CN117802011A - Composite probiotics for promoting iron absorption and application thereof - Google Patents
Composite probiotics for promoting iron absorption and application thereof Download PDFInfo
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- CN117802011A CN117802011A CN202410225116.9A CN202410225116A CN117802011A CN 117802011 A CN117802011 A CN 117802011A CN 202410225116 A CN202410225116 A CN 202410225116A CN 117802011 A CN117802011 A CN 117802011A
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- iron
- strain
- probiotic
- bbr60
- promoting
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Abstract
The invention relates to a method for promoting ironAbsorbed composite probiotics and application thereof, wherein the composite probiotics for promoting iron absorption are prepared from bacillus coagulansBacillus coagulans BC99 strain and Bifidobacterium breveBifidobacterium breve BBr60 strain. The invention develops a probiotic compound mode and a strategy for improving the bioavailability of iron by organisms, namely, the BC99 strain and the BBr60 strain are combined, and the effects of the BC99 strain and the BBr60 strain on promoting iron absorption can be mutually matched, mutually promoted and synergistically enhanced, and under the condition that the using bacterial amounts are consistent, compared with the single BC99 strain or the single BBr60 strain intervening mode, the effects of the two strains on promoting iron absorption are obviously improved.
Description
Technical Field
The invention belongs to the technical field of probiotics, and relates to a composite probiotic for promoting iron absorption and application thereof.
Background
Iron is one of the first trace elements found in humans, accounting for about 0.006% of the body weight, 3-5 g of iron is contained in adults, 60-70% of which is present in hemoglobin, 3% in myoglobin, less than 1% of iron in various enzyme systems, and the remaining 26-36% is present in the form of iron transporting substances and iron reserves. Iron is an important component of hemoglobin molecules, which can absorb oxygen in the lungs, bringing it along with the blood flow to the tissue cells of the body. When iron is supplied insufficiently, the hemoglobin content in blood gradually decreases, so that iron deficiency anemia occurs, and people feel tired and weak, etc.
The most important functions ensured by the proper amount of iron in the body are related to the transport and storage of oxygen, electron transfer, regulation of redox reactions, synthesis of hormones, replication of DNA, restoration and control of cell cycle, fixation of nitrogen and antioxidation. In the case of iron deficiency, even slight discomfort may impair the normal function of the human body. Common supplementation methods are food fortification and supplementation.
Ferrous sulfate (FeSO) 4 ) Is a common oral iron supplement, but studies have shown useFeSO 4 Or other forms of redox active iron, may present problems with gastric and intestinal intolerance in a large number of Iron Deficiency Anemia (IDA) patients, requiring less reactive adjuvant supplements (e.g. probiotics) as adjuvants and substitutes. The probiotics have the effect of protecting intestinal tracts, and can be used as an effective, safe and long-term dietary supplement. Further studies have shown that lactic acid produced by lactobacillus increases the dietary bioavailability of iron and that probiotics can be used as a way of optimizing the bioavailability of iron. And more studies have linked the bioavailability and absorption of iron to the microbial activity in the intestinal tract. Researchers have suggested that iron absorption and utilization may be promoted and improved by modulating intestinal microecology.
Therefore, how to provide a microbial preparation capable of effectively improving iron absorption, solving the problems of malnutrition caused by iron deficiency to patients and reducing adverse reactions in the iron supplementing process, and becoming a urgent need for solving.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a composite probiotics for promoting iron absorption and application thereof.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the invention provides a composite probiotic for promoting iron absorption, which is prepared from bacillus coagulans with a preservation number of CGMCC No. 21801Bacillus coagulans BC99 strain and bifidobacterium breve with preservation number of CGMCC No. 12915Bifidobacterium breve BBr60 strain.
The invention creatively develops a brand new probiotic compound mode and a brand new strategy for improving the bioavailability of iron by organisms, namely bacillus coagulansBacillus coagulans BC99 strain and Bifidobacterium breveBifidobacterium breve The BBr60 strain is compounded and combined, and the BBr60 strain and the BC99 strain can be mutually matched, mutually promoted and synergistically enhanced in the effect of promoting iron absorption, and can be matched with a single BC99 strain or a single BBr60 strain under the condition of consistent using bacterial loadCompared with the intervention mode, the compound of the two bacteria has obviously improved effect on promoting iron absorption, and is specifically expressed in: raise the level of hemoglobin, ferritin and serum iron in blood and raise the level of non-heme iron in liver. Therefore, the compound probiotics have good prospect in preparing products for improving the bioavailability of the iron supplement or medicines for preventing, relieving or treating related diseases caused by iron ion deficiency. Meanwhile, both bacteria are probiotics, the product safety is high, and the resistance is not easy to generate.
