CN101416681B - Selenium-rich Enterococcus faecium cernelle three-layer microcapsule and preparation method and use thereof - Google Patents
Selenium-rich Enterococcus faecium cernelle three-layer microcapsule and preparation method and use thereof Download PDFInfo
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Abstract
The invention provides a selenium-enriched lactic acid enterococcus faecalis three-layer microcapsule and a preparation method thereof. The microcapsule sequentially comprises a selenium-enriched lactic acid enterococcus faecalis, nutritive layer, an anti-acid anoxybiotic layer and a compression-resisting and breakage-proof layer from the inside to the outside. Wherein culture conditions of the selenium-enriched lactic acid enterococcus faecalis are 25-35 mg/l of selenium, 5-15 g/l of inoculum size, 28-32 h of fermentation time and 4.5-6.3 of pH when fermenting in an anaerobic jar; based on a double-layer embedding, the embedding method of the three-layer microcapsule is mainly mixing the three-layer microcapsule together with 2.0-2.5% of glutin aqueous solution and 0.1-0.3% of sodium alginate aqueous solution to make a suspension; an improved low-temperature spray drying method is adopted to form the compression-resisting and breakage-proof layer and to make the embedded selenium-enriched lactic acid enterococcus faecalis three-layer microcapsule have gastric acid-resisting, storage wearing, thermal stabilizing and compression-resisting and breakage-proof functions based on theoriginal biological function and organic selenium supplementing function. So the microcapsule is widely applied to producing feeds.
Description
Technical field
The present invention relates to a kind of selenium-rich Enterococcus faecium cernelle three-layer microcapsule and its production and application, be used for livestock and poultry and wildlife and culture the supply of organoselenium and prevent digestive tract diseases, improve the balance of intestinal microecology, belong to the microbial forage additive field.
Background technology
The lactic acid bacteria class biological fermentation goods that in feed, add, mainly contain bifidus bacillus (bifidobacterium breve, bifidus longum bb, bifidumbacterium bifidum and bifidobacterium adolescentis), milk-acid bacteria (Lactobacterium acidophilum and class lactobacillus johnsonii), faecalis (urine enterococcus and enterococcus faecalis) etc.1. its mechanism of action is mainly reflected in keeps archenteric flora normalizing, keeps the superiority of beneficial bacteria flora; 2. regulate the animal digestion metabolism; 3. prevent to produce harmful product, strengthen as intestinal bacteria in the intestines are movable, thereby cause protein transduction to turn to ammonia and amine, the both has pungency and toxicity.The lactic acid bacteria class additive of feeding can make the ammonia amount in enteron aisle, ight soil and the portal vein blood reduce, stop in the enteron aisle bacterium produce amine and in and enterogenous endotoxin etc.
Rich selenium lactic acid enterococcus faecalis is to be carrier with the lactic acid enterococcus faecalis, inorganic selenium is converted into the biological fermentation product of organoselenium.Under the situation of strictness control sulphur concentration, be electron donor in the fermentation, utilize luminous energy and heat energy to be power, assimilation small molecular organic acid and CO with selenium
2Etc. a series of biochemical fermentation processes, inorganic selenium is transformed into (sulfur-containing amino acid) on the organic macromolecule, have the big and high characteristics of inorganic selenium transformation efficiency of biosynthesizing output, be formed with organoselenium than high biological activity and low toxicity.At present, adopt the anaerobic fermentation tank fermentation process, the rich selenium cultivation and fermentation of lactic acid enterococcus faecalis be yet there are no report.
Rich selenium lactic acid enterococcus faecalis has the unique biological effect; the function of the biological function of organoselenium and lactic acid enterococcus faecalis is combined, reach the 26S Proteasome Structure and Function, the defence peroxidation that excite animal body immune globulin bletilla production of antibodies, protection cytolemma and macromole physiologically active substances such as karyomit(e) and Yeast Nucleic Acid and avoid the effect of the infringement of superoxide to safeguard human body.Hence one can see that, rich selenium lactic acid enterococcus faecalis preparation is a kind of more satisfactory nutrient feed additive, be mainly used in prevention digestive tract diseases, relevant with selenium deficiency more than 40 kind of disease and as a supplement organic selenium source fodder additives and be with a wide range of applications.
