Compared with Example 3, the only difference between this comparative example 3 is that step (2) aeration treatment operation and step (3) ultrasonic treatment operation are omitted, and other method steps are the same.

In order to compare the effect of the present invention, the peel pectin is used as the raw material, and then the degradation treatment is carried out by the method corresponding to the above-mentioned Example 3 and Comparative Examples 1 to 3, and then the product is subjected to the determination of the components (molar percentage content), and the specific comparison data are as follows. Table 1 shows:

Table 1

As can be seen from the above Table 1, the degradation material prepared by the method of the present invention contains high-purity GalA, which is an effective method for extracting and preparing oligogalacturonic acid and corresponding subsequent research, and has great popularization and application value.

Claims (10)

1. a degradation process of cloverleaf pectin, is characterized in that, comprises the steps: (1) Hydrolysis treatment: The peel pectin is put into the acid solution for acid hydrolysis treatment, the hydrolysis time is controlled to be 5-6h, and the acid hydrolysis solution is obtained after completion; (2) Ventilation treatment: The compound gas is introduced into the acid hydrolyzate obtained in step (1), and the duration of the controlled introduction is 6～8h; (3) Ultrasonic treatment: The probe of the ultrasonic instrument is immersed in the acid hydrolyzate after the treatment in step (2), and then ultrasonic treatment is performed for 50 to 55 minutes for standby; (4) Centrifugal treatment: The acid hydrolyzate treated in step (3) is centrifuged, and the centrifugal precipitate is obtained after filtering the filtrate for subsequent use; (5) Enzymatic hydrolysis treatment: First, the centrifugal precipitate obtained in step (4) is mixed with sodium acetate buffer, and then pectinase is added for enzymolysis treatment, followed by saponification, neutralization and demineralization, and finally freeze-drying to obtain pectin oligosaccharides. sugar mixture. 2. the degradation process of a kind of clover pericarp pectin according to claim 1, is characterized in that, the acid solution described in step (1) is the phosphoric acid solution that concentration is 1～1.5mol/L, described When the peel pectin is put into the acid solution, the corresponding weight ratio of the two is 1:95-100; the temperature of the acid solution is always maintained at 70-75° C. during the acid hydrolysis treatment. 3. the degradation process of a kind of clover pericarp pectin according to claim 1, it is characterised in that the composite gas described in the step (2) is corresponding by oxygen and argon according to volume ratio 2～3: 1 is mixed. 4. the degradation process of a kind of clover pericarp pectin according to claim 1, is characterized in that, the frequency of controlling ultrasonic wave is 300～340kHz, the pulse time is controlled to be 2 during the ultrasonic treatment described in the step (3) ~3s, the intermittent time was controlled to 2s, and the intensity was 260 to 280W/cm ² . 5. the degradation process of a kind of cloverleaf pericarp pectin according to claim 1, is characterized in that, during the centrifugation described in step (4), the rotating speed of controlled centrifugation is 2200～2400rpm. 6. The degradation process of a kind of cloverleaf pectin according to claim 1, is characterized in that, the pH value of the sodium acetate buffer described in step (5) is 4～5. 7. the degradation technique of a kind of cloverleaf pectin according to claim 1, is characterized in that, the corresponding weight ratio when the centrifugal sediment described in step (5) and sodium acetate buffer solution is blended is 1 : 80 to 85. 8. the degradation process of a kind of clover pericarp pectin according to claim 1, is characterized in that, the condition control during saponification treatment described in the step (5) is: the sodium hydroxide of 1～1.2mol/L The solution is saponified for 30 to 40 minutes. 9 . The degradation process of Trifolium pratense pectin according to claim 8 , wherein the temperature of the control solution is 30-33° C. during the saponification treatment. 10 . 10 . The degradation process of Trifolium pratense pericarp pectin according to claim 1 , wherein the temperature for freezing is controlled to be -25～-30° C. during the freeze-drying treatment described in the step (5). 11 .