CN112715964B - Spirulina oligosaccharide and application thereof in preparation of preparation for regulating intestinal health function - Google Patents
Spirulina oligosaccharide and application thereof in preparation of preparation for regulating intestinal health function Download PDFInfo
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- CN112715964B CN112715964B CN202011487323.XA CN202011487323A CN112715964B CN 112715964 B CN112715964 B CN 112715964B CN 202011487323 A CN202011487323 A CN 202011487323A CN 112715964 B CN112715964 B CN 112715964B
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- spirulina
- oligosaccharide
- preparation
- enzymolysis
- glycosidase
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- A—HUMAN NECESSITIES
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Abstract
The invention belongs to the technical field of functional nutritional foods, and particularly discloses spirulina oligosaccharide and application thereof in preparation of a functional preparation for regulating intestinal health. The spirulina oligosaccharide is extracted from spirulina powder by processes of repeated freeze thawing, ultrasonic-assisted hot water extraction, alcohol precipitation, deproteinization, decolorization, degradation, ultrafiltration and the like, wherein marine microorganism glycosidase is used for degradation, the average molecular weight of the obtained spirulina oligosaccharide is concentrated in 300-plus 2000Da, the spirulina oligosaccharide mainly comprises alpha-glucose, has the activity of simultaneously promoting the proliferation of bifidobacterium and lactobacillus, is superior to spirulina polysaccharide, can also obviously improve the diversity of intestinal flora and the abundance of beneficial bacteria, reduces the abundance of harmful bacteria, and can be applied to functional food and health care products for regulating the intestinal health.
Description
Technical Field
The invention belongs to the field of functional nutritional foods, and particularly relates to spirulina oligosaccharide and application thereof in preparation of a functional preparation for regulating intestinal health.
Background
The intestinal flora is closely related to the health and diseases of human bodies, pathogenic bacteria are prevented from invading epithelial cells of the intestinal tract by competing with the pathogenic bacteria for binding sites and generating antibacterial substances, and the healthy dynamic microecological balance of the intestinal tract is established. The functional oligosaccharide can selectively proliferate beneficial bacteria, and competitively exclude and inhibit pathogenic bacteria through biological barrier effect and fermentation to produce acid. When the oligosaccharide is taken under the condition of unbalanced intestinal flora, the nutrition-deficient intestinal beneficial bacteria can utilize the oligosaccharide to carry out metabolic growth, if the oligosaccharide is continuously taken, the beneficial bacteria can be massively propagated in a short period, so that the growth of harmful flora is inhibited, and the intestinal tract is gradually restored to balance. Functional oligosaccharides have been widely used as a microecological modulator for prevention and first-line treatment of microecological imbalance, and can regulate intestinal microecological imbalance, maintain microecological balance, improve host health level or promote health status.
The invention patent (202010126018.1) discloses a preparation method of xanthan gum oligosaccharide, which is obtained by performing acid hydrolysis dialysis and freeze-drying, and the invention patent (201811270639.6) discloses an application of bletilla striata oligosaccharide and a composition thereof in improving intestinal microecology, wherein the bletilla striata oligosaccharide is degraded by strong acid or glucoside hydrolase, and chemical reagents are introduced, so that certain potential pollution is brought to the environment.
Disclosure of Invention
In order to overcome the defects and shortcomings in the prior art, the invention provides a spirulina oligosaccharide which can be used as a functional factor to be applied to the development of health foods or health care products for patients with intestinal cancer, metabolic syndrome, cardiovascular diseases, enteritis, obesity or diabetes mellitus and the like.
Seaweed and microalgae polysaccharide and oligosaccharide are important material sources for finding prebiotic candidates, and the polysaccharide is degraded into oligosaccharide, so that the prebiotic is more favorable for intestinal microorganism fermentation and utilization. Spirulina is an edible microalgae, has long been used as a health and functional food, and has various biological activities of resisting oxidation, bacteria, virus, cancer, inflammation, and blood sugar. Research shows that spirulina and its water extract polysaccharide have no growth promoting effect on pathogenic bacteria such as Escherichia coli, etc., and have promoting effect on intestinal beneficial bacteria such as bifidobacterium, lactobacillus, etc., and we find that the activity of promoting bifidobacterium and lactobacillus is superior to polysaccharide in oligosaccharide obtained by degrading spirulina polysaccharide with microbial glycosidase.
