Method and application of hydrophilic barrier layer-assisted probiotics embedding tributyrin

Abstract

The present invention discloses a method and application for encapsulating tributyrin with a hydrophilic barrier layer to aid probiotics in encapsulating tributyrin, belonging to the field of oil microencapsulation technology. The method comprises the following steps: 1) mixing probiotic cells with tributyrin and washing with an aqueous solution to obtain a first complex containing probiotics encapsulating tributyrin; and 2) mixing the first complex with a hydrophilic solution to obtain a second complex containing probiotics encapsulating tributyrin with a hydrophilic barrier layer. The second complex (tributyrin-encapsulated complex) prepared using the present method exhibits high loading capacity, lacks surface oil, and completely masks the odor of tributyrin. It can release tributyrin in digestive fluids and exert intestinal health benefits. It can be used in food, medicine, and feed, as well as in oral or parenteral formulations.

Description

Technical Field

[0001] The present invention belongs to the technical field of oil microencapsulation, and particularly relates to a method for embedding tributyrin with the assistance of a hydrophilic barrier layer for probiotics and an application thereof. Background Art

[0002] Butyrate is an important substance for maintaining the health of the body. It plays a key role in intestinal function, immune regulation and metabolic balance. It is the main energy source for colon epithelial cells and has the functions of promoting intestinal mucosal repair and regeneration, maintaining the integrity of the intestinal barrier, and preventing "leaky gut". Butyrate, as an important member of short-chain fatty acids (SCFA), is mainly produced by probiotics in the lower digestive tract, especially in the colon, through the fermentation of dietary fiber. However, factors such as unbalanced diet, insufficient dietary fiber, and intestinal flora disorders lead to insufficient butyrate production in the intestine. The lack of intestinal butyrate is widely associated with a series of diseases, such as inflammation, metabolic disorders, cancer, and neurodegenerative diseases. Supplementing butyrate can become a new strategy for the prevention and treatment of these diseases.

[0003] However, butyric acid is a liquid with a strong rancid odor and is volatile, making it very inconvenient to use. It is generally replaced by butyrate. However, butyrate dissociates in the stomach, and the resulting butyric acid is absorbed by the stomach wall and cannot effectively reach the intestines to take effect.

[0004] Tributyrin, a precursor of butyric acid, is an oily liquid that is extremely insoluble in water. This solves the problem of butyric acid's volatility and partially improves its odor. However, tributyrin is easily hydrolyzed by intestinal lipase to produce butyric acid and glycerol, which are rapidly absorbed in the upper small intestine and cannot effectively reach the lower digestive tract to exert their effects. In addition, tributyrin itself has a bitter taste and a certain odor, resulting in poor palatability. Its oily nature means that when it is added to other components as an additive, it has a significant impact on the physical properties of the product.

[0005] In recent years, many studies have focused on the loading and encapsulation of tributyrin. For example, CN 102187945A uses white carbon black as a carrier to adsorb tributyrin, with a loading capacity of 3-15%; Application Publication No. CN 1044509701A uses fatty acids, emulsifiers, and calcium hydroxide as additives to convert tributyrin into solid particles, with a loading capacity of 22-44%; Application Publication No. CN 1088642105A uses starch as an additive and uses an enzymatic method to encapsulate tributyrin, with a loading capacity of 15-35.2%; 113080457A uses gum arabic, guar gum and xanthan gum as wall materials, microcrystalline cellulose, hydroxypropyl methylcellulose and maltodextrin as auxiliary wall materials, combined with emulsifiers and flavoring agents (strawberry powder, apple powder, orange powder, steviol glycosides and aspartame), through high-pressure homogenization emulsification and centrifugal spray drying to prepare a tributyrin microcapsule powder with a loading capacity of 45-55% and a surface oil content of 0.8-7.8%. CN113950325A mixes tributyrin or a tributyrin derivative with at least one odor-masking component (dextrin complex, lipid encapsulating agent) to encapsulate the mixture. The encapsulated complex formed can be combined with probiotics such as bifidobacteria and administered simultaneously or separately. It can also be used in combination with other additives such as superoxide dismutase, arabinoxylan, inulin, etc. In this patent application, tributyrin exists independently because it is encapsulated in the odor-masking component (dextrin complex, lipid encapsulating agent). When used, it can be administered separately or simultaneously with probiotics such as bifidobacteria or other additives.

