CN112159827B - Production process of agar oligosaccharide - Google Patents
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Abstract
The invention discloses a production process of agar oligosaccharide, which relates to the field of marine carbohydrate compound processing, and is characterized in that rotten asparagus is selected to screen and purify strains, the strains are subjected to seed expansion culture, the strains are inoculated into sterilized fresh asparagus to be subjected to fermentation treatment, and finally the fermented asparagus is dried in the sun, subjected to gel extraction, subjected to acid treatment, subjected to high-temperature treatment, crushed and sieved to obtain the required agar oligosaccharide. The invention has the beneficial effects that: by selecting rotten asparagus to screen and purify the strains, the production environment requirement, the production cost and the industrialization difficulty of the strains are reduced, and the stability and the yield of the product are improved by combining three methods, namely a biological method, a chemical method and a physical method in the process.
Description
Technical Field
The invention relates to the field of processing of marine carbohydrate compounds, and particularly relates to a production process of agar oligosaccharides.
Background
In recent decades, the development of new marine drugs by using marine saccharides from a wide variety of sources as raw materials and by chemical modification and degradation methods to enhance the biological activity of the marine saccharides has become an important research direction for the marine saccharides.
The oligosaccharide refers to a straight chain or branched chain low-degree polymerization saccharide substance consisting of 2-20 same or different monosaccharides, is also called oligosaccharide, and comprises two major types of common oligosaccharide and functional oligosaccharide. The common oligosaccharides are known as sucrose, maltose, lactose and the like, can be digested and absorbed by the body, have no growth promotion effect on beneficial intestinal bacteria, and the other type of oligosaccharides are functional oligosaccharides, such as fructo-oligosaccharide, xylo-oligosaccharide, soybean oligosaccharide, chitosan oligosaccharide, agar oligosaccharide, algin oligosaccharide and the like, which cannot be digested and absorbed by the body, but have various physiological activities, so that the oligosaccharide has wide market space in the fields of food, agriculture, medicine and the like.
Algal oligosaccharides have been found to have important physiological activities in recent years, and among them, agar oligosaccharides derived from red algae such as fence and gelidium amansii have been widely studied. For example, agar oligosaccharide claimed in chinese patent CN1593433A has prebiotics effect, agar oligosaccharide mixture claimed in chinese patent CN1485044A can be used for preparing medicines or food additives for lowering blood sugar, and agar oligosaccharide claimed in chinese patent CN1171592C has fibroblast growth promoting effect and ultraviolet ray oxidation injury protecting effect.
However, the application of agar oligosaccharides is still less so far, and the bottleneck factor influencing the application and development of agar oligosaccharides is mainly the preparation method. At present, agar is extracted from red algae such as hedgerow, agar and the like in the preparation process of agar oligosaccharide, and then the agar is degraded into oligosaccharide with smaller molecular weight by a chemical method or an enzymatic method. For example, chinese patents CN1394879A, CN1467226A, CN1513860A, etc. respectively claim that different chemical methods are used to degrade agar to obtain oligosaccharides; chinese patents CN1460716A, CN1460717A, CN1546500A, etc. claim that agarase preparations obtained from natural or genetically engineered bacteria are used for preparation of agaro-oligosaccharides.
China patents CN102827899A, CN104498563A, CN101955895A and the like respectively adopt Pacific thermochromatic bacilli, Bat vibrio and roseobacter to degrade to obtain agar oligosaccharides, and strains in the methods have the problems of high production environment requirement, high production cost, great industrialization difficulty and the like.
Disclosure of Invention
Aiming at the problems of high production environment requirement, high production cost, great industrialization difficulty and the like of strains in the prior art, the invention discloses a production process of agar oligosaccharide, and the specific scheme is as follows:
screening and culturing bacterial strains, namely selecting rotten asparagus to shake and culture in a culture solution, diluting the shake culture solution, coating the diluted shake culture solution on a flat plate of a screening culture medium, carrying out inverted culture on the flat plate at the temperature of 25-30 ℃ for 24-60 hours, and observing colony forms and formed pits;
secondly, selecting colonies with larger depressions and larger colony diameters, spot-planting the colonies into a fresh screening culture medium for culturing for 24-60 hours, and observing colony morphology and formed depressions;
step three, repeating the step two for screening until a purified strain is separated; putting the screened strains into a culture solution for expanding culture;
and step four, adding the strain cultured in the step three into the sterilized asparagus for fermentation, drying the fermented asparagus in the sun, extracting the gel, performing acid treatment, performing high-temperature treatment, crushing and sieving to obtain the required agar oligosaccharide.
Preferably, the components and their concentrations in the culture solution before shaking in step one are respectively: agarose 1-3g/L, peptone 3-8g/L, yeast powder 0.5-2.5g/L, ferric phosphate 0.01-0.02g/L, tap water as solvent, culture solution pH 7.2-7.6, and sterilizing at 130 deg.C of 115-.
Preferably, in the step one, the components and concentrations of the screening culture medium are respectively as follows: 10-20g/L of agarose, 3-8g/L of peptone, 0.5-2.5g/L of yeast powder, 0.01-0.02g/L of ferric phosphate, tap water as a solvent, 7.2-7.6 of culture solution pH, and sterilizing at 130 ℃ of 115-.
Preferably, in the first step, the shaking culture temperature is 25-30 ℃, and the shaking rotation speed is 150-200 rpm.
Preferably, in the step one, more than one dilution of the culture solution after shaking is coated on a plate of the screening culture medium, and each dilution is used for making more than three parallel samples.
Preferably, the asparagus sterilization in the fourth step adopts the steps of placing the asparagus in a sterilization pot and sterilizing for 8 hours at 105 ℃.
Preferably, the asparagus sterilized in the fourth step is cooled to normal temperature, and is sprayed with strains for fermentation for 1 week.
Preferably, the gel extraction process in the fourth step comprises alkali treatment, bleaching, gel boiling and filtering;
wherein the alkali treatment is to put the fermented and dried asparagus into alkali solution, the concentration of the alkali solution is 6-8%, the temperature of the alkali treatment is 70-90 ℃, and the time of the alkali treatment is 100-;
bleaching, namely putting the asparagus subjected to alkali treatment into bleaching liquid prepared by reacting sodium hypochlorite with effective chlorine concentration of 0.10% and pH value of about 9 with hydrochloric acid, bleaching for 10min, removing the bleaching liquid, soaking for 5min with 2.5% hydrochloric acid, immediately pouring out the acidification liquid, and washing to be neutral with distilled water;
the step of boiling and filtering is to put the washed asparagus into a boiling pot for boiling, and filter aid is added for filtering and extracting the gel after boiling.
Preferably, the acid treatment in the fourth step is to hydrolyze the agar solution obtained by extracting the agar by stirring with 0.5 percent sulfuric acid in a constant temperature pot at 60 ℃ for 4-5h, and adjusting the pH value to be neutral.
Preferably, the high-temperature treatment in the fourth step is to pour the agar oligosaccharide solution obtained after the acid treatment into a 130-140 ℃ roller for drying after the reduced-pressure concentration.
Preferably, the crushing and screening in the fourth step is to crush and screen the product discharged from the roller through a 80-mesh screen.
Has the advantages that:
the technical scheme of the invention has the following beneficial effects:
(1) by selecting the rotten asparagus to culture and purify the strains, the production environment requirement, the production cost and the industrialization difficulty of the strains are reduced.
