A low-carbon probiotic preparation and a method for preparing the same

Abstract

The application provides a low-carbon probiotic preparation and a preparation method thereof, and belongs to the technical field of probiotics. Carbon dioxide is introduced into a low-eutectic solvent, immobilized Mannheim succinic acid producing bacteria are added, fermentation culture is carried out, separation is performed, probiotics are inoculated in a liquid, fermentation culture is carried out, and then the low-carbon probiotic preparation is prepared through polylactic acid embedding. The low-carbon probiotic preparation prepared by the application firstly utilizes a CO2-resistant strain to convert carbon dioxide into an organic carbon source, thereby promoting the proliferation of probiotics. After embedding in polylactic acid, the low-carbon probiotic preparation can well resist gastric acid and is released in an intestinal alkaline environment, appropriately releases CO2 gas, utilizes CO2 to promote intestinal peristalsis, the probiotic can also inhibit inflammatory factors, thereby can inhibit intestinal inflammation, has a good promoting effect on postoperative rehabilitation, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of probiotic technology, specifically to a low-carbon probiotic preparation and its preparation method. Background Technology

[0002] Strengthen industrial restructuring, transformation and upgrading, and energy conservation and efficiency improvement; accelerate the low-carbon and zero-carbon transformation of energy; and increase carbon sinks, namely through afforestation, forest management, vegetation restoration and other measures, to absorb carbon dioxide in the atmosphere through plant photosynthesis and fix it in vegetation and soil, thereby achieving net-zero emissions of greenhouse gases from human activities by reducing the concentration of greenhouse gases in the atmosphere.

[0003] Carbon sequestration, also known as carbon storage, includes physical carbon sequestration and biological carbon sequestration. Physical carbon sequestration involves the long-term storage of carbon dioxide in extracted oil and gas wells, coal seams, and the deep sea. Biological carbon sequestration improves the carbon absorption and storage capacity of ecosystems by controlling carbon flux, making it the cheapest and least invasive method for fixing atmospheric carbon dioxide, and also the most economical, safe, and effective. Some probiotics can utilize carbon dioxide to react with hydrogen, producing organic compounds such as formic acid that they can use, thus exhibiting better physiological activity. Summary of the Invention

[0004] The purpose of this invention is to propose a low-carbon probiotic preparation and its preparation method. First, CO2-resistant strains are used to convert carbon dioxide into an organic carbon source, thereby promoting the proliferation of probiotics. After being embedded in polylactic acid, it can withstand gastric acid well and be released in the alkaline environment of the intestine, releasing CO2 gas appropriately. CO2 promotes intestinal peristalsis. Probiotics can also inhibit inflammatory factors, thereby inhibiting intestinal inflammation. It has a good promoting effect on postoperative recovery and has broad application prospects.

[0005] The technical solution of this invention is implemented as follows:

[0006] This invention provides a method for preparing a low-carbon probiotic preparation, which involves passing carbon dioxide into a eutectic solvent, adding immobilized Mannheim-producing succinic acid bacteria, fermenting and culturing, separating, inoculating probiotics in the liquid, fermenting and culturing, and then encapsulating with polylactic acid to obtain the low-carbon probiotic preparation.

[0007] As a further improvement to the present invention, the following steps are included:

[0008] S1. Preparation of eutectic solvent: Modified hydrogen bond acceptor and glycerol are mixed, heated and stirred until homogeneous to obtain eutectic solvent;

[0009] S2. Preparation of carbon dioxide-rich solution: Carbon dioxide is passed into a eutectic solvent to obtain a carbon dioxide-rich solution;

[0010] S3. Fermentation: Add immobilized transforming bacteria to a carbon dioxide-rich solution, heat and stir to ferment, filter to separate the immobilized transforming bacteria, wash the solid, freeze-dry, and keep the liquid for later use;

[0011] S4. Re-fermentation: Inoculate the liquid from step S3 with probiotic seed liquid, ferment and culture to obtain fermented liquid;

[0012] S5. Immobilization: Polylactic acid and surfactant are dissolved in dichloromethane to obtain an oil phase; fermentation broth is added dropwise to the oil phase, emulsified, the organic solvent is evaporated by ventilation, centrifuged, washed, and dried to obtain a low-carbon probiotic preparation.

[0013] As a further improvement of the present invention, the preparation method of the modified hydrogen bond acceptor in step S1 is as follows: amino acids and oleic acid are added to water, EDC and NHS are added, an amino compound is added, the mixture is stirred and reacted, dialyzed, and dried to obtain the modified hydrogen bond acceptor; the molar ratio of the modified hydrogen bond acceptor to glycerol is 1:1-3, the heating and stirring temperature is 40-50℃, and the time is 1-3h.