[0006] The above-mentioned existing encapsulation technologies can be summarized into two categories: inorganic adsorption and organic material encapsulation. However, when the inorganic adsorbent adsorbs more tributyrin, the tributyrin is not coated inside, and its surface also contains a large amount of tributyrin, resulting in the bitterness and odor of tributyrin cannot be masked, and it is easy to lose. The conventional encapsulation method of organic materials is also difficult to achieve the purpose of zero surface oil encapsulation, so it is still not possible to mask the bitterness and odor of tributyrin well, and often requires the addition of flavoring agents and fragrances to cover it. In addition, in these tributyrin encapsulation technologies, the use of organic solvents, emulsifiers, etc. is difficult to avoid. At the same time, the encapsulation process also involves high-energy consumption processes, such as high-pressure homogenization. More importantly, under the action of gastric juice and small intestinal fluid, the adsorption effect between tributyrin and the adsorption carrier material of the tributyrin complexes prepared by these encapsulation technologies is easily destroyed, and the sealing of the organic encapsulation material is also challenged and destroyed by digestion and degradation of the digestive fluid, resulting in tributyrin being easily released in the upper gastrointestinal tract and still unable to effectively reach the lower gastrointestinal tract to exert its effect.

[0007] Therefore, it is urgent to develop a tributyrin technology and product with high oil loading, zero surface oil rate, and no odor, which can be released at a specific point in the lower digestive tract, especially the colon, to solve the existing problems of tributyrin preparations. Summary of the Invention

[0008] In view of this, the present invention aims to overcome the shortcomings of the existing technology and provide a method for efficiently encapsulating tributyrin and its application. On the one hand, this method solves the problem that existing tributyrin products have difficulty in targeted release in the colon. On the other hand, it effectively solves the bitterness and odor issues of tributyrin, provides a more convenient use of tributyrin, and avoids the use of organic solvents, emulsifiers, and high-energy consumption processes during the preparation process.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides a method for embedding tributyrin with a hydrophilic barrier layer to assist probiotics, which comprises the following steps:

[0011] 1) uniformly mixing the probiotic cells and tributyrin to allow the tributyrin to enter the probiotic cells to obtain a first complex of probiotic-encapsulated tributyrin, and washing with an aqueous solution to remove surface oil;

[0012] 2) mixing the first complex from which the floating oil has been removed with a hydrophilic solution, wherein the hydrophilic substance in the hydrophilic solution is one or more of a polysaccharide, a protein, or a small molecule-mediated assembly; when the hydrophilic substance is a small molecule-mediated assembly, the small molecule mediates self-assembly of the components onto the surface of the first complex to obtain a second complex in which the hydrophilic barrier layer assists the probiotics in encapsulating tributyrin; when the hydrophilic substance comprises a polysaccharide or a protein, gelling the mixture of the first complex and the hydrophilic solution, and dispersing the obtained gel to obtain a second complex in which the hydrophilic barrier layer assists the probiotics in encapsulating tributyrin.

[0013] Preferably, the probiotic cells are in the form of dry powders, and the probiotic cells may be living or dead. Preferably, the probiotic cells are selected from the group consisting of Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactobacillus muciniphilus, Lactobacillus plantarum, Lactobacillus unifying, Lactobacillus euchromatus, Lactococcus, Streptococcus, Propionibacterium, Propionibacterium, Leuconostoc, Pediococcus, Weizmannia, Zoococcus, Staphylococcus, Saccharomyces, or Akkermansia muciniphila.

[0014] According to a preferred embodiment of the present invention, the mass ratio of the probiotic cells to tributyrin is 1:01-100.

[0015] According to a preferred embodiment of the present invention, the aqueous solution is an aqueous solution having a density higher than that of tributyrin, selected from a salt solution, an acid solution, an alkaline solution or an aqueous solution of small organic molecules; the salt is an organic acid salt, a halide salt, a nitrate, a sulfate, a phosphate or a carbonate; the acid is an organic acid, sulfuric acid, hydrochloric acid, nitric acid or phosphoric acid; the alkali is sodium hydroxide or potassium hydroxide; and the small organic molecules are monosaccharides, disaccharides, oligosaccharides of three to six monosaccharides, glycerol or ethylene glycol; and the washing process is as follows: adding the aqueous solution to a mixture of probiotic cells and tributyrin, removing free tributyrin floating oil in the upper layer of the liquid, then centrifuging the aqueous phase and removing the supernatant, and the resulting precipitate is the first complex for removing floating oil.

[0016] According to a preferred embodiment of the present invention, the mass ratio of the probiotic cells to the hydrophilic substance in the hydrophilic substance solution is 1:0.001-1.

[0017] According to a preferred embodiment of the present invention, the gelation treatment is as follows: changing the temperature and pH of the mixture of the first complex and the hydrophilic solution, or adding salt ions or enzymes to the mixture of the first complex and the hydrophilic solution to induce cross-linking of the hydrophilic substances, so that the mixture forms a gel; and further dispersing the obtained gel in water to obtain a second complex in which the hydrophilic barrier layer assists the probiotics in embedding tributyrin.