(2) The culture medium of the strain has simple composition and easy preparation, and improves the stability and yield of products by combining biological, chemical and physical methods.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the embodiments of the present invention will be described in detail and completely with reference to the examples of the present invention, and it is apparent that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. Thus, the following detailed description of the embodiments of the present invention, provided in the examples, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The embodiment realizes the process for preparing the agar oligosaccharide from the asparagus by combining biological, chemical and physical technologies, and the process not only reduces the production cost, but also improves the stability and the yield of the product. The specific scheme is as follows:
step one, screening and culturing strains, and preparing the following components and concentrations thereof: agarose 1-3g/L, peptone 3-8g/L, yeast powder 0.5-2.5g/L, ferric phosphate 0.01-0.02g/L, tap water as solvent, pH 7.2-7.6, and sterilizing at 130 deg.C of 115-. Selecting rotten asparagus, carrying out shake culture in a culture solution with the temperature of 25-30 ℃ and the rotation speed of 150-200rpm, diluting the shake culture solution, coating the diluted shake culture solution on a flat plate for screening a culture medium, carrying out inverted culture on the flat plate for 24-60 hours at the temperature of 25-30 ℃, and observing the colony morphology and the formed depression;
selecting a colony with a larger depression and a larger colony diameter, and point-planting the colony with the following components and concentrations: 10-20g/L of agarose, 3-8g/L of peptone, 0.5-2.5g/L of yeast powder, 0.01-0.02g/L of ferric phosphate, a solvent of tap water, a pH value of a mixed solution of 7.2-7.6, culturing for 24-60 hours in a fresh screening culture medium sterilized at the temperature of 115 ℃ and 130 ℃ for 10-30min, and observing colony morphology and formed pits;
step three, repeating the step two for screening until a purified strain is separated; putting the screened strains into a culture solution for expanding culture;
and step four, placing the asparagus in a sterilization pot, sterilizing for 8 hours at 105 ℃, cooling the sterilized asparagus to normal temperature, adding the strain cultured in the step three into the sterilized asparagus for fermentation treatment for 1 week, drying the fermented asparagus in the sun, extracting the gum, performing acid treatment, performing high-temperature treatment, crushing and sieving to obtain the required agar oligosaccharide.
In a preferred embodiment, more than one dilution of the culture solution after shaking is spread on the plate of the screening medium in the first step, and each dilution is used for making more than three parallel samples.
As a preferred embodiment, the gel extraction process in the fourth step comprises alkali treatment, bleaching, gel boiling and filtering;
as a preferred embodiment, the gel extraction process in the fourth step comprises alkali treatment, bleaching, gel boiling and filtering;
the alkali treatment is to put the fermented and dried asparagus into alkali solution, the concentration of the alkali solution is 6-8%, the temperature of the alkali treatment is 70-90 ℃, and the time of the alkali treatment is 100-;
bleaching, namely putting the asparagus subjected to alkali treatment into bleaching liquid prepared by reacting sodium hypochlorite with effective chlorine concentration of 0.10% and pH value of about 9 with hydrochloric acid, bleaching for 10min, removing the bleaching liquid, soaking for 5min with 2.5% hydrochloric acid, immediately pouring out the acidification liquid, and washing to be neutral with distilled water;
the step of boiling and filtering is to put the washed asparagus into a boiling pot for boiling, and filter aid is added for filtering and extracting the gel after boiling.
In a preferred embodiment, the acid treatment in the fourth step is to hydrolyze the agar solution obtained by extracting the agar with 0.5% sulfuric acid in a constant temperature pot at 60 ℃ for 4-5h under stirring, and adjust the pH to be neutral.
In a preferred embodiment, the high temperature treatment in the fourth step is to concentrate the agar oligosaccharide solution obtained after the acid treatment under reduced pressure, and then pour the concentrated solution into a roller at the temperature of 130 ℃ and 140 ℃ for drying.
As a preferred embodiment, the crushing and screening in the fourth step is to crush the product discharged from the roller and pass through a 80-mesh sieve.
The beneficial effects of the agar oligosaccharides obtained by the technical scheme of the embodiment are further illustrated by several groups of examples and comparative examples.
The first embodiment is as follows:
the preparation method of agar oligosaccharide in the embodiment comprises the following specific steps:
step one, screening and culturing strains, and preparing the following components and concentrations thereof: agarose 2g/L, peptone 5g/L, yeast powder 1g/L, and ferric phosphate 0.01g/L, wherein the solvent is tap water, the pH of the mixed solution is 7.2, and the culture solution is sterilized at 121 ℃ for 20 min. Selecting rotten asparagus, carrying out shake culture in a culture solution with the temperature of 28 ℃ and the rotation speed of 180rpm, taking the shake culture solution, diluting the culture solution by more than one dilution, coating the diluted culture solution on a flat plate for screening a culture medium, making more than three parallel samples for each dilution, carrying out inverted culture on the flat plate for 48 hours at the temperature of 25-30 ℃, and observing the colony morphology and the formed depression;
selecting a colony with a larger depression and a larger colony diameter, and point-planting the colony with the following components and concentrations: 15g/L of agarose, 5g/L of peptone, 1g/L of yeast powder, 0.01g/L of ferric phosphate, a solvent of tap water, wherein the pH value of a mixed solution is 7.2, the mixed solution is cultured in a fresh screening culture medium sterilized at 121 ℃ for 20min for 48 hours, and the colony morphology and the formed pits are observed;
step three, repeating the step two for screening until a purified strain is separated; putting the screened strains into a culture solution for expanding culture;
step four, placing the asparagus in a sterilization pot, sterilizing for 8 hours at 105 ℃, cooling the sterilized asparagus to normal temperature, adding the strain cultured in the step three into the sterilized asparagus for fermentation treatment, fermenting for 1 week, and drying the fermented asparagus in the sun; then putting the fermented and dried asparagus into an alkali solution, wherein the concentration of the alkali solution is 7%, the alkali treatment temperature is 80 ℃, the alkali treatment time is 110min, putting the asparagus subjected to alkali treatment into a bleaching solution prepared by reacting sodium hypochlorite with the effective chlorine concentration of 0.10% and the pH value of 9 with hydrochloric acid, bleaching for 10min, removing the bleaching solution, soaking for 5min by using 2.5% hydrochloric acid, immediately pouring the dry acidified solution, and washing to be neutral by using distilled water; putting the washed asparagus into a gel boiling tank for gel boiling, and adding a filter aid for filtering and extracting gel after gel boiling; stirring and hydrolyzing the agar solution obtained by extracting the agar with 0.5 percent sulfuric acid in a constant temperature pot at 60 ℃ for 4 hours, and adjusting the pH value to be neutral; decompressing and concentrating the agar oligosaccharide solution obtained after acid treatment, and pouring the agar oligosaccharide solution into a 130-140 ℃ roller for drying; and (3) crushing the product discharged from the roller and sieving the crushed product with a 80-mesh sieve to obtain the required agar oligosaccharide.
