CN109700032B - Probiotics microcapsule based on full-aqueous-phase complex coacervation and preparation method thereof - Google Patents
Probiotics microcapsule based on full-aqueous-phase complex coacervation and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a probiotic microcapsule based on full-aqueous phase complex coacervation and a preparation method thereof, wherein the method comprises the following steps: (1) obtaining probiotic bacteria mud; (2) respectively preparing an A type gelatin solution and a sodium caseinate solution with mass concentration of 0.5-5%, and preserving heat at 30-60 ℃ for later use; (3) mixing the bacterial sludge, an A-type gelatin solution and a sodium caseinate solution at 30-60 ℃, adjusting the pH, and inducing complex coacervation reaction to prepare wet microcapsules; the addition amount of the bacterial sludge is as follows: adding 10 per 1g gelatin8～1012The mass ratio of the A-type gelatin to the sodium caseinate is (0.25-4): 1, the pH range is 5.5-6.0; the method is based on the complex coacervation of the A type gelatin/sodium caseinate system, does not need the emulsification step, simplifies the method, saves the materials and shortens the preparation time of the microcapsule; improves the embedding rate and the storage survival rate of the probiotics.
Description
Technical Field
The invention relates to the technical field of food biotechnology and medicine, in particular to a probiotic microcapsule based on full-aqueous-phase complex coacervation and a preparation method thereof.
Background
The probiotics is the most important functional food component, has the functions of maintaining the balance of intestinal flora, enhancing the immunity of the organism, controlling cholesterol, blood fat, blood pressure and the like, and is widely applied to foods such as fermented products, dairy products, milk beverages and the like. Only after reaching the intestinal tract of a human body with enough viable bacteria, the probiotics can exert the probiotic function, but the probiotics are highly sensitive to environmental factors such as temperature, humidity, oxygen, acid and the like and are easy to die in the processes of processing, storage, digestion and the like, and microencapsulation is often taken as an important means for improving the problem. Probiotic microencapsulation methods commonly used in the food industry include spray drying, fluidized bed methods, emulsification methods, extrusion methods, and the like. However, the preparation conditions of the methods are strict and are not environment-friendly, and the prepared microcapsules have poor environmental tolerance and controllable release capacity.
Complex coacervation is a concept in polymer physics, which means that electrostatic interaction occurs between polyelectrolytes with opposite charges under a certain pH value, and finally desolvation is carried out to form an enrichment phase and a dilution phase with greatly different polyelectrolyte contents. The complex coacervation method is commonly used for microencapsulation of fat-soluble active components such as grease, essential oil, fat-soluble vitamins, fat-soluble antioxidants and the like, and is based on the principle that oil/water emulsion is prepared through emulsification according to incompatibility of water and oil phases, and then polyelectrolyte with opposite charges is triggered to form a compound on an oil-water interface through external conditions, so that fat-soluble components in oil drops are stabilized. The research of embedding lipophilic bioactive components in the complex coacervation hair is extensive and deep, and has better industrial application. However, the complex coacervation method has less research on embedding hydrophilic bioactive components, and a considerable part of embedding is based on a water/oil/water emulsion system, so that the preparation process is complex and the load capacity is low.
Chinese patent CN102580638A discloses a microencapsulation method for preparing hydrophilic substances as core materials by a complex coacervation method, which comprises the steps of firstly adding an oil phase substance and an emulsifier into the hydrophilic core materials, dispersing at high speed to form an emulsion, then adding the emulsion into a wall material solution in which specific protein gelatin and polysaccharide are mixed according to a certain proportion, adjusting the pH value to 3.9-4.2 for complex coacervation reaction, cooling, then adjusting the pH value to 6.0, adding glutamine transaminase for solidification to obtain a microcapsule solution, and drying and filtering the obtained wet microcapsules to obtain a powdery microcapsule product. The process of preparing the microcapsule by the complex coacervation method in the patent comprises an emulsification step, and the embedding is to embed the active ingredients by an oil/water or water/oil/water system, so the preparation process is complex and the embedding rate is low. The traditional complex coacervation method generally needs to add an oil phase, and forms oil/water or water/oil/water emulsion through emulsification to realize the embedding of active ingredients.