[0018] According to a preferred embodiment of the present invention, the polysaccharide can be selected from one or more of agar, carrageenan, pectin, alginic acid, inulin, konjac glucomannan, gellan gum, gum arabic, cellulose derivatives, chitosan, hyaluronic acid, xanthan gum, xyloglucan, chondroitin sulfate, and locust bean gum, and can be a polysaccharide of animal, plant, or microbial origin; the protein can be one or more of gelatin, collagen, whey protein, fibroin, casein, soy protein, wheat gluten, zein, ovalbumin, and fibrinogen, and can be a protein of animal, plant, or microbial origin; the small molecule of the small molecule-mediated assembly is a polyphenol, which forms a polyphenol-metal ion assembly, a polyphenol-protein assembly, or a polyphenol-polysaccharide assembly with one or more of metal ions, polysaccharides, or proteins; specifically, the polyphenol-metal assembly The compound can be selected from one or more of tannic acid, catechin, caffeic acid, ellagic acid, proanthocyanidin, gallic acid and iron ion, calcium ion, zinc ion, magnesium ion, copper ion complexes; the polyphenol-protein assembly can be selected from one or more of gelatin, collagen, whey protein, fibroin, casein, soy protein, wheat gluten, zein, ovalbumin, fibrinogen and tannic acid, catechin, caffeic acid, ellagic acid, proanthocyanidin, gallic acid complexes; the polyphenol-polysaccharide assembly can be selected from one or more of tannic acid, catechin, caffeic acid, ellagic acid, proanthocyanidin, gallic acid and agar, carrageenan, pectin, alginic acid, inulin, konjac glucomannan, gellan gum, gum arabic, cellulose derivatives, chitosan, hyaluronic acid, xanthan gum, xyloglucan, chondroitin sulfate, locust bean gum complexes.

[0019] According to a preferred embodiment of the present invention, in step 2), the hydrophilic substance forms a hydrophilic barrier layer on the surface of the probiotic cells, and the surface of the second complex is free of surface oil; within the second complex, the embedding amount of tributyrin is 60-90 wt %. With the assistance of the hydrophilic barrier layer, tributyrin is completely embedded in the probiotics.

[0020] The present invention also provides a second complex of tributyrin prepared by the method and comprising a hydrophilic barrier layer assisting probiotics in embedding.

[0021] The present invention also provides the use of the second complex comprising a hydrophilic barrier layer to assist probiotics in encapsulating tributyrin in the preparation of a pharmaceutical or feed. The pharmaceutical, food, or feed may be in the form of an oral or parenteral formulation. Preferably, the pharmaceutical is a drug for treating diseases caused by intestinal butyrate deficiency, including but not limited to inflammatory bowel disease (such as ulcerative colitis), irritable bowel syndrome, and leaky gut syndrome.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The present invention uses dried probiotic cells as carriers and, based on capillary action, efficiently absorbs tributyrin and embeds tributyrin inside the cells. Hydrophilic substances are then used to form a barrier layer on the surface of the probiotic cells to prevent tributyrin from overflowing from the probiotic cells to the surface.

[0024] The present invention can regulate the release of tributyrin in the digestive juice, and exert the inhibitory effect of tributyrin on intestinal cancer cells and its growth-promoting effect on normal intestinal cells. The multi-layer structure of the hydrophilic barrier layer and the double probiotic cells of the present invention synergistically delays the release of tributyrin from the complex in the upper digestive tract, and has the ability to release tributyrin in the lower digestive tract. Specifically, on the one hand, the hydrophilic barrier layer inhibits the direct action of gastric juice and small intestinal juice on tributyrin, reducing the release of tributyrin from the upper digestive tract. On the other hand, the hydrophilic barrier layer is gradually decomposed under the action of digestive enzymes and intestinal flora, realizing directional release in the lower digestive tract, especially the colon. The butyric acid generated after the hydrolysis of the released tributyrin is rapidly utilized by colon cells, promoting the repair and regeneration of the intestinal mucosa and maintaining the integrity of the intestinal barrier. The probiotics synergistically enhance the targeted release effect while encapsulating tributyrin. The probiotics used in the present invention have intestinal health benefits. Bifidobacterium, Lactobacillus and Akkermansia muciniphila have the ability to colonize the intestinal epithelial mucus layer. After the surface hydrophilic barrier layer is digested and decomposed, the bacterial cells have the ability to adhere to the mucus layer and release tributyrin in a targeted manner.