[0014] As a further improvement of the present invention, the amino acid is selected from at least one of L-glycine, L-alanine, and L-arginine, the amino compound is selected from at least one of triethylamine, ethylenediamine, and diethylamine, and the molar ratio of the amino acid, oleic acid, EDC, NHS and the amino compound is 2-4:1-2:3-4:3-4:1-2.

[0015] As a further improvement of the present invention, the carbon dioxide aeration rate in step S2 is 2-4 mL / min, and the solubility of carbon dioxide in the carbon dioxide-rich solution is 1.5-1.8 mol CO2 / kg DESs.

[0016] As a further improvement of the present invention, the solid-liquid ratio of the immobilized transforming bacteria and the carbon dioxide-rich solution in step S3 is 1:3-5 g / mL, the heating and stirring fermentation temperature is 38-40℃, 100-200 r / min, and the fermentation time is 120-168 h. The preparation method of the immobilized transforming bacteria is as follows:

[0017] Preparation of T1.UiO-66-NH2: Zirconium tetrachloride and 2-aminoterephthalic acid were added to a solvent, ultrasonically dispersed, hydrothermally reacted, cooled, centrifuged, washed, and dried to obtain UiO-66-NH2.

[0018] T2. Preparation of modified UiO-66-NH2: Oleic acid was added to toluene, EDC and NHS were added, the mixture was stirred and activated, UiO-66-NH2 was added, the mixture was stirred and reacted, centrifuged, washed and dried to obtain modified UiO-66-NH2;

[0019] T3. Immobilization: Modified UiO-66-NH2 was added to water, along with Mannheim succinic acid-producing bacteria, NHS, and EDC. The mixture was stirred, centrifuged, washed, and dried to obtain immobilized transformant bacteria.

[0020] As a further improvement of the present invention, in step T1, the molar ratio of zirconium tetrachloride and 2-aminoterephthalic acid is 1:1, the solvent is a mixture of N,N-dimethylformamide and acetic acid with a volume ratio of 38-40:1-2, the hydrothermal reaction temperature is 120-130℃, and the time is 20-24h; in step T2, the mass ratio of oleic acid, EDC, NHS, and UiO-66-NH2 is 3-5:2-3:2-3:6-8, the stirring activation time is 20-30min, and the stirring reaction time is 10-12h; in step T3, the mass ratio of modified UiO-66-NH2, Mannheim succinic acid-producing bacteria, NHS, and EDC is 10-15:3-5:1-3:1-3, and the stirring reaction time is 12-15h.

[0021] As a further improvement of the present invention, the probiotic seed solution in step S4 includes *Lactobacillus plantarum* seed solution, *Lactobacillus rhamnosus* seed solution, and *Bifidobacterium longum* seed solution, with inoculation amounts of 1-2 v / v%, 0.5-1.5 v / v%, and 1-3 v / v%, respectively, and the bacterial count of the seed solution is 10. 8 -10 9 The concentration of cfu / mL was determined by fermentation at a temperature of 35-38℃, a speed of 100-200 r / min, and a fermentation time of 24-36 h.

[0022] As a further improvement of the present invention, the mass ratio of polylactic acid, surfactant and fermentation broth in step S5 is 12-15:0.5-1:40-60, and the surfactant is selected from at least one of Span-20, Span-40, Span-60, Span-80 and Span-85.

[0023] This invention further protects a low-carbon probiotic preparation obtained by the above-described preparation method.

[0024] The present invention has the following beneficial effects:

[0025] This invention prepares a modified hydrogen bond acceptor by undergoing a dehydration condensation reaction of amino acids with oleic acid and amino compounds. Increasing the alkyl chain length improves CO2 solubility. Simultaneously, coupling with amino compounds promotes the binding of the amino structure with carbon dioxide, further enhancing CO2 solubility. This results in a eutectic solvent with excellent carbon dioxide solubility, achieving a carbon dioxide-rich solution with a solubility of 1.5-1.8 mol CO2 / kg DESs after carbon dioxide is introduced. This effectively absorbs carbon dioxide from the environment, contributing to environmental protection. For example, carbon dioxide produced from straw burning can be significantly reduced by first introducing this eutectic solvent, thus protecting the environment.