The complex coacervation method is very deficient in the embedding aspect of probiotics. As a new embedding method, a complex coacervation microencapsulation method is rarely reported in China about application in the field of probiotic embedding. How to develop a complex coacervation method suitable for probiotics to prepare the probiotic microcapsules becomes a problem to be solved urgently.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a probiotic microcapsule based on full-aqueous phase complex coacervation and a preparation method thereof, the method is based on the complex coacervation of an A-type gelatin/acidic protein system, an emulsification step is not needed, the method is simplified, materials are saved, and the microcapsule preparation time is shortened; the probiotic microcapsule prepared by the method has high survival rate and good stability in storage, digestion, heat treatment and the like.
The invention is realized by the following steps:
one purpose of the invention is to provide a preparation method of probiotic microcapsules based on full-water-phase complex coacervation, which is characterized by comprising the following steps:
step 1, culturing the frozen probiotic, centrifuging, and removing supernatant to obtain bacterial sludge for later use;
step 2, weighing a certain mass of type A gelatin, dissolving the type A gelatin in water to prepare a type A gelatin solution with the mass concentration of 0.5-5%, and preserving heat at 30-60 ℃ for later use;
step 3, weighing a certain mass of sodium caseinate solution, dissolving in water to prepare a sodium caseinate solution with the mass concentration of 0.5-5%, and preserving heat at 30-60 ℃ for later use;
step 4, mixing the bacterial sludge, the A-type gelatin solution and the sodium caseinate solution at the temperature of 30-60 ℃, adjusting the pH, inducing a complex coacervation reaction, and preparing wet microcapsules(ii) a The addition amount of the bacterial sludge is as follows: adding 10 per 1g gelatin8～1012cfu bacterial mud, the mass ratio of the type A gelatin to the sodium caseinate is as follows: 0.25-4: 1, the pH range is 5.5-6.0;
and 5, drying the wet microcapsule obtained in the step 4 to obtain the dry powder of the probiotic microcapsule.
Preferably, the temperature adopted in the steps 2 to 4 is 35 to 45 ℃.
The most preferable mass ratio of the type A gelatin to the sodium caseinate is 2: 1; the pH in step 4 is most preferably 6.0.
Preferably, after the type A gelatin solution, the sodium caseinate solution and the bacterial sludge in the step 4 are mixed, 0-5% of micromolecular sugar powder by mass is added, and the pH is adjusted after the mixture is uniformly stirred.
Specifically, the small molecular sugar comprises one or more of glucose, sucrose, fructose, trehalose, maltose, fructo-oligosaccharide and xylo-oligosaccharide; the stirring speed is 100-1000 r/min, and the time is 10-60 min.
Preferably, in the step 4, the bacterial sludge, the type A gelatin solution and the sodium caseinate solution are mixed, stirred for 10-60 min at a speed of 100-1000 r/min, and then cooled for 5-30 min in an ice-water bath.
Preferably, the drying in step 5 is spray drying or freeze drying; the conditions of the spray drying are as follows: controlling the inlet temperature to be 100-250 ℃ and the outlet temperature to be 50-150 ℃; the conditions of freeze drying are as follows: the temperature is controlled to be-20 ℃ to-80 ℃, and the freezing time is 6-48 h.
The second purpose of the invention is to provide the probiotic microcapsule prepared by the method.
Compared with the prior art, the invention has the following advantages and effects:
1. the preparation method of the probiotic microcapsule based on full-water-phase complex coacervation provided by the invention adopts an A-type gelatin/sodium caseinate complex coacervation system, and in the neutral pH range (5.5-6.0), the A-type gelatin is positively charged, the sodium caseinate is negatively charged, and the probiotic is negatively charged by adjusting the pH, and a liquid drop structure is formed by electrostatic complexation and microphase separation, and then the powdery microcapsule product can be obtained after cooling, solidification and drying. Firstly, an emulsification step is not needed, so that the method is simplified, the material is saved, and the microcapsule preparation time is shortened; secondly, the embedding rate and the storage survival rate of the probiotics are effectively improved, and the probiotics microcapsule has good stability of storage, digestion, heat treatment and the like; thirdly, the operation is simple and convenient, and the industrial production is easy to realize.
