CN100372542C - Use of algin oligocauharide as prebiotics - Google Patents
Use of algin oligocauharide as prebiotics Download PDFInfo
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- CN100372542C CN100372542C CNB2004100239269A CN200410023926A CN100372542C CN 100372542 C CN100372542 C CN 100372542C CN B2004100239269 A CNB2004100239269 A CN B2004100239269A CN 200410023926 A CN200410023926 A CN 200410023926A CN 100372542 C CN100372542 C CN 100372542C
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Abstract
The present invention uses an algin oligosaccharide as a prebiotic. Animal tests and human body tests prove that the algin oligosaccharide is a non-digestibility oligosaccharide which can obviously promote the growth of intestinal tract probiotics, such as bacillus bifidus, lactobacillus and the like, inhibit the growth of harmful bacteria, such as pathogenic bacteria, putrefaction bacteria and the like, perform the functions for improving the microecology environment of the intestinal tract and purifying the intestinal tract, and increase the immunizing power of a human body, improve the endocrine system and promote the absorption of nutritive substances. Thereby, the subhealth state of the human body is improved, and the algin oligosaccharide has the functions for preventing and treating tumors, enteric infection and the like, prolonging life, beautifying the face, etc. Thus, the algin oligosaccharide is a novel prebiotic, medicine, health product, food additive or feed supplement.
Description
Technical field:
The present invention relates to the application of algin oligosaccharide as prebiotics.
Background technology:
Prebiotics (Prebiotic) is not by host's pipe intestinal digesting, can optionally stimulate one or more probioticss to grow in host's intestinal, and then promotes the material of host health.Nineteen ninety-five Gibson proposes the notion of prebiotics first, and now prebiotics has obtained extensive studies and application as microecologic regulator a kind of.Prebiotics can significantly promote beneficial bacteria of intestinal tract propagation such as bacillus bifidus, and these a large amount of lactic acid of generation in the process of self-reproduction of bacillus bifidus, lactic acid can stimulate enterokinesia, discharges harmful substance in the body, promotes the absorption to calcium ion of vitamin B1, B2, B6 and human body.Prebiotics suppresses the growth of harmful bacterium such as pathogen, putrefaction bacteria simultaneously, reduces the generation of harmful substance in the intestinal.By the microenvironment of above-mentioned improved effect intestinal, so the two-ways regulation function of human body, improve body immunity, improve hormonal system, promote the absorption of nutrient substance, improve human body sub-health status thereby reach, play effects such as life lengthening, beauty treatment; And can prevent the generation of diseases such as curing oncoma, intestinal infection, safeguard health.But have not yet to see the report of algin oligosaccharide as prebiotics.
Summary of the invention:
The objective of the invention is algin oligosaccharide is used as prebiotics, it can remedy the above-mentioned deficiency of prior art.
Algin oligosaccharide is as prebiotics.
Animal and human's body evidence algin oligosaccharide is a kind of non-digestible oligosaccharide, can significantly promote the growth of beneficial bacteria of intestinal tract such as bacillus bifidus and lactobacillus, suppress the growth of harmful bacterium such as pathogen, putrefaction bacteria, played the effect that improves the intestinal microecology environment and purify intestinal, can improve body immunity, improve hormonal system, promote the absorption of nutrient substance, improve human body sub-health status thereby reach, bring into play effects such as diseases such as preventing curing oncoma, intestinal infection and life lengthening, beauty treatment.Therefore, algin oligosaccharide is a kind of novel prebiotics, and it is a kind of new medicine, health product, food additive or feed additive.
The specific embodiment:
(1) preparation of algin oligosaccharide:
Application number prepares the method for algin oligosaccharide for a kind of chemical oxidization method is provided in 02135571.1 the Chinese patent.Application number prepares the method for algin oligosaccharide for a kind of acid-hydrolysis method is provided in 01107952.5 the Chinese patent.
The invention provides the method that a kind of enzymatic isolation method prepares algin oligosaccharide, the molecular structure of Zhi Bei algin oligosaccharide is compared with the structure of the algin oligosaccharide of above-mentioned two kinds of methods preparation in this way, main chain all is by α-L-guluronic acid and two kinds of sugar units of beta-D-mannuronic acid continuously or the linear molecule that alternately is formed by connecting, mean molecule quantity is in the 500-2000 scope, the degree of polymerization is in the scope of 2-10, and difference is to have unsaturated double-bond between 5 in 4 in the carbon of its reduction end and the carbon.