In order to achieve the purpose, the invention adopts the following technical scheme:
a spirulina oligosaccharide with the average molecular weight of 300-2000Da, which is obtained by the following method:
s1, adding water into spirulina powder, repeatedly freezing and thawing, and then ultrasonically extracting spirulina protein liquid;
s2, carrying out alcohol precipitation on the spirulina protein solution obtained in the step S1, adding papain for enzymolysis, and carrying out deproteinization and decoloration to obtain a spirulina polysaccharide solution;
s3, adding marine microorganism glycosidase into the spirulina polysaccharide solution obtained in the step S2, carrying out enzymolysis for 5-8h at 30-50 ℃, carrying out ultrafiltration on an enzymolysis solution through a 5kDa ultrafiltration membrane, concentrating a filtrate, and freeze-drying to obtain coarse spirulina oligosaccharide dry powder;
s4, passing the crude spirulina oligosaccharide dry powder obtained in the step S3 through an anion exchange column to obtain neutral spirulina oligosaccharide.
As a preferable technical scheme, in the step S1, 20-40 times of volume of water is added into the spirulina powder, after repeated freeze thawing is carried out for three times, the spirulina powder is subjected to ultrasonic treatment and then reacts for 2-5 hours at the temperature of 60-90 ℃ to obtain the spirulina protein liquid.
As a preferable technical scheme, 3-4 times volume of absolute ethyl alcohol is added in the step S2 for alcohol precipitation.
As a preferable technical proposal, the marine microorganism glycosidase 500-2000U/g is added in the step S3.
As a preferable technical scheme, the nucleotide sequence of the marine microorganism glycosidase is shown in SEQ ID NO: 1, and the amino acid sequence of the encoded zymoprotein is shown as SEQ ID NO: 2, respectively.
As a preferred technical solution, in step S1, the ultrasonic extraction conditions are: ultrasonic power of 50% and ultrasonic treatment at 80 deg.C for 30 min.
As a preferred technical scheme, in step S2, 1% -2% of papain is added for enzymolysis, deproteinization is carried out, and 1% -1.5% of activated carbon is adopted for decoloration.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the invention adopts marine microorganism glycosidase to degrade spirulina, the obtained spirulina oligosaccharide has high yield which reaches more than 80 percent, simultaneously the activity of promoting the proliferation of Bifidobacterium and lactobacillus is better than that of spirulina polysaccharide, the diversity of intestinal microorganisms can be improved, the abundance of microorganisms beneficial to human bodies such as Bacteroides, Escherichia-Shigella, Megamonas, Bifidobacterium, Lactobacillus and the like can be improved, and the content of intestinal short-chain fatty acids, in particular isobutyric acid and isovaleric acid, can be improved, thereby showing that the spirulina oligosaccharide provided by the invention can well regulate the structure of intestinal flora and has better application prospect.
Drawings
FIG. 1 is a separation map of Spirulina oligosaccharide obtained in example 1 with DEAE-cellulose-52.
FIG. 2 is a graph showing the molecular weight distribution of Spirulina oligosaccharide SPO-1.
FIG. 3 is an IR chart of Spirulina oligosaccharide SPO-1.
FIG. 4 shows monosaccharide analysis of Spirulina oligosaccharide SPO-1.
FIG. 5 shows the difference analysis of microbial structures (a) wien diagram analysis, (b) principal component analysis, (C) principal coordinate analysis, (d) cluster analysis and (e) beta diversity thermogram analysis, C0 is fermentation broth 0h, C24 is glucose group fermentation 24h, and S24 is spirulina oligosaccharide SPO-1 group fermentation 24 h.
FIG. 6 shows the composition of the microorganisms C0, C24 and S24 at the phylum level (a), the thermographic analysis at the genus level (b) and the changes in the characteristic microorganisms (C). C0 is fermentation liquid 0h, C24 is glucose group fermentation 24h, S24 is spirulina oligosaccharide SPO-1 group fermentation 24 h.
