CN116103345A - Biological nano selenium synthesized by preset lactic acid bacteria and synthesis preparation process thereof - Google Patents
Biological nano selenium synthesized by preset lactic acid bacteria and synthesis preparation process thereof Download PDFInfo
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- CN116103345A CN116103345A CN202310049899.5A CN202310049899A CN116103345A CN 116103345 A CN116103345 A CN 116103345A CN 202310049899 A CN202310049899 A CN 202310049899A CN 116103345 A CN116103345 A CN 116103345A
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Abstract
The application provides a biological nano selenium synthesized by preset lactobacillus and a synthesis preparation process thereof, which are applied to the technical field of microorganism synthesis biology, and the method comprises the following steps: taking 50kg of selenium-enriched seawater, placing for 48 hours, taking 15kg of seawater supernatant, placing into a fermentation barrel, adding 1kg of brown sugar, and uniformly stirring; adding 3kg of medical soluble starch after the brown sugar is completely dissolved, fully and uniformly stirring, and carrying out sealed anaerobic fermentation; placing a small amount of bacterial liquid into a reaction vessel, placing a shaking table for shaking for 48 hours, and placing a high-power microscope for observing that the bacterial form accords with the characteristics of lactic acid bacteria; weighing sodium selenite with different weights, respectively placing into conical flasks, adding fermented bacterial liquid and enzyme preparation with the same weight into each conical flask, and performing stirring anaerobic culture; observing the color change in each flask during the stirred anaerobic culture; detecting the particle size of the biological nano selenium by utilizing a micro spectrum; the nano selenium synthesized by lactic acid bacteria has low toxicity, stability and high biological activity.
Description
Technical Field
The application relates to the technical field of microorganism synthesis biology, in particular to a biological nano selenium synthesized by preset lactic acid bacteria and a synthesis preparation process thereof.
Background
Selenium (Se) is taken as one of microelements necessary for life and living of organisms, plays an important role in maintaining redox homeostasis, regulating an immune system, protecting DNA and chromosomes from oxidative damage and the like, and mainly takes the forms of inorganic compound selenium, organic selenium and nano selenium, wherein the inorganic compound selenium has the highest toxicity and the nano selenium has the weakest toxicity; the lactobacillus is used as the most widely used fermentation strain in the food field, when people eat selenium-enriched food or health-care food prepared by lactobacillus fermentation, not only trace element selenium can be supplemented, but also the probiotics effect brought by the lactobacillus can be obtained, so the lactobacillus can become a good carrier and a transformant for synthesizing nano-selenium, and a new selenium-supplementing product is prepared by adding the lactobacillus and the food, but the following problems exist in the preparation process of the synthesized nano-selenium:
(1) The physical method can cause high-energy and high-consumption pollution in the process of manufacturing the nano selenium, and the preparation process is complex;
(2) The synthesis of nano-selenium by a chemical method is limited by conditions such as high temperature, high pressure, catalyst and the like, so that the purity of the synthesized nano-selenium is insufficient and the content of the synthesized nano-selenium is very low.
In order to solve the problems, the optimal scheme is to synthesize nano selenium by adopting microorganisms, based on the following steps:
reference patent application number CN 202210667076.4-a method for culturing biological nano-selenium producing bacteria based on biological nano-selenium preparation discloses: inoculating biological nano selenium-producing bacteria into a preset fermentation culture medium; introducing carbon dioxide into the fermentation medium to enable the biological nano-selenium-producing bacteria to adapt to an anaerobic environment, and carrying out anaerobic culture on the biological nano-selenium-producing bacteria; selecting a carbon source as a culture solution for culture, dripping the culture solution, and waiting for a preset time until the biological nano selenium producing bacteria have growth change; judging whether the growth change is matched with a preset strain change condition or not; wherein the predetermined strain variation includes strain division; if yes, the biological nano selenium-producing bacteria are cultivated; according to the invention, the growth change of the nano-selenium synthesized by the sodium selenite through microbial metabolism reduction is utilized, so that the synthesis time of the biological nano-selenium synthesized by the microorganism is shortened, and the synthesis purity of the biological nano-selenium is effectively improved.
