Patent Application
20240277784
The present disclosure relates to a strain of highly organic zinc-enriched Bifidobacterium animalis capable of promoting growth and reproductive development, belonging to the technical field of microorganisms.
Zinc is a trace element necessary to maintain normal growth and development of the human body. The human body contains 2-3 g of zinc, nearly 90% of which is found in muscles and bones. Healthy adults get about 10-15 mg of zinc daily from their diet, with a typical absorption rate of 20-30%. Zinc deficiency is considered to be a widespread nutritional deficiency worldwide, affecting approximately 31% of the global population, and is prevalent in both developed and developing countries, especially in developing countries. Zinc deficiency will lead to growth retardation and hypogonadism. Mild to moderate zinc deficiency is common all over the world. At present, in some poverty-stricken areas of China, due to the low intake of animal-based food, the access to zinc-rich food is limited, and plant-based food is the main food after birth. Phytate in the vegetable diet will inhibit the absorption of zinc, resulting in long-term zinc deficiency during growth and development, and further affecting growth and development.
Due to the inability of zinc to be stored in the human body, it is necessary to supplement zinc daily to maintain the normal functioning of the body, which may easily cause zinc deficiency in the human body. Zinc supplementation is currently mainly achieved through the additional intake of products containing organic or inorganic zinc. There are three types of zinc supplements on the market: inorganic zinc (ZnO, ZnSO4, ZnCl2, etc.), simple organic zinc (zinc gluconate, zinc acetate, zinc propionate, etc.), and organic zinc (amino acid chelated zinc, protein complexed zinc, etc.). Different zinc supplements have different absorption efficiencies. Studies have shown that organic zinc is more easily absorbed by the human body than inorganic zinc. The absorption rate of inorganic zinc is low, and the side effects thereof are obvious. The absorption rate of organic zinc alone is higher than that of inorganic zinc, but there are still some side effects such as gastrointestinal stimulation. Organic zinc is mainly synthesized artificially, which is relatively safe, but is more complicated in synthesis.
Microbial enrichment of zinc has been a research hotspot in recent decades. Adding inorganic salts to media enables microorganisms to enrich mineral ions, enrich trace elements on the cell surfaces or transport same into cells for storage. They exist in a form of complexes with amino acids, proteins, lipids and polysaccharides, thus realizing the transformation of inorganic trace elements into organic trace elements, and providing a good dietary source for the human body to supplement organic trace elements. At present, most of the strains used to enrich zinc by microorganisms are yeasts, and bifidobacteria are rarely used. In contrast, there are few reports on zinc-rich bifidobacteria, while bifidobacteria, as a common and beneficial microorganism to human health, also have the function of enriching metal ions. Moreover, bifidobacteria have more probiotic properties, and enriching zinc with bifidobacteria may have higher value than enriching zinc with yeasts. Zinc-rich probiotics can also regulate intestinal microbiota while supplementing zinc. As a new type of dietary zinc source containing active probiotics, the zinc-rich probiotics have more advantages than probiotics or other zinc supplements, which is worth exploring. Compared to supplementing inorganic zinc alone, it is necessary to study efficiently enriching inorganic zinc with the zinc-rich probiotics and converting same into organic zinc, and develop a zinc supplement that is inexpensive, simple to prepare, safe and reliable, and more efficient in supplementing zinc.
The present disclosure provides a probiotic preparation. The probiotic preparation contains B. animalis CCFM1230 or a product of the B. animalis CCFM1230 subjected to zinc enrichment culture; and the B. animalis CCFM1230 has been preserved in Guangdong Microbial Culture Collection Center (GDMCC) on Feb. 11, 2022, with a preservation number of GDMCC No: 62248.
In one embodiment, the product obtained after zinc enrichment culture includes cells from the B. animalis CCFM1230 subjected to zinc enrichment culture, or cell lysates containing organic zinc obtained from the B. animalis CCFM1230 subjected to zinc enrichment culture.