[0025] In prior art such as CN113950325A, tributyrin or a tributyrin derivative is mixed with at least one odor-masking component (dextrin complex, lipid encapsulating agent) and encapsulated by the dextrin complex or lipid encapsulating agent. However, the structure of dextrin includes a hydrophilic surface, a hydrophobic cavity, and two ports. Tributyrin adsorbed in the dextrin complex is easily dissolved and digested and absorbed by gastric juice, bile salts, and pancreatic juice. Liposomes are closed vesicles formed by the self-assembly of amphiphilic lipid molecules. The liposome structure is easily disturbed and destroyed by gastric lipase, bile salts, and pancreatic juice, resulting in the rapid release of tributyrin in the stomach and upper gastrointestinal tract. Therefore, tributyrin is unable to exert its inhibitory effects on lower gastrointestinal cancer cells and its growth-promoting effects on normal colon cells.

[0026] (2) The raw materials used in the present invention are probiotics, polysaccharides, proteins, polyphenols, and mineral elements, all of which are natural, safe, edible, and beneficial to the human body. This overcomes the technical limitations of the prior art in using emulsifiers, aspartame, and other flavoring agents, thereby achieving a natural, safe, and healthy effect. The probiotics and hydrophilic substances used in the present invention are all natural, safe, and beneficial to health, and can be used in oral or parenteral preparations.

[0027] (3) The embedding method of the present invention adopts a two-stage blending technology. By combining oil-bacteria powder and the first complex-hydrophilic aqueous solution step by step, it overcomes the high energy consumption technology path represented by high-pressure homogenization emulsification in the existing technology, thereby achieving a green and low-energy invention effect.

[0028] (4) The present invention uses a hydrophilic barrier layer to assist the probiotic cells in completely embedding tributyrin inside the cells, overcoming the problem that the prior art cannot completely embed tributyrin, thereby achieving the invention effect of zero surface oil in the embedding system; the present invention uses a hydrophilic barrier layer to assist the probiotic cells in completely embedding tributyrin inside the cells, overcoming the problem that the prior art cannot completely mask the odor and bitterness of tributyrin, thereby achieving the invention effect of zero flavoring and flavoring agents; the present invention uses a hydrophilic barrier layer to assist the probiotic cells in completely embedding tributyrin inside the cells, overcoming the problem that the prior art cannot deliver tributyrin to the lower digestive tract, especially the colon, thereby achieving the invention effect of releasing tributyrin in a targeted manner in the lower digestive tract, especially the colon, and supplementing butyric acid to promote colon health. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the loading amount of tributyrin (TB) in the first complex of Example 1.

[0030] Figure 2 This is the change pattern of tributyrin in the first complex of Example 1 with embedding time.

[0031] Figure 3 These are scanning electron microscope images and laser confocal microscopy images of the composites in Examples 1 to 3.

[0032] Figure 4 ATR-FTIR spectra of the complexes in Examples 1 to 3.

[0033] Figure 5 1 is the ATR-FTIR spectrum of the composite samples after grinding in Examples 1 to 3.

[0034] Figure 6 C is the XPS spectrum of the composite in Examples 1 to 3 1S Peak fitting diagram and O 1S Peak fitting plot.

[0035] Figure 7 : is the surface water contact angle of the composite in Examples 1 to 3. The picture above the data column in the figure is a photograph of the surface water contact angle of the composite.

[0036] Figure 8 is the water dispersibility of the composites in Examples 1 to 3.

[0037] Figure 9is the TGA curve of the composite in Examples 1 to 3 ( Figure 9 A) and DTG curve ( Figure 9 B in ).

[0038] Figure 10 Odor evaluation of the complexes in Examples 1 to 3 (electronic nose detection): compared with butyric acid (BA) ( Figure 10 A in ); compared with tributyrin (TB) ( Figure 10 B in ).

[0039] Figure 11 The complexes in Examples 1 to 3 act on colorectal cancer cells Caco-2 ( Figure 11 A) and HT-29 ( Figure 11 B), and normal intestinal epithelial cells IPEC-J2 ( Figure 11 C) Effects on growth performance.

[0040] Figure 12 It is the ratio of the butyric acid concentration in the mouse colon content to the butyric acid concentration in the in vitro fecal bacteria fermentation liquid after the complexes in Examples 1 to 3 are digested in mice and fermented by intestinal fecal bacteria in vitro.

[0041] Figure 13 Effect of adding the complexes in Examples 1 to 3 on chocolate processing performance ( Figure 13 A in), and chewing properties including hardness ( Figure 13 B in), elasticity ( Figure 13 C in), cohesion ( Figure 13 D in), rebound force ( Figure 13 E) and chewiness ( Figure 13 F) in the impact.