2. In the prior art, an A-type gelatin/sodium caseinate complex coacervation system is not adopted to successfully embed probiotics, because the complex coacervation method has a plurality of difficulties, the most difficult is how to regulate and control microphase separation; the preparation method of the probiotic microcapsule based on full-water-phase complex coacervation provided by the invention adopts the temperature of 30-60 ℃, the pH of 5.5-6.0, the concentrations of the A-type gelatin solution and the sodium caseinate solution are 0.5-5%, and the mass ratio of the A-type gelatin to the sodium caseinate is 0.25-4: 1 hour, so that microphase separation can be manually regulated and controlled, and further complete aqueous phase complex coacervation of probiotics is successfully carried out, which needs to be obtained by the applicant through a large amount of innovative exploration.
3. According to the preparation method of the probiotic microcapsule based on full-water-phase complex coacervation, the A-type gelatin solution and the sodium caseinate solution are used as the wall material of the complex coacervation, so that the embedding and protecting effects of the microcapsule can be improved, and the edible microcapsule can provide nutrition for a human body; the A-type gelatin is a typical basic protein, the isoelectric point of the A-type gelatin is usually in the range of pH 8.0-9.0, the A-type gelatin is cheap and easy to obtain and has no antibacterial activity, and the A-type gelatin can improve the pH of complex coacervation to a neutral range, so that the survival rate of thalli in the processes of drying, storage and the like is obviously improved.
Drawings
FIG. 1 is an optical morphology of complex coacervates of different examples and comparative examples;
fig. 2 is a result of the storage survival rate of probiotic microcapsules; wherein A is 11% RH, measured at 25 ℃; b is 33% RH, measured at 25 ℃; c is 11% RH, measured at 30 ℃; d is 33% RH, measured at 30 ℃.
Detailed Description
Example 1
The preparation method of the probiotic microcapsule based on full-water-phase complex coacervation comprises the following steps:
step 1, culturing the frozen probiotic, centrifuging, removing supernatant to obtain 1 × 109CFU/ml bacterial sludge for later use;
step 2, weighing a certain mass of type A Gelatin (GE) powder, dissolving the type A Gelatin (GE) powder in ultrapure water to prepare a GE solution with the concentration of 2% (w/w), and keeping the temperature at 40 ℃ for later use;
step 3, weighing a certain mass of sodium caseinate (NaCaS) powder, dissolving the powder in ultrapure water to prepare a NaCaS solution with the concentration of 2% (w/w), and preserving heat at 40 ℃ for later use;
step 4, 0.3ml of bacterial suspension (1X 10) is added at 40 DEG C9CFU/ml), 40g of 2% (w/w) GE solution, 20g of 2% (w/w) NaCaS solution and 1.2g of sucrose (final concentration 2% w/w) powder, and uniformly mixing at 400rpm for 20 min; adjusting pH to 5.5 with 10% acetic acid, and continuously stirring at 400rpm for 15min to completely complex and coagulate; and cooling in ice water bath for 15min to obtain microcapsule solution, and spray drying at inlet temperature of 110 deg.C and outlet temperature of 70 deg.C to obtain probiotic microcapsule.
Example 2
This example is the same as example 1 except that the pH of the mixed coagulation is 5.6.
Example 3
This example is the same as example 1 except that the pH of the mixed coagulation is 5.8.
Example 4
This example is the same as example 1 except that the pH of the mixed coagulation is 6.0.
Example 5
This example was carried out in the same manner as example 4 except that the pH of the mixed coagulation was 6.0 and the ratio of GE/NaCaS was 0.25.
Example 6
This example was carried out in the same manner as example 4 except that the pH of the mixed coagulation was 6.0 and the ratio of GE/NaCaS was 0.5.
Example 7
This example was carried out in the same manner as example 4 except that the pH of the mixed coagulation was 6.0 and the ratio of GE/NaCaS was 1.
