Preparation method of bile salt activated lipase microcapsules

Abstract

The invention discloses a method for preparing bile salt-activated lipase microcapsules, comprising: fully dissolving the wall material in water to obtain a wall material aqueous solution; uniformly mixing the enzyme solution with the wall material aqueous solution, then dropwise adding it to a CaCl2 solution, and then fixing it for 30-40 minutes to form beads; filtering the beads, washing and removing the residual CaCl2 solution and the residual enzyme solution to obtain wet microcapsules; placing the wet microcapsules at 80-20°C overnight, and vacuum freeze-drying to obtain dry microcapsules. The present invention uses sodium alginate and sodium alginate-whey protein as wall materials to prepare bile salt-activated lipase microcapsules, so that it can resist the strong acid effect of the stomach and smoothly reach the intestines, promote fat absorption of infants, and the microcapsules have a certain thermal stability, and BSSL in the microcapsules can also retain more than 70% after pasteurization.

Description

Technical Field

[0001] The present invention relates to the field of food, and more specifically, to a method for preparing bile salt-activated lipase microcapsules. Background Art

[0002] Bile salt stimulated lipase (BSSL), also known as carboxylic ester lipase (CEL) and cholesterol esterase (Cholesterol esterase), is an esterase that is active on various lipids, including glycerides, cholesterol and fat-soluble vitamins, but unlike other lipases, it works in the intestine and requires bile salt stimulation. Breast milk bile salt-activated lipase not only promotes fat absorption, has antibacterial and antiviral effects, but also plays a potential positive role in regulating intestinal flora and immune regulation. It is well known that newborns need to drink high-quality fats and essential nutrients for healthy and rapid growth. However, the exocrine pancreas of infants is immature at birth, and the amount of BSSL secreted is insufficient to support adequate fat absorption. However, this phenomenon of fat malabsorption almost only occurs in formula-fed infants, and this situation does not occur in breast-fed infants; therefore, it is particularly important for some infants who cannot consume breast milk to supplement bile salt-activated lipase. However, bile salt-activated lipase is not resistant to high temperatures and can be completely inactivated by pasteurization. Ensuring its activity during processing and storage is a huge challenge in product production. Summary of the invention

[0003] The purpose of the present invention is to provide a method for preparing bile salt-activated lipase microcapsules. The bile salt-activated lipase microcapsules are prepared with sodium alginate or sodium alginate-whey protein as wall materials, so that the microcapsules can resist the strong acid action of the stomach and smoothly reach the intestines, thereby promoting fat absorption in infants. The microcapsules have a certain thermal stability, and after pasteurization (62-65° C., 30 min), more than 70% of BSSL in the microcapsules can be retained.

[0004] In order to achieve these objects and other advantages of the present invention, a method for preparing bile salt-activated lipase microcapsules is provided, comprising:

[0005] Step 1: fully dissolving the wall material in water to obtain a wall material aqueous solution;

[0006] Step 2: Evenly mix the enzyme solution and the wall material aqueous solution at a volume ratio of 1:1-3, then add dropwise to a CaCl2 solution with a mass fraction of 2-4%, and then fix for 30-40 minutes to form beads;

[0007] Step 3, filtering the beads obtained in step 2, washing to remove the residual CaCl2 solution and the residual enzyme solution, and obtaining wet microcapsules;

[0008] Step 4: Place the wet microcapsules obtained in step 3 at -80 to -20°C overnight and freeze-dry them in vacuum to obtain dry microcapsules.

[0009] Preferably, in the method for preparing bile salt-activated lipase microcapsules, the wall material is sodium alginate, and the mass fraction of sodium alginate in the wall material aqueous solution is 2-3%.

[0010] Preferably, in the method for preparing bile salt-activated lipase microcapsules, the mass fraction of sodium alginate in the wall material aqueous solution is 2.5%.

[0011] Preferably, in the method for preparing bile salt-activated lipase microcapsules, the wall materials are sodium alginate and whey protein powder, the mass fraction of sodium alginate in the wall material aqueous solution is 2-3%, and the mass fraction of whey protein is 1.2-2.4%.

[0012] Preferably, in the method for preparing bile salt-activated lipase microcapsules, the mass fraction of sodium alginate in the wall material aqueous solution is 2.5%, and the mass fraction of whey protein is 1.2%.

[0013] Preferably, in the method for preparing bile salt-activated lipase microcapsules, in step 2, the enzyme solution and the wall material aqueous solution are evenly mixed in a volume ratio of 1:2.

[0014] Preferably, in the method for preparing bile salt-activated lipase microcapsules, in step 2, the enzyme solution and the wall material aqueous solution are uniformly mixed and then added dropwise to a 2% by mass CaCl2 solution.

[0015] Preferably, in the method for preparing bile salt-activated lipase microcapsules, the microcapsules are formed by fixing for 35 minutes in step 2.

[0016] Preferably, in the method for preparing bile salt-activated lipase microcapsules, the fixed temperature in step 2 is 4°C.