[0042] Figure 14 Effects of adding the complexes in Examples 1 to 3 to chocolate on the chocolate smell: 1 day ( Figure 14 A in ); 2 days ( Figure 14 B in ); 3 days ( Figure 14 C in ). DETAILED DESCRIPTION

[0043] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It will be understood that the specific embodiments described herein are merely for explaining the technical solutions and inventive concepts of the present invention, and are not intended to limit the present invention. In addition, it should be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings. Other parts not explicitly noted should be understood as conventional means or solutions in the prior art, which, in combination with the technical features shown in the present invention, can achieve the technical effects of the present invention.

[0044] It should be noted that, in the absence of conflict, the specific additional technical features in the embodiments and examples of the present invention may be combined or replaced with each other. The present application will be described in detail below with reference to the accompanying drawings, attached tables and in combination with examples.

[0045] Example 1

[0046] Add 5 mg of dried Bifidobacterium (Bif) powder to 200 μL of tributyrin, let it stand for 1 hour, then add saturated sodium chloride solution for washing, centrifuge at 6000 rpm at 25°C for 3 minutes, collect the precipitate, repeat twice, transfer the powder to a clean centrifuge tube, add water for washing, and centrifuge under the same centrifugation conditions as above, repeat 1-2 times to obtain the first complex A of probiotics-encapsulated tributyrin (TB@Bif).

[0047] 1 mg of the first complex A was mixed with 1 mL of acetonitrile and ultrasonicated in a water bath for 30 min. 0.5 mL of the sample solution was filtered through a 0.22 μm organic membrane and then subjected to high performance liquid chromatography analysis to quantify the amount of tributyrin loaded.

[0048] Example 2

[0049] Add 5 mg of dry Bifidobacterium powder to 200 μL of tributyrin, let it stand for 1 hour, then add saturated sodium chloride solution for washing, centrifuge at 6000 rpm at 25°C for 3 minutes, collect the precipitate, repeat twice, transfer the powder to a clean centrifuge tube, add water for washing, and centrifuge under the same centrifugation conditions as above, repeat 1-2 times to obtain the first complex A of probiotics-encapsulated tributyrin (TB@Bif).

[0050] The first complex A was added to 5 mL of water, and 7.5 mL of tannic acid (TA) solution (1 mg / mL) and 2.5 mL of CaCl2 solution (1 mg / mL) were added to the water at the same time. The mixture was quickly mixed and magnetically stirred at 200 rpm for 30 min. The mixture was then centrifuged at 6000 rpm for 3 min and washed twice with pure water to obtain the tannic acid-calcium-coated second complex B (TB@Bif@TA-Ca).

[0051] Example 3

[0052] Add 5 mg of dry Bifidobacterium powder to 200 μL of tributyrin, let it stand for 1 hour, then add saturated sodium chloride solution for washing, centrifuge at 6000 rpm at 25°C for 3 minutes, collect the precipitate, repeat twice, transfer the powder to a clean centrifuge tube, add water for washing, and centrifuge under the same centrifugation conditions as above, repeat 1-2 times to obtain the first complex A of probiotics-encapsulated tributyrin (TB@Bif).

[0053] Inulin (INU) powder and water were mixed in a mass ratio of 1:2 and heated to 80°C. After the powder was completely dissolved, the mixture was cooled to about 50°C at room temperature. 200 μL of inulin solution was taken and mixed evenly with the first complex A. The mixture was quickly placed in cold water and cooled until solidified to obtain the inulin gel-coated second complex B (TB@Bif@INU).

[0054] Experimental results

[0055] Figure 1 The content of tributyrin in the first composite of Example 1 is shown. The mass ratio of tributyrin to Bifidobacterium powder is 2.064-8.256, and the content of tributyrin is 74.5%-83.8 wt%. After conversion, 3.2-5.2 mg of tributyrin is embedded per mg of bacterial powder. Figure 2 The tributyrin entrapped capacity in the first complex prepared at different entrapment times in Example 1 is shown. The amount of tributyrin entrapped per milligram of bacterial powder gradually increases with entrapment time, reaching a maximum at 1 hour. The tributyrin entrapped capacity in the first complex then decreases with further entrapment time. The results indicate that within 1 hour, the dry bacterial powder rapidly absorbs tributyrin into the cells through capillary action and physical adsorption. After saturation, the high density of tributyrin causes the absorbed tributyrin to gradually escape from the cells, resulting in a decrease in the tributyrin entrapped capacity.

[0056] Figure 3 Scanning electron microscopy (SEM) and confocal laser scanning microscopy (LCS) images of the complexes in Examples 1-3 are presented. The SEM images demonstrate that tributyrin encapsulation does not affect bacterial morphology; the morphology of the first complex, A, is highly consistent with that of the bacteria. The second complex, B, exhibits a certain degree of adhesion, as the hydrophilic barrier layer on the bacterial surface binds the different bacteria together. Confocal laser scanning microscopy confirms that Nile red-stained tributyrin is encapsulated within the bacterial cells.

