CN107686824B - Preparation method of feeding lactobacillus microcapsule - Google Patents
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Abstract
The invention discloses a preparation method of a feeding lactobacillus microcapsule, which comprises the following steps: (1) activating lactobacillus strains, and fermenting and culturing to obtain fermentation liquor; (2) centrifuging the fermentation liquor prepared in the step (1), and collecting bacterial sludge; (3) adding the bacterial sludge obtained in the step (2) into a microcapsule wall material solution; (4) preparing microcapsules from the solution prepared in the step (3) through spray drying; wherein the temperature of an air inlet of the spray drying is 60-70 ℃, the temperature of an air outlet is 50-60 ℃, the vacuum degree is 0.03-0.04 MPa, and the feeding speed is 40-45 rpm; (5) the microcapsule product was collected. The viable count of the lactic acid bacteria microcapsule prepared by the method is 1010The preparation method has the advantages of more than cfu/g, preparation survival rate of more than or equal to 85.0%, particle size of 5-8 mu m, water content of the product of 4.80%, high acid resistance, cholate resistance and storage resistance, simple preparation process operation, low production cost and suitability for continuous production.
Description
Technical Field
The invention belongs to the technical field of microcapsule coating, and particularly relates to a preparation method of a feeding lactobacillus microcapsule.
Background
Lactic acid bacteria are normal flora of intestinal tracts of human beings and animals, and a large number of researches prove that the lactic acid bacteria preparation has positive effects on promoting the growth of swinery, improving the composition of the intestinal flora, improving the immunity of organisms and the like. However, as the lactic acid bacteria can not form spores in the growth process, viable bacteria which can successfully reach the intestinal tract are reduced due to the influence of gastric acid and intestinal bile salt after the bacteria enter the animal digestive tract. In addition, certain damage can be caused to the thalli during the processes of production, processing, transportation and the like of the products, and the exertion of the probiotic effect of the lactic acid bacteria and the high-efficiency utilization of the lactic acid bacteria in animal production are limited. The microcapsule technology is a method of coating a wall material with lactic acid bacteria as a core material to protect the bacteria from the external environment, and is currently considered to be one of the most practical methods for protecting the bacteria.
The spray drying technology is that under the condition of high temperature, the core material substance is dispersed in the wall material solution, mixed evenly and then sprayed and atomized in hot air flow, so that the solvent dissolving the wall material is quickly evaporated, and finally the microcapsule powder product is obtained. The microcapsule technology has the advantages of high coating speed, good product dispersibility and dissolubility, low production cost, suitability for continuous production and the like, and the method is widely applied to the production of functional foods. There are also reports on the research on the preparation of lactic acid bacteria microcapsules by using spray drying technology, such as: the application patents with publication numbers of CN106148314A and CN1103283975A and the Wang-Yao (2016) and the like all adopt a universal spray drying method to prepare the microcapsules, and the temperature of an air inlet is generally 130-170 ℃ in the spray drying granulation process. The invention patents with publication numbers CN1569043A and CN104706678A also disclose a fluidized bed spray drying granulation process, respectively, but the invention also sprays the lactobacillus concentrated solution under the condition that the pelleting auxiliary materials are in a boiling state (100 ℃). Zhangzhang (2012) indicates that the survival rate of lactobacillus casei spray-dried powder is only 2.6% when the air inlet temperature is 100 ℃. Since lactic acid bacteria cannot produce spores, the high temperature resistance of the cells is poor, and most of the cells are inactivated when the temperature exceeds 85 ℃. When the method is used for preparing the lactobacillus microcapsule, the concentration of viable bacteria in the prepared lactobacillus product can be reduced due to the high temperature generated in the process, the cell structure and physiological damage of the viable bacteria can be caused in the high-temperature process, the growth, reproduction and fermentation capacity of the bacteria in the animal body is reduced, and the exertion of the probiotic effect and the application cost in the culture production are influenced. The application No. 201510105655.X discloses that lactobacillus sakei is filtered and concentrated for 2-3 times by adopting a hollow fiber membrane concentration method, and then a microcapsule product is prepared by using a low-temperature spray drying method, wherein the survival rate of spray drying is 52.77%. The survival rate of the prepared lactobacillus microcapsule is yet to be further improved, and the preparation process of the lactobacillus microcapsule, which is simple to operate in the preparation process, low in production cost and suitable for pilot-scale production, is not yet provided at present.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preparation method of a feeding lactobacillus microcapsule, in particular to a method for preparing the feeding lactobacillus microcapsule by low-temperature spray drying, which can effectively reduce the damage of thalli in the spray drying process and improve the survival rate of live bacteria.
