An alginate gel microsphere embedding chicken bile enzyme conjugate and its preparation method

Abstract

The present invention provides a sodium alginate gel microsphere encapsulating a chicken bile enzyme conjugate and a preparation method thereof, which relates to the food field. In the present invention, Lactobacillus plantarum and Bacillus subtilis are used to co-ferment the aqueous extract of Dendrobium officinale to obtain the fermented polysaccharide of Dendrobium officinale, and the fermented polysaccharide of Dendrobium officinale and sodium alginate are used as a composite wall material to encapsulate the chicken bile enzyme conjugate. Then, the prepared sodium alginate gel microsphere is re-coated to form a sodium alginate gel microsphere with a stable encapsulation structure. The fermented polysaccharide of Dendrobium officinale with a moderate molecular weight obtained by fermentation treatment can interact with sodium alginate more efficiently, making the sodium alginate gel microsphere firmer and denser. At the same time, the prepared sodium alginate gel microsphere is re-coated, which further prevents the dissolution of the chicken bile enzyme conjugate in the gel microsphere. This multi-layer encapsulation structure provides good encapsulation protection for the chicken bile enzyme conjugate and effectively solves the problem of the bitter and fishy smell of the chicken bile enzyme conjugate.

Description

Technical Field

[0001] The present invention relates to the field of food, and particularly to an alginate gel microsphere encapsulating chicken bile enzyme-converted product and a preparation method thereof. Background Art

[0002] Chicken bile, as a by-product of chicken processing, has a huge output. Chicken bile contains rich bile acids, usually in the form of conjugated bile salts. After biological enzymatic hydrolysis, ursodeoxycholic acid in it can be converted into chenodeoxycholic acid, which has the effects of treating cholesterol stones, cholestatic diseases, primary biliary cirrhosis, etc. Although experimental studies have proved that chicken bile enzyme-converted product (or chicken bile enzymatic hydrolysate) has various pharmacological effects, its current clinical and food applications are still not extensive. Therefore, the development value of chicken bile enzyme-converted product needs to be studied more deeply and widely.

[0003] Chicken bile tastes bitter and has a strong fishy smell, and the same problem exists with chicken bile enzyme-converted product. Most of the commercially available chicken bile-related products are made into oral liquids and other beverages by means of sweeteners and bitter suppressants, or made into chicken bile powder capsules by encapsulation with gelatin capsules. For example, patent document CN101176737A discloses a pharmaceutical composition of chicken bile, which contains chicken bile paste, taste masking agent, sweetener and aromatic flavoring agent, and solves the problem of the specific fishy smell and bitterness of chicken bile through the way of pharmaceutical combination. This composition can be prepared into granules, capsules, tablets and dispersible tablets. However, such methods are not suitable for processing chicken bile enzyme-converted product into food, which limits its application in the food field.

[0004] At present, there is no report on using gel encapsulation technology to improve the fishy and bitter taste of chicken bile enzyme-converted product. The common gel encapsulation technology usually encapsulates some functional factors by compounding sodium alginate with high molecular materials such as proteins, polysaccharides and lipids, so as to achieve the effects of stabilizing active ingredients, masking bad flavors and improving bioavailability. In the prior art, the wall materials for making microsphere gels are mostly sodium alginate compounded with natural biopolymers. The simple compounding has the disadvantages of weak gel strength and poor mechanical properties of the calcified membrane, resulting in problems such as unstable release of the content and easy rupture of the microcapsules during the application of sodium alginate microcapsules.

[0005] Therefore, preparing an alginate gel microsphere with high hardness and strength and effectively reducing the dissolution of fishy and bitter components of chicken bile enzyme-converted product has important practical significance for promoting the application of chicken bile enzyme-converted product in the food field. Summary of the Invention

[0006] The present invention aims to provide a preparation method of an alginate gel microsphere with high hardness and strength and effectively reducing the dissolution of fishy and bitter components of chicken bile enzyme-converted product.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing alginate gel microspheres encapsulating chicken bile enzyme conjugate, comprising the following steps:

[0009] (1) Take Dendrobium officinale powder, extract it with water, centrifuge, and take the supernatant, then inoculate Lactobacillus plantarum and Bacillus subtilis for fermentation. After fermentation is completed, take the fermentation broth, concentrate and freeze-dry it to obtain Dendrobium officinale fermented polysaccharide;

[0010] (2) Mix sodium alginate, β-cyclodextrin, and the Dendrobium officinale fermented polysaccharide with water, heat, add the chicken bile enzyme conjugate thereto, and dissolve to obtain a chicken bile enzyme conjugate - sodium alginate solution;

[0011] (3) Drop the chicken bile enzyme conjugate - sodium alginate solution into calcium chloride solution for solidification to obtain alginate gel microspheres;

[0012] (4) Mix sodium alginate with water and heat to obtain a re - coating solution;

[0013] (5) Filter out the alginate gel microspheres from the calcium chloride solution, put them into the re - coating solution, stir, and filter to obtain the alginate gel microspheres encapsulating chicken bile enzyme conjugate.