In one embodiment, the B. animalis CCFM1230 has the following characteristics:
In one embodiment, the cells include, but are not limited to living cells or dead cells.
In one embodiment, the dead cells include, but are not limited to naturally inactive cells or inactivated cells.
In one embodiment, the probiotic preparation per gram or per milliliter contains the ≥1×1010 CFU/g or ≥1×1010 CFU/mL B. animalis CCFM1230, or cells obtained after zinc enrichment culture.
In one embodiment, the zinc enrichment culture is to culture the B. animalis CCFM1230 in a zinc-rich medium until the number of bacterial cells is greater than or equal to 1×108 CFU/mL.
In one embodiment, the zinc enrichment culture is to culture the B. animalis CCFM1230 in the zinc-rich medium for a period of time; and the zinc ion concentration in the zinc-rich medium is 200-700 mg/L.
In one embodiment, after being subjected to zinc enrichment culture, the B. animalis CCFM1230 is also subjected to drying process; and the drying process includes but is not limited to: vacuum freeze drying, spray drying, vacuum drying or fluidized bed drying.
The present disclosure further provides a preparation method of zinc-enriched B. animalis CCFM1230. The method includes the following steps:
In one embodiment, the zinc ion concentration in the zinc-rich liquid medium in step (2) is 200-700 mg/L.
In one embodiment, the zinc-rich liquid medium contains: 20-30 g/L glucose, a 15-25 g/L nitrogen source (a mass ratio of yeast extract powder to peptone is 1:2), 2 g/L anhydrous sodium acetate, 2 g/L diammonium hydrogen citrate, 2.6 g/L K2HPO4·H2O, 0.1 g/L MgSO4·7H2O, 0.05 g/L MnSO4·H2O, 1 g/L Tween-80, 0.5 g/L cysteine, and zinc sulfate (added according to the conversion of zinc ion concentration from 200 to 700 mg/L).
In one embodiment, the bacterial slurry of the zinc-enriched B. animalis is also subjected to drying process, so that bacterial powder of highly organic zinc-enriched B. animalis is obtained.
In one embodiment, the bacterial slurry of the zinc-enriched B. animalis is also arbitrarily subjected to drying process; and the drying process includes but is not limited to spray drying, vacuum drying, fluidized bed drying, or vacuum freeze drying.
In one embodiment, the bacterial slurry of the zinc-enriched B. animalis is inactivated and then arbitrarily subjected to drying process to obtain bacterial powder of highly organic zinc-enriched B. animalis CCFM1230 without cellular activity; and the drying process uses protein or dextrin as a filling agent, or does not use any filling agent.
The present disclosure also provides application of the B. animalis CCFM1230 or the probiotic preparation in the preparation of products capable of promoting the growth and reproductive development of young mammals.
In one embodiment, the products include food, medicines or health care products.
The present disclosure provides a strain of highly organic zinc-enriched B. animalis, which can enrich and absorb inorganic zinc and convert same into biological zinc in bacteria. After the strain is subjected to zinc enrichment culture, the zinc content in bacterial powder per gram can reach 3.8 mg or above, and the organic zinc content can reach 95.3%. The number of viable cells in the bacterial powder can reach 4.73×109 CFU/g or above. Regardless of whether having activity or not, the organic zinc enriched by the strain can be better absorbed and utilized by organisms, which can effectively promote the growth and reproductive development of mammals.
The B. animalis CCFM1230, classified and named Bifidobacterium animalis, has been preserved in Guangdong Microbial Culture Collection Center (GDMCC) on Feb. 11, 2022, with a preservation number of GDMCC No: 62248. The preservation address is 5th floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou.
The present disclosure will be further elaborated below in conjunction with specific examples.
Zinc sulfate (product code: 10024018, CAS: 7446-20-0) involved in the following examples was purchased from Sinopharm Chemical Reagent Co., Ltd.; and nitric acid (product code: yb2-308, CAS: 7697-37-2) was purchased from Sinopharm Chemical Reagent Co., Ltd.