[0017] Preferably, in the method for preparing the bile salt-activated lipase microcapsules, the method for preparing the enzyme solution is: using yeast strain KM71 as a host cell and pPIC9k as an exogenous expression vector to construct a recombinant yeast expression system for human bile salt-activated lipase, using a BMMY culture medium with a pH of 6.5 to concentrate and ferment the recombinant yeast in a shaker at 28°C and 250rpm, and using 0.5% methanol by volume to induce it to secrete and express human bile salt-activated lipase, and obtaining an enzyme solution after high-speed centrifugation to remove the bacterial precipitate.

[0018] The present invention has at least the following beneficial effects:

[0019] The present invention obtains the optimal preparation process conditions of microcapsules with sodium alginate as wall material through single factor and orthogonal test: the mass fraction of sodium alginate is 2.5%, the mass fraction of calcium chloride is 2%, the ratio of enzyme solution to wall material is 1:2 (v / v), and the fixing time is 35 minutes. Under the above conditions, the embedding rate of microcapsules prepared can reach 59%. The optimal preparation process conditions of microcapsules with sodium alginate-whey protein as wall material are: the mass fraction of sodium alginate is 2.5%, the mass fraction of calcium chloride is 2%, the ratio of enzyme solution to wall material is 1:2 (v / v), the fixing time is 35 minutes, and the mass fraction of whey protein is 1.2%. Under the above conditions, the embedding rate of microcapsules prepared can reach 82%. Through digestion stability test and thermal stability test, it is found that both wall materials have good protective effect on BSSL, and the sodium alginate-whey protein powder has better gastric digestion resistance effect. The results of the thermal stability test showed that after pasteurization, the bile salt-activated lipase in the two bile salt-activated lipase microcapsules could reach more than 70%.

[0020] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the effect of the mass fraction of sodium alginate on the microcapsule embedding rate and average particle size;

[0022] Figure 2 This is the effect of CaCl2 mass fraction on microcapsule embedding efficiency and average particle size;

[0023] Figure 3 This is the effect of fixed time on microcapsule embedding efficiency and average particle size;

[0024] Figure 4 This is the effect of the ratio of enzyme solution to wall material on the microcapsule embedding efficiency and average particle size;

[0025] Figure 5 This is the effect of whey protein quality fraction on microcapsule embedding efficiency and average particle size;

[0026] Figure 6 These are two types of microcapsule morphology diagrams; A1 represents wet microcapsules prepared with sodium alginate as the wall material; B1 represents wet microcapsules prepared with sodium alginate-whey protein powder as the wall material; A2 represents freeze-dried microcapsules prepared with sodium alginate as the wall material; B2 represents freeze-dried microcapsules prepared with sodium alginate-whey protein powder as the wall material;

[0027] Figure 7 These are two scanning electron micrographs of dry microcapsules; A3 represents a scanning electron micrograph of dry microcapsules prepared with sodium alginate as the wall material; B3 represents a scanning electron micrograph of dry microcapsules prepared with sodium alginate-whey protein powder as the wall material;

[0028] Figure 8 : is the tolerance graph of two microcapsules in simulated adult and infant gastric juice; wherein, A represents the tolerance graph of two microcapsules in simulated adult gastric juice, and B represents the tolerance graph of two microcapsules in simulated infant gastric juice;

[0029] Fig. 9 It is a graph showing the effect of storage at 60°C and 70°C on the retention rate of BSSL in the two microcapsules; wherein, A represents the graph showing the effect of storage at 60°C on the retention rate of BSSL in the two microcapsules, and B represents the graph showing the effect of storage at 70°C on the retention rate of BSSL in the two microcapsules. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with embodiments and drawings so that those skilled in the art can implement the invention with reference to the description.

[0031] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0032] Example 1

[0033] The method for preparing bile salt activated lipase microcapsules comprises:

[0034] Step 1: fully dissolving the wall material in water to obtain a wall material aqueous solution;

[0035] Step 2: Mix the enzyme solution and the wall material aqueous solution in a volume ratio of 1:2, then add dropwise to a 2% CaCl2 solution, and then fix for 35 minutes to form beads;

[0036] Step 3, filtering the beads obtained in step 2, washing to remove the residual CaCl2 solution and the residual enzyme solution, and obtaining wet microcapsules;

[0037] Step 4: Place the wet microcapsules obtained in step 3 at -20°C overnight and freeze-dry them in vacuum to obtain dry microcapsules.

[0038] In another technical solution, in the method for preparing bile salt-activated lipase microcapsules, the wall material is sodium alginate, and the mass fraction of sodium alginate in the wall material aqueous solution is 2.5%.

[0039] In another technical solution, in the method for preparing bile salt-activated lipase microcapsules, the fixed temperature in step 2 is 4°C.

[0040] In another technical scheme, in the preparation method of the bile salt-activated lipase microcapsules, yeast strain KM71 is used as a host cell and pPIC9k is used as an exogenous expression vector to construct a recombinant yeast expression system of human bile salt-activated lipase. The recombinant yeast is concentrated and fermented in a shaker at 28°C and 250rpm using a BMMY culture medium with a pH of 6.5, and is induced to secrete and express human bile salt-activated lipase using methanol with a volume fraction of 0.5%. The enzyme solution is obtained by high-speed centrifugation to remove the bacterial precipitate.