The preparation method of the composite probiotics adopts a technical method conventional in the field, and can be exemplified by: activating the BC99 strain or the BBr60 strain, and then respectively inoculating the activated BC99 strain or the BBr60 strain into a culture medium for culture to obtain a culture solution; centrifuging the culture solution, and re-suspending the bacteria to obtain bacterial suspension; mixing the two bacterial suspensions according to the ratio of the viable bacteria number. Or further adding a protective agent for freeze drying to obtain a freeze-dried bacterial powder product.
Preferably, the ratio of the number of viable bacteria of the BC99 strain to the BBr60 strain is (1-10): 1, such as 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, etc., and other specific values within the numerical range can be selected, and will not be described in detail herein.
Based on bacillus coagulansBacillus coagulans BC99 strain and Bifidobacterium breveBifidobacterium breve The potential interaction relation of BBr60 strain, the invention also discovers that when two strains are compounded in the specific ratio of the number of the living bacteria, the effect of promoting iron absorption is more remarkable.
In a second aspect, the present invention provides a probiotic for promoting iron absorption, the strain of which comprises the complex probiotic of the first aspect.
Preferably, the live bacteria content of the BC99 strain and the BBr60 strain in the probiotic is not less than 1 multiplied by 10 8 CFU/g or 1X 10 8 CFU/mL, e.g. 1X 10 8 CFU/g(CFU/mL)、2×10 8 CFU/g(CFU/mL)、5×10 8 CFU/g(CFU/mL)、8×10 8 CFU/g(CFU/mL)、1×10 9 CFU/g(CFU/mL)、5×10 9 CFU/g(CFU/mL)、1×10 10 CFU/g(CFU/mL)、1×10 11 CFU/g(CFU/mL)、1×10 12 CFU/g (CFU/mL), etc., other specific values within the numerical range may be selected, and will not be described in detail herein.
Preferably, the formulation of the probiotic agent comprises freeze-dried powder, capsules, tablets or granules.
The formulation of the probiotics related to the invention is not limited, and comprises the most commonly used freeze-dried powder, or further prepared capsules, tablets or granules. The lyophilized powder can be prepared by the following method:
activating the BC99 strain or the BBr60 strain, and then respectively inoculating the activated BC99 strain or the BBr60 strain into a culture medium for culture to obtain a culture solution; centrifuging the culture solution to obtain thalli; re-suspending the thalli by using a freeze-drying protective agent to obtain re-suspension; freeze-drying the heavy suspension, and mixing according to a proportion to obtain the finished product.
Preferably, the medium includes an MRS medium.
Preferably, the MRS medium includes, in concentration: 8-12 g/L peptone, 8-12 g/L beef extract, 15-25 g/L glucose, 10-20 g/L lactose, 3-7 g/L yeast powder, 1-3 g/L, K diammonium hydrogen citrate 2 PO 4 ·3H 2 O 2-3 g/L、MgSO 4 ·7H 2 O 0.05-0.2 g/L、MnSO 4 0.01-0.1 g/L, tween 80 0.5-2 mL/L, cysteine hydrochloride 0.1-1 g/L.
Preferably, the lyophilization is by vacuum freezing.
Preferably, the probiotic agent further comprises a protective agent and/or a functional auxiliary agent.
Preferably, the protective agent comprises any one or a combination of at least two of skim milk, gelatin, dextrin, acacia, dextran, sodium alginate, polyvinylpyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol.
The functional auxiliary agent comprises any one or a combination of at least two of fructo-oligosaccharide, galacto-oligosaccharide, xylo-oligosaccharide, isomalto-oligosaccharide, soybean oligosaccharide, inulin, spirulina, arthrospira, coriolus versicolor polysaccharide, stachyose, polydextrose, alpha-lactalbumin or lactoferrin.
In a third aspect, the present invention provides the use of a complex probiotic according to the first aspect or a probiotic according to the second aspect in the manufacture of a product for improving the bioavailability of an iron supplement.
In a fourth aspect, the present invention provides the use of a complex probiotic according to the first aspect or a probiotic according to the second aspect in the manufacture of a medicament for the prevention, alleviation or treatment of a disease associated with iron ion deficiency.
Preferably, the related diseases caused by iron ion deficiency include iron deficiency anemia, iron deficiency arteriosclerosis, iron deficiency neuropathy or iron deficiency immune function decline.
Preferably, the medicament further comprises an iron supplement.