The step that enzymatic isolation method prepares algin oligosaccharide is:
At first extract alginate lyase (seeing that application number is 03112273.6 patent), again it being joined 1 liter weight percent concentration according to the ratio of 10-50U is in the alginate aqueous solution of 0.5%-1%, carry out enzymolysis, hydrolysis temperature is 25-38 ℃, time is 6-8 hour, by centrifugal or remove by filter undegradable macromole Algin, carries out chromatography with Sephadex G25 gel column at last, collect the algin oligosaccharide component, concentrate drying.
(2) prebiotic function of algin oligosaccharide (to the regulating action in the intestinal microbial population):
(1) zoopery:
1. experimental animal and grouping: select healthy Kunming mouse for use, male and female half and half, body weight 18-20g purchases the moving institute that examines in Qingdao.Method by random packet is divided into six groups with mice, 8 every group.First group (A group) is the blank group, second group (B group) is the soybean oligo saccharide positive controls, the 3rd group (C group) is Algin administration group, the 4th group (D group) is low dosage algin oligosaccharide administration group, the 5th group (E group) is middle dosage algin oligosaccharide administration group, and the 6th group (F group) is high dose algin oligosaccharide administration group.
2. dosage and method: Algin is the commercially available prod.Soybean oligo saccharide is a colourless transparent liquid, is produced by Linyi, Shandong illawarra mountain pine biological product company limited.The amount that B group is irritated the stomach soybean oligo saccharide is: 3.33 milliliters/(kg body weight) (be equivalent to Coming-of-Age Day that manufacturer recommends take 20 times of suggestion amount).The C group is irritated the alginate aqueous solution of stomach 0.3% (percentage by weight).The D group is irritated the algin oligosaccharide of stomach 15.0-18.7mg/ml.The E group is irritated the algin oligosaccharide of stomach 20.0-37.5mg/ml.The F group is irritated the algin oligosaccharide of stomach 50.0-75.0mg/ml.C, D, E, F group are irritated the gastric liquid total amount by 0.02 milliliter/(gram body weight) calculating.The A group is irritated the distilled water of stomach equivalent.Each organized the continuous irrigation stomach 14 days, irritated that stomach is intact gets the analysis of accounts that mice caecum inclusions and feces carry out intestinal.
3. culture medium: bacillus bifidus adopts BL culture medium or BLb culture medium; Lactobacillus adopts the LBs culture medium; Enterococcus is adopted cholate one Esculin one sodium azide culture medium; Bacteroid is adopted the Bds culture medium; Enterobacteria is adopted the EMB culture medium; The fecal specimens diluent adopts thioglycollate medium; Caecum inclusions diluent adopts KH
2PO
44.5g, Na
2HPO
16.0g, L-cysteine hydrochloride 0.5g, tween 80 0.5ml, agar 1g, adding distil water is diluted to 1000ml, 115 ℃ of sterilization 20min.
4. the obtaining and measuring of mice test material: get stool in mice 0.2-0.4g, add thioglycollate medium, gradient dilution, making its weight percent concentration is 10 of stock solution
-1, 10
-2, 10
-3, 10
-1, 10
-5, 10
-6, 10
-8, 10
-9, to choose suitable dilution factor and be applied on the various selective mediums, each dilution factor is made three flat boards, count (viable count/gram stool sample) after the cultivation, average, select the characteristic bacterium colony simultaneously, carry out form and biochemical SHAPE DETECTION and identify, determine its Pseudomonas.EMB culture medium wherein, 37 ℃ of cholate seven-leaf glycosides-sodium azide culture medium, aerobic cultivation 24-48 hour, all the other anaerobe anaerobism were cultivated 48-72 hour.The result is as shown in table 1.
The caecum inclusions is washed out with diluent, gradient dilution, making its weight percent concentration is 10 of stock solution
-1, 10
-2, 10
-3, 10
-1, 10
-5, 10
-6, 10
-8, 10
-9, to choose suitable dilution factor and be applied on the various selective mediums, each dilution factor is made three flat boards, count (viable count/gram stool sample) after the cultivation, average, select the characteristic bacterium colony simultaneously, carry out form and biochemical SHAPE DETECTION and identify, determine its Pseudomonas.EMB culture medium wherein, 37 ℃ of cholate-Esculin-sodium azide culture medium, aerobic cultivation 24-48 hour, all the other anaerobe anaerobism were cultivated 48-72 hour.The result is as shown in table 2.