Detailed Description
The following further describes embodiments of the present invention with reference to the drawings. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The marine microorganism glycosidase used in this example had a sequenced nucleotide sequence shown in SEQ ID NO: 1, and the amino acid sequence of the encoded zymoprotein is shown as SEQ ID NO: 2, respectively.
Example 1
Preparation of spirulina oligosaccharide: weighing a certain amount of Spirulina powder, adding 30 times volume of distilled water, mixing, freezing at-20 deg.C, thawing at normal temperature, circulating for 3 times, extracting with ultrasonic wave (ultrasonic condition: power 50%, time 30min, temperature 80 deg.C), and stirring at 80 deg.C for 2 hr. Centrifuging at 10000r/min for 30min to remove precipitate, performing rotary evaporation concentration on supernate, adding 4 times volume of absolute ethyl alcohol, refrigerating at 4 ℃ for overnight, centrifuging to obtain precipitate, adding 2 times volume of distilled water for redissolving, adding 1% of papain, performing enzymolysis at 50 ℃ for 2h, performing deproteinization by a Sevag method, adding 1.5% (w/v) of activated carbon, decoloring at 40 ℃ for 1h, performing suction filtration to remove the activated carbon, adjusting pH to 8.0, adding 500U/g of marine microorganism glycosidase, performing hydrolysis at 40 ℃ for 8h, performing enzyme deactivation in 100 ℃ water bath for 10min, cooling, performing ultrafiltration with a 5kDa ultrafiltration membrane for 48h, concentrating filtrate, and freeze-drying to obtain the spirulina oligosaccharide.
Example 2
Preparation of spirulina oligosaccharide: weighing a certain amount of Spirulina powder, adding 40 times volume of distilled water, mixing, freezing at-20 deg.C, thawing at normal temperature, circulating for 3 times, extracting with ultrasonic wave (ultrasonic condition: power 50%, time 30min, temperature 80 deg.C), and stirring at 60 deg.C for 5 hr. Centrifuging at 10000r/min for 30min to remove precipitate, performing rotary evaporation concentration on supernate, adding 4 times volume of absolute ethyl alcohol, refrigerating at 4 ℃ for overnight, centrifuging to obtain precipitate, adding 2 times volume of distilled water for redissolving, adding 1% of papain, performing enzymolysis at 50 ℃ for 2h, performing deproteinization by a Sevag method, adding 1.5% (w/v) of activated carbon, decoloring at 40 ℃ for 1h, performing suction filtration to remove the activated carbon, adjusting pH to 8.0, adding 2000U/g of marine microorganism glycosidase, performing hydrolysis at 50 ℃ for 5h, performing enzyme deactivation in 100 ℃ water bath for 10min, cooling, performing ultrafiltration with a 5kDa ultrafiltration membrane for 48h, concentrating filtrate, and freeze-drying to obtain the spirulina oligosaccharide.
Example 3
Preparation of spirulina oligosaccharide: weighing a certain amount of Spirulina powder, adding 20 times volume of distilled water, mixing, freezing at-20 deg.C, thawing at normal temperature, circulating for 3 times, extracting with ultrasonic wave (ultrasonic condition: power 50%, time 30min, temperature 80 deg.C), and stirring at 90 deg.C for 2 hr. Centrifuging at 10000r/min for 30min to remove precipitate, performing rotary evaporation concentration on supernate, adding 3 times volume of absolute ethyl alcohol, refrigerating at 4 ℃ for overnight, centrifuging to obtain precipitate, adding 2 times volume of distilled water for redissolving, adding 1% of papain, performing enzymolysis at 50 ℃ for 2h, performing deproteinization by a Sevag method, adding 1.5% (w/v) of activated carbon, decoloring at 40 ℃ for 1h, performing suction filtration to remove the activated carbon, adjusting pH to 8.0, adding 1000U/g of marine microorganism glycosidase, hydrolyzing at 30 ℃ for 8h, performing enzyme deactivation in 100 ℃ water bath for 10min, cooling, performing ultrafiltration with a 5kDa ultrafiltration membrane for 48h, concentrating filtrate, and freeze-drying to obtain the spirulina oligosaccharide.