In order to achieve the purpose, the invention adopts the following technical scheme that:
a preparation method of a feeding lactobacillus microcapsule comprises the following steps:
(1) activating lactobacillus strains, and fermenting and culturing to obtain fermentation liquor;
(2) centrifuging the fermentation liquor prepared in the step (1), and collecting bacterial sludge;
(3) adding the bacterial sludge obtained in the step (2) into a microcapsule wall material solution;
(4) preparing microcapsules from the solution prepared in the step (3) through spray drying; wherein the temperature of an air inlet of the spray drying is 60-70 ℃, the temperature of an air outlet is 50-60 ℃, the vacuum degree is 0.03-0.04 MPa, and the feeding speed is 40-45 rpm;
(5) the microcapsule product was collected.
In the above-described production method, preferably, the solvent of the microcapsule solution is: 0.85% physiological saline; the solute comprises 13 to 15 weight percent of skim milk, 1 to 3 weight percent of cane sugar, 1 to 3 weight percent of starch, 2 to 4 weight percent of talcum powder, 1 to 2 weight percent of soybean protein isolate and 0.1 to 0.3 volume percent of tween-60.
In the above-described production method, preferably, the solvent of the microcapsule solution is: 0.85% physiological saline; the solute comprises 14% of skim milk, 2% of cane sugar, 3% of modified starch, 3% of talcum powder, 1% of isolated soy protein and 0.2% of tween-60 by volume percentage.
In the preparation method, preferably, the temperature of the air inlet is 60-65 ℃, the temperature of the air outlet is 50-55 ℃, and the vacuum degree is 0.04 MPa.
In the preparation method, preferably, the air inlet temperature is 65 ℃, the air outlet temperature is 55 ℃, and the feeding speed is 45 rpm.
The preparation method as described above, preferably, the lactic acid bacterial strain is: lactobacillus reuteri of porcine originLactobacillus reuteriZLR003，CGMCC No.11530。
In the preparation method, the centrifugation condition in the step (2) is preferably 6000 to 9000rpm for 5 to 15 minutes.
The viable count content of the obtained lactic acid bacteria microcapsule preparation is more than or equal to 1010cfu/g。
The application of the lactobacillus microcapsule prepared by the method in preparing feed additives.
The invention has the beneficial effects that:
the lactobacillus reuteri is a resident flora of animal intestinal tracts and one of feeding microbial strains which are allowed to be used in No. 2045 announced by Ministry of agriculture in China, and the strain also has wide application prospects. The invention discloses a low-temperature spray drying preparation process suitable for lactobacillus reuteri. The preparation process is simple to operate, the preparation survival rate is high, and good product fluidity, high yield and the like can be ensured. The method has positive significance for the production of the feeding lactobacillus microcapsule preparation and the popularization and application in animal production.