[0014] Further, in step (1),

[0015] the mass ratio of the Dendrobium officinale powder to water is 1:25 - 40;

[0016] the extraction temperature is 90 - 92 °C; the extraction time is 2 - 3 h;

[0017] the fermentation temperature is 25 - 30 °C; the fermentation time is 24 - 96 h;

[0018] Based on the mass of the supernatant, the viable count of the Lactobacillus plantarum inoculated is 10 8 ~10 10 cfu / g, and the viable count of the Bacillus subtilis inoculated is 10 8 ~10 10 cfu / g.

[0019] Further, in step (1), the mass ratio of the Dendrobium officinale powder to water is 1:30; the extraction temperature is 90 °C; the extraction time is 2 h; the fermentation temperature is 30 °C; the fermentation time is 72 h;

[0020] Based on the mass of the supernatant, the viable count of the Lactobacillus plantarum inoculated is 10 10 cfu / g, and the viable count of the Bacillus subtilis inoculated is 10 10cfu / g.

[0021] Further, in step (2),

[0022] The mass ratio of the sodium alginate, β-cyclodextrin, fermented polysaccharide of Dendrobium officinale, water and the enzyme-transformed product of chicken bile is 10:4:1 to 4:400:10, preferably 10:4:3:400:10;

[0023] The heating temperature is 50 - 53 °C; the heating time is 30 - 35 min.

[0024] Preferably, the heating temperature is 50 °C; the heating time is 30 min.

[0025] Further, in step (3),

[0026] The mass concentration of the calcium chloride solution is 1.11%;

[0027] The solidification is carried out under magnetic stirring, the rotation speed is 350 - 500 rpm, preferably 500 rpm, and the time is 30 min.

[0028] Further, in step (4),

[0029] The mass concentration of sodium alginate in the recoating solution is 0.5% - 1.25%;

[0030] The heating temperature is 50 - 53 °C, and the heating time is 30 - 35 min.

[0031] Preferably, the heating temperature is 50 °C, and the heating time is 30 min.

[0032] Further, in step (4),

[0033] The raw materials of the recoating solution further include at least one of maltodextrin, arabinose and fruit juice.

[0034] Further, in step (4),

[0035] The raw materials of the recoating solution further include maltodextrin, arabinose and fruit juice, and the mass ratio of sodium alginate, maltodextrin, arabinose, water and fruit juice is 4 - 12:4:48:800:64, preferably 8:4:48:800:64.

[0036] Further, the preparation method of the fruit juice includes: taking fruit pulp, adding pectinase and cellulase for enzymatic hydrolysis, and filtering the enzymatically hydrolyzed pulp to obtain the fruit juice, and the fruit pulp includes passion fruit pulp and / or mango pulp.

[0037] Furthermore, the raw materials of the fruit juice include passion fruit puree and mango puree, and the mass ratio of the passion fruit puree to the mango puree is 1-2:1-2;

[0038] Based on the mass of the fruit puree, the addition amount of pectinase is 0.6-0.85 g / kg, preferably 0.6 g / kg, and the addition amount of cellulase is 0.6-0.8 g / kg, preferably 0.6 g / kg.

[0039] In a second aspect, the present invention provides sodium alginate gel microspheres encapsulating the chicken bile enzyme conjugate obtained by the preparation method described above.

[0040] The technical solution of the present invention has the following advantages:

[0041] 1. The present invention provides a preparation method of sodium alginate gel microspheres encapsulating chicken bile enzyme conjugate. Lactobacillus plantarum and Bacillus subtilis are used for mixed fermentation of the aqueous extract of Dendrobium officinale to obtain Dendrobium officinale fermented polysaccharide, and the chicken bile enzyme conjugate is encapsulated with Dendrobium officinale fermented polysaccharide and sodium alginate as a compound wall material. Then, the prepared sodium alginate gel microspheres are re-coated to form sodium alginate gel microspheres with a stable encapsulation structure.

[0042] The Dendrobium officinale polysaccharide with a moderate molecular weight obtained by fermentation treatment can interact with sodium alginate more efficiently, making the sodium alginate gel microspheres firmer and denser. Compared with the compound of unfermented Dendrobium officinale polysaccharide and without adding Dendrobium officinale fermented polysaccharide, the thermal stability and storage stability of the microspheres are significantly improved, effectively improving the leaching problem of the chicken bile enzyme conjugate. At the same time, the prepared sodium alginate gel microspheres are re-coated, further preventing the dissolution of the chicken bile enzyme conjugate in the gel microspheres, improving the thermal stability and storage stability of the microspheres. This multi-layer encapsulation structure provides good encapsulation protection for the chicken bile enzyme conjugate, effectively solving the problem of bitter and fishy smell of the chicken bile enzyme conjugate, thereby broadening the application of the chicken bile enzyme conjugate in the food field.