Table 1: the various intestinal microbial population quantity of mice testing result behind the algin oligosaccharide continuous irrigation stomach (
N=8)
| Bacillus bifidus | Lactobacillus | Escherichia coli | Enterococcus | Bacteroid | ||
| The A group | Before irritating stomach | 7.40±0.10 | 7.85±0.63 | 7.91±1.13 | 5.58±1.02 | 7.86±0.58 |
| After irritating stomach | 7.50±0.34 | 7.95±0.29 | 7.82±0.86 | 5.60±0.41 | 7.84±0.16 | |
| The B group | Before irritating stomach | 7.58±0.38 | 7.89±0.30 | 7.89±0.88 | 5.11±0.75 | 7.74±0.29 |
| After irritating stomach | 7.94±0.16 | 8.54±0.42 | 7.80±0.11 | 5.42±0.23 | 7.92±0.25 | |
| The C group | Before irritating stomach | 7.57±0.25 | 7.85±0.15 | 7.88±0.76 | 5.45±0.59 | 7.73±0.13 |
| After irritating stomach | 7.66±0.12 | 8.58±0.43 | 7.79±0.13 | 5.52±0.65 | 7.85±0.45 | |
| The D group | Before irritating stomach | 7.41+0.76 | 7.83±0.38 | 7.90±0.75 | 5.07±0.18 | 7.86±0.42 |
| After irritating stomach | 8.61±0.35 1.3 | 8.62±0.55 2.3 | 7.58±0.21 1.3 | 5.65±0.43 | 7.47±0.42 1.3 | |
| The E group | Before irritating stomach | 7.52±0.31 | 7.86±0.60 | 7.91±0.30 | 5.40±0.21 | 7.89±0.24 |
| After irritating stomach | 8.43±1.12 | 8.48±0.20 2.3 | 7.67±0.38 | 5.69±0.85 | 7.74±0.28 | |
| The F group | Before irritating stomach | 7.48±0.65 | 7.84±0.25 | 7.89±0.82 | 5.26±0.20 | 7.64±0.29 |
| After irritating stomach | 8.53±0.11 1 | 8.33±0.14 | 7.94±0.32 | 5.31±0.55 | 7.84±0.14 | |
Table 2: mice caecum flora quantity testing result behind the brown alga oligose continuous irrigation stomach (
N=8)
| The A group | The B group | The C group | The D group | The E group | The F group | |
| Bacillus bifidus | 7.18± 0.22 | 7.48± 0.19 | 7.32± 1.07 | 7.7± 1.25 * | 7.65± 0.34 | 7.38± 1.13 |
| Lactobacillus | 8.12± 0.29 | 8.38± 0.18 | 8.34± 0.22 | 8.52± 0.25 | 8.5± 0.16 * | 8.43± 0.17 |
| Escherichia coli | 7.65± 0.35 | 7.72± 0.27 | 7.26± 0.31 | 6.8± 0.91 * | 7.03± 0.64 | 7.18± 0.28 |
| Enterococcus | 4.58± 0.71 | 5.11± 0.59 | 4.91± 0.77 | 5.07± 1.05 | 5.40± 0.92 | 5.26± 0.84 |
| Bacteroid | 7.75± 0.33 | 7.81± 0.27 | 7.70± 0.30 | 7.3± 0.49 * | 7.4± 0.38 * | 7.42± 0.18 |
Annotate:
*Compare (P＜0.05) with matched group
5.. experimental result:
The form of bacillus bifidus and biochemical characteristic: forming diameter on the BL flat board after cultivating in 48-72 hour is projection, the neat in edge of 0.5～1mm, becomes milky opaque colony; Microscopy is G
+, be forms such as Y-shaped, V-shape, bending.
Biochemical reaction: catalase test (-), indole test (-), the real test of gelatin liquefaction (-), nitrate reduction test (-), urease test (-).
Table 1 is the various intestinal microbial population quantity of a mice testing result behind the algin oligosaccharide continuous irrigation stomach, and table 2 is mice caecum flora quantity testing results behind the algin oligosaccharide continuous irrigation stomach.Numerical value in the table is the logarithm value of viable count/gram stool sample.Method of counting is: the bacterial population CFUg of every 1g feces
-1=clump count * 10
n* 40 (10
nFor with the corresponding dilution factor of bacterium colony; 40 is conversion constant), the result is with the logarithm value log CFUg of the bacterial clump in every gram feces
-1Expression.In table 1 and the table 2 listed numerical value be with average (
) be expressed as with standard deviation (s)
N is the quantity of each group mice, n=8.