Compared with the prior art, the invention has the following beneficial effects:
the invention creatively develops a brand new probiotic compound mode and a brand new strategy for improving the bioavailability of iron by organisms, namely bacillus coagulansBacillus coagulans BC99 strain and Bifidobacterium breveBifidobacterium breve The BBr60 strain is compounded and combined, and the two strains can be matched and promoted mutually, and the effects of promoting iron absorption are synergistically enhanced, so that under the condition of consistent using bacterial load, compared with the single BC99 strain or single BBr60 strain intervention mode, the effects of the two strains on promoting iron absorption are obviously improved, and the method is specifically characterized in that: raise the level of hemoglobin, ferritin and serum iron in blood and raise the level of non-heme iron in liver. Therefore, the compound probiotics have good prospect in preparing products for improving the bioavailability of the iron supplement or medicines for preventing, relieving or treating related diseases caused by iron ion deficiency. Meanwhile, both bacteria are probiotics, the product safety is high, and the resistance is not easy to generate.
The BC99 strain related to the invention is classified and named as bacillus coagulansBacillus coagulansThe preservation unit is China microorganismThe common microorganism center of the culture preservation management committee has the preservation time of 2021, 2 months and 1 day, the preservation number of CGMCC No. 21801 and the address of: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
The BBr60 strain of the invention is classified and named as Bifidobacterium breveBifidobacterium breveThe preservation unit is China general microbiological culture Collection center, the preservation time is 8 months and 29 days of 2016, the preservation number is CGMCC No. 12915, and the address is: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
Drawings
FIG. 1 is a graph showing the results of measurement of hemoglobin levels in blood of mice in each group;
FIG. 2 is a graph showing the results of serum ferritin levels of mice in each group;
FIG. 3 is a graph showing the results of serum iron content measurement of mice in each group;
FIG. 4 is a graph showing the results of non-heme iron level measurements in the liver of each group of mice;
FIG. 5 is a graph of Chao1 index results from intestinal flora analysis of mice in each group;
FIG. 6 is a graph showing the results of shannon index analysis of intestinal flora of mice in each group.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
Peptone, beef extract, glucose, lactose, yeast powder, diammonium hydrogen citrate, K, as referred to in the examples below 2 PO 4 ·3H2O、MgSO 4 ·7H 2 O、MnSO 4 Tween 80 and cysteine hydrochloride were purchased from national pharmaceutical group chemical Co.
The following examples relate to the following media:
MRS Medium (g/L): 10g/L peptone, 10g/L beef extract, 15g/L glucose, 15g/L lactose, 5g/L yeast powder, 2g/L, K diammonium hydrogen citrate 2 PO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.1g/L、MnSO 4 0.05g/L, tween 801 mL/L, cysteine amino acid salt 0.5g/L.
The BC99 strain classification referred to in the examples below is designated Bacillus coagulansBacillus coagulansThe preservation unit is China general microbiological culture Collection center, the preservation time is 2021, 2 months and 1 day, the preservation number is CGMCC No. 21801, and the address is: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
The class of BBr60 strains according to the following examples is designated as Bifidobacterium breveBifidobacterium breveThe preservation unit is China general microbiological culture Collection center, the preservation time is 8 months and 29 days of 2016, the preservation number is CGMCC No. 12915, and the address is: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
The bacterial suspensions referred to in the following examples: inoculating the required strain into MRS liquid culture medium, culturing at 37deg.C for 24 hr for activation, and continuously activating for 2 times to obtain activating solution; inoculating the activating solution into MRS liquid culture medium according to the inoculum size of 2% (v/v), and culturing at 37 ℃ for 24 hours to obtain bacterial solution; centrifuging the bacterial liquid at 6000g for 10min, and re-suspending the bacterial body by using PBS.
Experimental results data were statistically analyzed using ggplot2 in R language, representing p <0.001, p <0.01, p <0.05, ns representing no significant difference compared to CTL group.
Examples
The compound probiotics related to the invention are explored in the embodiment as an auxiliary supplement, and the regulation and control of the hemoglobin level, the serum ferritin level and the serum iron content of a low-iron dietary mouse and the influence on the intestinal flora of the mouse are carried out on the basis of supplementing ferrous sulfate solution:
(1) Test animals
CD1 mice of 3 weeks of age were selected and purchased from Shanghai Chengxi Biotechnology Co., ltd, and have been subjected to ethical approval by the Shanghai laboratory animal research center animal ethical Committee (ethical number: 2023082001). All animals were housed under specific pathogen-free barrier conditions and were housed in independent cages for one week with adaptation. Animal experiments strictly follow the guidelines for laboratory animal care and use.