[0043] 2. In the preparation method provided by the present invention, the raw materials of the re-coating solution can further include maltodextrin, arabinose and fruit juice. Maltodextrin and arabinose act as sweeteners, and the addition of fruit juice can provide fruity aroma, further improving the edible taste and flavor of the microspheres. Preferably, the fruit juice is obtained by enzymatic hydrolysis of mango puree and passion fruit puree. Through experiments, it is proved that the enzymatic hydrolysis addition ratio of passion fruit puree and mango puree has a greater impact on the odor and taste of the chicken bile enzyme conjugate - sodium alginate gel microspheres, and has no obvious impact on the sensory evaluation of color and adhesiveness. The prepared gel microspheres have a sweet and fruity smell, beautiful color, no obvious adhesion phenomenon, moderate soft hardness, and excellent overall flavor and taste. Description of the Drawings

[0044] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings required for the description of the specific embodiments or the prior art. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0045] Figure 1 Process flow chart for the preparation of sodium alginate gel microspheres embedding chicken bile enzyme transformant in Example 1 of the present invention;

[0046] Figure 2 Effect of fermentation time of Dendrobium officinale polysaccharide on the thermal stability of gel microspheres in Experimental Example 1 of the present invention;

[0047] Figure 3 Effect of fermentation time of Dendrobium officinale polysaccharide on the storage stability of gel microspheres in Experimental Example 1 of the present invention;

[0048] Figure 4 Effect of addition amount of Dendrobium officinale fermented polysaccharide on the thermal stability of gel microspheres in Experimental Example 2 of the present invention;

[0049] Figure 5 Effect of addition amount of Dendrobium officinale fermented polysaccharide on the storage stability of gel microspheres in Experimental Example 2 of the present invention;

[0050] Figure 6 Effect of addition amount of Dendrobium officinale fermented polysaccharide on the hardness of gel microspheres in Experimental Example 2 of the present invention;

[0051] Figure 7 Effect of addition amount of sodium alginate in the recoating solution on the hardness and recoating thickness of gel microspheres in Experimental Example 4 of the present invention;

[0052] Figure 8 Effect of addition amount of sodium alginate in the recoating solution on the storage stability of gel microspheres in Experimental Example 4 of the present invention;

[0053] Figure 9 Effect of addition amount of sodium alginate in the recoating solution on the thermal stability of gel microspheres in Experimental Example 4 of the present invention. Specific Embodiments

[0054] The following embodiments are provided to better further understand the present invention, which is not limited to the best embodiment, and does not limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior art features falls within the protection scope of the present invention.

[0055] Source of Raw Materials

[0056] Dendrobium officinale: Dendrobium officinale from Huoshan County, Anhui Province;

[0057] Passion fruit: Golden passion fruit from Beiliu City, Guangxi Zhuang Autonomous Region;

[0058] Mango: Tianyang Fragrant Mango from Tianyang County, Guangxi Zhuang Autonomous Region;

[0059] Bacillus subtilis BS-15: This strain has been disclosed in patent document CN112940960A and its deposit number is CGMCC No.20851.

[0060] Lactobacillus plantarum P-8: This strain has been disclosed in patent document CN102994422A and its deposit number is CGMCC No.6312.

[0061] Chicken bile enzymatic conversion product: provided by Zhongshan Ligao Biopharmaceutical Co., Ltd., the taurochenodeoxycholic acid in chicken bile is converted into tauroursodeoxycholic acid using an enzymatic hydrolysis process.

[0062] If specific experimental procedures or conditions are not specified in the Examples or Experimental Examples, the procedures or conditions described in the literature in the field can be followed. All raw materials and instruments used are commercially available, including but not limited to those used in the Examples or Experimental Examples of this application.

[0063] Example 1

[0064] This embodiment provides a method for preparing sodium alginate gel microspheres encapsulating chicken bile enzyme conversion products. The process flow is as follows: Figure 1 The specific steps are as follows:

[0065] (1) Preparation of Dendrobium officinale fermentation stock solution:

[0066] Weigh 100 g of Dendrobium officinale powder, add 3000 g of distilled water, mix, stir evenly, place in a constant temperature water bath, extract at 90°C for 2 h, and centrifuge to obtain the supernatant, which is the Dendrobium officinale fermentation stock solution;

[0067] (2) Fermented Dendrobium officinale stock solution:

[0068] 0.2 g of plant lactobacillus P-8 and Bacillus subtilis BS-15 powder were weighed respectively and activated in 200 g of normal saline for 30 min. 2000 g of Dendrobium officinale fermentation stock solution was taken and 60 g of activated bacterial solution was inoculated (based on the mass of Dendrobium officinale fermentation stock solution, the number of viable Lactobacillus plantarum was 10). 10 cfu / g, the number of viable bacteria of Bacillus subtilis was 10 10 cfu / g), placed in a constant temperature incubator at 30°C for 72 hours, concentrated and freeze-dried to obtain Dendrobium officinale fermented polysaccharide;

[0069] (3) Preparation of chicken bile enzyme-transformed product-sodium alginate solution:

[0070] Weigh 10 g of sodium alginate, 4 g of β-cyclodextrin, and 3 g of fermented polysaccharide of Dendrobium officinale, add 400 g of distilled water, mix evenly, place it in a water bath at 50 °C for 30 min, then add 10 g of chicken bile enzyme-transformed product to it, and stir until completely dissolved to obtain chicken bile enzyme-transformed product-sodium alginate solution;

[0071] (4) Preparation of sodium alginate gel microspheres:

[0072] Weigh 22.2 g of calcium chloride, add it to 2000 g of distilled water and stir to dissolve to obtain calcium chloride solution. Drop the chicken bile enzyme-transformed product-sodium alginate solution into the calcium chloride solution, and stir magnetically at a speed of 500 rpm for 30 min for solidification to obtain chicken bile enzyme-transformed product-sodium alginate gel microspheres;

[0073] (5) Preparation of fruit juice:

[0074] After cutting the passion fruit in half, take out the pulp to obtain passion fruit puree. Take fresh mangoes, peel and pit them, and then make the pulp into mango puree. Weigh 22 g of passion fruit puree and 44 g of mango puree, add pectinase and cellulase for enzymatic hydrolysis (based on the total mass of passion fruit puree and mango puree, the addition amount of pectinase is 0.6 g / kg, and the addition amount of cellulase is 0.8 g / kg). The enzymatic hydrolysis temperature is 32 °C and the enzymatic hydrolysis time is 2.5 h. Filter the enzymatically hydrolyzed puree to obtain fruit juice;

[0075] (6) Preparation of the re-wrapping solution:

[0076] Weigh 8 g of sodium alginate, 4 g of maltodextrin, and 48 g of arabinose, add 800 g of distilled water, mix evenly, place it in a water bath at 50 °C for 30 min, then add 64 g of fruit juice to it, and stir evenly to obtain the re-wrapping solution;

[0077] (7) Preparation of re-wrapped sodium alginate gel microspheres:

[0078] Filter out the solidified chicken bile enzyme-transformed product-sodium alginate gel microspheres from the calcium chloride solution, disperse them into the re-wrapping solution, stir magnetically at a speed of 300 rpm for 20 min, and then filter to obtain re-wrapped chicken bile enzyme-transformed product-sodium alginate gel microspheres.

[0079] Example 2

[0080] This example provides a method for preparing sodium alginate gel microspheres encapsulating chicken bile enzyme-transformed product. The specific steps refer to Example 1, and the difference is only that in step (2) of the fermentation of Dendrobium officinale stock solution, the fermentation time is adjusted from 72 h to 24 h.

[0081] Example 3

[0082] This example provides a method for preparing sodium alginate gel microspheres encapsulating chicken bile enzyme transformant. The specific steps refer to Example 1, with the only difference being that in step (2) the fermentation of Dendrobium officinale original solution, the fermentation time is adjusted from 72 h to 48 h.

[0083] Example 4

[0084] This example provides a method for preparing sodium alginate gel microspheres encapsulating chicken bile enzyme transformant. The specific steps refer to Example 1, with the only difference being that in step (2) the fermentation of Dendrobium officinale original solution, the fermentation time is adjusted from 72 h to 96 h.

[0085] Example 5

[0086] This example provides a method for preparing sodium alginate gel microspheres encapsulating chicken bile enzyme transformant. The specific steps refer to Example 1, with the only difference being that in step (3) the addition amount of Dendrobium officinale fermented polysaccharide is adjusted from 3 g to 1 g.

[0087] Example 6

[0088] This example provides a method for preparing sodium alginate gel microspheres encapsulating chicken bile enzyme transformant. The specific steps refer to Example 1, with the only difference being that in step (3) the addition amount of Dendrobium officinale fermented polysaccharide is adjusted from 3 g to 2 g.

[0089] Example 7

[0090] This example provides a method for preparing sodium alginate gel microspheres encapsulating chicken bile enzyme transformant. The specific steps refer to Example 1, with the only difference being that in step (3) the addition amount of Dendrobium officinale fermented polysaccharide is adjusted from 3 g to 4 g.