[0026] *Mannheim succinic acid-producing bacteria* is another facultative anaerobic, CO2-loving Gram-negative bacterium isolated and screened from the bovine rumen. It can grow under CO2-rich anaerobic conditions and produce organic carbon sources that can be utilized by common probiotics. Simultaneously, this invention prepares a carrier with an amino structure, UiO-66-NH2, which has a large specific surface area and can effectively immobilize the loaded bacterial cells. Furthermore, the amino structure can dehydrate and condense with oleic acid, thereby coupling oleic acid to the carrier and greatly increasing the carrier's CO2 adsorption capacity. Additionally, the *Mannheim succinic acid-producing bacteria* cell membrane contains many protein peptides, which can also be immobilized on the carrier through the condensation of EDC and NHS, thus producing immobilized transformant bacteria. This not only efficiently converts carbon dioxide in solution into organic matter but also releases many factors beneficial to probiotic growth, such as succinic acid, thus providing a favorable environment for subsequent probiotic growth and proliferation.

[0027] Probiotics are added to the liquid, and after fermentation, the mixture is encapsulated in polylactic acid (PLA). PLA itself has good acid resistance and exhibits good stability in acidic environments. Therefore, PLA-coated microcapsules can maintain their structural integrity for a relatively long time in acidic environments such as gastric acid. However, their stability is relatively poor under alkaline conditions, and hydrolysis may occur in the alkaline environment of the intestine, leading to the destruction of the microcapsule structure. This releases the probiotics from the microcapsules. At the same time, the released probiotics can utilize the PLA shell material to release CO2 gas appropriately, which promotes intestinal peristalsis. The probiotics can also inhibit inflammatory factors, thereby suppressing intestinal inflammation and promoting postoperative recovery.

[0028] The low-carbon probiotics prepared in this invention first utilize CO2-resistant strains to convert carbon dioxide into an organic carbon source, thereby promoting the proliferation of probiotics. After being embedded in polylactic acid, they can effectively resist gastric acid and be released in the alkaline environment of the intestine, appropriately releasing CO2 gas. The CO2 promotes intestinal peristalsis. The probiotics can also inhibit inflammatory factors, thereby suppressing intestinal inflammation and having a good promoting effect on postoperative recovery, showing broad application prospects. Detailed Implementation

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Mannheim-derived succinic acid bacteria, 10 billion CFU / g; Lactobacillus plantarum, 10 billion CFU / g; Lactobacillus rhamnosus, 10 billion CFU / g; Bifidobacterium longum, 10 billion CFU / g.

[0031] Preparation of inoculum seed solution: Inoculate the inoculum into slant agar medium, incubate at 35-38℃ and 150 r / min for 24-36 h to activate the culture, and obtain a culture with a bacterial count of 10. 8 -10 9 CFU / mL bacterial seed solution.

[0032] Preparation Example 1: Preparation of Immobilized Transformant Bacteria

[0033] The method is as follows:

[0034] Preparation of T1.UiO-66-NH2: 0.1 mol zirconium tetrachloride and 0.1 mol 2-aminoterephthalic acid were added to 200 mL of solvent, ultrasonically dispersed at 1200 W for 15 min, hydrothermally reacted at 120 °C for 20 h, cooled, centrifuged, washed, and dried to obtain UiO-66-NH2.

[0035] The solvent is a mixture of N,N-dimethylformamide and acetic acid in a volume ratio of 38:2.

[0036] T2. Preparation of modified UiO-66-NH2: 3g of oleic acid was added to 150mL of toluene, 2g of EDC and 2g of NHS were added, the mixture was stirred and activated for 20min, 6g of UiO-66-NH2 was added, the mixture was stirred and reacted for 10h, centrifuged, washed and dried to obtain modified UiO-66-NH2;

[0037] T3. Immobilization: 10g of modified UiO-66-NH2 was added to water, along with 3g of Mannheim succinic acid-producing bacteria, 1g of NHS and 1g of EDC. The mixture was stirred and reacted for 12h, then centrifuged, washed and dried to obtain the immobilized transformant bacteria.

[0038] Preparation Example 2: Preparation of Immobilized Transformant Bacteria

[0039] The method is as follows:

[0040] Preparation of T1.UiO-66-NH2: 0.1 mol zirconium tetrachloride and 0.1 mol 2-aminoterephthalic acid were added to 200 mL of solvent, ultrasonically dispersed at 1200 W for 15 min, hydrothermally reacted at 130 °C for 24 h, cooled, centrifuged, washed, and dried to obtain UiO-66-NH2.

[0041] The solvent is a mixture of N,N-dimethylformamide and acetic acid in a volume ratio of 40:1.

[0042] T2. Preparation of modified UiO-66-NH2: 5g oleic acid was added to 150mL toluene, 3g EDC and 3g NHS were added, and the mixture was stirred and activated for 30min. 8g UiO-66-NH2 was added, and the mixture was stirred and reacted for 12h. After centrifugation, washing and drying, modified UiO-66-NH2 was obtained.

[0043] T3. Immobilization: 15g of modified UiO-66-NH2 was added to water, along with 5g of Mannheim succinic acid-producing bacteria, 3g of NHS and 3g of EDC. The mixture was stirred and reacted for 15h, then centrifuged, washed and dried to obtain the immobilized transformant bacteria.