[0057] Figure 4 Figure 2 is the ATR-FTIR spectrum of the complexes in Examples 1-3. The spectra show that a small amount of tributyrin is present on the surface of the first complex (TB@Bif), while the surfaces of the second complexes (TB@Bif@TA-Ca and TB@Bif@INU) have no tributyrin absorption peak at all and exhibit spectra similar to those of their hydrophilic barrier layers. This confirms that the hydrophilic barrier layer has good barrier properties for tributyrin embedded in the bacteria, preventing it from escaping. Figure 5The ATR-FTIR spectra of the complexes in Examples 1-3 are obtained from ground samples. A clear tributyrin absorption peak is observed in both the first and second complexes. Grinding disrupts the bacterial cells in the complexes, releasing tributyrin. Combined with analysis of the infrared spectra of the samples after different treatments, complete encapsulation of tributyrin is confirmed.

[0058] XPS spectroscopy was used to further precisely analyze the chemical composition of the sample surfaces. Table 1 shows the XPS results for the complexes in Examples 1-3. As can be seen from the table, the surface carbon content of the first complex (TB@Bif) was significantly higher than that of Bifidobacterium bifidum, even exceeding that of tributyrin-containing TB. This indicates that in addition to tributyrin on the surface of the first complex, hydrophobic components of the bacteria were also exposed during the tributyrin encapsulation process, resulting in an increase in carbon content and a decrease in the polar elements N, O, P, and S. The presence of calcium on the surface of the second complex (TB@Bif@TA-Ca) in Example 2, along with a significant decrease in the N, O, P, and S content, confirms the presence of tannic acid-calcium as the hydrophilic coating. The surface carbon and oxygen compositions of the second complex (TB@Bif@INU) in Example 3 were close to those of the hydrophilic gel inulin, demonstrating that the inulin gel hydrophilic coating of the first complex was achieved.

[0059] Table 1. Elemental composition of sample surface in XPS spectra

[0060]

[0061] In Table 1, * indicates that the element content of the data is derived from the theoretical molecular formula, and # indicates that the data is reported in the reference literature (Journal of Building Engineering, 2023, 80, 108121).

[0062] The XPS C and O element peaks were separated, and the results were as follows: Figure 6 , as shown in Table 2 and Table 3. C 1S The peak separation results revealed that the sample surface was composed of four C-containing groups: CC, CO(N), C=O and OC=O bonds, and O 1SPeak separation revealed that the sample surface is composed of three types of O-containing groups: C=O, OH, and CO-OC bonds. After tributyrin loading and TA-Ca and INU coating, the surface C-C bond content increased. The proportion of C=O bonds on the surface of the first composite (TB@Bif) also increased, due to the overflow of tributyrin and the fact that each tributyrin molecule contains three C=O bonds. The surface OH bond content decreased after tributyrin encapsulation. After the second composite was blocked by the TA-Ca hydrophilic layer, the OH bond content recovered and increased to 39.91%. After treatment with the INU hydrophilic barrier layer, the OH bond content increased significantly to 94.31%. XPS results further confirmed the presence of the hydrophilic barrier layer on the bacterial cell surface and its effectiveness in blocking intracellular tributyrin.

[0063] Table 2. C 1S The content of functional groups on the sample surface in the spectrum

[0064]

[0065] Table 3. 1S The content of functional groups on the sample surface in the spectrum

[0066]

[0067] Figure 7 The surface water contact angle results showed that the water contact angles of Bif, TB@Bif, TB@Bif@TA-Ca, and TB@Bif@INU were 27.7°, 87.2°, 46.9°, and 0°, respectively. This indicates that the first complex, TB@Bif, has surface oil, which increases the surface hydrophobicity and the water contact angle. The second complexes, TB@Bif@TA-Ca and TB@Bif@INU, lack surface oil. The hydrophilic barrier layer on the bacterial surface imparts excellent hydrophilicity, resulting in a decrease in the water contact angle. The high hydrophilicity of inulin makes the second complex, TB@Bif@INU, extremely hydrophilic.

[0068] Figure 8The water dispersibility of each complex in Examples 1-3 is shown. Bifidobacterium powder exhibits good water dispersibility in water. The first complex, TB@Bif, quickly sinks after dispersion in water. This is due to the greater density of tributyrin compared to water and the high tributyrin entrapment in the first complex. The escaped tributyrin renders the bacteria surface hydrophobic, resulting in rapid sedimentation. Tannic acid exhibits excellent hydrophilicity. Coated with the tannic acid-calcium hydrophilic composition, the second complex, TB@Bif@TA-Ca, regains a relatively uniform dispersion in water. Inulin gel exhibits extremely strong hydrophilicity. After dispersion in water, the second complex, TB@Bif@INU, exhibits a milky white color with no precipitation observed at the bottom of the solution, demonstrating good water dispersibility.