The invention provides a preparation method of a feeding lactobacillus microcapsule, which mainly adopts low-temperature spray drying to prepare the lactobacillus microcapsule, wherein the temperature of an air inlet is lower than 60-70 ℃ and the temperature of an air outlet is 50-60 ℃ in the spray drying process. The viable count of the lactic acid bacteria microcapsule prepared by the method reaches 1010More than cfu/g, particle size of 5-8 mu m, water content of 4.80 percent, high acid resistance, cholate resistance and storage resistance, simple preparation process operation, low production cost and suitability for continuous production. The adopted microcapsule matrix and wall material can effectively reduce the damage of the thalli in the spray drying process and can protect the thalli in animalsThe growth, reproduction and fermentation capability in vivo ensures the exertion of the probiotic effect and the application in the culture production.
Drawings
FIG. 1 is a heat resistance curve of the strain of the present invention.
FIG. 2 shows the results of screening for protective substrates used by the strains of the present invention.
FIG. 3 shows selection results of skim milk used for the strain of the present invention.
FIG. 4 shows the results of selection of saccharides used by the strain of the present invention.
FIG. 5 shows the selection results of the starch substance used by the strain of the present invention.
FIG. 6 shows the selection results of gel-like substances used for the strains of the present invention.
FIG. 7 shows the effect of air inlet temperature in low temperature spray drying on viable count of the product.
FIG. 8 shows the effect of low temperature spray drying vacuum on viable count of the product.
FIG. 9 is a graph showing the effect of low temperature spray drying feed rate on viable count of a product.
FIG. 10 shows the results of single factor analysis of soy protein isolate.
Fig. 11 shows the results of single factor analysis of talc.
FIG. 12 shows the results of single factor assay for Tween 60.
Fig. 13 shows the morphology of the lactic acid bacteria microcapsule product observed under an electron microscope.
FIG. 14 shows the acid resistance of the lactobacillus microcapsule product.
FIG. 15 shows the determination of the bile salt resistance of the lactobacillus microcapsule product.
FIG. 16 shows the effect of room temperature storage conditions on viable count of lactic acid bacteria microcapsules.
Detailed Description
The following examples are intended to further illustrate the invention but should not be construed as limiting it. Modifications and substitutions may be made thereto without departing from the spirit and scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1 study of the Heat resistance of Lactobacillus reuteri of porcine origin
The invention firstly treats the porcine lactobacillus reuteriLactobacillus reuteriZLR003, measuring heat resistance, selecting air inlet temperature in spray drying process according to heat resistance of strain, and selecting microcapsule matrix and wall material based on principle of improving heat resistance of strain to reduce damage of thallus in spray drying process. Wherein, the strain used in the invention is the Lactobacillus reuteri of porcine originLactobacillus reuteriZLR003 is obtained by separation and purification of the inventor, and the method can be seen in the application number of 201010295637.X and is preserved in China General Microbiological Culture Collection Center (CGMCC), and the preservation number is as follows: CGMCC No. 11530.
1.1 measurement of Heat resistance
(1) Activating the swine lactobacillus reuteri twice, inoculating the swine lactobacillus reuteri in an MRS liquid culture medium by 1% of inoculation amount, culturing for 24h, centrifuging to obtain bacterial sludge, placing the bacterial sludge in 0.85% of sterile physiological saline with the same volume as that before centrifugation, respectively placing the 0.85% of sterile physiological saline in 55 ℃, 60 ℃, 65 ℃ and 70 ℃ for preheating before inoculation, respectively measuring the heat-resistant survival rates of the swine lactobacillus reuteri at 55 ℃, 30s, 55 ℃, 60 ℃, 30s, 65 ℃, 60s, 70 ℃ and 60s, respectively repeating the treatment for 3 times, and drawing the lg value of the viable count as a horizontal coordinate, wherein the result is shown in figure 1 to obtain a strain heat-resistant curve. As a result, the influence on the viable bacteria is greatest when the temperature reaches 70 ℃, particularly when the duration reaches 60s, so that the temperature for microencapsulating the strain is determined to be not more than 70 ℃.