Comparative example 1
Preparation of spirulina oligosaccharide: weighing a certain amount of Spirulina powder, adding 30 times volume of distilled water, mixing, freezing at-20 deg.C, thawing at normal temperature, circulating for 3 times, extracting with ultrasonic wave (ultrasonic condition: power 50%, time 30min, temperature 80 deg.C), and stirring at 80 deg.C for 2 hr. Centrifuging at 10000r/min for 30min to remove precipitate, performing rotary evaporation concentration on supernate, adding 3 times volume of absolute ethyl alcohol, refrigerating at 4 ℃ for overnight, centrifuging to obtain precipitate, adding 2 times volume of distilled water for redissolving, adding 1% of papain, performing enzymolysis at 50 ℃ for 2h, performing deproteinization by a Sevag method, adding 1.5% (w/v) of activated carbon, decoloring at 40 ℃ for 1h, performing suction filtration to remove the activated carbon, adjusting pH to 4.5, adding 1000U/g of Viscozyme, hydrolyzing at 50 ℃ for 8h, performing enzyme deactivation in 100 ℃ water bath for 10min, cooling, performing ultrafiltration with a 5kDa ultrafiltration membrane for 48h, concentrating filtrate, and freeze-drying to obtain the spirulina oligosaccharide.
Comparative example 2
Preparation of spirulina oligosaccharide: weighing a certain amount of Spirulina powder, adding 30 times volume of distilled water, mixing, freezing at-20 deg.C, thawing at normal temperature, circulating for 3 times, extracting with ultrasonic wave (ultrasonic condition: power 50%, time 30min, temperature 80 deg.C), and stirring at 80 deg.C for 2 hr. Centrifuging at 10000r/min for 30min to remove precipitate, performing rotary evaporation concentration on supernate, adding 4 times volume of absolute ethyl alcohol, refrigerating in a refrigerator at 4 ℃ overnight, centrifuging to obtain precipitate, adding 2 times volume of distilled water for redissolving, adding 1% of papain, performing enzymolysis at 50 ℃ for 2h, performing deproteinization by a Sevag method, adding 1.5% (w/v) of activated carbon, decoloring at 40 ℃ for 1h, performing suction filtration to remove the activated carbon, adjusting pH to 5.0, adding AMG 1000U/g, performing hydrolysis at 60 ℃ for 8h, performing enzyme deactivation in a water bath at 100 ℃ for 10min, cooling, performing ultrafiltration with a 5kDa ultrafiltration membrane for 48h, concentrating filtrate, and freeze-drying to obtain the spirulina oligosaccharide.
Comparative example 3
Preparation of spirulina oligosaccharide: weighing a certain amount of Spirulina powder, adding 30 times volume of distilled water, mixing, freezing at-20 deg.C, thawing at normal temperature, circulating for 3 times, extracting with ultrasonic wave (ultrasonic condition: power 50%, time 30min, temperature 80 deg.C), and stirring at 80 deg.C for 2 hr. Centrifuging at 10000r/min for 30min to remove precipitate, performing rotary evaporation concentration on supernate, adding 3 times volume of absolute ethyl alcohol, refrigerating in a refrigerator at 4 ℃ overnight, centrifuging to obtain precipitate, adding 2 times volume of distilled water for redissolving, adding 1% of papain, performing enzymolysis at 50 ℃ for 2h, performing deproteinization by a Sevag method, adding 1.5% (w/v) of activated carbon, decoloring at 40 ℃ for 1h, performing suction filtration to remove the activated carbon, adjusting pH to 4.5, adding Celluclast 1000U/g, performing hydrolysis at 50 ℃ for 8h, performing enzyme deactivation in a water bath at 100 ℃ for 10min, cooling, performing ultrafiltration with a 5kDa ultrafiltration membrane for 48h, concentrating filtrate, and freeze-drying to obtain the spirulina oligosaccharide.