[0041] Example 2

[0042] The method for preparing bile salt activated lipase microcapsules comprises:

[0043] Step 1: fully dissolving the wall material in water to obtain a wall material aqueous solution;

[0044] Step 2: Mix the enzyme solution and the wall material aqueous solution in a volume ratio of 1:1, then add dropwise to a 2% CaCl2 solution, and then fix for 30 minutes to form beads;

[0045] Step 3, filtering the beads obtained in step 2, washing to remove the residual CaCl2 solution and residual enzyme solution, and obtaining wet microcapsules;

[0046] Step 4: Place the wet microcapsules obtained in step 3 at -20°C overnight and freeze-dry them in vacuum to obtain dry microcapsules.

[0047] In another technical solution, in the method for preparing bile salt-activated lipase microcapsules, the wall material is sodium alginate, and the mass fraction of sodium alginate in the wall material aqueous solution is 2%.

[0048] In another technical solution, in the method for preparing bile salt-activated lipase microcapsules, the fixed temperature in step 2 is 4°C.

[0049] In another technical scheme, in the preparation method of the bile salt-activated lipase microcapsules, yeast strain KM71 is used as a host cell and pPIC9k is used as an exogenous expression vector to construct a recombinant yeast expression system of human bile salt-activated lipase. The recombinant yeast is concentrated and fermented in a shaker at 28°C and 250rpm using a BMMY culture medium with a pH of 6.5, and is induced to secrete and express human bile salt-activated lipase using methanol with a volume fraction of 0.5%. The enzyme solution is obtained by high-speed centrifugation to remove the bacterial precipitate.

[0050] Example 3

[0051] The method for preparing bile salt activated lipase microcapsules comprises:

[0052] Step 1: fully dissolving the wall material in water to obtain a wall material aqueous solution;

[0053] Step 2: Mix the enzyme solution and the wall material aqueous solution in a volume ratio of 1:3, then add dropwise to a 4% CaCl2 solution, and then fix for 40 minutes to form beads;

[0054] Step 3, filtering the beads obtained in step 2, washing to remove the residual CaCl2 solution and the residual enzyme solution, and obtaining wet microcapsules;

[0055] Step 4: Place the wet microcapsules obtained in step 3 at -20°C overnight and freeze-dry them in vacuum to obtain dry microcapsules.

[0056] In another technical solution, in the method for preparing bile salt-activated lipase microcapsules, the wall material is sodium alginate, and the mass fraction of sodium alginate in the wall material aqueous solution is 3%.

[0057] In another technical solution, in the method for preparing bile salt-activated lipase microcapsules, the fixed temperature in step 2 is 4°C.

[0058] In another technical scheme, in the preparation method of the bile salt-activated lipase microcapsules, yeast strain KM71 is used as a host cell and pPIC9k is used as an exogenous expression vector to construct a recombinant yeast expression system of human bile salt-activated lipase, and the recombinant yeast is concentrated and fermented in a shaker at 28°C and 250rpm using a BMMY culture medium with a pH of 6.5, and is induced to secrete and express human bile salt-activated lipase using methanol (volume fraction of 0.5%), and the enzyme solution is obtained after high-speed centrifugation to remove the bacterial precipitate.

[0059] Example 4

[0060] The method for preparing bile salt activated lipase microcapsules comprises:

[0061] Step 1: fully dissolving the wall material in water to obtain a wall material aqueous solution;

[0062] Step 2: Mix the enzyme solution and the wall material aqueous solution in a volume ratio of 1:2, then add dropwise to a 2% CaCl2 solution, and then fix for 35 minutes to form beads;

[0063] Step 3, filtering the beads obtained in step 2, washing to remove the residual CaCl2 solution and the residual enzyme solution, and obtaining wet microcapsules;

[0064] Step 4: Place the wet microcapsules obtained in step 3 at -20°C overnight and freeze-dry them in vacuum to obtain dry microcapsules.

[0065] In another technical solution, in the method for preparing bile salt-activated lipase microcapsules, the wall materials are sodium alginate and whey protein powder, the mass fraction of sodium alginate in the wall material aqueous solution is 2.5%, and the mass fraction of whey protein is 1.2%.

[0066] In another technical solution, in the method for preparing bile salt-activated lipase microcapsules, the fixed temperature in step 2 is 4°C.

[0067] In another technical scheme, in the preparation method of the bile salt-activated lipase microcapsules, yeast strain KM71 is used as a host cell and pPIC9k is used as an exogenous expression vector to construct a recombinant yeast expression system of human bile salt-activated lipase. The recombinant yeast is concentrated and fermented in a shaker at 28°C and 250rpm using a BMMY culture medium with a pH of 6.5, and is induced to secrete and express human bile salt-activated lipase using methanol with a volume fraction of 0.5%. The enzyme solution is obtained by high-speed centrifugation to remove the bacterial precipitate.