Modified MRS liquid medium: 10 g/L peptone, 10 g/L beef extract, a 5 g/L yeast extract, 20 g/L glucose, 2 g/L anhydrous sodium acetate, 2 g/L diammonium hydrogen citrate, 2.6 g/L K2HPO4·3H2O, 0.1 g/L MgSO4·7H2O, 0.05 g/L MnSO4·H2O, 1 g/L Tween-80, 0.5 g/L cysteine, and 1000 g/L distilled water.
Modified MRS solid medium: 10 g/L peptone, 10 g/L beef extract, a 5 g/L yeast extract, 20 g/L glucose, 2 g/L anhydrous sodium acetate, 2 g/L diammonium hydrogen citrate, 2.6 g/L K2HPO4·3H2O, 0.1 g/L MgSO4·7H2O, 0.05 g/L MnSO4·H2O, 1 g/L Tween-80, 0.5 g/L cysteine, 20 g/L agar, and 1000 g/L distilled water.
Zinc-rich liquid medium: 20-30 g/L glucose, a 15-25 g/L nitrogen source (a mass ratio of yeast extract powder to peptone was 1:2), 2 g/L anhydrous sodium acetate, 2 g/L diammonium hydrogen citrate, 2.6 g/L K2HPO4·3H2O, 0.1 g/L MgSO4·7H2O, 0.05 g/L MnSO4·H2O, 1 g/L Tween-80, 0.5 g/L cysteine, 1000 g/L distilled water, and zinc sulfate (added according to the conversion of zinc ion concentration from 200 to 700 mg/L).
Infant feces from Shanghai were taken as samples, and 10-fold gradient dilution was carried out with sterile normal saline until the samples were diluted to 10−6; after that, 100 μL of dilution solutions with dilution factors of 10−4, 10−5 and 10−6 were respectively taken and coated on a modified MRS solid medium by a spread plate method, and then were cultured at 37° C. for 48 h; and colony morphology was observed and recorded. Colonies of different morphologies on the modified MRS solid medium were selected for streaking isolation; and after the colonies were cultured at 37° C. for 48 h, single colonies of different morphologies on the modified MRS solid medium were selected again for streaking isolation until the pure single colonies of consistent morphologies were obtained. The pure colonies on the modified MRS solid medium were selected and inoculated into a zinc-rich liquid medium containing zinc sulfate, and then were cultured at 37° C. for 18 h; the bacterial liquid was transferred to a sterile centrifuge tube, and centrifuged at 8000 g for 10 min; and the superstratum medium was discarded, and the obtained bacterial slurry was rinsed for 2 times and then freeze-dried to obtain zinc-rich bacterial powder. An atomic absorption spectrophotometer was used to detect the zinc content in the bacterial powder, and the strains with stronger zinc enrichment ability were selected.
The isolated strain with stronger zinc enrichment ability was subjected to PCR amplification of 16S rDNA, and the PCR product was sent to Invitrogen Trading (SHANGHAI) Co., Ltd. for sequencing. The sequencing results were compared with nucleic acid sequences in NCBI, and finally a strain of B. animalis was obtained, named Bifidobacterium animalis CCFM1230.
The B. animalis CCFM1230 was inoculated into a modified MRS liquid medium and cultured at 37° C. for 18 h; 1 mL of bacterial liquid was taken into a sterile centrifuge tube, centrifuged at 8000 g for 10 min, and then the superstratum medium was discarded; and the bacterial slurry was resuspended in a 30% glycerol solution and preserved at −80° C.
Optionally, bacterial powder may also be prepared by inactivating and drying the zinc-enriched B. animalis, and the drying method may be selected from spray drying, vacuum drying, fluidized bed drying, or vacuum freeze drying.