(2) Low iron diet mouse model establishment
Low iron diet mice models were induced using low iron feed (iron concentration: 3mg/kg, formulation TD 80396, harlan-Teklad, USA) for 3 weeks of feeding. All mice were given ad libitum throughout the experiment.
(3) Grouping and intervention mode for animals
After 3 weeks of feeding with the low iron diet described above, 48 mice were randomly divided into 6 groups (8 per group): low iron diet model group (CTL), low iron diet mice group treated with ferrous sulfate solution (FeSO) 4 ) Low iron diet mice groups treated with ferrous sulfate solution and BC99 strain (FeSO 4 +bc 99), low iron diet mice group treated with ferrous sulfate solution and BBr60 strain (FeSO 4 +bbr 60), low iron diet mice group treated with ferrous sulfate solution and BC99 strain and BBr60 strain (FeSO 4 +Bc99+BBr60, ratio of viable count of two strains 4:1), low iron diet mice group treated with ferrous sulfate solution and ATCC7050 strain (commercial Bacillus coagulans strain) and BBr60 strain (FeSO 4 +ATCC7050+BBr60, the ratio of the number of viable bacteria of the two strains is 4:1).
The intervention modes of each group are as follows:
CTL: low iron feed (iron concentration 3 mg/kg);
FeSO 4 : treatment of low-iron feed (iron concentration 3 mg/kg) +ferrous sulfate solution (iron concentration 60mg/L,10 ml/L);
FeSO 4 +bc99: low iron feed (iron concentration 3 mg/kg) +ferrous sulphate solution (iron concentration 60mg/L,10 ml/min.)) +BC99 (5×10) 8 CFU/day/only) treatment;
FeSO 4 +bbr60: low-iron feed (iron concentration 3 mg/kg) +ferrous sulfate solution (iron concentration 60mg/L,10 ml/min.)) +BBr60 (5×10) 8 CFU/day) treatment;
FeSO 4 +bc99+bbr60: low iron feed (iron concentration 3 mg/kg) +ferrous sulphate solution (iron concentration 60mg/L,10 ml/min.)) +Bc99+BBr60 (total 5×10) 8 CFU/day) treatment;
FeSO 4 +atcc7050+bbr60: low iron feed (iron concentration 3 mg/kg) +ferrous sulphate solution (iron concentration 60mg/L,10 ml/stock) +ATCC7050+BBr60 (total 5×10) 8 CFU/day) treatment;
each group was given daily oral low iron feed (iron concentration: 3 mg/kg) and maintained a low iron diet. All mice were given ad libitum throughout the experiment.
(4) Index analysis
After 10 consecutive days of dosing, blood samples were collected, mice were euthanized, tissue collected, liver samples and stool samples were taken, and flash frozen in liquid nitrogen for subsequent analysis.
(4.1) measurement of hemoglobin: after adding 5. Mu.L of whole blood to the Drabkin reagent and centrifuging at 13000 rpm for 5 minutes, the hemoglobin concentration was calculated from the change in optical density of 540 and nm. All experimental procedures were performed strictly according to the instructions of the apparatus and kit. The results are shown in FIG. 1.
(4.2) determination of ferritin: ferritin was determined using the Spectro ferritin MT enzyme-linked immunosorbent assay (ELISA) kit. Serum ferritin concentrations were deduced from standard curves described in the manufacturer's protocol. All experimental procedures were performed strictly according to the instructions of the apparatus and kit. The results are shown in FIG. 2.
(4.3) serum iron content determination: serum iron concentration quantitative colorimetric iron determination was performed using a QuantiChrom iron determination kit (DIFE-250, bioassay Systems, USA) at wavelength 590 nm. All experimental procedures were performed strictly according to the instructions of the apparatus and kit. The results are shown in FIG. 3.
Hemoglobin is the most commonly used index for diagnosing iron deficiency anemia. Serum ferritin is positively correlated with the amount of iron stored in the body, and reflects a sensitive index of iron storage change in the body. Serum iron mainly refers to iron present in serum that binds transferrin, and is reduced in response to iron levels in plasma, iron deficiency and iron deficiency anemia.
As can be seen from the results of fig. 1, 2 and 3: feSO compared with CTL group mice 4 Hemoglobin, serum ferritin and serum iron content of group miceThe amount is increased. At the same time take FeSO 4 Based on the above, the three indexes of the mice are compared with the single FeSO administration after the dry prognosis of the probiotics BC99, BBr60 and the compound probiotics BC99+BBr60 4 The group effect is better, and the effect of promoting iron absorption is optimal for the composite probiotics BC99+BBr60 group.