Example two:
the preparation method of agar oligosaccharide in the embodiment comprises the following specific steps:
step one, screening and culturing strains, and preparing the following components and concentrations thereof: agarose 2g/L, peptone 8g/L, yeast powder 1g/L, and ferric phosphate 0.02g/L, wherein the solvent is tap water, the pH of the mixed solution is 7.6, and the culture solution is sterilized at 121 ℃ for 20 min. Selecting rotten asparagus, carrying out shake culture in a culture solution with the temperature of 28 ℃ and the rotation speed of 180rpm, taking the shake culture solution, diluting the culture solution by more than one dilution, coating the diluted culture solution on a flat plate for screening a culture medium, making more than three parallel samples for each dilution, carrying out inverted culture on the flat plate for 48 hours at the temperature of 25-30 ℃, and observing the colony morphology and the formed depression;
selecting a colony with a larger depression and a larger colony diameter, and point-planting the colony with the following components and concentrations: 15g/L of agarose, 8g/L of peptone, 1g/L of yeast powder, 0.02g/L of ferric phosphate, a solvent of tap water, wherein the pH value of a mixed solution is 7.6, the mixed solution is cultured in a fresh screening culture medium sterilized at 121 ℃ for 20min for 48 hours, and the colony morphology and the formed pits are observed;
step three, repeating the step two for screening until a purified strain is separated; putting the screened strains into a culture solution for expanding culture;
step four, placing the asparagus in a sterilization pot, sterilizing for 8 hours at 105 ℃, cooling the sterilized asparagus to normal temperature, adding the strain cultured in the step three into the sterilized asparagus for fermentation treatment, fermenting for 1 week, and drying the fermented asparagus in the sun; then putting the fermented and dried asparagus into an alkali solution, wherein the concentration of the alkali solution is 7%, the alkali treatment temperature is 80 ℃, the alkali treatment time is 110min, putting the asparagus subjected to alkali treatment into a bleaching solution prepared by reacting sodium hypochlorite with the effective chlorine concentration of 0.10% and the pH value of 9 with hydrochloric acid, bleaching for 10min, removing the bleaching solution, soaking for 5min by using 2.5% hydrochloric acid, immediately pouring the dry acidified solution, and washing to be neutral by using distilled water; putting the washed asparagus into a gel boiling tank for gel boiling, and adding a filter aid for filtering and extracting gel after gel boiling; stirring and hydrolyzing the agar solution obtained by extracting the agar with 0.5 percent sulfuric acid in a constant temperature pot at 60 ℃ for 4 hours, and adjusting the pH value to be neutral; decompressing and concentrating the agar oligosaccharide solution obtained after acid treatment, and pouring the agar oligosaccharide solution into a 130-140 ℃ roller for drying; and (3) crushing the product discharged from the roller and sieving the crushed product with a 80-mesh sieve to obtain the required agar oligosaccharide.
Example three:
the preparation method of agar oligosaccharide in the embodiment comprises the following specific steps:
step one, screening and culturing strains, and preparing the following components and concentrations thereof: agarose 1g/L, peptone 5g/L, yeast powder 1g/L, and ferric phosphate 0.01g/L, wherein the solvent is tap water, the pH of the mixed solution is 7.2, and the culture solution is sterilized at 121 ℃ for 20 min. Selecting rotten asparagus, carrying out shake culture in a culture solution with the temperature of 28 ℃ and the rotation speed of 180rpm, taking the shake culture solution, diluting the culture solution by more than one dilution, coating the diluted culture solution on a flat plate for screening a culture medium, making more than three parallel samples for each dilution, carrying out inverted culture on the flat plate for 48 hours at the temperature of 25-30 ℃, and observing the colony morphology and the formed depression;
selecting a colony with a larger depression and a larger colony diameter, and point-planting the colony with the following components and concentrations: 10g/L of agarose, 5g/L of peptone, 1g/L of yeast powder, 0.01g/L of ferric phosphate, a solvent of tap water, wherein the pH value of a mixed solution is 7.2, the mixed solution is cultured in a fresh screening culture medium sterilized at 121 ℃ for 20min for 60 hours, and the colony morphology and the formed pits are observed;
step three, repeating the step two for screening until a purified strain is separated; putting the screened strains into a culture solution for expanding culture;
step four, placing the asparagus in a sterilization pot, sterilizing for 8 hours at 105 ℃, cooling the sterilized asparagus to normal temperature, adding the strain cultured in the step three into the sterilized asparagus for fermentation treatment, fermenting for 1 week, and drying the fermented asparagus in the sun; then putting the fermented and dried asparagus into an alkali solution, wherein the concentration of the alkali solution is 7%, the alkali treatment temperature is 80 ℃, the alkali treatment time is 110min, putting the asparagus subjected to alkali treatment into a bleaching solution prepared by reacting sodium hypochlorite with the effective chlorine concentration of 0.10% and the pH value of 9 with hydrochloric acid, bleaching for 10min, removing the bleaching solution, soaking for 5min by using 2.5% hydrochloric acid, immediately pouring the dry acidified solution, and washing to be neutral by using distilled water; putting the washed asparagus into a gel boiling tank for gel boiling, and adding a filter aid for filtering and extracting gel after gel boiling; stirring and hydrolyzing the agar solution obtained by extracting the agar with 0.5 percent sulfuric acid in a constant temperature pot at 60 ℃ for 4 hours, and adjusting the pH value to be neutral; decompressing and concentrating the agar oligosaccharide solution obtained after acid treatment, and pouring the agar oligosaccharide solution into a 130-140 ℃ roller for drying; and (3) crushing the product discharged from the roller and sieving the crushed product with a 80-mesh sieve to obtain the required agar oligosaccharide.
Example four:
the preparation method of agar oligosaccharide in the embodiment comprises the following specific steps:
step one, screening and culturing strains, and preparing the following components and concentrations thereof: agarose 2g/L, peptone 5g/L, yeast powder 1g/L, and ferric phosphate 0.01g/L, wherein the solvent is tap water, the pH of the mixed solution is 7.2, and the culture solution is sterilized at 121 ℃ for 20 min. Selecting rotten asparagus, carrying out shake culture in a culture solution with the temperature of 28 ℃ and the rotation speed of 180rpm, taking the shake culture solution, diluting the culture solution by more than one dilution, coating the diluted culture solution on a flat plate for screening a culture medium, making more than three parallel samples for each dilution, carrying out inverted culture on the flat plate for 48 hours at the temperature of 25-30 ℃, and observing the colony morphology and the formed depression;
selecting a colony with a larger depression and a larger colony diameter, and point-planting the colony with the following components and concentrations: 15g/L of agarose, 5g/L of peptone, 1g/L of yeast powder, 0.01g/L of ferric phosphate, a solvent of tap water, wherein the pH value of a mixed solution is 7.2, the mixed solution is cultured in a fresh screening culture medium sterilized at 121 ℃ for 20min for 24 hours, and the colony morphology and the formed pits are observed;
step three, repeating the step two for screening until a purified strain is separated; putting the screened strains into a culture solution for expanding culture;
step four, placing the asparagus in a sterilization pot, sterilizing for 8 hours at 105 ℃, cooling the sterilized asparagus to normal temperature, adding the strain cultured in the step three into the sterilized asparagus for fermentation treatment, fermenting for 1 week, and drying the fermented asparagus in the sun; then putting the fermented and dried asparagus into an alkali solution, wherein the concentration of the alkali solution is 8%, the alkali treatment temperature is 70 ℃, the alkali treatment time is 120min, putting the asparagus subjected to alkali treatment into a bleaching solution prepared by the reaction of sodium hypochlorite and hydrochloric acid, the effective chlorine concentration of which is 0.10% and the pH value of which is 9, bleaching for 10min, removing the bleaching solution, soaking for 5min by using 2.5% hydrochloric acid, immediately pouring the dry acidified solution, and washing to be neutral by using distilled water; putting the washed asparagus into a gel boiling tank for gel boiling, and adding a filter aid for filtering and extracting gel after gel boiling; stirring and hydrolyzing the agar solution obtained by extracting the agar with 0.5 percent sulfuric acid in a constant temperature pot at 60 ℃ for 4 hours, and adjusting the pH value to be neutral; decompressing and concentrating the agar oligosaccharide solution obtained after acid treatment, and pouring the agar oligosaccharide solution into a 130-140 ℃ roller for drying; and (3) crushing the product discharged from the roller and sieving the crushed product with a 80-mesh sieve to obtain the required agar oligosaccharide.