Optionally, the bacterial powder of the inactivated highly zinc-enriched B. animalis can be prepared according to the following method: after the fermentation of the aforementioned step (2) was completed, the bacterial liquid was centrifuged at 8000 g for 20 min at 4° C., the wet bacterial cells were rinsed for 2 times with pure water, and the washed wet bacterial cells were subjected to spray drying, vacuum drying, fluidized bed drying, or vacuum freeze drying; protein or dextrin was used as a filling agent (the mass ratio of bacterial slurry to a filling agent solution was 1:1, and the filling agent solution was whey protein, collagen, soybean protein, or a dextrin solution with a mass fraction of 13%); and the bacterial powder of the inactive highly zinc-enriched B. animalis was obtained, and the content of organic zinc in the bacterial powder per gram can reach 3605.45 μg or above.
1. Detection of Zinc Content in Zinc-Enriched B. animalis
Referring to the method in Example 2, bacterial liquid of B. animalis CCFM1230 was obtained by fermentation in a zinc-rich liquid medium with zinc ion concentration of 200 mg/L, and bacterial powder thereof was prepared. 0.1 g-0.15 g of a bacterial powder sample was weighed and put into a microwave digestion tank, and 5 mL of nitric acid was added for microwave digestion. After cooling, the digestion tank was taken out, and the acid was removed on an electric heating plate at 140° C.-160° C. until being about 1 mL. After the digestion tank was cooled off, a digestion solution was transferred to a 25 mL volumetric flask, the digestion tank was washed 2-3 times with a small amount of water, a washing solution was combined into the volumetric flask, the volume was set to scale by using water, and the obtained product was mixed well for later use. At the same time, a reagent blank test was carried out.
The zinc content in the sample was measured by flame atomic absorption spectrometry with reference to the first method in the national standard GB 5009.14-2017 of the People's Republic of China, and the detection result was 3.8 mg/g.
2. Analysis on Organic Zinc in Zinc-Enriched B. animalis
0.5 g of bacterial powder of zinc-enriched B. animalis CCFM1230 was accurately weighed and put into a beaker, 45 mL of distilled water was added, and the pH of the distilled water was adjusted to 6.5 with dilute acid or alkali. After that, a 50 mL volumetric flask was used to determine the volume, and the pH of the distilled water with the determined volume was adjusted to 6.5. After the volume was determined, a zinc-enriched B. animalis solution was transferred to a beaker, and then slowly and uniformly stirred with a glass rod for 5-10 min. After stirring, the solution was centrifuged at 8000 g for 15 min at room temperature. The supernatant obtained by centrifugation was collected for measuring the water-soluble zinc (i.e., inorganic zinc) content on the cell surfaces of the zinc-enriched B. animalis. 45 mL of a 10 mmol/L EDTA solution was added into the precipitate, and the pH of the EDTA solution was adjusted to 6.5 with dilute acid or alkali. After that, a 50 mL volumetric flask was used to determine the volume, and the pH of the EDTA solution with the determined volume was adjusted to 6.5. After the volume was determined, a zinc-enriched B. animalis solution was transferred to a beaker, and then slowly and uniformly stirred with a glass rod for 5-10 min. After stirring, the solution was centrifuged at 8000 g for 15 min at room temperature. The supernatant obtained by centrifugation was collected for measuring the content of zinc complexed by cell wall polysaccharides and proteins. The second centrifugation precipitation was used to measure the contents of organic macromolecules or small molecules bound to zinc in the cells of the zinc-enriched B. animalis.