[0069] Figure 9 is the TGA curve of each composite in Examples 1 to 3 ( Figure 9 A) and DTG curve ( Figure 9 The results showed that encapsulation significantly improved the thermal stability of tributyrin. Under thermal conditions, tributyrin gradually escaped from the first complex, while the hydrophilic barrier layer of tannic acid-calcium and inulin gel in the second complex prevented the escape of tributyrin, further improving the thermal stability of TB.

[0070] Figure 10 Odor evaluation (electronic nose detection) of the complexes in Examples 1-3 revealed that the odors of both the first and second complexes were significantly different from those of butyric acid (BA) and tributyrin (TB), demonstrating that the complexes effectively masked the odors of tributyrin and the butyric acid that may be produced by its decomposition. The odor of tributyrin released from the first complex was relatively low, but it was the furthest from the air compared to the second complex, indicating a slight odor.

[0071] Figure 11 The effects of the complexes in Examples 1 to 3 on the growth performance of colorectal cancer cells Caco-2 and HT-29, as well as normal intestinal epithelial cells IPEC-J2, are shown. The results show that the first and second complexes have the same anti-cancer cell proliferation properties as unencapsulated tributyrin, with no significant difference (p > 0.05), and exhibit significant cancer cell toxicity (p < 0.0001). Furthermore, the first and second complexes have the same growth-promoting properties as unencapsulated tributyrin, with no significant difference (p > 0.05). The tributyrin encapsulation system of the present invention has no effect on the intestinal health benefits of tributyrin. After digestion in simulated gastric and intestinal fluids, tributyrin is released from the encapsulation system and exerts its efficacy.

[0072] Figure 12 The ratio of the butyric acid content in the colon contents to the butyric acid content in the fermentation broth in vitro after the complexes in Examples 1 to 3 were digested in mice and fermented by fecal bacteria in vitro. As can be seen from the figure, the butyric acid content in the colon contents in vivo is within 3% of the butyric acid content in the fecal bacteria fermentation broth in vitro, indicating that butyric acid is rapidly absorbed and utilized by the intestinal epithelium. Figure 11 The cell growth results shown are consistent, with only a small amount of butyric acid remaining in the colon contents without being absorbed. Among them, the ratio is the smallest in the group without embedded butyric acid and tributyrin, indicating that most of the released butyric acid has been absorbed by the intestine starting from the upper digestive tract, and its concentration is extremely low when it reaches the colon. However, the presence of bifidobacterium cells and a hydrophilic barrier layer in each complex in Examples 1 to 3 slows down or prevents the release of tributyrin in the upper digestive tract, thereby allowing more release in the lower digestive tract, especially the colon. The butyric acid concentrations in the colon contents of the TB@Bif, TB@Bif@TA-Ca, and TB@Bif@INU groups are 4.5, 6.1, and 6.7 times that of the TB group without embedded tributyrin. This shows that each complex in the present invention has the ability to increase colon-directed butyric acid release, which is beneficial to promoting colon health.

[0073] Figure 13 The effects of adding each complex to chocolate in Examples 1-3 on its processing and chewing properties are shown. Unencapsulated tributyrin increased chocolate adhesion, making demolding difficult and resulting in the highest chewing energy requirement. The first and second complexes did not significantly affect chocolate processing, but reduced hardness and chewiness parameters. However, they had no significant effect on cohesion and elasticity, demonstrating that they can optimize oral processing energy consumption while maintaining chocolate structural integrity. Figure 14 The effects of adding the complexes in Examples 1-3 to chocolate on its aroma are shown. The results show that neither tributyrin nor the complexes in Examples 1-3 affect the chocolate's aroma, significantly different from the aroma of chocolate added with butyric acid. Adding the complexes in Examples 1-3 to chocolate neither affects its aroma nor its processing. It should be noted that while tributyrin added directly to chocolate does not affect its aroma, its bitterness still compromises its palatability.

[0074] In summary, the present invention provides a method and application of using a hydrophilic barrier layer to assist probiotics in embedding tributyrin. Traditional methods for embedding tributyrin have problems such as surface oil exposure and incomplete odor masking, and require the addition of flavoring agents and flavor correctives. At the same time, the preparation process requires the use of organic solvents and emulsifiers, and also involves a high-energy embedding process. The method of using a hydrophilic barrier layer to assist probiotics in embedding tributyrin proposed in the present invention can achieve complete embedding of tributyrin at room temperature and low energy consumption. The embedding materials are green, natural, and healthy, and have a high loading capacity. The odor of tributyrin is completely shielded, but it does not affect the health benefits of tributyrin in the intestine, indicating that the tributyrin embedding complex can be normally released in the intestine and exert its efficacy. Since the odor and bitterness of tributyrin are completely shielded, the tributyrin embedding complex is added to foods represented by chocolate without affecting the smell of the chocolate itself or the processing of chocolate. This is of great significance to the application of tributyrin in food, medicine, feed and other products, and provides a method for developing oral functional products for supplementing the body with short-chain fatty acids.