The expression of the upper right side label " 1 " of some numerical value is compared (P＜0.05) with own control in table 1 and the table 2; Label " 2 " expression is compared (P＜0.01) with own control; Label " 3 " expression is compared (P＜0.05) with matched group.P is a significant difference, adopts SPSS 10.0 for Windows statistical softwares to draw.P＜0.01 is that difference is very remarkable, and P＜0.05 is a significant difference, P〉0.05 there is not significance for difference.
By table 1 as seen, after per os gave the mice algin oligosaccharide, 5 kinds of bacterial numbers in the mouse intestinal changed.Soybean oligo saccharide positive controls (B group) is compared with blank group (A group), and the quantity of bacillus bifidus has increase trend, and difference does not have significance (P〉0.05), and the quantity of lactobacillus obviously increases, significant difference (P＜0.05); Separately Algin administration group (C group) is compared with blank group (A group), and the quantity of bacillus bifidus has increase trend, and difference does not have significance (P〉0.05), and the quantity of lactobacillus obviously increases, significant difference (P＜0.05); Low dosage algin oligosaccharide administration group (D group) is compared with blank group (A group), the quantity of bacillus bifidus obviously increases (P＜0.05), the quantity of lactobacillus significantly increases (P＜0.05), and the quantity of escherichia coli and bacteroid obviously reduces (P＜0.05).Middle dosage algin oligosaccharide administration group (E group) is compared with blank group (A group), the quantity of bacillus bifidus significantly increases (P＜0.05), the quantity of lactobacillus significantly increases (P＜0.05), high dose algin oligosaccharide administration group (F group) is compared with blank group (A group), the quantity of bacillus bifidus obviously increases (P＜0.05), the quantity of lactobacillus has increase trend, and difference does not have significance (P〉0.05).
The quantity of B group bacillus bifidus has increased by 2.29 times, and the quantity of lactobacillus has increased by 5.25 times.
The quantity of C group bacillus bifidus has increased by 1.23 times, and the quantity of lactobacillus has increased by 5.37 times.
The quantity of D group bacillus bifidus has increased by 15.85 times, and the quantity of lactobacillus has increased by 6.16 times.
The quantity of E group bacillus bifidus has increased by 7.58 times, and the quantity of lactobacillus has increased by 4.17 times.
The quantity of F group bacillus bifidus has increased by 11.22 times, and the quantity of lactobacillus has increased by 3.09 times.
The B group is lower than the D group to the increase of bacillus bifidus quantity more as can be known through above, that is, algin oligosaccharide is better than soy oligosaccharide to the quantity increase degree of bacillus bifidus and lactobacillus.The increase of bacillus bifidus and lactobacillus quantity is not because the reason of Algin polysaccharide.
By table 2 as seen, after per os gave the mice algin oligosaccharide, 5 kinds of bacterial numbers in the mice caecum changed.Compare with matched group, the quantity of D group (low dosage algin oligosaccharide administration group) bacillus bifidus obviously increases (P＜0.05), and the quantity of lactobacillus significantly increases (P＜0.05), and the quantity of escherichia coli and bacteroid obviously reduces (P＜0.05).The quantity of E group (middle dosage algin oligosaccharide administration group) bacillus bifidus increases (P＜0.05), and the quantity of lactobacillus significantly increases (P＜0.05).In comparison sheet data as can be known, B group is lower than the D group to the increase of bacillus bifidus quantity, that is, algin oligosaccharide is better than soy oligosaccharide to the quantity increase degree of bacillus bifidus.
In summary, algin oligosaccharide is not by host's pipe intestinal digesting, and optionally these the two kinds of probioticss of stimulation of bifidobacteria and lactobacillus are grown in host's intestinal, suppresses the growth of escherichia coli and bacteroid, and then the enhancement host health, be a kind of novel prebiotics.The effective dose that the algin oligosaccharide prebiotics is used to regulate intestinal microbial population is 15.0-75.0mg/ml, is optimum with the dosage range of 15.0-30.0mg/ml.The effect that the quantity of bacillus bifidus is increased is better than commercially available soybean oligo saccharide.