[0068] Example 5

[0069] The method for preparing bile salt activated lipase microcapsules comprises:

[0070] Step 1: fully dissolving the wall material in water to obtain a wall material aqueous solution;

[0071] Step 2: Mix the enzyme solution and the wall material aqueous solution in a volume ratio of 1:1, then add dropwise to a 2% CaCl2 solution, and then fix for 30 minutes to form beads;

[0072] Step 3, filtering the beads obtained in step 2, washing to remove the residual CaCl2 solution and the residual enzyme solution, and obtaining wet microcapsules;

[0073] Step 4: Place the wet microcapsules obtained in step 3 at -20°C overnight and freeze-dry them in vacuum to obtain dry microcapsules.

[0074] In another technical solution, in the method for preparing bile salt-activated lipase microcapsules, the wall materials are sodium alginate and whey protein powder, the mass fraction of sodium alginate in the wall material aqueous solution is 2%, and the mass fraction of whey protein is 1.2%.

[0075] In another technical solution, in the method for preparing bile salt-activated lipase microcapsules, the fixed temperature in step 2 is 4°C.

[0076] In another technical scheme, in the preparation method of the bile salt-activated lipase microcapsules, yeast strain KM71 is used as a host cell and pPIC9k is used as an exogenous expression vector to construct a recombinant yeast expression system of human bile salt-activated lipase. The recombinant yeast is concentrated and fermented in a shaker at 28°C and 250rpm using a BMMY culture medium with a pH of 6.5, and is induced to secrete and express human bile salt-activated lipase using methanol with a volume fraction of 0.5%. The enzyme solution is obtained by high-speed centrifugation to remove the bacterial precipitate.

[0077] Example 6

[0078] The method for preparing bile salt activated lipase microcapsules comprises:

[0079] Step 1: fully dissolving the wall material in water to obtain a wall material aqueous solution;

[0080] Step 2: Mix the enzyme solution and the wall material aqueous solution in a volume ratio of 1:3, then add dropwise to a 4% CaCl2 solution, and then fix for 40 minutes to form beads;

[0081] Step 3, filtering the beads obtained in step 2, washing to remove the residual CaCl2 solution and the residual enzyme solution, and obtaining wet microcapsules;

[0082] Step 4: Place the wet microcapsules obtained in step 3 at -20°C overnight and freeze-dry them in vacuum to obtain dry microcapsules.

[0083] In another technical solution, in the method for preparing bile salt-activated lipase microcapsules, the wall materials are sodium alginate and whey protein powder, the mass fraction of sodium alginate in the wall material aqueous solution is 3%, and the mass fraction of whey protein is 2.4%.

[0084] In another technical solution, in the method for preparing bile salt-activated lipase microcapsules, the fixed temperature in step 2 is 4°C.

[0085] In another technical scheme, in the preparation method of the bile salt-activated lipase microcapsules, yeast strain KM71 is used as a host cell and pPIC9k is used as an exogenous expression vector to construct a recombinant yeast expression system of human bile salt-activated lipase. The recombinant yeast is concentrated and fermented in a shaker at 28°C and 250rpm using a BMMY culture medium with a pH of 6.5, and is induced to secrete and express human bile salt-activated lipase using methanol with a volume fraction of 0.5%. The enzyme solution is obtained by high-speed centrifugation to remove the bacterial precipitate.

[0086] 1 Materials and methods

[0087] 1.1 Materials and reagents

[0088] Bile salt-activated lipase (BSSL): homemade in the laboratory; whey protein powder: Yuanye Company; sodium alginate: chemically pure; calcium chloride: analytically pure; pepsin: Beijing Solebow Company; p-nitrophenol (standard): Aladdin; 4-nitrophenyl myristate: Aladdin.

[0089] 1.2 Instruments and Equipment

[0090] LGJ-18C freeze dryer, Beijing Sihuan Furui Technology Co., Ltd.; Model 680 microplate reader, American, Bio-Rad; SU3500 scanning electron microscope, Hitachi, Japan.

[0091] 1.3 Experimental methods

[0092] 1.3.1 Preparation of bile salt-activated lipase

[0093] The yeast strain KM71 was used as the host cell and pPIC9k as the exogenous expression vector to construct a recombinant yeast expression system of human bile salt-activated lipase. The recombinant yeast was concentrated and fermented in a shaker at 250 rpm at 28°C using BMMY culture medium at pH 6.5, and 0.5% methanol was used to induce the secretion and expression of human bile salt-activated lipase. The enzyme solution was obtained by high-speed centrifugation to remove the bacterial precipitate.

[0094] 1.3.2 Preparation of bile salt-activated lipase microcapsules using sodium alginate as wall material

[0095] Weigh sodium alginate and dissolve it in water to obtain a sodium alginate aqueous solution with a mass fraction of 2-3%, which is set aside. Evenly mix the enzyme solution and the sodium alginate aqueous solution in a volume ratio of 1:1-3, then add it dropwise into a CaCl2 solution with a mass fraction of 2-4% using a syringe, and then place it at 4°C for 30-40 minutes to form beads, and then filter and wash to remove the residual CaCl2 solution and residual enzyme solution to obtain wet microcapsules, place the wet microcapsules at -80 to -20°C overnight, and obtain dry microcapsules by vacuum freeze drying.