Comparative example 4
Preparation of spirulina oligosaccharide: weighing a certain amount of Spirulina powder, adding 30 times volume of distilled water, mixing, freezing at-20 deg.C, thawing at normal temperature, circulating for 3 times, extracting with ultrasonic wave (ultrasonic condition: power 50%, time 30min, temperature 80 deg.C), and stirring at 80 deg.C for 2 hr. Centrifuging at 10000r/min for 30min to remove precipitate, performing rotary evaporation concentration on supernate, adding 4 times volume of absolute ethyl alcohol, refrigerating in a refrigerator at 4 ℃ overnight, centrifuging to obtain precipitate, adding 2 times volume of distilled water for redissolving, adding 1% of papain, performing enzymolysis at 50 ℃ for 2H, performing deproteinization by a Sevag method, adding 1.5% (w/v) of activated carbon, decoloring at 40 ℃ for 1H, performing suction filtration to remove the activated carbon, freezing to obtain spirulina polysaccharide, adding 100 times volume of 3% H2O2 solution for dissolving, performing reaction at 80 ℃ for 4H, performing ultrafiltration with a 5kDa ultrafiltration membrane for 48H, concentrating filtrate, and freeze-drying to obtain the spirulina oligosaccharide.
Comparative example 5
Preparation of spirulina oligosaccharide: weighing a certain amount of Spirulina powder, adding 30 times volume of distilled water, mixing, freezing at-20 deg.C, thawing at normal temperature, circulating for 3 times, extracting with ultrasonic wave (ultrasonic condition: power 50%, time 30min, temperature 80 deg.C), and stirring at 80 deg.C for 2 hr. Centrifuging at 10000r/min for 30min to remove precipitate, performing rotary evaporation concentration on supernate, adding 4 times volume of absolute ethyl alcohol, refrigerating in a refrigerator at 4 ℃ overnight, centrifuging to obtain precipitate, adding 2 times volume of distilled water for redissolving, adding 1% of papain, performing enzymolysis at 50 ℃ for 2h, performing deproteinization by a Sevag method, adding 1.5% (w/v) of activated carbon, decoloring at 40 ℃ for 1h, performing suction filtration to remove the activated carbon, freezing to obtain spirulina polysaccharide, adding 100 times volume of 0.1M HCl solution for dissolving, performing reaction at 80 ℃ for 4h, adding 1M NaOH to adjust pH to 7.0, performing ultrafiltration with a 5kDa ultrafiltration membrane for 48h, concentrating filtrate, and performing freeze-drying to obtain the spirulina oligosaccharide.
Application example 1:
example 1 separation of spirulina oligosaccharides: passing through DEAE-cellulose-52 ion column (2.6 × 40cm), eluting with distilled water, 0.1 mol/L sodium chloride and 0.3mol/L sodium chloride, separating to obtain SPO-1, SPO-2 and SPO-3, respectively, and obtaining three purified components as shown in FIG. 1. Of these, the probiotic activity of SPO-1 is highest, as shown in Table 1 in example 10. The SPO-1 has better probiotic activity on Lactobacillus paracasei and Bifidobacterium animalis, and is obviously improved compared with the probiotic activity of the spirulina crude polysaccharide, the average molecular weight of the spirulina oligosaccharide SPO-1 is concentrated in 300-2000Da and mainly contains alpha-glucose, and the formula is shown in figure 2.
Application example 2:
evaluation of in vitro probiotic activity of spirulina oligosaccharides:
adding 1% (v/v) of 2% (w/v) glucose solution or spirulina oligosaccharide solution into the bacterial liquid of L.paracasei and B.animalis, then respectively inoculating the bacterial liquid on MRS and BBL culture media, culturing for 24h under anaerobic conditions at 37 ℃, sampling for 0h and 48h, respectively counting on MRS and BBL agar plates by a dilution method, using physiological saline as a blank control, and repeating the experiment at least three times, wherein the result is expressed by CFU mL-1.
② adding 1% (v/v) of Escherichia coli liquid into M9 culture medium containing 2% (w/v) of glucose solution or spirulina oligosaccharide solution, culturing for 24h at 37 ℃ under aerobic condition, counting on LB agar plate by dilution method, using distilled water as blank control, repeating the experiment at least three times, and the result is expressed as CFU mL-1.
Probiotic activity score ═ e (24h sample group probiotic logCFU mL-1-0h sample group probiotic logCFU mL-1)/(24h glucose group probiotic log CFU mL-1-0h glucose group probiotic log CFU mL-1) - (24h sample group escherichia coli logCFU mL-1-0h sample group escherichia coli logCFU mL-1)/(24h glucose group escherichia coli log CFU mL-1 h glucose group escherichia coli logCFU mL-1). As shown in Table 1, the yield of the spirulina oligosaccharide provided by the invention is high, reaches more than 80%, and the activity of promoting the proliferation of bifidobacteria and lactobacilli is better than that of spirulina polysaccharide and the oligosaccharide prepared by other methods.