[0044] Preparation Example 3: Preparation of Immobilized Transformant Bacteria

[0045] The method is as follows:

[0046] Preparation of T1.UiO-66-NH2: 0.1 mol zirconium tetrachloride and 0.1 mol 2-aminoterephthalic acid were added to 200 mL of solvent, ultrasonically dispersed at 1200 W for 15 min, hydrothermally reacted at 125 °C for 22 h, cooled, centrifuged, washed, and dried to obtain UiO-66-NH2.

[0047] The solvent is a mixture of N,N-dimethylformamide and acetic acid in a volume ratio of 39:1.

[0048] T2. Preparation of modified UiO-66-NH2: 4g of oleic acid was added to 150mL of toluene, 2.5g of EDC and 2.5g of NHS were added, and the mixture was stirred and activated for 25min. Then, 7g of UiO-66-NH2 was added, and the mixture was stirred and reacted for 11h. After centrifugation, washing and drying, modified UiO-66-NH2 was obtained.

[0049] T3. Immobilization: 12g of modified UiO-66-NH2 was added to water, along with 4g of Mannheim succinic acid-producing bacteria, 2g of NHS and 2g of EDC. The mixture was stirred and reacted for 13 hours. After centrifugation, washing and drying, immobilized transformant bacteria were obtained.

[0050] Comparative Preparation Example 1

[0051] The difference compared to preparation example 3 is that step T2 was not performed.

[0052] Specifically as follows:

[0053] Preparation of T1.UiO-66-NH2: 0.1 mol zirconium tetrachloride and 0.1 mol 2-aminoterephthalic acid were added to 200 mL of solvent, ultrasonically dispersed at 1200 W for 15 min, hydrothermally reacted at 125 °C for 22 h, cooled, centrifuged, washed, and dried to obtain UiO-66-NH2.

[0054] The solvent is a mixture of N,N-dimethylformamide and acetic acid in a volume ratio of 39:1.

[0055] T2. Immobilization: 12g UiO-66-NH2 was added to water, along with 4g Mannheim succinic acid-producing bacteria, 2g NHS and 2g EDC. The mixture was stirred and reacted for 13 hours. After centrifugation, washing and drying, immobilized transformant bacteria were obtained.

[0056] Preparation Example 4: Preparation of Modified Hydrogen Bond Acceptors

[0057] The method is as follows: 0.2 mol L-glycine and 0.1 mol oleic acid were added to 200 mL of water, along with 0.3 mol EDC and 0.3 mol NHS. The mixture was stirred and activated for 30 min. Then, 0.1 mol ethylenediamine was added, and the mixture was stirred and reacted for 12 h. The mixture was dialyzed for 72 h using a dialysis bag with a pore size of 5000 Da. The unpermeable solution was dried to obtain the modified hydrogen bond acceptor.

[0058] Preparation Example 5: Preparation of Modified Hydrogen Bond Acceptor

[0059] The method is as follows: 0.4 mol L-alanine and 0.2 mol oleic acid were added to 200 mL of water, followed by 0.4 mol EDC and 0.4 mol NHS. The mixture was stirred and activated for 30 min. Then, 0.2 mol ethylenediamine was added, and the mixture was stirred and reacted for 12 h. The mixture was dialyzed for 72 h using a dialysis bag with a pore size of 5000 Da. The unpermeable solution was dried to obtain the modified hydrogen bond acceptor.

[0060] Preparation Example 6: Preparation of Modified Hydrogen Bond Acceptor

[0061] The method is as follows: 0.3 mol L-arginine and 0.15 mol oleic acid were added to 200 mL of water, along with 0.35 mol EDC and 0.35 mol NHS. The mixture was stirred and activated for 30 min. Then, 0.15 mol ethylenediamine was added, and the mixture was stirred and reacted for 12 h. The mixture was dialyzed for 72 h using a dialysis bag with a pore size of 5000 Da. The unpermeable solution was dried to obtain the modified hydrogen bond acceptor.

[0062] Comparative Preparation Example 2

[0063] The difference compared to Preparation Example 6 is that oleic acid was not added.

[0064] The method is as follows: 0.45 mol L-arginine was added to 200 mL of water, along with 0.35 mol EDC and 0.35 mol NHS. The mixture was stirred and activated for 30 min. Then, 0.15 mol ethylenediamine was added, and the mixture was stirred and reacted for 12 h. The mixture was dialyzed for 72 h using a dialysis bag with a pore size of 5000 Da. The unpermeable solution was dried to obtain the modified hydrogen bond acceptor.