Comparative example one:
the preparation method of the agar oligosaccharide in the comparative example comprises the following specific steps:
step one, strain obtaining and culturing, namely obtaining Pacific Marmoreus H2 from the China center for type culture Collection, and using asparagus powder sieved by 80 meshes to the Pacific Marmoreus H21 percent, 0.2 percent of yeast extract, 3 percent of NaCl and 1.5 percent of agar powder, is prepared by distilled water, is activated by an activation medium plate sterilized at the temperature of 121 ℃ for 20min, and then a single colony is selected and inoculated on a medium plate containing CMC-Na21.0 percent, yeast extract 0.2 percent and NaCl 3 percent, prepared by distilled water, and is cultured in a seed culture medium sterilized at the temperature of 121 ℃ for 20min in a shaking way to OD under the condition of 30 DEG C600＝1.0；
Step two, adding 3 percent of asparagus powder sieved by a 80-mesh sieve and 3 percent of MgCl into the fermentation liquor according to the inoculation proportion of 3 percent20.25 percent of the total sugar content, prepared by distilled water, and continuously carrying out shake fermentation culture for 48 hours at the temperature of 30 ℃ in a sugar production culture medium sterilized at the temperature of 121 ℃ for 20 min;
centrifuging the fermentation liquor for 15min by 10000X g, and collecting supernatant;
and step four, sequentially using a membrane filtration device to sequentially use a 0.45-micron filtration membrane, a 0.22-micron filtration membrane, a 30000D ultrafiltration membrane and a 300D nanofiltration membrane on the supernatant to obtain a filtrate, namely the asparagus agar oligosaccharide.
Comparative example two:
the preparation method of the agar oligosaccharide in the comparative example comprises the following specific steps:
step one, strain obtaining and culturing, namely obtaining vibrio alginolyticus from a Chinese typical culture collection center, inoculating the vibrio alginolyticus into a seed culture medium which consists of 35g/L of NaCl, 5g/L of peptone, 10g/L of yeast powder, 2.0g/L of agar and 1g/L of ferrous sulfate heptahydrate, adjusting the pH to 7.0, preparing tap water, sterilizing at 121 ℃ for 20min, and performing shake culture at 25 ℃ and 140rpm for 24 hours after inoculation to obtain seed fermentation liquor;
adding fermentation liquor into a fermentation medium which is prepared from 35g/L of NaCl, 5g/L of peptone, 10g/L of yeast powder, 2.0g/L of agar and 1g/L of ferrous sulfate heptahydrate according to an inoculation proportion of 11%, adjusting the pH to 7.0, preparing tap water, and culturing the fermentation medium at the temperature of 121 ℃ for 20min for 48h to obtain fermentation liquor containing agarase;
and step three, adding the agar into distilled water, heating and boiling to dissolve the agar, quickly cooling to 40 ℃, adding the fermentation liquor containing the agarase, and carrying out enzymolysis reaction for 40 hours at 40 ℃. Centrifuging the enzymolysis liquid at 10000r/min for 20min, taking supernate and filtering on a 10kDa ultrafiltration membrane, collecting filtrate, and freeze-drying to obtain the agar oligosaccharide.
Comparative example three:
the preparation method of the agar oligosaccharide in the comparative example comprises the following specific steps:
step one, strain obtaining and culturing, namely obtaining Paenibacillus WL-15 from the China center for type culture collection, inoculating the Paenibacillus WL-15 to agar 20g/L, glucose 1g/L, yeast extract powder 5g/L, NaCl 20g/L and MgSO 24·7H2O 5g/L，KCl 1g/L，FeSO4·7H2O 0.02g/L，CaCl20.2g/L，NaH2PO40.78g/L, and culturing in a slant culture medium with water as solvent and pH of 7.5 at 28 deg.C for 36 h.
Step two, selecting a single colony to be inoculated in agar of 2g/L, yeast extract powder of 5g/L, peptone of 10g/L, NaCl of 20g/L and FeSO4·7H2O 0.02g/L，NaH2PO40.6g/L, water is used as a solvent, and the pH value is 7.0, wherein the liquid seed culture is carried out for 24 hours under the oscillating condition of 200r/min at 28 ℃ to obtain seed liquid;
thirdly, inoculating the seed solution into 2.5g/L agar and NaNO in a volume ratio of 9%3 6g/L，NaCl 15g/L，NaH2PO40.6g/L，MgSO4·7H2O 5g/L，KCl 1g/L，FeSO4·7H2O 0.02g/L，CaCl20.2g/L，MnCl2·4H2O0.15 g/L, culturing for 24h at 30 ℃ and 250r/min in a liquid fermentation culture medium with the pH of 7.0 by taking water as a solvent to obtain fermentation liquor containing agarase, centrifuging the fermentation liquor containing the agarase for 20min at 4 ℃ and 8000r/min, pouring out supernatant, and discarding precipitates to obtain clear liquid, namely crude agarase liquid;
step four, Na with pH of 7.0 and concentration of 0.2mol/L is used2HPO4-NaH2PO4Preparing 3g/L agar substrate solution (heating for dissolving) by using buffer solution, wherein the agar substrate solutionAdding the crude agarase liquid into the liquid, sealing, performing enzymolysis reaction for 120min at 45 ℃ under the condition of oscillation at 200r/min, taking supernate, filtering on a 10kDa ultrafiltration membrane, collecting filtrate, and freeze-drying to obtain the agaro-oligosaccharide.
Comparative example four:
the preparation method of the agar oligosaccharide in the comparative example comprises the following specific steps:
step one, screening and culturing strains, and preparing the following components and concentrations thereof: agarose 2g/L, peptone 5g/L, yeast powder 1g/L, and ferric phosphate 0.01g/L, wherein the solvent is tap water, the pH of the mixed solution is 8.0, and the culture solution is sterilized at 121 ℃ for 20 min. Selecting rotten asparagus, carrying out shake culture in a culture solution with the temperature of 28 ℃ and the rotation speed of 180rpm, taking the shake culture solution, diluting the culture solution by more than one dilution, coating the diluted culture solution on a flat plate for screening a culture medium, making more than three parallel samples for each dilution, carrying out inverted culture on the flat plate for 48 hours at the temperature of 25-30 ℃, and observing the colony morphology and the formed depression;
selecting a colony with a larger depression and a larger colony diameter, and point-planting the colony with the following components and concentrations: 15g/L of agarose, 5g/L of peptone, 1g/L of yeast powder, 0.01g/L of ferric phosphate, a solvent of tap water, wherein the pH value of a mixed solution is 8.0, the mixed solution is cultured in a fresh screening culture medium sterilized at 121 ℃ for 20min for 48 hours, and the colony morphology and the formed pits are observed;
step three, repeating the step two for screening until a purified strain is separated; putting the screened strains into a culture solution for expanding culture;
step four, placing the asparagus in a sterilization pot, sterilizing for 8 hours at 105 ℃, cooling the sterilized asparagus to normal temperature, adding the strain cultured in the step three into the sterilized asparagus for fermentation treatment, fermenting for 1 week, and drying the fermented asparagus in the sun; then putting the fermented and dried asparagus into an alkali solution, wherein the concentration of the alkali solution is 7%, the alkali treatment temperature is 80 ℃, the alkali treatment time is 110min, putting the asparagus subjected to alkali treatment into a bleaching solution prepared by reacting sodium hypochlorite with the effective chlorine concentration of 0.10% and the pH value of 9 with hydrochloric acid, bleaching for 10min, removing the bleaching solution, soaking for 5min by using 2.5% hydrochloric acid, immediately pouring the dry acidified solution, and washing to be neutral by using distilled water; putting the washed asparagus into a gel boiling tank for gel boiling, and adding a filter aid for filtering and extracting gel after gel boiling; stirring and hydrolyzing the agar solution obtained by extracting the agar with 0.5 percent sulfuric acid in a constant temperature pot at 60 ℃ for 4 hours, and adjusting the pH value to be neutral; decompressing and concentrating the agar oligosaccharide solution obtained after acid treatment, and pouring the agar oligosaccharide solution into a 130-140 ℃ roller for drying; and (3) crushing the product discharged from the roller and sieving the crushed product with a 80-mesh sieve to obtain the required agar oligosaccharide.