[0075] Finally, it should be noted that the above embodiments are merely experimental examples to demonstrate the inventive concept of the present invention and are not intended to limit the present invention. All variations that can be directly derived or imagined by those skilled in the art from the contents disclosed herein should be considered to be within the scope of protection of the present invention.

Claims

1. A method for embedding tributyrin with a hydrophilic barrier layer to assist probiotics, characterized in that: The following steps are involved: 1) uniformly mixing probiotic cells and tributyrin to allow the tributyrin to enter the probiotic cells to obtain a first complex of probiotics-encapsulated tributyrin, and washing with an aqueous solution to remove surface oil; the probiotic cells are in the form of a dry powder; 2) mixing the first complex from which the floating oil has been removed with a hydrophilic solution, wherein the hydrophilic substance in the hydrophilic solution is one or more of a polysaccharide, a protein, or a small molecule-mediated assembly; when the hydrophilic substance is a small molecule-mediated assembly, the small molecule mediates self-assembly of the components onto the surface of the first complex to obtain a second complex in which the hydrophilic barrier layer assists the probiotics in encapsulating tributyrin; when the hydrophilic substance comprises a polysaccharide or a protein, gelling the mixture of the first complex and the hydrophilic solution, and dispersing the obtained gel to obtain a second complex in which the hydrophilic barrier layer assists the probiotics in encapsulating tributyrin.

2. The method according to claim 1, wherein: The probiotic cells are selected from the group consisting of Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactobacillus muciniphilus, Lactobacillus plantarum, Lactobacillus consolidans, Lactobacillus euchromatus, Lactococcus, Streptococcus, Propionibacterium, Leuconostoc, Pediococcus, Weizmannella, Zoococcus, Staphylococcus, Saccharomyces or Akkermansia muciniphila.

3. The method according to claim 1, wherein: The mass ratio of the probiotic cells to tributyrin is 1:01-100.

4. The method according to claim 1, wherein: The aqueous solution is an aqueous solution with a density higher than that of tributyrin, and is selected from a salt solution, an acid solution, an alkaline solution or an organic small molecule aqueous solution; the salt is an organic acid salt, a halide salt, a nitrate, a sulfate, a phosphate or a carbonate; the acid is an organic acid, sulfuric acid, hydrochloric acid, nitric acid or phosphoric acid; the alkali is sodium hydroxide or potassium hydroxide; and the organic small molecule is a monosaccharide, a disaccharide, an oligosaccharide of three to six monosaccharides, glycerol or ethylene glycol; and the washing process is as follows: adding the aqueous solution to a mixture of probiotic cells and tributyrin, removing free tributyrin floating oil in the upper layer of the liquid, then centrifuging the aqueous phase and removing the supernatant, and the resulting precipitate is the first complex for removing floating oil.

5. The method according to claim 1, wherein The mass ratio of the probiotic cells to the hydrophilic substance in the hydrophilic substance solution is 1:0.001-1.

6. The method according to claim 1, wherein: The gelation treatment comprises: changing the temperature and pH of the mixture of the first complex and the hydrophilic solution, or adding salt ions or enzymes to the mixture of the first complex and the hydrophilic solution to induce cross-linking of the hydrophilic substances so that the mixture forms a gel; and further dispersing the obtained gel in water to obtain a second complex in which the hydrophilic barrier layer assists the probiotics in embedding tributyrin.

7. The method according to claim 1, wherein: The small molecule of the small molecule-mediated assembly is polyphenol, which forms a polyphenol-metal ion assembly, a polyphenol-protein assembly or a polyphenol-polysaccharide assembly with one or more of metal ions, polysaccharides or proteins.

8. The method according to claim 1, wherein: In step 2), the hydrophilic substance forms a hydrophilic barrier layer on the surface of the probiotic cells, and the surface of the second complex is free of surface oil. In the second complex, the content of tributyrin is 60-90 wt %. With the assistance of the hydrophilic barrier layer, the tributyrin is completely embedded in the probiotics.

9. A second complex of tributyrin with a hydrophilic barrier layer to assist probiotics in encapsulating the tributyrin, characterized in that The method according to any one of claims 1 to 8 is used to prepare the present invention.

10. Use of the second compound according to claim 9, characterized in that: Used for preparing medicines or feed.

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