At present, lactic acid bacteria class is generally taked the biological embedding techniques (Chen Jiankai of bilayered microcapsule, Weng Wen, milk-acid bacteria double-layer embedment technique and the bioactive research of embedding bacterium, northwest agricultural journal, 2005,14 (6): 191-194), to prevent that extraneous adverse environmental factors such as thalline and oxygen from contacting, better at performance beneficial microorganism biological action.By the micro encapsulation embedding can make product store and food processing process in, prevent that thalline from being destroyed by high temperature and improve number of viable, bilayered microcapsule biology embedding techniques can also improve the destruction function of thalline stomach juice-resistant and cholic acid and be applied in the pharmaceutical products.Common microcapsule embedded technology mainly adopt methods such as interfacial polymerization, complex coacervation, orifice coacervation, spray-drying process (Li Ying, the application of microcapsulary and wall material commonly used [J] thereof. processing of farm products .2008,01.).But microcapsule double-layer embedment technique in the past is only applicable to human food prods and pharmaceutical products, as be applied in the production of sour milk, cheese etc., can be by its stomach juice-resistant of microcapsule embedded realization and the stable effect of heat resistanceheat resistant, but can not be useful for the process of manufacture of preparation animal pellet feed.Its obvious defects is because the extruding and the high temperature of feed processing, can make the capsule fragmentation behind the double-layer embedment, and microorganism is heated and loses biological activity.
At present, and the existing report of feeding probiotic bacterium three microcapsule embedding techniqueses (Harbin Mei Hua biotech company, embedding of probiotic bacterium multilayer and intestinal-specific field planting technology [Z] .2007,10.).The beneficial microorganism that is adopted is divergent bifidus bacillus, Lactobacterium acidophilum, thermophilus streptococcus, lactobacillus delbruockii subspecies bulgaricus and yeast saccharomyces cerevisiae, the first layer protective layer of its probiotic bacterium three microcapsules forms primary microcapsules for the protein after adopting trans-glutaminases crosslinked with the probiotic bacterium embedding; Second layer protective layer be with primary microcapsules with after the suitable dilution agent mixes, coat at 30 ℃～40 ℃ hydrogenated oil and fat with fusing point and to form secondary microcapsule; The three-layer protection layer is a release controlling coating material.This three microcapsule embeddings be for the problem that solves target and sustained-release and controlled release to be applied among the human food prods; its three-layer protection layer still can not solve anti-extrusion and pyritous problem in the animal-feed course of processing; the controlled release wall material that is adopted costs an arm and a leg; the compressive property that forms the 3rd layer of embedding cyst wall is little, heat resistance is poor; exist the defective of pressurized friability, can not be applied in the animal pellet feed production.
We adopt natural polymer colloidal solution is the equal-volume mixing solutions of aqueous gelatin solution and sodium alginate aqueous solution, high density aqueous gelatin solution and lower concentration sodium alginate aqueous solution or high density sodium alginate aqueous solution and lower concentration aqueous gelatin solution carry out mutual mix and match in the colloid concentration scope that sets, utilize the good characteristics of support property of the good and gelatin of the film forming properties of sodium alginate, solved the breakable problem of microcapsule resistance to compression.
Summary of the invention
In order to overcome the defective of above-mentioned bilayer and three microcapsules and embedding method thereof, the object of the present invention is to provide a kind of selenium-rich Enterococcus faecium cernelle three-layer microcapsule and preparation method thereof, by the selenium-rich Enterococcus faecium cernelle three-layer microcapsule embedding, make rich selenium lactic acid enterococcus faecalis microcapsule after the embedding on basis, have the function of anti-hydrochloric acid in gastric juice, anti-storage, thermally-stabilised and resistance to compression anti-crushing concurrently, so that selenium-rich Enterococcus faecium cernelle three-layer microcapsule is applied in fodder production with original biological function and additional organoselenium function.
For achieving the above object, the invention provides a kind of selenium-rich Enterococcus faecium cernelle three-layer microcapsule, comprise rich selenium lactic acid enterococcus faecalis, nutritive layer, antiacid non-oxygen shaft floor and resistance to compression spall shield from inside to outside successively, described nutritive layer is the sterilization skimmed milk, described antiacid non-oxygen shaft floor is kappa-carrageenan and Viscogum BE, and described resistance to compression spall shield is the natural polymer colloid.