[0096] 1.3.3 Preparation of bile salt-activated lipase microcapsules using sodium alginate-whey protein powder as wall material

[0097] Weigh sodium alginate and whey protein powder and dissolve them in water to make the mass fraction of sodium alginate 2-3% and the mass fraction of whey protein 1.2-2.4% for later use. Evenly mix the enzyme solution and the above aqueous solution in a volume ratio of 1:1-3, then add it dropwise into a CaCl2 solution with a mass fraction of 2-4% by a syringe, then place it at 4°C for 30-40 minutes to form beads, then filter and wash to remove the residual CaCl2 solution and residual enzyme solution to obtain wet microcapsules, place the wet microcapsules at -80 to -20°C overnight, and obtain dry microcapsules by vacuum freeze drying.

[0098] 1.3.4 Determination of microcapsule embedding efficiency

[0099] Weigh 0.5g of the dried microcapsules, add 5mL of PBS solution, and shear at high speed for 3min. Centrifuge at 4℃ and 5000r / min for 5min to release all the BSSL embedded in the microcapsules. Then determine the BSSL activity by the p-nitrophenol method to determine the embedding rate. The embedding rate is calculated as follows:

[0100]

[0101] 1.3.5 Average particle size of microcapsules

[0102] Take 30 unfreeze-dried microcapsules, measure them with a vernier caliper and take the average value. Repeat the measurement 3 times for each microcapsule and take the average value.

[0103] 1.3.6 Scanning Electron Microscope (SEM)

[0104] The surface morphology of the sample was observed using a scanning electron microscope. The sample was evenly fixed on the sample stage, sprayed with gold for 90 seconds, placed in the sample chamber and observed at different magnifications.

[0105] 1.3.7 Tolerance of microcapsules in simulated gastric fluid

[0106] Preparation of adult gastric juice stock solution: Dissolve 2 g of sodium chloride in 800 mL of deionized water, adjust the pH to 1 with 0.1 mol / L HCl, and then make up to 1000 mL.

[0107] Preparation of children's gastric juice stock solution: Dissolve 2g sodium chloride in 800ml deionized water, adjust the pH to 3 with 0.1mol / L HCl, and then make up to 1000mL.

[0108] Before the formal digestion, 0.5 g of each type of BSSL microcapsule was taken and added to two 4.5 mL gastric juice stock solutions respectively. Pepsin was then added to reach 2000 U / mL. The shaker temperature was set to 37°C, the speed was 100 r / min, the digestion time was 1 h, and the microcapsule precipitates were collected at 5 min, 10 min, 15 min, 30 min, and 60 min respectively. After washing, the encapsulation solution was added and the BSSL activity in the microcapsules was determined, and the unencapsulated BSSL was used as a control.

[0109] 1.3.8 Thermal stability test

[0110] The two dried BSSL microcapsule samples were stored at 60℃ and 70℃ for 1 hour, 0.5g of microcapsule samples were taken out every 10 minutes, and the BSSL activity in the microcapsules was measured. The retention rate (%) was used as the evaluation index.

[0111]

[0112] 1.4 Experimental design

[0113] 1.4.1 Single-factor experimental design

[0114] In order to obtain the best encapsulation rate process, the effects of four factors, namely, sodium alginate mass fraction (%), enzyme solution to wall material ratio (v / v), fixation time (min), and CaCl2 mass fraction (%) on the BSSL microcapsule encapsulation rate were compared, and a single-factor experiment was used for experimental design, as shown in Table 1.

[0115] Table 1 Single factor level table for preparation of bile salt activated lipase microcapsules

[0116]

[0117] The CaCl2 mass fraction was set to 3.0%, the fixation time was 30min, and the ratio of enzyme solution to wall material was 1:3 (v / v); the sodium alginate mass fraction was set to 2.5%, the fixation time was 30min, and the ratio of enzyme solution to wall material was 1:3 (v / v); the sodium alginate mass fraction was set to 2.5%, the CaCl2 mass fraction was 3.0%, and the ratio of enzyme solution to wall material was 1:3 (v / v); the sodium alginate mass fraction was set to 2.5%, the CaCl2 mass fraction was 3.0%, and the fixation time was 30min; the effects of the sodium alginate mass fraction, the CaCl2 mass fraction, the fixation time and the ratio of enzyme solution to wall material on the microcapsule embedding rate were investigated respectively. Preparation of microcapsules: First, the enzyme solution and wall material were mixed and dripped into calcium chloride solution, and then placed at 4℃ to form a gel, then filtered and washed to obtain wet microcapsules, placed at -20℃ to fix and then freeze-dried to obtain microcapsules.