Comparative example five:
the preparation method of the agar oligosaccharide in the comparative example comprises the following specific steps:
step one, screening and culturing strains, and preparing the following components and concentrations thereof: agarose 2g/L, peptone 5g/L, yeast powder 1g/L, and ferric phosphate 0.01g/L, wherein the solvent is tap water, the pH of the mixed solution is 7.2, and the culture solution is sterilized at 121 ℃ for 20 min. Selecting rotten asparagus, carrying out shake culture in a culture solution with the temperature of 28 ℃ and the rotation speed of 180rpm, taking the shake culture solution, diluting the culture solution by more than one dilution, coating the diluted culture solution on a flat plate for screening a culture medium, making more than three parallel samples for each dilution, culturing for 48 hours at the temperature of 25-30 ℃, and observing the colony morphology and the formed depression;
selecting a colony with a larger depression and a larger colony diameter, and point-planting the colony with the following components and concentrations: 15g/L of agarose, 5g/L of peptone, 1g/L of yeast powder, 0.01g/L of ferric phosphate, a solvent of tap water, wherein the pH value of a mixed solution is 7.2, the mixed solution is cultured in a fresh screening culture medium sterilized at 121 ℃ for 20min for 48 hours, and the colony morphology and the formed pits are observed;
step three, repeating the step two for screening until a purified strain is separated; putting the screened strains into a culture solution for expanding culture;
step four, placing the asparagus in a sterilization pot, sterilizing for 8 hours at 105 ℃, cooling the sterilized asparagus to normal temperature, adding the strain cultured in the step three into the sterilized asparagus for fermentation treatment, fermenting for 1 week, and drying the fermented asparagus in the sun; then putting the fermented and dried asparagus into an alkali solution, wherein the concentration of the alkali solution is 7%, the alkali treatment temperature is 80 ℃, the alkali treatment time is 110min, putting the asparagus subjected to alkali treatment into a bleaching solution prepared by reacting sodium hypochlorite with the effective chlorine concentration of 0.10% and the pH value of 9 with hydrochloric acid, bleaching for 10min, removing the bleaching solution, soaking for 5min by using 2.5% hydrochloric acid, immediately pouring the dry acidified solution, and washing to be neutral by using distilled water; putting the washed asparagus into a gel boiling tank for gel boiling, and adding a filter aid for filtering and extracting gel after gel boiling; stirring and hydrolyzing the agar solution obtained by extracting the agar with 0.5 percent sulfuric acid in a constant temperature pot at 60 ℃ for 4 hours, and adjusting the pH value to be neutral; decompressing and concentrating the agar oligosaccharide solution obtained after acid treatment, and pouring the agar oligosaccharide solution into a 130-140 ℃ roller for drying; and (3) crushing the product discharged from the roller and sieving the crushed product with a 80-mesh sieve to obtain the required agar oligosaccharide.
Comparative example six:
the preparation method of the agar oligosaccharide in the comparative example comprises the following specific steps:
step one, screening and culturing strains, and preparing the following components and concentrations thereof: agarose 2g/L, peptone 5g/L, yeast powder 1g/L, and ferric phosphate 0.01g/L, wherein the solvent is tap water, the pH of the mixed solution is 7.2, and the culture solution is sterilized at 121 ℃ for 20 min. Selecting rotten asparagus, carrying out shake culture in a culture solution with the temperature of 28 ℃ and the rotation speed of 180rpm, taking the shake culture solution, diluting the culture solution by more than one dilution, coating the diluted culture solution on a flat plate for screening a culture medium, making more than three parallel samples for each dilution, carrying out inverted culture on the flat plate for 48 hours at the temperature of 25-30 ℃, and observing the colony morphology and the formed depression;
selecting a colony with a larger depression and a larger colony diameter, and point-planting the colony with the following components and concentrations: 15g/L of agarose, 5g/L of peptone, 1g/L of yeast powder, 0.01g/L of ferric phosphate, a solvent of tap water, wherein the pH value of a mixed solution is 7.2, the mixed solution is cultured in a fresh screening culture medium sterilized at 121 ℃ for 20min for 48 hours, and the colony morphology and the formed pits are observed;
step three, repeating the step two for screening until a purified strain is separated; putting the screened strains into a culture solution for expanding culture;
step four, adding the strains cultured in the step three into the asparagus for fermentation treatment, fermenting for 1 week, and drying the fermented asparagus in the sun; then putting the fermented and dried asparagus into an alkali solution, wherein the concentration of the alkali solution is 7%, the alkali treatment temperature is 80 ℃, the alkali treatment time is 110min, putting the asparagus subjected to alkali treatment into a bleaching solution prepared by reacting sodium hypochlorite with the effective chlorine concentration of 0.10% and the pH value of 9 with hydrochloric acid, bleaching for 10min, removing the bleaching solution, soaking for 5min by using 2.5% hydrochloric acid, immediately pouring the dry acidified solution, and washing to be neutral by using distilled water; putting the washed asparagus into a gel boiling tank for gel boiling, and adding a filter aid for filtering and extracting gel after gel boiling; stirring and hydrolyzing the agar solution obtained by extracting the agar with 0.5 percent sulfuric acid in a constant temperature pot at 60 ℃ for 4 hours, and adjusting the pH value to be neutral; decompressing and concentrating the agar oligosaccharide solution obtained after acid treatment, and pouring the agar oligosaccharide solution into a 130-140 ℃ roller for drying; and (3) crushing the product discharged from the roller and sieving the crushed product with a 80-mesh sieve to obtain the required agar oligosaccharide.