[0091] Example 8

[0092] This example provides a method for preparing sodium alginate gel microspheres encapsulating chicken bile enzyme transformant. The specific steps refer to Example 1, with the only difference being that in step (5) the addition amount of fruit puree is adjusted from 22 g of passion fruit puree and 44 g of mango puree to 33 g of passion fruit puree and 33 g of mango puree.

[0093] Example 9

[0094] This example provides a method for preparing sodium alginate gel microspheres encapsulating chicken bile enzyme transformant. The specific steps refer to Example 1, with the only difference being that in step (5) the addition amount of fruit puree is adjusted from 22 g of passion fruit puree and 44 g of mango puree to 44 g of passion fruit puree and 22 g of mango puree.

[0095] Example 10

[0096] This example provides a method for preparing alginate gel microspheres encapsulating chicken bile enzyme transformant. The specific steps refer to Example 1, with the only difference being that in step (5), the addition amount of fruit puree is adjusted from 22 g of passion fruit puree and 44 g of mango puree to 66 g of mango puree.

[0097] Example 11

[0098] This example provides a method for preparing alginate gel microspheres encapsulating chicken bile enzyme transformant. The specific steps refer to Example 1, with the only difference being that in step (5), the addition amount of fruit puree is adjusted from 22 g of passion fruit puree and 44 g of mango puree to 66 g of passion fruit puree.

[0099] Example 12

[0100] This example provides a method for preparing alginate gel microspheres encapsulating chicken bile enzyme transformant. The specific steps refer to Example 1, with the only difference being that in step (6), the addition amount of sodium alginate is adjusted from 8 g to 4 g.

[0101] Example 13

[0102] This example provides a method for preparing alginate gel microspheres encapsulating chicken bile enzyme transformant. The specific steps refer to Example 1, with the only difference being that in step (6), the addition amount of sodium alginate is adjusted from 8 g to 6 g.

[0103] Example 14

[0104] This example provides a method for preparing alginate gel microspheres encapsulating chicken bile enzyme transformant. The specific steps refer to Example 1, with the only difference being that in step (6), the addition amount of sodium alginate is adjusted from 8 g to 10 g.

[0105] Comparative Example 1

[0106] This comparative example provides a method for preparing alginate gel microspheres encapsulating chicken bile enzyme transformant. The specific steps refer to Example 1, with the only difference being that step (2) is omitted, and the polysaccharide of Dendrobium officinale obtained by concentrating and freeze-drying the original fermentation solution of Dendrobium officinale prepared in step (1) is used. In step (3), an equal amount of polysaccharide of Dendrobium officinale is used to replace the fermented polysaccharide of Dendrobium officinale, denoted as the original fermentation solution of Dendrobium officinale for 0 h.

[0107] Comparative Example 2

[0108] This comparative example provides a method for preparing alginate gel microspheres encapsulating chicken bile enzyme transformant. The specific steps refer to Example 1, with the only difference being that in step (3), the addition amount of the fermented polysaccharide of Dendrobium officinale is adjusted from 3 g to 0 g.

[0109] Comparative Example 3

[0110] This comparative example provides a method for preparing sodium alginate gel microspheres encapsulating chicken bile enzyme transformant. The specific steps refer to Example 1, with the difference being that steps (5)-(7) are omitted, and the amount of sodium alginate added in the recoating solution is recorded as 0 g.

[0111] Experimental Example 1 Effect of Fermentation Time of Dendrobium officinale Polysaccharide on the Stability of Gel Microspheres

[0112] This experimental example aims to explore the effect of the fermentation time of Dendrobium officinale polysaccharide on the thermal stability and storage stability of gel microspheres. The experimental samples are the sodium alginate gel microspheres encapsulating chicken bile enzyme transformant prepared in Examples 1-4 and Comparative Example 1.

[0113] The method for measuring the thermal stability of gel microspheres is as follows: Heat beakers containing 50 mL of normal saline to 37 °C, 45 °C, 50 °C, and 55 °C respectively, then add 10 g of the experimental sample, keep for 5 min, and then place the beakers in an ice-water bath to cool for 10 min, and measure the concentration of bile acids in the normal saline.

[0114] The method for measuring the storage stability of gel microspheres is as follows: Place 10 g of the experimental sample in a beaker containing 50 mL of sterile normal saline and store at 4 °C for 6 d. Take samples at 0 d, 2 d, 4 d, and 6 d of storage respectively, and measure the concentration of bile acids in the normal saline.

[0115] Method for measuring the concentration of bile acids: After concentrating and freeze-drying the leached bile acid solution, weigh a certain amount of the sample, add it to mass spectrometry water, and vortex to mix evenly to obtain a diluted sample; take 100 μL of the sample, add 300 μL of the precipitant (acetonitrile: methanol = 8:2) of the mixed internal standard solution, vortex to mix evenly, let it stand on ice for 30 min, centrifuge at 12000 rpm at 4 °C for 10 min, and take all the supernatant for LC-MS analysis.