Described natural polymer colloid can be in gelatin, gum arabic or the sodium alginate at least a.Be preferably the mixture of gelatin and sodium alginate.
The present invention also provides a kind of method for preparing described selenium-rich Enterococcus faecium cernelle three-layer microcapsule, comprises the cultivation and the selenium-rich Enterococcus faecium cernelle three-layer microcapsule embedding of rich selenium lactic acid enterococcus faecalis; The zymotechnique of wherein said rich selenium lactic acid enterococcus faecalis is: fermented liquid-sterilization-cooling-inoculation (adds lactic acid enterococcus faecalis kind and Na
2SeO
3)-fermentation culture-check-rich selenium lactic acid enterococcus faecalis product.
Lactic acid enterococcus faecalis kind in the preferred embodiment of the present invention is provided by Institute of Microbiology, Heilongjiang Academy of Sciences.
The culture condition of described rich selenium lactic acid enterococcus faecalis is: described fermented liquid can be the fermented liquid commonly used or the substratum of any lactic acid enterococcus faecalis; The employing ventilation anaerobic jar fermentation of bleeding, selenium addition 25～35mg/L, inoculum size 5～15g/L, fermentation time 28～32h, pH4.5～6.3.The fermentation culture of selecting for use in the preferred embodiment of the present invention is: peptone 10.0g, extractum carnis 10.0g, yeast extract 5.0g, glucose 20.0g, K
2HPO
42.0g, tween 80 1ml, Triammonium citrate 2.0g, sodium acetate 5.0g, MgSO
47H
2O0.58g, MnSO
44H
2O0.25g, distilled water 1L; Selenium addition in the preferred embodiment of the present invention is that 35mg/L, inoculum size are that 10g/L, fermentation time are that 28h, pH value are 4.5～6.3.
Described selenium-rich Enterococcus faecium cernelle three-layer microcapsule embedding may further comprise the steps:
I layer (nutritive layer) embedding: rich selenium lactic acid enterococcus faecalis is mixed with the sterilization skimmed milk, make 40%～60% (w/v) thalline skimmed milk suspension, spray is (aqueous solution of the present invention is the aqueous solution that distilled water is mixed with) in the aseptic aqueous hydrochloric acid of pH4, forms the thalli granule of skimmed milk embedding;
II layer (non-oxygen shaft floor) embedding: the thalli granule of skimmed milk embedding and 0.02%～0.03% (w/v) the kappa-carrageenan aqueous solution and 0.20%～0.25% (w/v) Viscogum BE aqueous solution (two kinds of colloid aqueous solution equal-volumes mix), make suspension (40%～60% (w/v) thalli granule colloidal suspension), spray forms embedded particles in 8 ℃～10 ℃ aseptic cold water;
III layer (resistance to compression spall shield) embedding: the small-particle of II layer embedding is mixed with the natural polymer colloid aqueous solution, make suspension (50%～70% (w/v) thalline colloidal suspension), adopt spray-dired low temperature to improve one's methods, obtain three microcapsule finished products.
Natural polymer colloid aqueous solution described in the embedding of described III layer is at least a in aqueous gelatin solution, the gum arabic aqueous solution or the sodium alginate aqueous solution.Preferred concentration is the aqueous gelatin solution of 2.0%～2.5% (w/v) and the arbitrary proportion mixing solutions of the sodium alginate aqueous solution that concentration is 0.1%～0.3% (w/v), and its high density aqueous gelatin solution and lower concentration sodium alginate aqueous solution or high density sodium alginate aqueous solution and lower concentration aqueous gelatin solution in described concentration range carry out mutual mix and match.More preferably concentration is the aqueous gelatin solution of 2.5% (w/v) and the equal-volume mixing solutions of the sodium alginate aqueous solution that concentration is 0.1% (w/v).Gelatin and sodium alginate aqueous solution are all through 115 ℃ of sterilization 15min.
Described spraying drying can adopt any spray-drier, for example the DS-1000 spray-drier; 95 ℃～105 ℃ of the dry inlet temperature of the spray drying method in low temperature after the improvement, sample size 180L/h～360L/h, 50 ℃～55 ℃ of temperature outs.