Comparative example seven:
the preparation method of the agar oligosaccharide in the comparative example comprises the following specific steps:
step one, screening and culturing strains, and preparing the following components and concentrations thereof: agarose 2g/L, peptone 5g/L, yeast powder 1g/L, and ferric phosphate 0.01g/L, wherein the solvent is tap water, the pH of the mixed solution is 7.2, and the culture solution is sterilized at 121 ℃ for 20 min. Selecting rotten asparagus, carrying out shake culture in a culture solution with the temperature of 28 ℃ and the rotation speed of 180rpm, taking the shake culture solution, diluting the culture solution by more than one dilution, coating the diluted culture solution on a flat plate for screening a culture medium, making more than three parallel samples for each dilution, carrying out inverted culture on the flat plate for 48 hours at the temperature of 25-30 ℃, and observing the colony morphology and the formed depression;
selecting a colony with a larger depression and a larger colony diameter, and point-planting the colony with the following components and concentrations: 15g/L of agarose, 5g/L of peptone, 1g/L of yeast powder, 0.01g/L of ferric phosphate, a solvent of tap water, wherein the pH value of a mixed solution is 7.2, the mixed solution is cultured in a fresh screening culture medium sterilized at 121 ℃ for 20min for 48 hours, and the colony morphology and the formed pits are observed;
step three, repeating the step two for screening until a purified strain is separated; putting the screened strains into a culture solution for expanding culture;
step four, placing the asparagus in a sterilization pot, sterilizing for 8 hours at 105 ℃, cooling the sterilized asparagus to normal temperature, adding the strain cultured in the step three into the sterilized asparagus for fermentation treatment, fermenting for 1 week, and drying the fermented asparagus in the sun; then putting the fermented and dried asparagus into an alkali solution, wherein the concentration of the alkali solution is 10%, the alkali treatment temperature is 80 ℃, the alkali treatment time is 110min, putting the asparagus subjected to alkali treatment into a bleaching solution prepared by reacting sodium hypochlorite with the effective chlorine concentration of 0.10% and the pH value of 9 with hydrochloric acid, bleaching for 10min, removing the bleaching solution, soaking for 5min by using 2.5% hydrochloric acid, immediately pouring the dry acidified solution, and washing to be neutral by using distilled water; putting the washed asparagus into a gel boiling tank for gel boiling, and adding a filter aid for filtering and extracting gel after gel boiling; stirring and hydrolyzing the agar solution obtained by extracting the agar with 0.5 percent sulfuric acid in a constant temperature pot at 60 ℃ for 4 hours, and adjusting the pH value to be neutral; decompressing and concentrating the agar oligosaccharide solution obtained after acid treatment, and pouring the agar oligosaccharide solution into a 130-140 ℃ roller for drying; and (3) crushing the product discharged from the roller and sieving the crushed product with a 80-mesh sieve to obtain the required agar oligosaccharide.
Comparative example eight:
the preparation method of the agar oligosaccharide in the comparative example comprises the following specific steps:
step one, screening and culturing strains, and preparing the following components and concentrations thereof: agarose 2g/L, peptone 5g/L, yeast powder 1g/L, and ferric phosphate 0.01g/L, wherein the solvent is tap water, the pH of the mixed solution is 7.2, and the culture solution is sterilized at 121 ℃ for 20 min. Selecting rotten asparagus, carrying out shake culture in a culture solution with the temperature of 28 ℃ and the rotation speed of 180rpm, taking the shake culture solution, diluting the culture solution by more than one dilution, coating the diluted culture solution on a flat plate for screening a culture medium, making more than three parallel samples for each dilution, carrying out inverted culture on the flat plate for 48 hours at the temperature of 25-30 ℃, and observing the colony morphology and the formed depression;
selecting a colony with a larger depression and a larger colony diameter, and point-planting the colony with the following components and concentrations: 15g/L of agarose, 5g/L of peptone, 1g/L of yeast powder, 0.01g/L of ferric phosphate, a solvent of tap water, wherein the pH value of a mixed solution is 7.2, the mixed solution is cultured in a fresh screening culture medium sterilized at 121 ℃ for 20min for 48 hours, and the colony morphology and the formed pits are observed;
step three, repeating the step two for screening until a purified strain is separated; putting the screened strains into a culture solution for expanding culture;
step four, placing the asparagus in a sterilization pot, sterilizing for 8 hours at 105 ℃, cooling the sterilized asparagus to normal temperature, adding the strain cultured in the step three into the sterilized asparagus for fermentation treatment, fermenting for 1 week, and drying the fermented asparagus in the sun; then putting the fermented and dried asparagus into an alkali solution, wherein the concentration of the alkali solution is 7%, the alkali treatment temperature is 80 ℃, the alkali treatment time is 110min, putting the asparagus subjected to alkali treatment into a bleaching solution prepared by reacting sodium hypochlorite with the effective chlorine concentration of 0.10% and the pH value of 9 with hydrochloric acid, bleaching for 10min, removing the bleaching solution, soaking for 5min by using 5.0% hydrochloric acid, immediately pouring the dry acidified solution, and washing to be neutral by using distilled water; putting the washed asparagus into a gel boiling tank for gel boiling, and adding a filter aid for filtering and extracting gel after gel boiling; stirring and hydrolyzing the agar solution obtained by extracting the agar with 0.5 percent sulfuric acid in a constant temperature pot at 60 ℃ for 4 hours, and adjusting the pH value to be neutral; decompressing and concentrating the agar oligosaccharide solution obtained after acid treatment, and pouring the agar oligosaccharide solution into a 130-140 ℃ roller for drying; and (3) crushing the product discharged from the roller and sieving the crushed product with a 80-mesh sieve to obtain the required agar oligosaccharide.
Comparative example nine:
the preparation method of the agar oligosaccharide in the comparative example comprises the following specific steps:
step one, screening and culturing strains, and preparing the following components and concentrations thereof: agarose 2g/L, peptone 5g/L, yeast powder 1g/L, and ferric phosphate 0.01g/L, wherein the solvent is tap water, the pH of the mixed solution is 7.2, and the culture solution is sterilized at 121 ℃ for 20 min. Selecting rotten asparagus, carrying out shake culture in a culture solution with the temperature of 28 ℃ and the rotation speed of 180rpm, taking the shake culture solution, diluting the culture solution by more than one dilution, coating the diluted culture solution on a flat plate for screening a culture medium, making more than three parallel samples for each dilution, carrying out inverted culture on the flat plate for 48 hours at the temperature of 25-30 ℃, and observing the colony morphology and the formed depression;
selecting a colony with a larger depression and a larger colony diameter, and point-planting the colony with the following components and concentrations: 15g/L of agarose, 5g/L of peptone, 1g/L of yeast powder, 0.01g/L of ferric phosphate, a solvent of tap water, wherein the pH value of a mixed solution is 7.2, the mixed solution is cultured in a fresh screening culture medium sterilized at 121 ℃ for 20min for 24 hours, and the colony morphology and the formed pits are observed;
step three, repeating the step two for screening until a purified strain is separated; putting the screened strains into a culture solution for expanding culture;
step four, placing the asparagus in a sterilization pot, sterilizing for 8 hours at 105 ℃, cooling the sterilized asparagus to normal temperature, adding the strain cultured in the step three into the sterilized asparagus for fermentation treatment, fermenting for 1 week, and drying the fermented asparagus in the sun; then putting the fermented and dried asparagus into an alkali solution, wherein the concentration of the alkali solution is 7%, the alkali treatment temperature is 80 ℃, the alkali treatment time is 110min, putting the asparagus subjected to alkali treatment into a bleaching solution prepared by the reaction of sodium hypochlorite and hydrochloric acid, the effective chlorine concentration of which is 0.20% and the pH value of which is 10, bleaching for 10min, removing the bleaching solution, soaking for 5min by using 2.5% hydrochloric acid, immediately pouring out the dry acidified solution, and washing to be neutral by using distilled water; putting the washed asparagus into a gel boiling tank for gel boiling, and adding a filter aid for filtering and extracting gel after gel boiling; stirring and hydrolyzing the agar solution obtained by extracting the agar with 0.5 percent sulfuric acid in a constant temperature pot at 60 ℃ for 4 hours, and adjusting the pH value to be neutral; decompressing and concentrating the agar oligosaccharide solution obtained after acid treatment, and pouring the agar oligosaccharide solution into a 130-140 ℃ roller for drying; and (3) crushing the product discharged from the roller and sieving the crushed product with a 80-mesh sieve to obtain the required agar oligosaccharide.