[0116] Chromatographic conditions: Chromatographic column: Waters ACQUITY UPLC BEH C18 (2.1×100 mm, 1.7 μm); Mobile phase: Phase A: 0.1% formic acid aqueous solution, Phase B: acetonitrile; Column temperature: 50 °C; Injection volume: 2 μL; Flow rate: 0.3 mL / min.

[0117] Mass spectrometry conditions: Electrospray ionization source (ESI), negative ion ionization mode. Ion source temperature 550 °C, ion source voltage -4500 V, curtain gas 35 psi, both nebulizing gas and auxiliary gas are 60 psi. Scanning is carried out using multiple reaction monitoring (MRM).

[0118] The results are as Figures 2 - 3 shown.

[0119] AsFigure 2 As shown, the thermal stability of the gel microspheres decreases with the increase in temperature. The thermal stability of the gel microspheres prepared in Examples 1-4 is better than that in Comparative Example 1. When the fermentation time of Dendrobium officinale polysaccharide is 72 h, its thermal stability is the best, and the bile acid dissolution amount is the lowest at each heating temperature.

[0120] As Figure 3 shown, the storage stability of the gel microspheres decreases with the increase in storage days. After 6 days of storage, the leaching concentration of bile acid is the highest for the unfermented Dendrobium officinale polysaccharide, which is 0.1949 g / L. When the fermentation time is 72 h, the storage stability of the gel microspheres is the best, and the bile acid dissolution amount is the lowest during the storage process.

[0121] After fermentation treatment, the molecular weight of Dendrobium officinale polysaccharide decreases, and its structure and composition change. The moderately molecular weight Dendrobium officinale polysaccharide obtained by fermentation treatment can interact with sodium alginate more efficiently to enhance the density of the gel. In addition, there are also studies showing that the activity of small molecule biological polysaccharides is better than that of macromolecular polysaccharides, and the activity of acidic polysaccharides is stronger than that of neutral polysaccharides. It can be seen that fermentation treatment of Dendrobium officinale polysaccharide can not only improve the embedding strength but also provide higher biological activity.

[0122] Experimental Example 2 Effect of the addition amount of fermented Dendrobium officinale polysaccharide on the stability and hardness of gel microspheres

[0123] This experimental example aims to explore the effects of the addition amount of fermented Dendrobium officinale polysaccharide on the thermal stability, storage stability, and hardness of gel microspheres. The experimental samples are sodium alginate gel microspheres embedding chicken bile enzyme transformant prepared in Examples 1, 5-7 and Comparative Example 2.

[0124] The measurement methods for the thermal stability and storage stability of the gel microspheres refer to Experimental Example 1.

[0125] The measurement method for the hardness of the gel microspheres is as follows: Use an XT Plus analyzer equipped with a cylindrical measurement probe (SMS P / 36R) to measure the hardness of the gel microspheres, and compress the gel microspheres to 25% of their original height at a speed of 40 mm / min.

[0126] The results are as Figures 4 - 6 shown.

[0127] As Figure 4 and 5 shown, with the increase in the addition amount of fermented Dendrobium officinale polysaccharide, the bile acid dissolution amount gradually decreases, indicating that the thermal stability and storage stability of the gel microspheres gradually increase, and the fermented Dendrobium officinale polysaccharide can effectively improve the leaching problem of chicken bile enzyme transformant. Among them, when the addition amounts of fermented Dendrobium officinale polysaccharide are 3 g and 4 g, the stability results are almost the same. Considering economic benefits and other factors, 3 g of fermented Dendrobium officinale polysaccharide is the optimal addition amount.

[0128] The hardness reflects the ability of the hydrogel beads to resist cracking during processing or storage. Excessive gel hardness causes the gel microspheres to be prone to breakage, while too low hardness makes the gel microspheres prone to deformation. As Figure 6 shown, when the addition amount of fermented polysaccharide from Dendrobium officinale is 3 g, the hardness of the gel microspheres is 40.3 g. With the increase of the addition amount of fermented polysaccharide from Dendrobium officinale, the change in its hardness is not significant.

[0129] Experimental Example 3 Effect of the ratio of passion fruit puree to mango puree on the sensory quality of gel microspheres

[0130] This experimental example aims to explore the effect of the ratio of passion fruit puree to mango puree on the thermal sensory quality (odor, color, morphology, adhesiveness, softness and hardness, taste) of gel microspheres. The experimental samples are sodium alginate gel microspheres embedding chicken bile enzyme-converted products prepared in Examples 1, 8 - 11.

[0131] Fifteen people were selected to form a sensory evaluation panel. The chicken bile enzyme-converted product - gel microspheres were scored from six aspects: odor, color, morphology, adhesiveness, softness and hardness, and taste, with a full score of 100 points. The scoring criteria for the sensory evaluation of gel microspheres are shown in Table 1. The result is the average score of the 15 panelists' scores as the final score.