[0065] Comparative preparation example 3

[0066] The difference compared to Preparation Example 6 is that ethylenediamine was not added.

[0067] The method is as follows: 0.45 mol L-arginine and 0.15 mol oleic acid were added to 200 mL of water, along with 0.35 mol EDC and 0.35 mol NHS. The mixture was stirred and activated for 30 min, and then stirred for 12 h. The mixture was dialyzed for 72 h using a dialysis bag with a pore size of 5000 Da. The unpermeable solution was dried to obtain the modified hydrogen bond acceptor.

[0068] Example 1

[0069] This embodiment provides a method for preparing a low-carbon probiotic preparation, including the following steps:

[0070] S1. Preparation of eutectic solvent: The modified hydrogen bond acceptor obtained in Preparation Example 4 was mixed with glycerol in a molar ratio of 1:1, heated to 40°C, and stirred for 1 hour to obtain the eutectic solvent.

[0071] S2. Preparation of carbon dioxide-rich solution: Carbon dioxide was introduced into a eutectic solvent at a flow rate of 2 mL / min to obtain a carbon dioxide-rich solution with a carbon dioxide solubility of 1.62 mol CO2 / kg DESs.

[0072] S3. Fermentation: Add 10g of the immobilized transformant bacteria prepared in Example 1 to 30mL of carbon dioxide-rich solution, ferment at 38℃ and 100r / min for 120h, filter to separate the immobilized transformant bacteria, wash the solid, freeze dry, and keep the liquid for use;

[0073] S4. Re-fermentation: Inoculate the liquid from step S3 with Lactobacillus plantarum seed solution, Lactobacillus rhamnosus seed solution, and Bifidobacterium longum seed solution, with inoculation amounts of 1v / v%, 0.5v / v%, and 1v / v%, respectively, at 35℃ and 100r / min for 24h to obtain fermentation liquid.

[0074] S5. Immobilization: Dissolve 12g polylactic acid and 0.5g Span-40 in 200mL dichloromethane to obtain an oil phase; add 40g fermentation broth to the oil phase, emulsify at 8000r / min for 15min, evaporate the organic solvent by ventilation, centrifuge, wash, and dry to obtain a low-carbon probiotic preparation.

[0075] Example 2

[0076] This embodiment provides a method for preparing a low-carbon probiotic preparation, including the following steps:

[0077] S1. Preparation of eutectic solvent: The modified hydrogen bond acceptor obtained in Preparation Example 5 was mixed with glycerol in a molar ratio of 1:3. The mixture was heated to 50°C and stirred for 3 hours to obtain the eutectic solvent.

[0078] S2. Preparation of carbon dioxide-rich solution: Carbon dioxide was introduced into a eutectic solvent at a flow rate of 4 mL / min to obtain a carbon dioxide-rich solution with a carbon dioxide solubility of 1.78 mol CO2 / kg DESs.

[0079] S3. Fermentation: Add 10g of the immobilized transformant bacteria prepared in Example 2 to 50mL of carbon dioxide-rich solution, ferment at 40℃ and 200r / min for 168h, filter to separate the immobilized transformant bacteria, wash the solid, freeze dry, and keep the liquid for use;

[0080] S4. Re-fermentation: Inoculate the liquid from step S3 with Lactobacillus plantarum seed solution, Lactobacillus rhamnosus seed solution, and Bifidobacterium longum seed solution, with inoculation amounts of 2v / v%, 1.5v / v%, and 3v / v%, respectively. Ferment at 38℃ and 200r / min for 36h to obtain fermentation liquid.

[0081] S5. Immobilization: Dissolve 15g polylactic acid and 1g Span-80 in 200mL dichloromethane to obtain an oil phase; add 60g fermentation broth to the oil phase, emulsify at 8000r / min for 15min, evaporate the organic solvent by ventilation, centrifuge, wash, and dry to obtain a low-carbon probiotic preparation.

[0082] Example 3

[0083] This embodiment provides a method for preparing a low-carbon probiotic preparation, including the following steps:

[0084] S1. Preparation of eutectic solvent: The modified hydrogen bond acceptor obtained in Preparation Example 6 was mixed with glycerol in a molar ratio of 1:2. The mixture was heated to 45°C and stirred for 2 hours to obtain the eutectic solvent.

[0085] S2. Preparation of carbon dioxide-rich solution: Carbon dioxide was introduced into a eutectic solvent at a flow rate of 3 mL / min to obtain a carbon dioxide-rich solution with a carbon dioxide solubility of 1.69 mol CO2 / kg DESs.