TABLE 1 evaluation of probiotic Activity of Spirulina oligosaccharides
Note: different letters show significant differences between different rows, p <0.05.
Application example 3:
the spirulina oligosaccharide regulates the activity of intestinal flora in vitro: fresh excrement (20-30 years old, ingested normally, without digestive diseases, and antibiotics, probiotics and prebiotics are not taken for at least 3 months) is collected and mixed in equal quantity for 3 healthy volunteers, 10% (v/v) Dulbecco's PBS is used for dilution to obtain 20% (w/w) excrement mixed liquor, then high-speed homogenization is carried out, the homogenized culture solution is filtered by four layers of gauze, and the filtered solution is quickly transferred to an anaerobic tank. 10mL of sterilized spirulina oligosaccharide solution (50mg/mL) or sterile ultrapure water (blank control) were mixed with 45mL of fecal culture medium and 45mL of culture medium, sealed in an anaerobic fermentation tube, and cultured in a shaker at 37 ℃ for 24 h. Sampling at 0, 6,12 and 24 time points respectively, performing bioinformatics analysis by adopting a high-throughput sequencing method to obtain the regulation effect on the intestinal flora, and detecting the content of short-chain fatty acid by using GC-MS. The results are shown in FIGS. 5 and 6, and tables 2 and 3, and show that the spirulina oligosaccharides can well regulate the intestinal flora structure by increasing the diversity of intestinal microorganisms, increasing the abundance of microorganisms Bacteroides, Escherichia-Shigella, Megamonas, Bifidobacterium and Lactobacillus beneficial to the human body, and increasing the content of intestinal short-chain fatty acids, particularly isobutyric acid and isovaleric acid.
TABLE 2 alpha diversity comparison of microorganisms
Note: p <0.05 in comparison with blank control and p <0.05 in comparison with group C24, #
TABLE 3 SCFAs concentrations at different time points of in vitro fermentation
Note: there was a significant difference between the different letter representations in each column (p <0.05).
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Sequence listing
<110> Nanhai ocean institute of Chinese academy of sciences
<120> spirulina oligosaccharide and application thereof in preparation of preparation for regulating intestinal health
<130> ZM201392CS
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atggtaaatc ttaaagtttt attgccagtg attttgatga tcacaatatc caaagctcag 60
gaaaaatata aagatgttct gttgcctaaa gaacttactc agataggaga ttggaaagta 120
gacaagaact tgtcagatga ttttaactat acaaccaaaa acaaaaaatt ctttaaaaaa 180
tggaaggata gttataccaa tgattggaca ggacccgggt taagtcattt ttcttcaaat 240
cattctatat taaaagacgg aaaccttgaa ataaaagctg aacgaaaacc accaaataaa 300
gtgtattgtg gagtaatatc atctagaaaa gaagtaattt atccggctta tatggaaatt 360
aaaatgaaaa ttagtggttt gaaattgtca tctaattttt ggtttattag taaagatcag 420
gttcttgaaa ttgatgtaaa tgaaacatat ggcaatgaac ctgacagaag caaaaaaatg 480
gggacgaatt accatatttt tcagagaaca ccttttaaag atcttacacc taataacgga 540
aagcactata ctgcaaaggg agctccattc ctaaaagacc aatttcatcg ttttggttgc 600
cattggaaag atgcgtatca cgcagacttt tatcttgatg gaacattggt gcgacaactc 660
actatagaag accctagaac atctggtgtt ggttttaatc aagggttact tatggttata 720
gatacggaag atcatgattg gcgatctaaa aaagggatta ctccaaccga tgatgaactt 780
ttggatgaaa ccataaatac tatgtatgta gattgggtac gtgtgtataa acccaaataa 840
<210> 2
<211> 279
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<213> Marine bacteria