The present invention also provides the application of described selenium-rich Enterococcus faecium cernelle three-layer microcapsule, it is added in the animal-feed or as the animal health product as fodder additives directly feed.Granulated feed is produced: add rich selenium lactic acid enterococcus faecalis capsule 60～120g/100kg feed in feedstuff raw material; Directly feed: in perfect compound feed, add rich selenium lactic acid enterococcus faecalis capsule 50～100g/100kg perfect compound feed and directly feed together, or separately with the amount of rich selenium lactic acid enterococcus faecalis capsule by 0.2～0.4g/kg body weight, after serveing on 31 time every day, alternate-day feeding is 1 time again, to play the animal health effect.
The outstanding advantage that the present invention is compared with prior art had is: 1. selenium-rich Enterococcus faecium cernelle three-layer microcapsule of the present invention has overcome the defective of double-deck in the past and three microcapsules, in the feed granulating course of processing, be difficult for being extruded broken and can tolerate the moment high temperature of granulating and producing, have anti-hydrochloric acid in gastric juice, anti-storage, characteristics such as thermally-stabilised, anti-extrusion; 2. lactic acid enterococcus faecalis behind the rich selenium is carried out three microcapsule embeddings, remedied double-layer embedment technique and can not be used for the difficult problem that granulated feed is produced, and the microorganism of embedding can be used in the animal-feed processing widely; 3. the preparation method is simple, and used wall material price is cheap and easy to get, is convenient to biological fermentation and operation, greatly reduces cost; 4. the present invention uses in the animal-feed processing, has supply animal organoselenium, prevents digestive tract diseases, improves the balance biological function of intestinal microecology.
Embodiment
1, the cultivation of rich selenium lactic acid enterococcus faecalis
Zymotechnique: fermented liquid-sterilization-cooling-inoculation (adds the lactic acid enterococcus faecalis kind of 10g/L and the Na of 35mg/L
2SeO
3)-fermentation culture-check-rich selenium lactic acid enterococcus faecalis product.
Lactic acid enterococcus faecalis kind is provided by Institute of Microbiology, Heilongjiang Academy of Sciences.
Cultivation and fermentation condition: fermentation culture: peptone 10.0g, extractum carnis 10.0g, yeast extract 5.0g, glucose 20.0g, K
2HPO
42.0g, tween 80 1ml, Triammonium citrate 2.0g, sodium acetate 5.0g, MgSO
47H
2O0.58g, MnSO
44H
2O0.25g, distilled water 1L; The employing anaerobic jar is cultivated, selenium addition 35mg/L, inoculum size 10g/L, fermentation time 28h, pH value 4.5～6.3.
The thalline selenium content unit mass of the rich selenium lactic acid of gained enterococcus faecalis is 0.615mg/g.
2, selenium-rich Enterococcus faecium cernelle three-layer microcapsule embedding techniques
Nutritive layer embedding (I layer): rich selenium lactic acid enterococcus faecalis mixes with the sterilization skimmed milk, and------fine needle spray sample injector---splashes in the aseptic hydrochloric acid distilled water solution of pH4---static 10min---collection small-particle---the sterile solution repetitive scrubbing several of pH4 to make suspension (50% thalline skimmed milk suspension);
Antiacid non-oxygen shaft floor embedding (II layer): the small-particle of I layer embedding is mixed (two kinds of colloid aqueous solution equal-volumes mix)------large needle spray sample injector (stirring)---splashes in 10 ℃ of aseptic cold water---collecting small-particle---with 10 ℃ of aseptic cold water repetitive scrubbings for several times to make thalline packaging material mixed solution (50% thalline colloidal suspension) with 0.03%K-carrageenin sterile water solution and 0.20% Viscogum BE sterile water solution;
Resistance to compression spall shield embedding (III layer): the small-particle that the embedding of II layer collected mixes (two kinds of colloid aqueous solution equal-volumes mixing)---suspension of making (60% thalline colloidal suspension) mixing---with the spray-dired low temperature spray drying of DS-1000 spray-drier (or other spraying equipment) employing (105 ℃ of the inlet temperature of improving one's methods with 2.5% gelatin sterile water solution and 0.1% sodium alginate sterile water solution (115 ℃ of sterilization 15min), sample size 360L/h, 54 ℃ of temperature outs)---microcapsule finished product (storing below 5 ℃).