[0086] S3. Fermentation: Add 10g of the immobilized transformant bacteria prepared in Example 3 to 40mL of carbon dioxide-rich solution, ferment at 39℃ and 150r / min for 144h, filter to separate the immobilized transformant bacteria, wash the solid, freeze dry, and keep the liquid for use;

[0087] S4. Re-fermentation: Inoculate the liquid from step S3 with Lactobacillus plantarum seed solution, Lactobacillus rhamnosus seed solution, and Bifidobacterium longum seed solution, with inoculation amounts of 1.5 v / v%, 1 v / v%, and 2 v / v%, respectively. Ferment at 37°C and 150 r / min for 30 h to obtain fermentation liquid.

[0088] S5. Immobilization: Dissolve 13g polylactic acid and 0.7g Span-85 in 200mL dichloromethane to obtain an oil phase; add 50g fermentation broth to the oil phase, emulsify at 8000r / min for 15min, evaporate the organic solvent by ventilation, centrifuge, wash, and dry to obtain a low-carbon probiotic preparation.

[0089] Comparative Example 1

[0090] Compared to Example 3, the difference is that the modified hydrogen bond acceptor is replaced by an equal amount of L-arginine, so the solubility of carbon dioxide in step S2 is 1.12 mol CO2 / kg DESs.

[0091] Comparative Example 2

[0092] The difference from Example 3 is that the modified hydrogen bond acceptor was prepared by Comparative Preparation Example 2, so the solubility of carbon dioxide in step S2 is 1.32 mol CO2 / kg DESs.

[0093] Comparative Example 3

[0094] The difference from Example 3 is that the modified hydrogen bond acceptor was prepared by Comparative Preparation Example 3, so the solubility of carbon dioxide in step S2 is 1.39 mol CO2 / kg DESs.

[0095] Comparative Example 4

[0096] The difference from Example 3 is that the immobilized transformant bacteria were prepared from Comparative Preparation Example 1.

[0097] Comparative Example 5

[0098] Compared with Example 3, the difference is that in step S4, no *Lactobacillus plantarum* seed solution was inoculated, and the inoculation amounts were 1 v / v% and 2 v / v, respectively.

[0099] Comparative Example 6

[0100] The difference from Example 3 is that in step S4, no Lactobacillus rhamnosus seed culture was inoculated, and the inoculation amounts were 1.5 v / v% and 2 v / v, respectively.

[0101] Comparative Example 7

[0102] Compared with Example 3, the difference is that no Bifidobacterium longum seed solution was inoculated in step S4, and the inoculation amount was 1.5 v / v% and 1 v / v, respectively.

[0103] Comparative Example 8

[0104] The difference from Example 3 is that step S5 was not performed.

[0105] Specifically as follows:

[0106] S1. Preparation of eutectic solvent: The modified hydrogen bond acceptor obtained in Preparation Example 6 was mixed with glycerol in a molar ratio of 1:2. The mixture was heated to 45°C and stirred for 2 hours to obtain the eutectic solvent.

[0107] S2. Preparation of carbon dioxide-rich solution: Carbon dioxide was introduced into a eutectic solvent at a flow rate of 3 mL / min to obtain a carbon dioxide-rich solution with a carbon dioxide solubility of 1.69 mol CO2 / kg DESs.

[0108] S3. Fermentation: Add 10g of the immobilized transformant bacteria prepared in Example 3 to 40mL of carbon dioxide-rich solution, ferment at 39℃ and 150r / min for 144h, filter to separate the immobilized transformant bacteria, wash the solid, freeze dry, and keep the liquid for use;

[0109] S4. Re-fermentation: Inoculate the liquid from step S3 with Lactobacillus plantarum seed solution, Lactobacillus rhamnosus seed solution, and Bifidobacterium longum seed solution, with inoculation amounts of 1.5 v / v%, 1 v / v%, and 2 v / v%, respectively. Ferment at 37°C and 150 r / min for 30 h, then freeze-dry to obtain the product.

[0110] Test Example 1

[0111] Five-week-old male BALB / c mice (22-28g) were selected and allowed free access to food and water at room temperature. The BALB / c mice were randomly divided into a control group, an ulcer group, Example 1-3 groups, and Comparative Examples 1-8 groups, with 10 mice in each group. After 7 days of acclimatization, the control group continued to be fed a basal diet, while the other groups were fed a diet containing 0.2% adenine for 6 weeks. Then, the control group received an intraperitoneal injection of dimethyl sulfoxide, while the ulcer group received a subcutaneous injection of 2.5g / L indomethacin NaHCO3 solution. Example 1-3 groups and Comparative Examples 1-8 groups were treated with 1g / kg of low-carbon probiotic preparation by gavage, followed by an injection of indomethacin 24 hours later.