[0118] 1.4.2 Orthogonal Experimental Design

[0119] The ratio of enzyme solution to wall material was set to 1:2 (v / v). Through single factor experiment, three factors, namely, mass fraction of sodium alginate, mass fraction of calcium chloride and fixation time, were selected. Three levels were set for each factor to prepare microcapsules: enzyme solution and wall material were mixed in a volume ratio of 1:2 and then dripped into calcium chloride solution, and then fixed at 4°C to form gel. After that, wet microcapsules were obtained by filtration and washing, and fixed at -20°C and freeze-dried to obtain microcapsules. The embedding rate was taken as the investigation index, and L9(3 3 ) The embedding conditions were optimized orthogonally, as shown in Table 2.

[0120] Table 2 Orthogonal factor level table

[0121]

[0122] 1.4.3 Effect of added whey protein mass fraction on BSSL microcapsule encapsulation efficiency

[0123] Taking the best orthogonal experimental results, whey protein mass fractions of 0.8%, 1.2%, 1.6%, 2.0%, and 2.4% were added. Sodium alginate and whey protein were evenly mixed in water and then dropped into a calcium chloride solution. After fixing at 4°C, the wet microcapsules were washed and fixed at -20°C and freeze-dried to obtain dry microcapsules.

[0124] 1.5 Data processing

[0125] Statistix 8.1 and SPSS 20.2 software were used for statistical analysis and significant difference analysis (P < 0.05); Origin 2017 software was used for drawing. The experiment was repeated 3 times, and each treatment group was paralleled 3 times. The data are expressed as mean ± standard deviation.

[0126] 2 Results and analysis

[0127] 2.1 Main factors affecting BSSL microcapsule embedding efficiency

[0128] 2.1.1 Effect of Sodium Alginate Mass Fraction on Microcapsule Encapsulation Efficiency and Particle Size

[0129] like Figure 1As shown in the figure, for microcapsules prepared with sodium alginate as the wall material, with the increase of sodium alginate concentration, the particle size of microcapsules showed a significant increase trend, and the embedding rate showed a trend of increasing first and then decreasing. When the mass fraction of sodium alginate was 2.5% and 3%, the embedding rates were 38.3% and 40.5%, respectively, which were significantly higher than the other three groups, and the particle sizes were 2.50mm and 2.84mm, respectively. When the mass fraction of sodium alginate was 3.5%, the embedding rate decreased significantly. This is because the embedding method of this experiment is the extrusion method. When the concentration of sodium alginate is too high, the viscosity increases, extrusion is more difficult, and the microcapsules formed are uneven and not round, which is also very likely to be the reason for the low embedding rate of microcapsules. Therefore, this experiment selected a sodium alginate mass fraction of 3% as the optimal wall material concentration.

[0130] 2.1.2 Effect of CaCl2 mass fraction on microcapsule embedding efficiency and particle size

[0131] like Figure 2 As shown in the figure, for the microcapsules prepared with sodium alginate as the wall material, with the increase of CaCl2 mass fraction, the particle size of the microcapsules showed an increasing trend. When the CaCl2 mass fraction increased from 2% to 4%, the increase of the microcapsule particle size was relatively slow; when the CaCl2 mass fraction increased from 1% to 3%, the embedding rate of the microcapsules increased significantly. When the CaCl2 mass fraction was 3%, the embedding rate was the highest, and the embedding rate was greater than 30%; when the CaCl2 mass fraction increased from 3% to 5%, the embedding rate decreased significantly. When the CaCl2 mass fraction was 5%, the embedding rate was the lowest, only 18%. When the CaCl2 mass fraction is too high, the particle size increases and the wall material of the microcapsule becomes thicker, which has a negative impact on the embedding effect of the microcapsule; when the CaCl2 mass fraction is too low, the enzyme solution and the wall material are not fixed in time and are dissolved in the CaCl2 solution, so the microcapsule particle size is small and the embedding rate is low; with the increase of the CaCl2 mass fraction, the wall material reacts rapidly with the CaCl2 solution to form a calcium alginate network structure, which quickly embeds the BSSL. Therefore, the CaCl2 mass fraction of 3% was selected as the optimal CaCl2 concentration in this experiment.

[0132] 2.1.3 Effect of fixation time on microcapsule embedding efficiency and particle size

[0133] like Figure 3As shown in the figure, for microcapsules prepared with sodium alginate as the wall material, with the extension of the fixation time, the particle size and embedding rate of BSSL microcapsules showed a trend of first increasing and then decreasing; when the fixation time was 40min, the embedding rate of the microcapsules was 36%, which was significantly higher than that of the other four groups. When the fixation time was 30min, the particle size of the microcapsules was the largest, which was 2.5mm. In the five groups of experiments, when the fixation time was 10min and 50min, the embedding rate was low. This is because the reaction of sodium alginate and CaCl2 is relatively easy, but it also takes a certain amount of time to complete. The low embedding rate of 10min fixation may be due to incomplete fixation. When the microcapsules were rinsed, the unembedded bile salt-activated lipase was washed away. The embedding rate decreased after 50min fixation. This may be because as the fixation time was extended, the microcapsule particles were immersed in the CaCl2 solution for a long time, and were affected by the ionic strength for a long time, resulting in a decrease in the activity of bile salt-activated lipase. Therefore, 40min was selected as the optimal fixation time in this experiment.