Comparative example ten:
the preparation method of the agar oligosaccharide in the comparative example comprises the following specific steps:
step one, screening and culturing strains, and preparing the following components and concentrations thereof: agarose 2g/L, peptone 5g/L, yeast powder 1g/L, and ferric phosphate 0.01g/L, wherein the solvent is tap water, the pH of the mixed solution is 7.2, and the culture solution is sterilized at 121 ℃ for 20 min. Selecting rotten asparagus, carrying out shake culture in a culture solution with the temperature of 28 ℃ and the rotation speed of 180rpm, taking the shake culture solution, diluting the culture solution by more than one dilution, coating the diluted culture solution on a flat plate for screening a culture medium, making more than three parallel samples for each dilution, carrying out inverted culture on the flat plate for 48 hours at the temperature of 25-30 ℃, and observing the colony morphology and the formed depression;
selecting a colony with a larger depression and a larger colony diameter, and point-planting the colony with the following components and concentrations: 15g/L of agarose, 5g/L of peptone, 1g/L of yeast powder, 0.01g/L of ferric phosphate, a solvent of tap water, wherein the pH value of a mixed solution is 7.2, the mixed solution is cultured in a fresh screening culture medium sterilized at 121 ℃ for 20min for 24 hours, and the colony morphology and the formed pits are observed;
step three, repeating the step two for screening until a purified strain is separated; putting the screened strains into a culture solution for expanding culture;
step four, placing the asparagus in a sterilization pot, sterilizing for 8 hours at 105 ℃, cooling the sterilized asparagus to normal temperature, adding the strain cultured in the step three into the sterilized asparagus for fermentation treatment, fermenting for 1 week, and drying the fermented asparagus in the sun; then putting the fermented and dried asparagus into an alkali solution, wherein the concentration of the alkali solution is 7%, the alkali treatment temperature is 80 ℃, the alkali treatment time is 110min, putting the asparagus subjected to alkali treatment into a bleaching solution prepared by reacting sodium hypochlorite with the effective chlorine concentration of 0.10% and the pH value of 9 with hydrochloric acid, bleaching for 10min, removing the bleaching solution, soaking for 5min by using 2.5% hydrochloric acid, immediately pouring the dry acidified solution, and washing to be neutral by using distilled water; putting the washed asparagus into a glue boiling tank for boiling glue, and filtering and extracting the glue after the glue is boiled; stirring and hydrolyzing the agar solution obtained by extracting the agar with 0.5 percent sulfuric acid in a constant temperature pot at 60 ℃ for 4 hours, and adjusting the pH value to be neutral; decompressing and concentrating the agar oligosaccharide solution obtained after acid treatment, and pouring the agar oligosaccharide solution into a 130-140 ℃ roller for drying; and (3) crushing the product discharged from the roller and sieving the crushed product with a 80-mesh sieve to obtain the required agar oligosaccharide.
Comparative example eleven:
the preparation method of the agar oligosaccharide in the comparative example comprises the following specific steps:
step one, screening and culturing strains, and preparing the following components and concentrations thereof: agarose 2g/L, peptone 5g/L, yeast powder 1g/L, and ferric phosphate 0.01g/L, wherein the solvent is tap water, the pH of the mixed solution is 7.2, and the culture solution is sterilized at 121 ℃ for 20 min. Selecting rotten asparagus, carrying out shake culture in a culture solution with the temperature of 28 ℃ and the rotation speed of 180rpm, taking the shake culture solution, diluting the culture solution by more than one dilution, coating the diluted culture solution on a flat plate for screening a culture medium, making more than three parallel samples for each dilution, carrying out inverted culture on the flat plate for 48 hours at the temperature of 25-30 ℃, and observing the colony morphology and the formed depression;
selecting a colony with a larger depression and a larger colony diameter, and point-planting the colony with the following components and concentrations: 15g/L of agarose, 5g/L of peptone, 1g/L of yeast powder, 0.01g/L of ferric phosphate, a solvent of tap water, wherein the pH value of a mixed solution is 7.2, the mixed solution is cultured in a fresh screening culture medium sterilized at 121 ℃ for 20min for 24 hours, and the colony morphology and the formed pits are observed;
step three, repeating the step two for screening until a purified strain is separated; putting the screened strains into a culture solution for expanding culture;
step four, placing the asparagus in a sterilization pot, sterilizing for 8 hours at 105 ℃, cooling the sterilized asparagus to normal temperature, adding the strain cultured in the step three into the sterilized asparagus for fermentation treatment, fermenting for 1 week, and drying the fermented asparagus in the sun; then putting the fermented and dried asparagus into an alkali solution, wherein the concentration of the alkali solution is 7%, the alkali treatment temperature is 80 ℃, the alkali treatment time is 110min, putting the asparagus subjected to alkali treatment into a bleaching solution prepared by reacting sodium hypochlorite with the effective chlorine concentration of 0.10% and the pH value of 9 with hydrochloric acid, bleaching for 10min, removing the bleaching solution, soaking for 5min by using 2.5% hydrochloric acid, immediately pouring the dry acidified solution, and washing to be neutral by using distilled water; putting the washed asparagus into a gel boiling tank for gel boiling, and adding a filter aid for filtering and extracting gel after gel boiling; stirring and hydrolyzing the agar solution obtained by extracting the agar with 0.6 percent sulfuric acid in a constant temperature pot at 60 ℃ for 4 hours, and adjusting the pH value to be neutral; decompressing and concentrating the agar oligosaccharide solution obtained after acid treatment, and pouring the agar oligosaccharide solution into a 130-140 ℃ roller for drying; and (3) crushing the product discharged from the roller and sieving the crushed product with a 80-mesh sieve to obtain the required agar oligosaccharide.