(2) experimenter's experiment:
The experimenter is a blood, urine, just routine examination and liver function test, heart rate, blood pressure, Electrocardioscopy, 15 of all normal volunteers of index (7 of men, 8 of woman), experimenter's mean age (42.3 ± 7.5) year, recent no obvious endocrine metabolism illness, do not used antibiotic etc, the duration of test normal diet does not have other lactobacillus beverages and uses.
Experimental technique is identical with zoopery, and the experimenter is preceding respectively at taking, take after 7 days and after taking 14 days, get the analysis that feces carries out intestinal microbial population.The experimenter takes and is tried thing after 7 days, the quantity of bacillus bifidus and lactobacillus obviously increases, and has increased 5-7 before the bacillus bifidus ratio is taken doubly, has increased 4.5-6 before the lactobacillus ratio is taken doubly, increase by 10 times before indivedual experimenter's bacillus bifidus ratios are taken, increased by 11 times before the lactobacillus ratio is taken.The quantity of escherichia coli and bacteroid obviously reduces, and has reduced 2-4 before the escherichia coli ratio is taken doubly, has reduced 3-4 before the bacteroid ratio is taken doubly, enterococcal quantity does not have significant change, and 4 people's constipation situations make moderate progress, and show as the defecation rule, the feces character is normal, and defecation is unobstructed.The experimenter has increased 6.5-7.5 doubly before taking and being tried thing the bacillus bifidus ratio is taken after 14 days, before taking, the lactobacillus ratio increased 5-6 doubly, reduced 2-4 before the escherichia coli ratio is taken doubly, reduced 3-4 before the bacteroid ratio is taken doubly, enterococcal quantity does not have significant change.3 people have appetite to increase phenomenon, and 2 people's aerofluxuss increase to some extent.Do not have diarrhoea, no colic symptoms occurs.Diet, sleep and the mental status are all good.
Used algin oligosaccharide is the algin oligosaccharide of enzymatic isolation method preparation in the present embodiment, when in above-mentioned experiment, it being used instead the algin oligosaccharide of the algin oligosaccharide of chemical oxidization method preparation and acid-hydrolysis method preparation, its experimental technique is identical with the algin oligosaccharide of enzymatic isolation method preparation, effect is also approximate, no longer repeats at this.The three all has optionally stimulation of bifidobacteria and these two kinds of probioticss of lactobacillus and grows in host's intestinal, suppresses the growth of escherichia coli and bacteroid, and then the effect of enhancement host health, is a kind of novel prebiotics.
The dosage of algin oligosaccharide of the present invention when the medicine is 5mg-10mg, and every day 2 times is oral; Dosage when the health product is 2.5mg-5mg, and every day 2 times is oral; Percetage by weight during as food additive is 0.1%-0.8%; Percetage by weight during as feed additive is 0.5%-1%.Described food is yoghourt, milk powder; Described feedstuff is chicken feedstuff, pig feed, cattle feedstuff.These four kinds with just obviously taking effect by way of 7-14 days.
Claims (2)
1. the application of algin oligosaccharide in the preparation prebiotics, it is characterized in that, described algin oligosaccharide has following feature: main chain is by α-L-guluronic acid and two kinds of sugar units of beta-D-mannuronic acid continuously or the linear molecule that alternately is formed by connecting, mean molecule quantity is in the 500-2000 scope, there is unsaturated double-bond in the degree of polymerization between 5 in 4 in carbon of its reduction end and the carbon in the scope of 2-10.
2. algin oligosaccharide is as feed additive, it is characterized in that, described algin oligosaccharide has following feature: main chain is by α-L-guluronic acid and two kinds of sugar units of beta-D-mannuronic acid continuously or the linear molecule that alternately is formed by connecting, mean molecule quantity is in the 500-2000 scope, there is unsaturated double-bond in the degree of polymerization between 5 in 4 in carbon of its reduction end and the carbon in the scope of 2-10.
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| CN104095183B (en) * | 2014-06-30 | 2016-05-18 | 中国科学院海洋研究所 | A kind of application of algal polysaccharides |
| GB201517639D0 (en) | 2015-10-06 | 2015-11-18 | Algipharma As | Use of alginate oligomers to treat or prevent microbial overgrowth in the intestinal tract |
| CN106011197A (en) * | 2016-05-20 | 2016-10-12 | 浙江省农业科学院 | Microorganism-derived algin and preparation and application thereof |
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| CN113397170B (en) * | 2021-06-02 | 2023-02-24 | 青岛海洋生物医药研究院股份有限公司 | Application of marine prebiotics composition for regulating human intestinal flora |
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