[0132] Table 1 Sensory scoring criteria for gel microspheres

[0133]

[0134] The sensory evaluation results are shown in Table 2.

[0135] Table 2 Sensory evaluation results of gel microspheres

[0136]

[0137]

[0138] As shown in Table 2, the enzymatic hydrolysis addition ratio of passion fruit puree to mango puree has a greater impact on the odor and taste of gel microspheres, and has no obvious effect on the sensory evaluation of color and adhesiveness. Among them, the sensory score when passion fruit puree and mango puree are compounded and added is higher than that of single components. When the enzymatic hydrolysis addition ratio of passion fruit puree to mango puree is 1:2, the sensory evaluation score is the highest, which is 88 points. The prepared gel microspheres have a sweet and fragrant fruit odor, beautiful color, no obvious adhesion phenomenon, moderate softness and hardness, and excellent overall flavor and taste.

[0139] Experimental Example 4 Effect of the addition amount of sodium alginate in the recoating solution on the hardness, recoating thickness and stability of gel microspheres

[0140] This experimental example aims to explore the effects of the addition amount of sodium alginate in the recoating solution on the hardness, recoating thickness, thermal stability, and storage stability of gel microspheres. The experimental samples are sodium alginate gel microspheres encapsulating chicken bile enzyme transformants prepared in Example 1, Examples 12 - 14, and Comparative Example 3.

[0141] The method for measuring the thermal stability and storage stability of gel microspheres refers to Experimental Example 1. The method for measuring the hardness of gel microspheres refers to Experimental Example 2.

[0142] The method for measuring the recoating thickness of gel microspheres is as follows:

[0143] Use a vernier caliper to measure the diameter of the gel microsphere. Remove the outer recoating layer of the microsphere and use a vernier caliper to measure the inner diameter of the sodium alginate gel microsphere. The outer recoating thickness of the microsphere is calculated using the following formula:

[0144] T = (d1 - d0) / 2

[0145] In the formula, d0 is the inner diameter of the sodium alginate gel microsphere, d1 is the diameter of the sodium alginate gel microsphere, and T is the outer recoating thickness of the microsphere.

[0146] The results are as Figures 7 - 9 shown.

[0147] After the gel microspheres are cured in calcium chloride solution for 30 minutes, the calcium ions adhered to the surface can recombine with sodium alginate in the recoating solution to form a gel film.

[0148] As Figure 7 shown, as the addition amount of sodium alginate in the recoating solution increases, the recoating thickness gradually increases. When the addition amount of sodium alginate exceeds 8 g, the increase in recoating thickness tends to level off. The greater the recoating thickness, the better the embedding and protection effect on chicken bile enzyme transformants. However, considering that the total diameter of the gel microspheres should meet the requirements of production application and edible quality, the coating thickness should not be too large. The recoating thickness of the gel microspheres prepared in Example 1 is 0.52 mm, which is the optimal thickness. The hardness of the gel microspheres decreases with the increase in the addition amount of sodium alginate. Gel microspheres with too small hardness are prone to soft collapse and deformation. Among them, the hardness of the gel microspheres with a sodium alginate addition amount of 10 g is 38.93 N, which is 10.35 N less than the hardness of the microspheres without sodium alginate addition.

[0149] As Figure 8 and 9As shown, with the increase of the placement time, the leaching concentration of bile acids gradually increases. Among them, in Comparative Example 3, no sodium alginate was added for re - coating. During the placement process, the leaching concentration of bile acids was the highest and the embedding effect was the worst. When the addition amount of sodium alginate exceeded 8 g (Example 1), the gel microspheres showed adhesion during the re - coating process, resulting in uneven secondary coating, which decreased its storage stability and increased the leaching concentration of bile acids. Therefore, the addition amount of sodium alginate in the re - coating solution in Example 1 is the optimal addition amount.

[0150] Obviously, the above - mentioned embodiments are only examples clearly described and not limitations on the implementation manners. For those of ordinary skill in the art, based on the above description, other different forms of changes or modifications can be made. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A preparation method of sodium alginate gel microspheres embedding chicken bile enzyme-converted products, characterized in that, It includes the following steps: (1) Take Dendrobium officinale powder, add water for extraction, centrifuge, take the supernatant, inoculate Lactobacillus plantarum and Bacillus subtilis for fermentation. After fermentation is completed, take the fermentation broth, concentrate and freeze-dry it to obtain Dendrobium officinale fermented polysaccharide; (2) Mix sodium alginate, β-cyclodextrin, and the Dendrobium officinale fermented polysaccharide with water, heat it, add the chicken bile enzyme conjugate to it, and dissolve it to obtain a chicken bile enzyme conjugate-sodium alginate solution; (3) Drop the chicken bile enzyme conjugate-sodium alginate solution into calcium chloride solution for solidification to obtain sodium alginate gel microspheres; (4) Mix sodium alginate with water and heat it to obtain a re-wrapping solution; (5) Filter out the sodium alginate gel microspheres from the calcium chloride solution, put them into the re-wrapping solution, stir, and filter to obtain the sodium alginate gel microspheres embedding the chicken bile enzyme conjugate.