Example 8
This example was carried out in the same manner as example 4 except that the pH of the mixed coagulation was 6.0 and the ratio of GE/NaCaS was 4.
Comparative example 1
This comparative example is the same as example 1 except that the pH of the mixture was 4.5.
Comparative example 2
This comparative example is the same as example 1 except that the pH of the mixture was 6.5.
Comparative example 3
This comparative example was NaCaS, pH6.0, and the remainder of the example 4.
Comparative example 4
The comparative example was GE, pH6.0, and the remainder of the example 4.
Comparative example 5
This comparative example was identical to example 4 except that the pH of the mixed coacervate was 6.0 and the ratio of GE/NaCaS was 6.
Experimental example 1
1. Microcapsule morphology determination: the morphology of the microcapsules of examples 1-4, above, and comparative examples 1-2 was monitored by the BT-1600 image particle analysis system. A small amount of wet microcapsules are dropped on a glass slide, observed under a microscope and photographed at a magnification of 10 times and 20 times.
2. And (3) determining the embedding rate: in each of the microcapsules of examples 1 to 4 and comparative examples 1 to 2, 0.01g of the microcapsule dry powder was added to 0.99g of a peptone salt solution (PS, 1.0g/L peptone, 8.5g/L NaCl, pH 6.8) and dispersed for 30 seconds with a vortex shaker to completely disperse the microcapsules. 0.1mL of the liquid was diluted to an appropriate ratio with physiological saline (9g/L NaCl), spread on MRS solid medium, and then subjected to static culture at 37 ℃ for 48 hours, followed by counting. Meanwhile, 1.0mL of the original bacterial liquid added into the wall material is taken, diluted and evenly coated on an MRS solid culture medium, and the count is carried out after the static culture at 37 ℃ for 48 hours.
The embedding rate (EY) of the microcapsules was calculated as follows:
EY＝N/N0×100％
wherein N represents the number of viable bacteria released from the microcapsule, and N0 represents the total number of bacteria used for microcapsule encapsulation.
3. The optical morphology and the embedding rate data of the microcapsules of examples 1 to 4 and comparative examples 1 to 2 are shown in table 2, and the optical morphology graph of the microcapsules is shown in fig. 1.
TABLE 1
As shown in Table 1, when the pH value is in the range of 5.5-6.0, which is the preferable pH value, the experimental groups 1-4 can exhibit better spherical particle morphology, and the embedding rate is high. When the pH of 4.5 was selected for comparative example 1 and the pH of 6.5 was selected for comparative example 2, the microcapsules could not be made spherical and the embedding rate was low.
Experimental example 2
The morphology of complex aggregates with different mixing ratios of GE/NaCaS was observed under a microscope by the method of Experimental example 1 and the embedding rate was calculated, i.e., the morphology characteristics and the embedding rate data of the microcapsules of examples 4 to 8 and comparative examples 3 to 5 are shown in Table 2.
TABLE 2
As can be seen from Table 2, when the mixing ratio of GE/NaCaS is 0.25-4: 1, the examples 4 to 8 all showed good spherical particle morphology and high embedding rate. The microcapsules of comparative examples 3 to 5 could not be formed into a spherical shape, and the embedding rate was low.
Experimental example 3 determination of storage survival Rate
1. GE/NaCaS (example 4), NaCaS (comparative example 3), and GE (comparative example 4) complex systems were prepared at complex coacervation ph6.0 and complex coacervation temperature of 40 ℃, and the storage survival rates of each group of probiotic microcapsules were measured. The storage stability of the microcapsules was evaluated by measuring the amount of the microcapsules surviving at different temperatures and Relative Humidities (RH). The probiotic microcapsules of example 4, comparative example 3 and comparative example 4 were hermetically stored under conditions of 11% RH (25 ℃, 30 ℃) and 33% RH (25 ℃, 30 ℃), respectively, and the saturated solutions corresponding to different relative humidities at 25 ℃ were: saturated lithium chloride solution (11% relative humidity); saturated solution of magnesium chloride (relative humidity 33%). The survival amount of the cells was measured every 4 days for the samples stored at 33% RH; the viable cell amount of the sample stored at 11% RH was measured at 7d intervals by plate counting method for 28d of experiment time. The relative survival logarithm of the thalli is plotted against the storage days, and the inactivation constant is obtained by linear fitting of a curve.