1.2 screening of Heat-resistant protective matrices
The heat-resistant activity of the porcine lactobacillus reuteri strain at 55 ℃ for 30s, 55 ℃ for 60s, 60 ℃ for 30s, 60 ℃ for 60s, 65 ℃ for 30s, 65 ℃ for 60s, 70 ℃ for 30s and 70 ℃ for 60s is researched by respectively using 0.85% physiological saline and pH 7.0 PBS buffer as protective matrixes, and each treatment is repeated for 3 times. The results are shown in fig. 2, and show that 0.85% physiological saline has a better protective effect, and therefore, 0.85% physiological saline is determined as a protective base.
1.3 choice of microcapsule wall Material
JMP statistical analysis software is adopted to respectively select 0-20% of skim milk, saccharides (glucose, sucrose and lactose), starch substances (modified starch, soluble starch and maltodextrin), gels (sodium alginate, gum and carrageenan) and other substances to carry out single-factor component and addition proportion screening, the result is shown in figure 3-figure 6, finally, a large number of tests prove that the heat-resistant protective agent of the lactobacillus reuteri of swine origin is 13-15% of skim milk, 1-3% of sucrose, 1-3% of modified starch and 2-4% of carrageenan, the percentages are weight percentages, the solution is adopted as a protective matrix and a protective agent component, and the viable bacteria number of the strain is improved by 2 orders of magnitude compared with that of the strain without the protective agent after the solution is subjected to 60s at 70 ℃.
Example 2 study of Process parameters for preparing lactic acid bacteria microcapsules by spray drying at Low temperature
The optimization research work of the spray drying process parameters of the lactobacillus reuteri of swine origin is carried out according to the preferable results of the embodiment 1, and the optimization research work is as follows:
2.1 optimization of Inlet and Outlet air temperature
Respectively selecting 55 deg.C, 60 deg.C, 65 deg.C, 70 deg.C and 75 deg.C as air inlet temperature, measuring the influence of the temperature on viable count of product and microcapsule particle size, and repeating each treatment for 3 times. The drying speed can be increased along with the increase of the temperature, the moisture content of the product is reduced, the particle size of the product is not obviously influenced at different temperatures under the observation of an electron microscope, but the viable count of the product is reduced when the temperature reaches 70 ℃ and 75 ℃. As shown in FIG. 7, the temperature of the inlet and outlet air in the spray drying process is determined to be 60-70 ℃, the corresponding temperature of the outlet air is preferably 50-60 ℃, and the particle size of the product is 8.74 +/-0.31 μm.
2.2 optimization of vacuum degree in spray drying Process
Selecting vacuum degree of 0.02MPa, 0.03MPa, 0.04MPa, and 0.05MPa, respectively, determining viable count of the microcapsule, repeating each treatment for 3 times, and observing microcapsule particle size with electron microscope as shown in FIG. 8. The result shows that the vacuum degree is increased, the particle size of the product is reduced, the number of viable bacteria in the product is the highest when the vacuum degree is preferably 0.03-0.04 MPa, and the particle size of the product is 8.25 +/-0.57 mu m.
2.3 optimization of the feed rate
According to the feeding speed range of spray drying, the feeding speeds of 20 rpm, 25rpm, 30rpm, 35rpm, 40rpm, 45rpm and 50rpm are respectively selected, the influence of the feeding speeds on the viable count of the product and the particle size of the microcapsule is measured, and each treatment is repeated for 3 times. As shown in FIG. 9, the time for the heat treatment of the cells was shortened with the increase of the feeding speed, the particle size was increased, and the water content was increased, and when the feeding speed was 40 to 50rpm, the particle size was 6.18. + -. 0.63. mu.m, and the water content was less than 5%.