The prior art optimizes the synthesis method of the biological nano-selenium, but aiming at the scheme, the synthesis time of the biological nano-selenium is shortened by matching the growth change of the biological nano-selenium with the change condition of the biological nano-selenium, and impurities possibly generated in the process are mixed into the biological nano-selenium.
Disclosure of Invention
The purpose of the application is to provide a biological nano-selenium synthesized by preset lactic acid bacteria and a synthesis preparation process thereof, and aims to solve the problems of high energy and high consumption pollution and complex preparation process in the process of synthesizing nano-selenium.
In order to achieve the above purpose, the present application provides the following technical solutions:
the application provides a biological nano selenium synthesized by preset lactobacillus and a synthesis preparation process thereof, comprising the following steps:
s1: taking 50kg of selenium-enriched seawater, placing for 48 hours, taking 15kg of seawater supernatant, placing into a fermentation barrel, adding 1kg of brown sugar, and uniformly stirring;
s2: adding 3kg of medical soluble starch after the brown sugar is completely dissolved, fully and uniformly stirring, and carrying out sealed anaerobic fermentation;
s3: placing a small amount of bacterial liquid into a reaction vessel, placing a shaking table for shaking for 48 hours, placing a high-power microscope to observe that the form of bacteria accords with the characteristics of lactic acid bacteria, using gram staining to the bacteria, and placing the bacteria under the microscope to observe the generated gram positive bacteria;
s4: weighing sodium selenite with different weights, respectively placing into conical flasks, adding fermented bacterial liquid and enzyme preparation with the same weight into each conical flask, and performing stirring anaerobic culture;
s5: observing the color change in each conical flask in the stirring anaerobic culture process, and judging that sodium selenite is completely synthesized and converted into biological nano-selenium in a preset bacterial liquid when the color appearing in the conical flask is completely brick red and no crystal is generated in the mixed liquid by using a microscope;
s6: detecting the particle size of the biological nano selenium by utilizing a micro spectrum.
Further, after the brown sugar is completely dissolved, 3kg of medical soluble starch is added, and the mixture is fully and uniformly stirred, and the step of sealing anaerobic fermentation comprises the following steps:
and in the sealed anaerobic fermentation process, stirring for 3-5 times per day, and fermenting at normal temperature for 15 days to obtain fermented bacterial liquid.
Further, the steps of placing a small amount of bacterial liquid in a reaction vessel, placing a shaking table for shaking for 48 hours, placing a high-power microscope to observe that the form of bacteria accords with the characteristics of lactic acid bacteria, using gram staining to the bacteria, and placing the bacteria under the microscope to observe the generated gram positive bacteria include:
the specific method for using gram dyeing comprises the steps of using ammonium oxalate crystal violet to dye for 1 minute, then using distilled water to wash, adding iodine solution to cover the surface to dye, then performing water washing, using absorbent paper to absorb water, adding alcohol to shake lightly to decolorize, then performing water washing, finally using the red tomato dye to dilute dye, and then performing distilled water washing, drying and microscopic examination.
Further, the steps of weighing sodium selenite with different weights, respectively placing the sodium selenite into conical flasks, adding fermented bacterial liquid and enzyme preparations with the same weight into each conical flask, and performing stirring anaerobic culture, wherein the steps comprise:
adding alkaline o-trisphenol, yellow phosphorus, metallic chromium and dilute sulfuric acid into a conical flask for chemical deoxidation.
Further, the step of detecting the particle size of the biological nano selenium by using a micro spectrum comprises the following steps: the particle size range of the biological nano selenium is 22-69nm through micro spectrum detection.
Further, the predetermined lactic acid bacteria are any one of streptococcus, leuconostoc, pediococcus, lactococcus, lactobacillus and bifidobacterium.