2. The inactivation of lactic acid bacteria during storage is expressed in log relative survival (log N/N0) and is formulated as follows:
logNt＝logN0+kTt
wherein N0 represents the number of original cells (CFU/g), Nt represents the number of viable cells (CFU/g) after storage for a certain period of time, T represents the storage time (d), and kT represents the inactivation constant (d-1) at temperature T.
3. As shown in FIG. 2, FIG. 2 shows the change of the relative logarithm of viable cells with storage time, and the slope of the curve represents the cell inactivation constant, and the lower the cell inactivation constant k, the lower the cell inactivation rate, and the better the storage stability. As shown in fig. 2, at the same temperature, relative humidity has a large influence on cell survival, the inactivation rate of cells increases as humidity increases, while at a relatively low humidity (RH 11%), temperature has a relatively small influence on cell survival, and high temperature and high humidity accelerate the death rate of lactic acid bacteria. It is shown that the inactivation constant of lactic acid bacteria, k (GE/NaCaS) < k (GE), was observed under each storage condition, indicating that sodium caseinate has better protection effect on the storage of lactic acid bacteria at room temperature than gelatin type A, and that after the two proteins are combined and coagulated, the inactivation constant k value of lactic acid bacteria is the smallest compared with the k value of the two groups of controls, indicating that the combined coagulation of gelatin type A and sodium caseinate has better protection effect on the storage of lactic acid bacteria at room temperature.
The invention is not to be considered as limited to the particular embodiments shown, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (7)
1. A preparation method of probiotic microcapsules based on full-aqueous phase complex coacervation is characterized by comprising the following steps:
step 1, culturing the frozen probiotic, centrifuging, and removing supernatant to obtain bacterial sludge for later use;
step 2, weighing a certain mass of type A gelatin, dissolving the type A gelatin in water to prepare a type A gelatin solution with the mass concentration of 0.5-5%, and preserving heat at 30-60 ℃ for later use;
step 3, weighing a certain mass of sodium caseinate powder, dissolving the sodium caseinate powder in water to prepare a sodium caseinate solution with the mass concentration of 0.5-5%, and preserving heat at 30-60 ℃ for later use;
step 4, mixing the bacterial sludge, the type A gelatin solution and the sodium caseinate solution at 30-60 ℃, uniformly mixing for 20min at 400rpm, adjusting the pH, continuously stirring for 15min at 400rpm, inducing complex coacervation reaction, cooling for 5-30 min in ice-water bath, and preparing wet microcapsules; the addition amount of the bacterial sludge is as follows: adding 10 per 1g gelatin8～1012The mass ratio of the A-type gelatin to the sodium caseinate is (0.25-4): 1, the pH range is 5.5-6.0;
step 5, drying the wet microcapsule obtained in the step 4 to obtain dry powder of the probiotic microcapsule;
the drying in the step 5 is spray drying or freeze drying.
2. The method of claim 1, wherein the mass ratio of type a gelatin to sodium caseinate is 2: 1.
3. the method according to claim 1, wherein the pH in the step 4 is 6.0.
4. The preparation method according to claim 1, wherein in the step 4, the type A gelatin solution, the sodium caseinate solution and the bacterial sludge are mixed, and then the powder of the small molecule sugar with the final concentration of 0-5 w/w% is added, and after uniform stirring, the pH is adjusted.
5. The method of claim 4, wherein the small molecule sugar comprises one or more of glucose, sucrose, fructose, trehalose, and maltose.
6. The method of claim 1, wherein the spray drying conditions are: controlling the inlet temperature to be 100-250 ℃ and the outlet temperature to be 50-150 ℃; the conditions of freeze drying are as follows: the temperature is controlled to be-20 ℃ to-80 ℃, and the freezing time is 6-48 h.
7. A probiotic microcapsule prepared by the method of any one of claims 1 to 6.
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