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Met Val Asn Leu Lys Val Leu Leu Pro Val Ile Leu Met Ile Thr Ile
1 5 10 15
Ser Lys Ala Gln Glu Lys Tyr Lys Asp Val Leu Leu Pro Lys Glu Leu
20 25 30
Thr Gln Ile Gly Asp Trp Lys Val Asp Lys Asn Leu Ser Asp Asp Phe
35 40 45
Asn Tyr Thr Thr Lys Asn Lys Lys Phe Phe Lys Lys Trp Lys Asp Ser
50 55 60
Tyr Thr Asn Asp Trp Thr Gly Pro Gly Leu Ser His Phe Ser Ser Asn
65 70 75 80
His Ser Ile Leu Lys Asp Gly Asn Leu Glu Ile Lys Ala Glu Arg Lys
85 90 95
Pro Pro Asn Lys Val Tyr Cys Gly Val Ile Ser Ser Arg Lys Glu Val
100 105 110
Ile Tyr Pro Ala Tyr Met Glu Ile Lys Met Lys Ile Ser Gly Leu Lys
115 120 125
Leu Ser Ser Asn Phe Trp Phe Ile Ser Lys Asp Gln Val Leu Glu Ile
130 135 140
Asp Val Asn Glu Thr Tyr Gly Asn Glu Pro Asp Arg Ser Lys Lys Met
145 150 155 160
Gly Thr Asn Tyr His Ile Phe Gln Arg Thr Pro Phe Lys Asp Leu Thr
165 170 175
Pro Asn Asn Gly Lys His Tyr Thr Ala Lys Gly Ala Pro Phe Leu Lys
180 185 190
Asp Gln Phe His Arg Phe Gly Cys His Trp Lys Asp Ala Tyr His Ala
195 200 205
Asp Phe Tyr Leu Asp Gly Thr Leu Val Arg Gln Leu Thr Ile Glu Asp
210 215 220
Pro Arg Thr Ser Gly Val Gly Phe Asn Gln Gly Leu Leu Met Val Ile
225 230 235 240
Asp Thr Glu Asp His Asp Trp Arg Ser Lys Lys Gly Ile Thr Pro Thr
245 250 255
Asp Asp Glu Leu Leu Asp Glu Thr Ile Asn Thr Met Tyr Val Asp Trp
260 265 270
Val Arg Val Tyr Lys Pro Lys
275
Claims (6)
1. A spirulina oligosaccharide, wherein the average molecular weight of the spirulina oligosaccharide is concentrated at 300-:
s1, adding water into spirulina powder, repeatedly freezing and thawing, and then ultrasonically extracting spirulina protein liquid;
s2, carrying out alcohol precipitation on the spirulina protein solution obtained in the step S1, adding papain for enzymolysis, and carrying out deproteinization and decoloration to obtain a spirulina polysaccharide solution;
s3, adding marine microorganism glycosidase into the spirulina polysaccharide solution obtained in the step S2, carrying out enzymolysis for 5-8h at 30-50 ℃, carrying out ultrafiltration on an enzymolysis solution through a 5kDa ultrafiltration membrane, concentrating a filtrate, and freeze-drying to obtain coarse spirulina oligosaccharide dry powder; the nucleotide sequence of the marine microorganism glycosidase is shown in SEQ ID NO: 1, and the amino acid sequence of the encoded zymoprotein is shown as SEQ ID NO: 2 is shown in the specification;
s4, passing the crude spirulina oligosaccharide dry powder obtained in the step S3 through an anion exchange column to obtain neutral spirulina oligosaccharide.
2. The spirulina oligosaccharide as claimed in claim 1, wherein the spirulina powder is added with 20-40 times of water volume in step S1, and after repeated freeze thawing for three times, the spirulina powder is reacted for 2-5h at 60-90 ℃ after ultrasonic treatment to obtain the spirulina protein liquid.
3. The spirulina oligosaccharide of claim 1, wherein step S2 is performed by adding 3-4 times volume of absolute ethanol for alcohol precipitation.
4. The spirulina oligosaccharide of claim 1, wherein the marine microorganism glycosidase is added at 500-2000U/g in step S3.
5. The spirulina oligosaccharide as claimed in claim 1, wherein in step S2, 1-2% of papain is added for enzymolysis and deproteinization, and 1-1.5% of activated carbon is used for decolorization.
6. Use of the spirulina platensis oligosaccharide of claim 1 in the preparation of a health food or health care product for regulating the function of intestinal bacteria.
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