The application provides a biological nano selenium synthesized by preset lactic acid bacteria and a synthesis preparation process thereof, and has the following beneficial effects:
(1) The preset lactobacillus synthesized biological nano selenium and the synthesis preparation process thereof overcome the defects that impurities are easy to introduce in the traditional physical and chemical method for preparing nano selenium, the energy consumption is high, the cost is high, and the particle size distribution of the product is uneven, and the method has wide popularization value;
(2) The method synthesizes nano-selenium by lactic acid bacteria with low toxicity, stability and high biological activity, and prepares the lactic acid bacteria which can endure high-concentration sodium selenite and synthesize biological nano-selenium by smelling seawater near soil with high selenium from Zhanjiang city and county and cultivating the seawater;
(3) The biological nano-selenium prepared by adopting the biological fermentation process has the characteristics of environmental friendliness, high yield, safety, high efficiency and the like, and the biological nano-selenium obtained by the production is used for selenium-rich fertilizers and selenium-rich feeds, and has remarkable selenium-rich effects on crops, melons, fruits, vegetables, meat, eggs and milk after fertilization or feeding, and meanwhile, the biological nano-selenium has low requirements on equipment, simple process, low cost and high purity of synthesized biological nano-selenium.
Drawings
FIG. 1 is a schematic flow chart of a process for synthesizing biological nano selenium by lactic acid bacteria and synthesizing and preparing the biological nano selenium according to an embodiment of the present application;
FIG. 2 is a photograph of a gram-positive bacterium observed under a microscope;
FIG. 3 is a photograph of multiple samples taken by a high power microscope to produce crystals;
FIG. 4 is a picture of observation during an anaerobic culture with agitation without any crystals produced;
FIG. 5 is a photograph of the result of gram stain.
The implementation, functional features and advantages of the present application will be further described with reference to the accompanying drawings in conjunction with the embodiments.
Detailed Description
It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application.
The following description of the embodiments of the present application will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all, of the embodiments of the present application. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments herein without making any inventive effort, are intended to be within the scope of the present application.
Referring to fig. 1, a schematic flow chart of a process for synthesizing biological nano selenium by lactic acid bacteria and synthesizing and preparing the biological nano selenium is provided;
the preparation method for synthesizing biological nano selenium by lactic acid bacteria comprises the following steps:
s1: after 50kg of selenium-enriched seawater is taken and placed for 48 hours, 15kg of seawater supernatant is taken and placed into a fermentation barrel, and 1kg of brown sugar is added for uniform stirring.
In the step, seawater is put into a beaker, then sterile phosphate buffer solution is added for suspension, glass beads are added, oscillation is carried out for 30min at normal temperature and 180r/min, standing is carried out, suspension is collected after gravel and large particles are settled, the suspension is put into a centrifuge for centrifugation for 10 min at 5000r/min, and the sediment is put into a refrigerator at 4 ℃ for preservation after being suspended by 5ml of sterile phosphate buffer solution; 2ml of the suspension is taken and respectively added into a triangular flask containing 150 mu g/mLNa2SeO3 liquid fermentation medium, shake culture is carried out for 2 days at 37 ℃ and 180r/min, after subculture for 4 times with 5% inoculum size, dilution plate culture and streak separation are combined, and a colony group of selenium-resistant microorganisms is obtained; wherein the selenium-rich soil is soil with selenium content enriched to be more than 0.4mg/kg, bacteria existing in seawater nearby the soil have high tolerance to selenium salt, the soil is easy to culture in nature, and 15kg of supernatant is taken and added into a fermentation barrel.
S2: adding 3kg of medical soluble starch after the brown sugar is completely dissolved, fully and uniformly stirring, and carrying out sealed anaerobic fermentation; and in the sealed anaerobic fermentation process, stirring for 3-5 times per day, and fermenting at normal temperature for 15 days to obtain fermented bacterial liquid.
In the step, 3kg of medical soluble starch is added after the brown sugar is dissolved and fully and uniformly stirred, the medical soluble starch is white or light yellow powder which is easy to dissolve in cold water and is not easy to freeze after being cooled, biological fermentation can be carried out, sealing anaerobic fermentation is carried out after uniform stirring, the anaerobic fermentation is a fermentation process carried out under the condition of isolating air and not contacting with molecular oxygen, the solution is fully stirred for 3-5 times each day in the sealing anaerobic fermentation process, normal-temperature fermentation is carried out for 15 days, and finally the fermented bacterial liquid is obtained.