[0112] After successful modeling, intestinal transit time was measured. Mice in each group were gavaged with 2 mL of 10% activated charcoal and the gavage time of each mouse was recorded. Each mouse was placed in a metabolic cage and the time of excretion of the first black feces of each mouse was observed and recorded. The time from gavage to excretion of the first black feces was the intestinal transit time.

[0113] After the modeling was completed, the mice in each group were placed in metabolic cages, and the feces of each group of mice were collected and counted over 24 hours. The weight of each feces was then measured using an electronic balance as wet weight (W1), and then dried to constant weight and measured as dry weight (W2). The percentage of water content in the feces was calculated using the formula below.

[0114] Fecal water content = [W1 - W2) / W1] × 100%

[0115] The results are shown in Table 1.

[0116] Table 1

[0117] Group Time to first black stool passage (min) Fecal moisture content (%) control group 56.2±11.3 59.6±8.9 Ulcer group 325.7±35.6 44.9±7.2 Example 1 124.1±21.2 55.2±8.5 Example 2 121.4±19.5 54.9±9.2 Example 3 120.7±17.8 56.6±8.0 Comparative Example 1 152.6±23.5 49.7±9.6 Comparative Example 2 144.7±24.9 52.3±10.1 Comparative Example 3 141.2±23.7 51.1±9.6 Comparative Example 4 147.8±24.1 50.6±11.3 Comparative Example 5 150.1±22.9 50.3±12.4 Comparative Example 6 153.5±26.7 48.7±11.9 Comparative Example 7 149.6±24.5 50.8±12.1 Comparative Example 8 192.5±33.2 46.5±13.6

[0118] Note: * indicates P<0.05 compared with the control group; # indicates P<0.05 compared with the ulcer group.

[0119] As shown in the table above, the low-carbon probiotic preparations obtained in Examples 1-3 of this invention can significantly improve the constipation symptoms in mice with ulcer constipation.

[0120] Twenty-four hours after successful modeling, mice in each group were sacrificed under general anesthesia, and gastric tissue was collected. The tissue was homogenized in 10% ice-cold phosphate buffer, centrifuged, and the supernatant was collected. The levels of total antioxidant state (TAS), total oxidized state (TOS), tumor necrosis factor α (TNF-α), and interleukin-6 (IL-6) in the gastric tissue were measured according to the ELISA kit instructions. The oxidative stress index was calculated as TOS / TAS.

[0121] The results are shown in Table 2.

[0122] Table 2

[0123] Group Oxidative stress index TNF-α content (ng / g) IL-6 content (ng / g) control group 0.19±0.06 6.23±0.75 2.59±0.27 Ulcer group 0.81±0.12 14.21±1.82 6.92±0.66 Example 1 0.33±0.09 5.55±0.84 2.42±0.34 Example 2 0.32±0.07 5.48±0.89 2.40±0.31 Example 3 0.30±0.05 5.32±0.81 2.37±0.29 Comparative Example 1 0.47±0.14 8.15±0.92 3.42±0.44 Comparative Example 2 0.41±0.12 7.67±0.89 3.10±0.39 Comparative Example 3 0.40±0.16 7.79±0.95 3.19±0.36 Comparative Example 4 0.45±0.13 7.92±0.90 3.24±0.37 Comparative Example 5 0.49±0.15 8.08±0.89 3.34±0.49 Comparative Example 6 0.46±0.17 8.12±0.82 3.39±0.46 Comparative Example 7 0.51±0.16 7.97±0.79 3.29±0.41 Comparative Example 8 0.67±0.22 9.45±0.94 5.21±0.65

[0124] Note: * indicates P<0.05 compared with the control group; # indicates P<0.05 compared with the ulcer group.

[0125] As shown in the table above, the low-carbon probiotic preparations obtained in Examples 1-3 of this invention can significantly improve the antioxidant and anti-inflammatory properties of mice with ulcer constipation.

[0126] In mice with gastric ulcers, the anti-inflammatory agent caused severe gastrointestinal damage and oxidative stress. At the same time, the adenine diet induced constipation in the mice, thus simulating the symptoms of constipation and increased inflammatory markers in postoperative patients. By taking the low-carbon probiotic preparation of this invention, CO2 gas can be released appropriately, which can promote intestinal peristalsis. The probiotics can also inhibit inflammatory factors, thereby inhibiting intestinal inflammation and promoting postoperative recovery.