3, the little glue ratio defective product of selenium-rich Enterococcus faecium cernelle three-layer, survival rate
Under the anaerobically fermenting environment to selenium addition 35mg/L, inoculum size 10g/L, fermentation time 28h, pH4.5～6.3, the rich selenium lactic acid intestines fecal bacteria of producing, by the biological embedding techniques of three microcapsules, microcapsule product rate, the survival rate detected result of rich selenium lactic acid enterococcus faecalis, as shown in table 1.
Rich selenium lactic acid enterococcus faecalis number adopts and improves the mensuration that MRS plate culture medium bacterium colony appearance method is carried out colony-forming unit (cfu/ml).
Primary colony number * 100% of survival rate=microcapsule embedded group of not embedding group of measuring of colony number/microcapsule
Ratio defective product=not embedding group of microcapsule embedded group of unit volume quality/microcapsule unit volume quality * 100%
Table 1 microcapsule product rate, survival rate
As can be seen from Table 1, adopting three microcapsule embedding techniqueses to obtain ratio defective product is 82.68%; Cultivating the microcapsule embedded back of counting thalline survival rate through the dilution plate bacterium colony is 72.15%; And because the quality of microcapsule embedded wall material increases, make thalline selenium content unit mass, be reduced to 0.571mg/g by original 0.615mg/g.
The thalline survival rate is all very high after this shows the product pick-up rate of rich selenium lactic acid intestines fecal bacteria three microcapsules and embedding.
4, the resistant proof of selenium-rich Enterococcus faecium cernelle three-layer microcapsule
1) anti-stomach acidity:
Under artificial hydrochloric acid in gastric juice (pH2.1) environment, through 2h, the survival rate of the rich selenium lactic acid intestines fecal bacteria of embedding reaches more than 96%, is not subjected to the influence of hydrochloric acid in gastric juice.
2) anti-storage:
The rich selenium lactic acid intestines fecal bacteria of embedding was stored 1 year at normal temperatures, by initial 5.7 * 10
8The cfu/ml viable count only reduces to 5.4 * 10
7Cfu/ml, the thalline survival rate remains on (embedding thalline survival rate only is not 0.003%) more than 19.5%, and the anti-shelf characteric of embedding thalline is good.
3) heat-resistant stability:
50 ℃, 30min thalline survival rate are that 89.71%, 100 ℃, 5min are that 86.91%, 120 ℃, 2min are 89.18%, and the rich selenium lactic acid enterococcus faecalis through three microcapsule embeddings is described, the heat-resistant stability of its microcapsule product is good.
4) the anti-fragility of resistance to compression:
Three microcapsules (feeding probiotic bacterium three microcapsules to selenium-rich Enterococcus faecium cernelle three-layer microcapsule and prior art, Harbin Mei Hua biotech company) and the anti-fragility of the resistance to compression of bilayered microcapsule (the milk-acid bacteria double-layer embedment technique according to people such as Chen Jiankai prepares) compare test, respectively at 3min, 30kg, 3min, 50kg and 3min, 80kg, pressure aperture area 5cm
2Condition under, the present invention's three microcapsules are significantly better than three layers and two layers of capsule product of prior art, its percentage of damage is as shown in table 2.
The breakage-proof comparison test of table 2 microcapsule resistance to compression
Claims (8)
1. selenium-rich Enterococcus faecium cernelle three-layer microcapsule, comprise rich selenium lactic acid enterococcus faecalis, nutritive layer, antiacid non-oxygen shaft floor and resistance to compression spall shield from inside to outside successively, it is characterized in that described nutritive layer is the sterilization skimmed milk, described antiacid non-oxygen shaft floor is kappa-carrageenan and Viscogum BE, described resistance to compression spall shield is the natural polymer colloid, and described natural polymer colloid is the mixture of gelatin and sodium alginate.