Comparative example twelve:
the preparation method of the agar oligosaccharide in the comparative example comprises the following specific steps:
step one, screening and culturing strains, and preparing the following components and concentrations thereof: agarose 2g/L, peptone 5g/L, yeast powder 1g/L, and ferric phosphate 0.01g/L, wherein the solvent is tap water, the pH of the mixed solution is 7.2, and the culture solution is sterilized at 121 ℃ for 20 min. Selecting rotten asparagus, carrying out shake culture in a culture solution with the temperature of 28 ℃ and the rotation speed of 180rpm, taking the shake culture solution, diluting the culture solution by more than one dilution, coating the diluted culture solution on a flat plate for screening a culture medium, making more than three parallel samples for each dilution, carrying out inverted culture on the flat plate for 48 hours at the temperature of 25-30 ℃, and observing the colony morphology and the formed depression;
selecting a colony with a larger depression and a larger colony diameter, and point-planting the colony with the following components and concentrations: 15g/L of agarose, 5g/L of peptone, 1g/L of yeast powder, 0.01g/L of ferric phosphate, a solvent of tap water, wherein the pH value of a mixed solution is 7.2, the mixed solution is cultured in a fresh screening culture medium sterilized at 121 ℃ for 20min for 24 hours, and the colony morphology and the formed pits are observed;
step three, repeating the step two for screening until a purified strain is separated; putting the screened strains into a culture solution for expanding culture;
step four, placing the asparagus in a sterilization pot, sterilizing for 8 hours at 105 ℃, cooling the sterilized asparagus to normal temperature, adding the strain cultured in the step three into the sterilized asparagus for fermentation treatment, fermenting for 1 week, and drying the fermented asparagus in the sun; then putting the fermented and dried asparagus into an alkali solution, wherein the concentration of the alkali solution is 7%, the alkali treatment temperature is 80 ℃, the alkali treatment time is 110min, putting the asparagus subjected to alkali treatment into a bleaching solution prepared by reacting sodium hypochlorite with the effective chlorine concentration of 0.10% and the pH value of 9 with hydrochloric acid, bleaching for 10min, removing the bleaching solution, soaking for 5min by using 2.5% hydrochloric acid, immediately pouring the dry acidified solution, and washing to be neutral by using distilled water; putting the washed asparagus into a gel boiling tank for gel boiling, and adding a filter aid for filtering and extracting gel after gel boiling; stirring and hydrolyzing the agar solution obtained by extracting the agar with 0.2 percent sulfuric acid in a constant temperature pot at 60 ℃ for 4 hours, and adjusting the pH value to be neutral; decompressing and concentrating the agar oligosaccharide solution obtained after acid treatment, and pouring the agar oligosaccharide solution into a 130-140 ℃ roller for drying; and (3) crushing the product discharged from the roller and sieving the crushed product with a 80-mesh sieve to obtain the required agar oligosaccharide.
The four groups of embodiments and the twelve groups of comparative proportions are compared in the aspects of process consumption, agar oligosaccharide extraction rate, agar oligosaccharide polymerization degree and production benefit, and specific data are as follows:
TABLE 1 data relating to the various groups of examples and comparative examples
The process cost is material cost, labor cost and water and electricity cost; the method for measuring the extraction rate of the agar oligosaccharide comprises the following steps: weighing fermented and sun-dried thallus Gracilariae, and recording the mass as m0Drying, crushing and sieving agar solution obtained by extracting agar, and recording the agar oligosaccharide powder as m1The extraction rate of agar oligosaccharide is m1/m0100% and the polymerization degree of the agar oligosaccharide are measured by a mass spectrum and nuclear magnetic resonance instrument, and the production benefit is the extraction amount of the agar oligosaccharide-the process consumption.
As can be seen from table 1, the strain is screened and cultured by using rotten asparagus, the strain is inoculated into the sterilized asparagus for fermentation treatment, and finally the fermented asparagus is dried in the sun, subjected to gel extraction, subjected to acid treatment, subjected to high-temperature treatment, crushed and sieved, so that compared with the process using other strains, the process consumption is reduced under the condition that the extraction amount of agar oligosaccharides is not much different, the production benefit of the process is improved, and in addition, the agar oligosaccharides with lower polymerization degree can be obtained; the flat plate of the post-strain screening culture medium is subjected to inverted culture at the temperature of 25-30 ℃, so that the growth of aerobic bacteria can be effectively inhibited, the interference of mixed strains and the influence on post-strain fermentation treatment are avoided, and the degradation of agar is facilitated; the fresh asparagus is sterilized, so that the growth of degradation bacteria can be prevented from being inhibited due to introduction of sundry bacteria in a fermentation process, and the degradation of agar is further influenced, so that the polymerization degree of agar oligosaccharide is greatly improved; in the gum extraction process, the concentration of alkali liquor or hydrochloric acid is increased, but the extraction rate of agar oligosaccharide is reduced, and the production benefit is influenced; by adding the food-grade filter aid, ash content can be effectively reduced, and pollution to products is avoided; during acid treatment, the extraction rate of agar oligosaccharide is improved along with the increase of the concentration of sulfuric acid, but when the concentration of the sulfuric acid is more than 0.5 percent, the influence on the extraction rate of the agar oligosaccharide is small.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A production process of agar oligosaccharides is characterized by comprising the following steps:
screening and culturing bacterial strains, namely selecting rotten asparagus to shake and culture in a culture solution, diluting the shake culture solution, coating the diluted shake culture solution on a flat plate of a screening culture medium, carrying out inverted culture on the flat plate at the temperature of 25-30 ℃ for 24-60 hours, and observing colony forms and formed pits; the screening culture medium comprises the following components in percentage by concentration: 10-20g/L of agarose, 3-8g/L of peptone, 0.5-2.5g/L of yeast powder, 0.01-0.02g/L of ferric phosphate, wherein the solvent is tap water, the pH value of a culture solution is 7.2-7.6, and the sterilization is carried out for 10-30min at the temperature of 115-;
secondly, selecting colonies with larger depressions and larger colony diameters, spot-planting the colonies into a fresh screening culture medium for culturing for 24-60 hours, and observing colony morphology and formed depressions;
step three, repeating the step two for screening until a purified strain is separated; putting the screened strains into a culture solution for expanding culture;
and step four, sterilizing the asparagus by placing the asparagus in a sterilization pot, sterilizing for 8 hours at 105 ℃, adding the strain cultured in the step three into the sterilized asparagus for fermentation, drying, extracting the gel, performing acid treatment on the fermented asparagus, performing reduced pressure concentration on the agar oligosaccharide solution obtained after the acid treatment, pouring the agar oligosaccharide solution into a 130-140 ℃ roller for drying, crushing and sieving to obtain the required agar oligosaccharide.
2. The production process of agar oligosaccharides according to claim 1, wherein the components and their concentrations in the culture solution before shaking in step one are respectively: agarose 1-3g/L, peptone 3-8g/L, yeast powder 0.5-2.5g/L, ferric phosphate 0.01-0.02g/L, tap water as solvent, culture solution pH 7.2-7.6, and sterilizing at 130 deg.C of 115-.
3. The process for producing agar oligosaccharides as claimed in claim 2, wherein the shaking culture temperature in step one is 25-30 ℃, and the shaking rotation speed is 150-.
4. The process for producing agar oligosaccharides according to claim 3, wherein the culture solution after shaking in the first step is spread on a plate of a screening medium at more than one dilution, and each dilution is used for making more than three parallel samples.
5. The production process of agar oligosaccharides according to claim 1, wherein the sterilized asparagus in step four is cooled to normal temperature, and is sprayed with strains for fermentation for 1 week.
6. The production process of agar oligosaccharides according to claim 1, wherein the gel extraction process in step four comprises alkali treatment, bleaching, gel boiling and filtration;
wherein the alkali treatment is to put the fermented and dried asparagus into alkali solution, the concentration of the alkali solution is 6-8%, the temperature of the alkali treatment is 70-90 ℃, and the time of the alkali treatment is 100-;
bleaching, namely putting the asparagus subjected to alkali treatment into bleaching liquid prepared by reacting sodium hypochlorite with effective chlorine concentration of 0.10% and pH value of 9 with hydrochloric acid, bleaching for 10min, removing the bleaching liquid, soaking for 5min with 2.5% hydrochloric acid, immediately pouring out the acidification liquid, and washing to be neutral with distilled water;
the step of boiling and filtering is to put the washed asparagus into a boiling pot for boiling, and filter aid is added for filtering and extracting the gel after boiling.
7. The process for producing agar oligosaccharides according to claim 1, wherein the acid treatment in step four is to hydrolyze the agar solution obtained by gum extraction with 0.5% sulfuric acid under stirring in a pot at a constant temperature of 60 ℃ for 4-5h, and adjust the pH to neutral.
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