(4.4) liver non-heme iron level determination: liver tissue samples were weighed and homogenized with 1ml glass homogenizer with 0.15M NaCl (1:5 w/v) in 10 mM NaOH-Hepes buffer (pH 7.0). Non-heme iron analysis was then performed on the homogenized aliquots. Iron values are expressed as concentrations (nmol iron/mg wet weight). The results are shown in FIG. 4.
The liver is one of the most prominent iron reservoirs in the body and is also the center for iron metabolism and regulation. When iron is needed by the body, the liver can release transferrin iron to meet the iron requirements of other tissues and organs. By measuring non-heme iron levels in the liver, the storage and distribution of iron in the body can be better understood.
As can be seen from the results of fig. 4: compared with the CTL group of mice, the liver of the other groups of mice has higher non-heme iron level than that of the CTL group, and FeSO is taken simultaneously 4 Based on the above, the dry prognosis of the probiotics BC99, BBr60 or the compound probiotics BC99+BBr60 is carried out, and the non-heme iron level in the liver of the mice is higher than that of the single FeSO administration 4 The group is higher, and the effect of the compound probiotics BC99+BBr60 group is optimal.
(5) Analysis of the intestinal microbiota of mice: modulation of alpha diversity of microbial communities
The V3-V4 region of the 16SrRNA gene was amplified from a mouse fecal sample by PCR using 341F and 805R primers. The PCR reaction was as follows: denaturation at 95℃for 3 min, denaturation at 94℃for 0.5 min, annealing at 58℃for 0.5 min, denaturation at 72℃for 0.5 min, 21 cycles total, and final extension at 72℃for 5 min. The products of the different samples were indexed and mixed in equal proportions according to the manufacturer's instructions and sequenced using the Illumina Miseq platform (2 x 300 bp).
As shown in fig. 5 and 6, the α -diversity of the microbial communities of the other groups, including the microbial abundance (expressed as the Chao1 index) and the microbial diversity (expressed as the shannon index), increased significantly compared to the CTL group, especially after the intervention of the complex probiotics bc99+bbr60 and the probiotic BC 99.
The applicant states that the technical solution of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above embodiments, i.e. it does not mean that the present invention must be implemented by the above embodiments. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Claims (10)
1. A composite probiotics for promoting iron absorption is characterized in that the composite probiotics for promoting iron absorption are prepared from bacillus coagulans with the preservation number of CGMCC No. 21801Bacillus coagulans BC99 strain and bifidobacterium breve with preservation number of CGMCC No. 12915Bifidobacterium breve BBr60 strain.
2. The composite probiotic bacteria promoting iron absorption according to claim 1, characterized in that the ratio of the number of viable bacteria of the BC99 strain to the BBr60 strain is (1-10): 1.
3. A probiotic agent that promotes iron absorption, characterized in that the strain in the probiotic agent comprises the complex probiotic of claim 1 or 2.
4. The probiotic agent for promoting iron absorption according to claim 3, characterized in that the live bacteria content of the BC99 strain and BBr60 strain in the probiotic agent is not less than 1 x 10 8 CFU/g or 1X 10 8 CFU/mL。
5. A probiotic agent for promoting iron absorption according to claim 3, characterized in that the formulation of the probiotic agent comprises a lyophilized powder, a capsule, a tablet or a granule;
the probiotic agent also comprises a protective agent and/or a functional auxiliary agent.
6. The probiotic agent for promoting iron absorption according to claim 5, characterized in that the protective agent comprises any one or a combination of at least two of skim milk, gelatin, dextrin, acacia, dextran, sodium alginate, polyvinylpyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol;
the functional auxiliary agent comprises any one or a combination of at least two of fructo-oligosaccharide, galacto-oligosaccharide, xylo-oligosaccharide, isomalto-oligosaccharide, soybean oligosaccharide, inulin, spirulina, arthrospira, coriolus versicolor polysaccharide, stachyose, polydextrose, alpha-lactalbumin or lactoferrin.
7. Use of a complex probiotic of claim 1 or 2 or a probiotic of any one of claims 3 to 6 in the manufacture of a product for improving the bioavailability of an iron supplement.
8. Use of a complex probiotic according to claim 1 or 2 or a probiotic according to any one of claims 3 to 6 for the manufacture of a medicament for the prevention, alleviation or treatment of a related disease caused by iron ion deficiency.
9. The use according to claim 8, wherein the related diseases caused by iron deficiency include iron deficiency anemia, iron deficiency arteriosclerosis, iron deficiency neuropathy or iron deficiency immune dysfunction.
10. The use of claim 8, wherein the medicament further comprises an iron supplement.
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