S3: placing a small amount of bacterial liquid into a reaction vessel, placing a shaking table for shaking for 48 hours, placing a high-power microscope to observe that the form of bacteria accords with the characteristics of lactic acid bacteria, using gram staining to the bacteria, and placing the bacteria under the microscope to observe the generated gram positive bacteria; the specific method for using gram dyeing comprises the steps of using ammonium oxalate crystal violet to dye for 1 minute, then using distilled water to wash, adding iodine solution to cover the surface to dye, then performing water washing, using absorbent paper to absorb water, adding alcohol to shake lightly to decolorize, then performing water washing, finally using the red tomato dye to dilute dye, and then performing distilled water washing, drying and microscopic examination.
In the step, a small amount of solution is put in a reaction vessel, a shaking table is placed for shaking for 48 hours, the shaking table can enable a substrate to transfer and play a role better in a system, the substrate is observed by using a high-power microscope and completely accords with the characteristics of lactobacillus, and for further demonstration, gram-positive bacteria generated by the reaction vessel can be observed under a microscope after the reaction vessel is dyed, the gram-positive bacteria are blue-violet, and the gram-positive bacteria are obtained by the following steps: primary dyeing: dripping a plurality of crystal violet dye liquor on the smear for a plurality of minutes, and washing with water; mordant dyeing: dripping iodine solution for mordant dyeing for several minutes, and washing with water; decoloring: dripping alcohol, gently shaking the slide to remove the dye liquor as much as possible, and washing with water after a few minutes until the color is basically removed; counterstaining: dripping diluted carbolic acid multiple red dye liquor into the smear for multiple dyeing for multiple minutes, washing with water, absorbing with water absorbing paper, observing with an oil lens and recording; finally, washing with distilled water, drying and microscopic examination are carried out after lean dyeing with the red dye solution.
S4: weighing sodium selenite with different weights, respectively placing into conical flasks, adding fermented bacterial liquid and enzyme preparation with the same weight into each conical flask, and performing stirring anaerobic culture; adding alkaline o-trisphenol, yellow phosphorus, metallic chromium and dilute sulfuric acid into a conical flask for chemical deoxidation.
In this step, tables of the experimental group and the control group are set as follows:
40g,60g,80g,100g and 120g of sodium selenite are weighed and put into a 1000ml conical flask respectively, meanwhile, enzyme preparation with the same weight is added into each conical flask, fermented bacterial liquid is added into each conical flask to 1000ml, stirring anaerobic culture is carried out at 37 ℃ for 3000r/min, anaerobic culture means that alkaline o-triphenol, yellow phosphorus, metal chromium and dilute sulfuric acid are added into the conical flask for chemical deoxidation, aerobic bacteria and germinated seeds consume oxygen through respiration, the color change of each sample can be observed in the process, the color change of each flask is checked, finally, the darkest appearance of 100g conical flask is found, the darkish appearance of 120g and 80g is lighter, other conical flasks are light red liquid, after stirring is stopped, 80g,100g and 120g of brick red precipitate can be observed, the other two are red liquid, the brick red precipitate can be taken under a microscope, 80g and 120g of brick red precipitate can be observed, or the brick red precipitate can be completely converted into 100g of selenium crystal in 100L, and no crystal can be completely converted into 100g of selenium crystal, and no full of selenium crystal can be completely converted into 100g of selenium crystal.
S5: observing the color change in each conical flask in the stirring anaerobic culture process, and judging that sodium selenite is completely synthesized and converted into biological nano-selenium in a preset bacterial liquid when the color appearing in the conical flask is completely brick red and no crystal is generated in the mixed liquid by using a microscope.