Organification degree=(content of zinc complexed by cell wall polysaccharides and proteins+content of organic macromolecules or small molecules bound to zinc in cells)/total zinc content
The zinc content in each component was measured by flame atomic absorption spectrometry with reference to the first method in the national standard GB 5009.14-2017 of the People's Republic of China. The detection results are as follows:
It can be seen that the total zinc content of the bacterial powder of the zinc-enriched B. animalis CCFM1230 reaches 3.8 mg/g, indicating that the B. animalis has a stronger ability to enrich zinc. The content of inorganic zinc is 4.7%, indicating that the assimilation effect of the B. animalis on the inorganic zinc is better. 8.0% of zinc is bound to macromolecules such as polysaccharides and proteins in the cell wall in a form of organisms; and 87.2% of zinc is bound to organic macromolecules or small molecules in the cells of the B. animalis. The same method was used to detect the organic zinc content in the bacterial powder of the inactive highly zinc-enriched B. animalis. The results show that the total zinc content in the bacterial powder prepared from the inactivated bacterial slurry is 3.8 mg/g, the inorganic zinc content is 4.7%, and the organic zinc content is 95.3%.
The zinc content of probiotics from different sources after zinc enrichment culture was retrieved and collected, where Bifidobacterium bifidum O4, Bifidobacterium adolescentis W5, Bifidobacterium adolescentis HuNan-2016 MRS 11-2, Bifidobacterium animalis HuNan-2016 22-3, Bifidobacterium breve HuNan-2016 49-7, Bifidobacterium breve GuXi-2016 6-7, Lactobacillus reuteri 138-1, and Lactobacillus bulgaricus MJ-1 were published in the paper Enrichment Characteristics of Lactic Acid Bacteria for Zinc and Relieving Effect of Zinc-rich Lactic Acid Bacteria on Colitis in Mice; Bifidobacterium breve WC 0421, Bifidobacterium breve WC 0480, Bifidobacterium breve WC 0481, Bifidobacterium infantis WC 0460, and Bifidobacterium pseudocatenulatum WC 0455 were published in the paper Zinc Uptake by Lactic Acid Bacteria; and Bacillus subtilis NZ56 was published in the patent with Publication No. CN 108220208 B.
B. animalis
B. bifidum
B. adolescentis
B. adolescentis
B. animalis
B. breve
B. breve
B. breve
B. breve
B. breve
B. infantis
B. pseudocatenulatum
Bacillus subtilis
The strains shown in Table 3 were cultured in the same manner as in Example 2, and the zinc content after 18 h of culture was measured. Bifidobacterium breve F-JS-ZJ-1-M5, Lactobacillus rhamnosus DG11-1, Lactobacillus plantarum NFM11, Lactobacillus casei RS-2-1, Lactobacillus fermentium NT65-2 were the zinc-enriched strains obtained by self-screening.
B. animalis
B. bifidum
B. adolescentis
B. breve
L. rhamnosus
L. reuteri
L. plantarum
L. casei
L. fermentium
L. bulgaricus
The above are the strains described in the existing documents or patents, which are relatively low in zinc enrichment content and organic zinc conversion rate, and thus cannot achieve the desired effects of high zinc enrichment and high organic zinc content of the strain provided by the present disclosure.
The rats in the inorganic zinc group were gavaged with zinc oxide suspension daily at a dose of 0.7 mg of Zn/rat while being fed with zinc-deficient feed;
the rats in the zinc-enriched B. animalis group were gavaged with bacterial suspension daily at a dose of 0.7 mg of Zn/rat while being fed with zinc-deficient feed (the bacterial powder prepared in Example 2 according to the zinc content was dissolved in normal saline);
the rats in zinc-deficient control group were fed with zinc-deficient feed and gavaged with an equal volume of normal saline; and
the rats in normal control group were fed with control feed and gavaged with an equal volume of normal saline for two weeks.
19.45 ± 0.24bcd
7.85 ± 0.19bc
19.43 ± 2.74ab
32.39 ± 2.41abc
B. animalis
Although the present disclosure has been disclosed as above in exemplary examples, it is not intended to limit the present disclosure. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present disclosure. Therefore, the protection scope of the present disclosure shall be as defined in the claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2022102031776 | Mar 2022 | CN | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2023/079598 | Mar 2023 | WO |
| Child | 18650203 | US |