[0127] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a low-carbon probiotic preparation, characterized in that, Includes the following steps: S1. Preparation of eutectic solvent: A modified hydrogen bond acceptor and glycerol are mixed, heated and stirred until homogeneous to obtain a eutectic solvent; the modified hydrogen bond acceptor is prepared as follows: amino acids and oleic acid are added to water, EDC and NHS are added, an amino compound is added, the mixture is stirred and reacted, dialyzed, and dried to obtain the modified hydrogen bond acceptor; the amino compound is selected from at least one of triethylamine, ethylenediamine, and diethylamine; S2. Preparation of carbon dioxide-rich solution: Carbon dioxide is passed into a eutectic solvent to obtain a carbon dioxide-rich solution; S3. Fermentation: Immobilized transforming bacteria are added to a carbon dioxide-rich solution, heated and stirred for fermentation, the immobilized transforming bacteria are separated by filtration, the solid is washed, freeze-dried, and the liquid is retained; the preparation method of the immobilized transforming bacteria is as follows: T1. Preparation of UiO-66-NH2: Zirconium tetrachloride and 2-aminoterephthalic acid were added to a solvent, ultrasonically dispersed, hydrothermally reacted, cooled, centrifuged, washed, and dried to obtain UiO-66-NH2; T2. Preparation of modified UiO-66-NH2: Oleic acid was added to toluene, EDC and NHS were added, the mixture was stirred and activated, UiO-66-NH2 was added, the mixture was stirred and reacted, centrifuged, washed and dried to obtain modified UiO-66-NH2; T3. Immobilization: Modified UiO-66-NH2 was added to water, along with Mannheim succinic acid-producing bacteria, NHS, and EDC. The mixture was stirred, centrifuged, washed, and dried to obtain immobilized transformant bacteria. S4. Re-fermentation: Inoculate the liquid from step S3 with probiotic seed solution, ferment and culture to obtain fermented bacterial solution; the probiotic seed solution includes Lactobacillus plantarum seed solution, Lactobacillus rhamnosus seed solution, and Bifidobacterium longum seed solution. S5. Immobilization: Polylactic acid and surfactant are dissolved in dichloromethane to obtain an oil phase; fermentation broth is added dropwise to the oil phase, emulsified, the organic solvent is evaporated by ventilation, centrifuged, washed, and dried to obtain a low-carbon probiotic preparation.

2. The preparation method according to claim 1, characterized in that, In step S1, the molar ratio of the modified hydrogen bond acceptor to glycerol is 1:1-3, and the heating and stirring temperature is 40-50℃ for 1-3 hours.

3. The preparation method according to claim 2, characterized in that, The amino acid is selected from at least one of L-glycine, L-alanine, and L-arginine, and the molar ratio of the amino acid, oleic acid, EDC, NHS, and amino compound is 2-4:1-2:3-4:3-4:1-2.

4. The preparation method according to claim 1, characterized in that, The carbon dioxide aeration rate in step S2 is 2-4 mL / min, and the solubility of carbon dioxide in the carbon dioxide-rich solution is 1.5-1.8 mol CO2 / kg DESs.

5. The preparation method according to claim 1, characterized in that, In step S3, the solid-liquid ratio of the immobilized transforming bacteria and the carbon dioxide-rich solution is 1:3-5 g / mL, and the heating and stirring fermentation temperature is 38-40℃, 100-200 r / min, and the fermentation time is 120-168 h.

6. The preparation method according to claim 1, characterized in that, In step T1, the molar ratio of zirconium tetrachloride and 2-aminoterephthalic acid is 1:1, the solvent is a mixture of N,N-dimethylformamide and acetic acid with a volume ratio of 38-40:1-2, and the hydrothermal reaction temperature is 120-130℃ for 20-24 h. In step T2, the mass ratio of oleic acid, EDC, NHS, and UiO-66-NH2 is 3-5:2-3:2-3:6-8, the stirring activation time is 20-30 min, and the stirring reaction time is 10-12 h. In step T3, the mass ratio of modified UiO-66-NH2, Mannheim succinic acid-producing bacteria, NHS, and EDC is 10-15:3-5:1-3:1-3, and the stirring reaction time is 12-15 h.

7. The preparation method according to claim 1, characterized in that, In step S4, the inoculation amounts of *Lactobacillus plantarum* seed solution, *Lactobacillus rhamnosus* seed solution, and *Bifidobacterium longum* seed solution in the probiotic seed solution are 1-2 v / v%, 0.5-1.5 v / v%, and 1-3 v / v, respectively, and the bacterial count of the seed solution is 10. 8 -10 9 The concentration of cfu / mL was determined by fermentation at a temperature of 35-38℃, a speed of 100-200 r / min, and a fermentation time of 24-36 h.

8. The preparation method according to claim 1, characterized in that, In step S5, the mass ratio of polylactic acid, surfactant, and fermentation broth is 12-15:0.5-1:40-60, and the surfactant is selected from at least one of Span-20, Span-40, Span-60, Span-80, and Span-85.

9. A low-carbon probiotic preparation prepared by the method according to any one of claims 1-8.