2. The preparation method of the sodium alginate gel microspheres embedding the enzyme-transformed chicken bile according to claim 1, characterized in that, In step (1), the mass ratio of the Dendrobium officinale powder to water is 1:25 - 40; the extraction temperature is 90 - 92 °C; the extraction time is 2 - 3 h; the fermentation temperature is 25 - 30 °C; the fermentation time is 24 - 96 h; Based on the mass of the supernatant, the viable count of the Lactobacillus plantarum inoculated is 10 8 ~10 10 cfu / g, and the viable count of the Bacillus subtilis inoculated is 10 8 ~10 10 cfu / g.

3. The preparation method of the sodium alginate gel microspheres embedding the chicken bile enzyme transformant according to claim 2, characterized in that, In step (1), the mass ratio of the Dendrobium officinale powder to water is 1:30; the extraction temperature is 90 °C; the extraction time is 2 h; the fermentation temperature is 30 °C; the fermentation time is 72 h; Based on the mass of the supernatant, the viable count of the Lactobacillus plantarum inoculated is 10 10 cfu / g, and the viable count of the Bacillus subtilis inoculated is 10 10 cfu / g.

4. The preparation method of the sodium alginate gel microspheres embedding the enzyme-converted product of chicken bile, characterized in that, In step (2), the mass ratio of sodium alginate, β-cyclodextrin, Dendrobium officinale fermented polysaccharide, water, and chicken bile enzyme conjugate is 10:4:1 - 4:400:10; the heating temperature is 50 - 53 °C; the heating time is 30 - 35 min.

5. The preparation method of the sodium alginate gel microspheres embedding the enzyme-converted product of chicken bile according to claim 4, characterized in that, In step (2), the mass ratio of sodium alginate, β-cyclodextrin, Dendrobium officinale fermented polysaccharide, water, and chicken bile enzyme conjugate is 10:4:3:400:

10.

6. The preparation method of the sodium alginate gel microspheres embedding chicken bile enzyme transformant according to claim 1, characterized in that, In step (3), the mass concentration of the calcium chloride solution is 1.11%; the solidification is carried out under magnetic stirring, the rotation speed is 350 - 500 rpm, and the time is 30 min.

7. The preparation method of the sodium alginate gel microspheres embedding chicken bile enzyme transformant according to claim 1, characterized in that, In step (4), the mass concentration of sodium alginate in the re-wrapping solution is 0.5% - 1.25%; the heating temperature is 50 - 53 °C, and the heating time is 30 - 35 min.

8. The preparation method of the sodium alginate gel microspheres embedding the enzyme-converted product of chicken bile, characterized in that, In step (4), the raw materials of the re-wrapping solution further include at least one of maltodextrin, arabinose, and fruit juice.

9. The preparation method of the sodium alginate gel microspheres embedding the chicken bile enzyme transformant according to claim 1, characterized in that, In step (4), the raw materials of the re-wrapping solution further include maltodextrin, arabinose, and fruit juice, and the mass ratio of sodium alginate, maltodextrin, arabinose, water, and fruit juice is 4 - 12:4:48:800:

64.

10. The preparation method of the sodium alginate gel microspheres embedding chicken bile enzyme-converted product according to claim 9, characterized in that, In step (4), the raw materials of the re-wrapping solution further include maltodextrin, arabinose, and fruit juice, and the mass ratio of sodium alginate, maltodextrin, arabinose, water, and fruit juice is 8:4:48:800:

64.

11. The preparation method of the sodium alginate gel microspheres embedding chicken bile enzyme-converted product according to any one of claims 8-10, characterized in that, The preparation method of the fruit juice includes: taking fruit puree, adding pectinase and cellulase for enzymatic hydrolysis, and filtering the hydrolyzed puree to obtain the fruit juice, and the fruit puree includes passion fruit puree and / or mango puree.

12. The preparation method of the sodium alginate gel microspheres embedding the chicken bile enzyme conjugate according to claim 11, characterized in that, The raw materials of the juice include passion fruit puree and mango puree, and the mass ratio of the passion fruit puree to the mango puree is 1-2:1-2; Based on the mass of the fruit puree, the addition amount of pectinase is 0.6-0.85 g / kg, and the addition amount of cellulase is 0.6-0.8 g / kg.

13. Sodium alginate gel microspheres of the entrapped chicken bile enzyme transformant obtained by the preparation method according to any one of claims 1-12.

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