2.4 perfection of the technique for preparing lactobacillus microcapsules by low-temperature spray drying
The spray drying machine test shows that when the carrageenan is added into the solution, the wall material is easily sticky and blocks the spray head. Therefore, according to the optimized optimal process parameters, on the basis of the previous period, JMP statistical software is adopted to carry out compound optimization screening on the soybean protein isolate, the talcum powder and the Tween 60 respectively, and the results are shown in figures 10, 11 and 12. The soybean protein isolate is a surfactant, can reduce the surface tension of water and oil, can reduce the surface tension of water and air, is easy to form stable emulsion, and has lower cost price. The talcum powder and the Tween-60 are beneficial to increasing the lubricating property and the emulsifying property of the wall material.
Through the computer test, the best microcapsule wall material is finally determined to be 13-15 percent of skim milk, 1-3 percent of cane sugar, 1-3 percent of modified starch, 2-4 percent of talcum powder, 1-2 percent of soybean protein isolate and 600.1-0.3 percent of tween (volume ratio), and the viable count reaches 1.60 × 10 after low-temperature spray drying10More than cfu/g, the particle size of the product is 5-8 mu m, and the water content of the product is 4.80%.
Example 3
The method for preparing the lactobacillus microcapsule by low-temperature spray drying comprises the following steps:
(1) activating lactobacillus strain (CGMCC No. 11530) twice, inoculating in MRS liquid culture medium at 1%, culturing at 37 deg.C for 24 hr to obtain fermentation broth with viable count of 3.5 × 109cfu/mL；
(2) Centrifuging the fermentation liquor prepared in the step (1) at 8000rpm for 10 minutes, and collecting bacterial sludge;
(3) adding the bacterial sludge into a prepared microcapsule wall material solution; wherein, the adopted microcapsule wall materials are as follows: 14.0% of skim milk, 2.0% of sucrose, 3.0% of modified starch, 3.0% of talcum powder, 1.0% of soybean protein isolate and 600.2% of tween, and the substances are dissolved in 0.85% of physiological saline to form a microcapsule wall material solution.
(4) Carrying out spray drying on the solution prepared in the step (3) with the air inlet temperature of 65 ℃, the air outlet temperature of 55 ℃, the vacuum degree of 0.04MPa and the feeding speed of 45rpm as technological parameters for preparing microcapsules;
(5) collecting microcapsule product, and determining the viable count of the product to be 1.6 × 1010cfu/g，The survival rate of the preparation is more than or equal to 85.0 percent.
Example 4 determination of lactic acid bacteria microcapsule Performance parameters
The lactic acid bacteria microcapsule product prepared in example 3 was subjected to the following measurement.
(1) Morphology electron microscope observation of lactic acid bacteria microcapsule product
The microcapsule product of lactic acid bacteria prepared in example 3 was subjected to electron microscope scanning, and fig. 13 (a) and (b) are the product morphology diagrams under the conditions of 12000 times and 7000 times, respectively.
(2) Acid resistance
The microcapsule product is incubated at pH2.0 and pH3.0 for 0, 2, and 4 hr respectively, and the viable count is determined as shown in FIG. 14. In FIG. 14, (1) shows the result at pH2.0, and (2) shows the result at pH 3.0. The result shows that the influence of pH2.0 and pH3.0 on the viable count of the lactobacillus microcapsule product is small after the microcapsule is coated.
(3) Resistance to bile salts
The lactic acid bacteria microcapsule product is respectively kept in culture medium containing 0.1%, 0.2% and 0.3% of pig bile salt for 0, 3, 6 and 8 hours, and then the viable count is determined. The results are shown in FIG. 15, in which (1) the results are obtained in the medium containing 0.2% porcine bile salt and (2) the results are obtained in the medium containing 0.3% porcine bile salt. The results show that under the above conditions, the lg values of the viable count of the lactobacillus microcapsule are respectively 9.42, 9.06 and 9.03 with the lowest viable countAll the amounts are 109cfu/g or more.
(4) Storage Properties
The lactobacillus microcapsule product is stored at room temperature under vacuum, viable count is measured at 0 th, 30 th and 60 th days respectively, and the result is shown in FIG. 16, which shows that the lactobacillus microcapsule product is stored at room temperature for 60 days, lg value is 10.10, and viable count is still 1010cfu/g or more.