S6: detecting the particle size of the biological nano selenium by utilizing a micro spectrum; the particle size range of the biological nano selenium is 22-69nm through micro spectrum detection.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, apparatus, article, or method that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, apparatus, article, or method. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, apparatus, article or method that comprises the element.
The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the scope of the claims, and all equivalent structures or equivalent processes using the descriptions and drawings of the present application, or direct or indirect application in other related technical fields are included in the scope of the claims of the present application.
Although embodiments of the present application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the application, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. The biological nano selenium synthesized by the preset lactic acid bacteria and the synthesis and preparation process thereof are characterized by comprising the following steps:
s1: taking 50kg of selenium-enriched seawater, placing for 48 hours, taking 15kg of seawater supernatant, placing into a fermentation barrel, adding 1kg of brown sugar, and uniformly stirring;
s2: adding 3kg of medical soluble starch after the brown sugar is completely dissolved, fully and uniformly stirring, and carrying out sealed anaerobic fermentation;
s3: placing a small amount of bacterial liquid into a reaction vessel, placing a shaking table for shaking for 48 hours, placing a high-power microscope to observe that the form of bacteria accords with the characteristics of lactic acid bacteria, using gram staining to the bacteria, and placing the bacteria under the microscope to observe the generated gram positive bacteria;
s4: weighing sodium selenite with different weights, respectively placing into conical flasks, adding fermented bacterial liquid and enzyme preparation with the same weight into each conical flask, and performing stirring anaerobic culture;
s5: observing the color change in each conical flask in the stirring anaerobic culture process, and judging that sodium selenite is completely synthesized and converted into biological nano-selenium in a preset bacterial liquid when the color appearing in the conical flask is completely brick red and no crystal is generated in the mixed liquid by using a microscope;
s6: detecting the particle size of the biological nano selenium by utilizing a micro spectrum.
2. The method for synthesizing biological nano selenium by preset lactic acid bacteria and the synthesis preparation process thereof according to claim 1, wherein the step of fully stirring and uniformly stirring 3kg of medical soluble starch after the brown sugar is completely dissolved and performing sealed anaerobic fermentation comprises the following steps:
and in the sealed anaerobic fermentation process, stirring for 3-5 times per day, and fermenting at normal temperature for 15 days to obtain fermented bacterial liquid.
3. The method for synthesizing biological nano selenium by using preset lactobacillus and the synthesis preparation process thereof according to claim 1, wherein the steps of placing a small amount of bacterial liquid in a reaction vessel, placing a shaking table for shaking for 48 hours, placing a high-power microscope to observe that the form of bacteria accords with the characteristics of lactobacillus, using gram staining for the bacteria, and placing the bacteria under the microscope to observe the generated gram positive bacteria comprise the following steps:
the specific method for using gram dyeing comprises the steps of using ammonium oxalate crystal violet to dye for 1 minute, then using distilled water to wash, adding iodine solution to cover the surface to dye, then performing water washing, using absorbent paper to absorb water, adding alcohol to shake lightly to decolorize, then performing water washing, finally using the red tomato dye to dilute dye, and then performing distilled water washing, drying and microscopic examination.
4. The method for synthesizing biological nano-selenium by using preset lactobacillus and the synthesis preparation process thereof according to claim 1, wherein the steps of weighing sodium selenite with different weights, respectively placing the sodium selenite into conical flasks, adding fermented bacterial liquid and enzyme preparation with the same weight into each conical flask, and performing stirring anaerobic culture comprise the following steps:
adding alkaline o-trisphenol, yellow phosphorus, metallic chromium and dilute sulfuric acid into a conical flask for chemical deoxidation.
5. The method for synthesizing biological nano-selenium by using preset lactic acid bacteria according to claim 1, wherein the step of detecting the particle size of the biological nano-selenium by using micro-spectrum comprises: the particle size range of the biological nano selenium is 22-69nm through micro spectrum detection.
6. The method for synthesizing biological nano-selenium by using preset lactic acid bacteria according to claim 1, wherein the preset lactic acid bacteria are any one of streptococcus, leuconostoc, pediococcus, lactococcus, lactobacillus and bifidobacterium.
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