2. method for preparing the described selenium-rich Enterococcus faecium cernelle three-layer microcapsule of claim 1, comprise the cultivation and the selenium-rich Enterococcus faecium cernelle three-layer microcapsule embedding of rich selenium lactic acid enterococcus faecalis, the culture condition that it is characterized in that wherein said rich selenium lactic acid enterococcus faecalis is: in the fermented liquid commonly used or substratum of lactic acid enterococcus faecalis, the employing ventilation anaerobic jar fermentation of bleeding, selenium addition 25～35mg/L, inoculum size 5～15g/L, fermentation time 28～32h, pH value 4.5～6.3; Wherein said selenium-rich Enterococcus faecium cernelle three-layer microcapsule embedding may further comprise the steps:
The embedding of I layer: i.e. the embedding of nutritive layer, rich selenium lactic acid enterococcus faecalis is mixed with the sterilization skimmed milk, make 40%～60% (w/v) thalline skimmed milk suspension, spray forms the thalli granule of skimmed milk embedding in the aseptic aqueous hydrochloric acid of pH4;
II layer embedding: the i.e. embedding of antiacid non-oxygen shaft floor, 0.02%～0.03% (w/v) the kappa-carrageenan aqueous solution and 0.20%～0.25% (w/v) Viscogum BE aqueous solution equal-volume are mixed, the thalli granule that adds the embedding of I layer, make 40%～60% (w/v) thalline colloidal suspension, spray forms the embedding small-particle in 8 ℃～10 ℃ aseptic cold water;
The embedding of III layer: i.e. the embedding of resistance to compression spall shield, the small-particle of II layer embedding is mixed with the natural polymer colloid aqueous solution, make 50%～70% (w/v) thalline colloidal suspension, adopt spray-dired low temperature to improve one's methods, obtain three microcapsule finished products.
3. method according to claim 2, it is characterized in that the culture condition of described rich selenium lactic acid enterococcus faecalis is: described fermented liquid is peptone 10.0g, extractum carnis 10.0g, yeast extract 5.0g, glucose 20.0g, K
2HPO
42.0g, tween 80 1ml, Triammonium citrate 2.0g, sodium acetate 5.0g, MgSO
47H
2O0.58g, MnSO
44H
2O 0.25g, distilled water 1L; The employing ventilation anaerobic jar fermentation of bleeding, selenium addition 35mg/L, inoculum size 10g/L, fermentation time 28h, pH value 4.5～6.3.
4. method according to claim 2 is characterized in that the natural polymer colloid aqueous solution is the mixing solutions of aqueous gelatin solution and sodium alginate aqueous solution described in the embedding of described III layer.
5. method according to claim 2, it is characterized in that natural polymer colloid aqueous solution described in the embedding of described III layer is the arbitrary proportion mixing solutions of sodium alginate aqueous solution of the aqueous gelatin solution and 0.1%～0.3% (w/v) of concentration 2.0%～2.5% (w/v), high density aqueous gelatin solution and lower concentration sodium alginate aqueous solution or high density sodium alginate aqueous solution and lower concentration aqueous gelatin solution carry out mutual mix and match in described concentration range.
6. method according to claim 5 is characterized in that described natural polymer colloid aqueous solution is that concentration is the equal-volume mixing solutions of sodium alginate aqueous solution of the aqueous gelatin solution and 0.1% (w/v) of 2.5% (w/v), and through 115 ℃ of sterilization 15min.
7. method according to claim 2 is characterized in that described spraying drying adopts any spray-drier, 95 ℃～105 ℃ of dry inlet temperature, sample size 180L/h～360L/h, 50 ℃～55 ℃ of temperature outs.
8. the application of the described selenium-rich Enterococcus faecium cernelle three-layer microcapsule of claim 1 adds it in animal-feed as additive and to feed or directly to feed separately as the animal health product.
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| IL199781A0 (en) | 2009-07-09 | 2010-05-17 | Yohai Zorea | Heat resistant probiotic compositions and healthy food comprising them |
| CN104431370A (en) * | 2014-12-15 | 2015-03-25 | 南京优帆生物科技有限公司 | Efficient probiotics microcapsule as well as preparation method and application thereof |
| CN113106030A (en) * | 2020-06-11 | 2021-07-13 | 扬州大学 | Method for preparing selenium-rich enterococcus faecium by using inorganic selenium and application thereof |
| CN113350382A (en) * | 2021-06-02 | 2021-09-07 | 成都仕康美生物科技有限公司 | Intestinal fecal bacteria microcapsule and preparation method thereof |
| CN114711430A (en) * | 2022-03-31 | 2022-07-08 | 杭州曼士元食品科技有限公司 | Food composition preparation for resisting helicobacter pylori |
| CN115844006A (en) * | 2022-12-19 | 2023-03-28 | 河南佳禾康生物食品科技有限公司 | Collard spirulina microcapsule, preparation method and application |
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