[0134] 2.1.4 Effect of the ratio of enzyme solution to wall material on microcapsule encapsulation efficiency and particle size

[0135] like Figure 4 As shown in the figure, for microcapsules prepared with sodium alginate as the wall material, when the ratio of enzyme solution to wall material (v / v) is 1:2, the optimal embedding rate is 34.7% and the particle size is 2.49 mm. When the ratio of enzyme solution to wall material is too high, the particle size increases significantly and the embedding rate shows a significant downward trend; the main reason for this phenomenon may be that when sodium alginate is excessive, the intermolecular force increases, the solution viscosity also increases, and the violent collision has a negative impact on the embedding rate and particle size. Therefore, this experiment selected the ratio of enzyme solution to wall material as 1:2 as the optimal ratio.

[0136] 2.1.5 Orthogonal experiment to optimize embedding process conditions

[0137] According to the single factor test, the three most significant factors affecting the microcapsule embedding rate were selected, namely, the mass fraction of sodium alginate, the mass fraction of CaCl2 and the fixation time. Each factor was set at three levels. The embedding rate was used as the evaluation index. The L9(3 3 ) The fixed conditions were optimized by orthogonal experiment, and the results are shown in Table 3.

[0138] It can be seen from Table 3 that the optimal combination is A1B2C2, that is, the mass fraction of CaCl2 is 2%, the mass fraction of sodium alginate is 2.5%, and the fixation time is 35min. From the range R, it can be seen that the order of influence on the BSSL microcapsule embedding efficiency is CaCl2 mass fraction (A) > sodium alginate mass fraction (B) > fixation time (C).

[0139] Table 3 Orthogonal test results

[0140]

[0141]

[0142] In order to study whether each factor has a significant effect on the embedding rate of BSSL microcapsules, variance analysis was performed on the data in Table 3. The results are shown in Table 4. From the F value, it can be seen that the order of the influence of the three factors on the embedding rate is: CaCl2 mass fraction (A) > sodium alginate mass fraction (B) > fixation time (C), which is consistent with the results of orthogonal test range analysis. The CaCl2 mass fraction has a significant effect on the embedding rate of BSSL microcapsules (P<0.05), while the sodium alginate mass fraction and fixation time have no significant effect on the embedding rate of BSSL microcapsules.

[0143] Table 4 Analysis of variance of test results

[0144]

[0145] Note: *.P<0.05, significant difference.

[0146] 2.2 Preparation of bile salt-activated lipase microcapsules using sodium alginate-whey protein as wall material

[0147] Microcapsules were prepared according to the method of Example 4. After adding 1.2% whey protein powder by mass in Example 4, Figure 5 As shown in the figure, the embedding rate of the microcapsules is 82% and the particle size is 2.52 mm; while in Example 1, the embedding rate of the microcapsules prepared with sodium alginate as the wall material is 59.26%. This shows that the embedding rate of BSSL microcapsules prepared with sodium alginate-whey protein powder as the composite wall material can be greatly improved compared with the BSSL microcapsules prepared with sodium alginate as the wall material. This may be because when sodium alginate is used as the wall material alone, the surface of sodium alginate has more pores, so the embedding rate of the prepared BSSL microcapsules is low. After adding whey protein, the whey protein can make up for the pores on the surface of sodium alginate, thereby improving the embedding rate of BSSL microcapsules.

[0148] 2.3 Evaluation of properties of BSSL microcapsules

[0149] 2.3.1 Morphological observation of microcapsules

[0150] like Figure 6 As shown in A1 and B1, the two wet lipase microcapsules prepared by the methods in Examples 1 and 4 are both spherical, evenly distributed and have no significant difference; Figure 6 As shown in A2 and B2, the freeze-dried microcapsule particles with sodium alginate-whey protein powder as the wall material are more delicate and uniform. Figure 7As shown in A3 and B3, the surface of the bile salt-activated lipase microcapsules prepared with a single sodium alginate as the wall material is not smooth and has a large number of pores, which is also the reason for the low embedding rate of the microcapsules prepared with a single sodium alginate; while the surface of the lipase microcapsules prepared with sodium alginate-whey protein powder as the wall material is uniform and the particles are spherical. Studies have shown that different drying methods can affect the structural characteristics of microcapsules, such as surface characteristics, size, water content and shape, and freeze drying usually produces softer, larger and more elliptical microcapsules.

[0151] 2.3.2 Tolerance of BSSL microcapsules in simulated gastric fluid of adults and children

[0152] Tolerance of the two BSSL microcapsules in simulated adult and infant gastric fluids Figure 8 As shown in the figure, in the process of simulated gastric digestion, due to the destruction of the microcapsule wall material, the retention rate of BSSL in the two microcapsules showed a continuous downward trend as the digestion time prolonged. Figure 8 As shown in Figure A, after 60 minutes of gastric digestion, the retention rate of BSSL in microcapsules prepared with sodium alginate as the wall material was 63%, while the retention rate of BSSL in microcapsules prepared with sodium alginate-whey protein as the wall material was 78%. Figure 8 As shown in Figure B, after 60 minutes of gastric digestion, the retention rate of BSSL in microcapsules prepared with sodium alginate as the wall material is 62%, while the retention rate of BSSL in lipase microcapsules prepared with sodium alginate-whey protein powder composite wall material is 80%; this is because in an acidic environment, the microcapsule wall material is destroyed and the core material is released. The longer the residence time in the acidic environment, the more serious the damage to the microcapsule wall material, and the stronger the acidity, the more serious the damage to the microcapsule wall material. The above results show that both wall materials have a certain gastric digestion resistance effect, among which sodium alginate-whey protein powder as the wall material has a better effect.

[0153] 2.3.3 Thermal stability

[0154] BSSL is a lipase that is extremely sensitive to high temperatures. Studies have shown that BSSL loses its activity completely after pasteurization. Fig. 9 The figure shows the change of BSSL retention rate in the microcapsule samples of BSSL prepared with sodium alginate as the wall material and BSSL microcapsules prepared with sodium alginate-whey protein powder as the wall material stored at 60℃ and 70℃ for 1 hour. Fig. 9 As shown in Figure A, with the extension of storage time, BSSL in the microcapsules is continuously dissolved, and the microcapsule wall material is seriously damaged. After 60 minutes of storage, the stability of the microcapsules prepared by sodium alginate-whey protein powder is slightly improved compared with the microcapsules prepared by single sodium alginate wall material; while the free BSSL has mostly lost its activity after 20 minutes of storage. Fig. 9As shown in Figure B, the activity of microcapsules prepared with two wall materials was almost completely lost after being stored at 70℃ for 1 hour, while the free BSSL was completely inactivated after being stored for 20 minutes. In summary, BSSL fixed in microcapsules has a certain stability, and after pasteurization (62-65℃, 30 minutes), BSSL in microcapsules can also retain more than 70%.

[0155] 3 Conclusion

[0156] Through single factor and orthogonal experiments, this study found that the optimal preparation process conditions for microcapsules with sodium alginate as wall material were as follows: sodium alginate mass fraction of 2.5%, calcium chloride mass fraction of 2%, enzyme solution and wall material ratio of 1:2 (v / v), fixation time of 35min, and the embedding rate of microcapsules prepared under these conditions could reach 59%. The optimal preparation process conditions for microcapsules with sodium alginate-whey protein as wall material were as follows: sodium alginate mass fraction of 2.5%, calcium chloride mass fraction of 2%, enzyme solution and wall material ratio of 1:2 (v / v), fixation time of 35min, and whey protein mass fraction of 1.2%. Under these conditions, the embedding rate of microcapsules prepared could reach 82%. Through digestion stability test and thermal stability test, it was found that both wall materials had good protective effect on BSSL, and sodium alginate-whey protein powder had better gastric digestion resistance. The results of thermal stability test showed that after pasteurization, the bile salt activated lipase in the two bile salt activated lipase microcapsules could reach more than 70%. This study achieved the purpose of BSSL being resistant to gastric acid and heat, which has certain significance for promoting fat digestion and absorption.

[0157] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the embodiments and illustrations shown and described herein.

Claims

1. A method for preparing bile salt-activated lipase microcapsules, characterized in that: include: Step 1, fully dissolving the wall material in water to obtain a wall material aqueous solution; the wall material is sodium alginate and whey protein powder, the mass fraction of sodium alginate in the wall material aqueous solution is 2.5%, and the mass fraction of whey protein is 1.2%; Step 2: Mix the enzyme solution and the wall material aqueous solution in a volume ratio of 1:2, then add dropwise to a 2% CaCl2 solution, and then fix for 35 minutes to form beads; Step 3, filtering the beads obtained in step 2, washing to remove the residual CaCl2 solution and residual enzyme solution, and obtaining wet microcapsules; Step 4: Place the wet microcapsules obtained in step 3 at -80 to -20°C overnight and freeze-dry them in vacuum to obtain dry microcapsules.

2. The method for preparing bile salt-activated lipase microcapsules according to claim 1, characterized in that: The fixed temperature in step 2 is 4°C.

3. The method for preparing bile salt-activated lipase microcapsules according to claim 1, characterized in that: The preparation method of the enzyme solution comprises the following steps: using yeast strain KM71 as a host cell and pPIC9k as an exogenous expression vector to construct a recombinant yeast expression system of human bile salt-activated lipase, using a BMMY culture medium with a pH of 6.5 to concentrate and ferment the recombinant yeast in a shaker at 28° C. and 250 rpm, inducing the yeast to secrete and express human bile salt-activated lipase using methanol with a volume fraction of 0.5%, and obtaining the enzyme solution after high-speed centrifugation to remove bacterial precipitates.

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