Tests on acid resistance, cholate resistance and storage resistance show that the capsule matrix and the wall material selected by the invention can effectively reduce the damage of thalli in the spray drying process, and show that when the lactobacillus microcapsule prepared by the invention is used as a feeding additive, the lactobacillus microcapsule can resist gastric juice with strong acidity and high-concentration cholate, can successfully reach the intestinal tract of animals, and has good activity to play the role.
In conclusion, the invention provides a method for preparing feeding lactobacillus microcapsules by using low-temperature spray drying, wherein the temperature of an air inlet is 60-70 ℃ and the temperature of an air outlet is 50-60 ℃ in the spray drying process. Under the condition, the viable count of the prepared lactic acid bacteria microcapsule reaches 10 by combining the optimized microcapsule wall material and spray drying process parameters10The particle size is 5-8 mu m, the water content of the product is 4.80%, and tests on acid resistance, cholate resistance and storage resistance show that the product has high acid resistance, cholate resistance and storage resistance, and the preparation process is simple to operate, low in production cost and suitable for application in the feed industry and livestock and poultry breeding production.
Compared with high-temperature spray drying, the high-temperature spray drying method has the advantages that the high water content is easily formed under the low-temperature condition, the viable count of the product in the storage process is reduced, and in addition, the good product flowability, high yield and the like are ensured. The method provides a wall material suitable for preparing the lactobacillus microcapsule under the low temperature condition and suitable spray drying process parameters to form a set of complete preparation process, which has positive significance for the production of the feeding lactobacillus microcapsule preparation and the popularization and application in animal production.
Claims (7)
1. A preparation method of a feeding lactobacillus microcapsule is characterized by comprising the following steps:
(1) activating lactobacillus strains, and fermenting and culturing to obtain fermentation liquor;
(2) centrifuging the fermentation liquor prepared in the step (1), and collecting bacterial sludge;
(3) adding the bacterial sludge obtained in the step (2) into a microcapsule wall material solution;
(4) preparing microcapsules from the solution prepared in the step (3) by spray drying; wherein the temperature of an air inlet of the spray drying is 60-70 ℃, the temperature of an air outlet is 50-60 ℃, the vacuum degree is 0.03-0.04 MPa, and the feeding speed is 40-45 rpm;
(5) collecting the microcapsule product;
the solvent of the microcapsule solution is: 0.85% physiological saline; the solute comprises 13 to 15 weight percent of skim milk, 1 to 3 weight percent of cane sugar, 1 to 3 weight percent of modified starch, 2 to 4 weight percent of talcum powder, 1 to 2 weight percent of soybean protein isolate and 0.1 to 0.3 volume percent of tween-60;
the lactobacillus strain is porcine lactobacillus reuteriLactobacillus reuteriZLR003，CGMCCNo.11530。
2. The method according to claim 1, wherein the solvent of the microcapsule solution is: 0.85% physiological saline; the solute comprises 14% of skim milk, 2% of cane sugar, 3% of modified starch, 3% of talcum powder, 1% of isolated soy protein and 0.2% of tween-60 by volume percentage.
3. The preparation method of claim 1, wherein the temperature of the air inlet is 60-65 ℃, the temperature of the air outlet is 50-55 ℃, and the vacuum degree is 0.04 MPa.
4. The method of claim 3, wherein the inlet air temperature is 65 ℃, the outlet air temperature is 55 ℃, and the feed rate is 45 rpm.
5. The method according to claim 1, wherein the centrifugation in step (2) is carried out at 6000 to 9000rpm for 5 to 15 minutes.
6. A microcapsule preparation of lactic acid bacteria prepared by the process according to any one of claims 1 to 5.
7. Use of a microcapsule of lactic acid bacteria according to claim 6 for the preparation of a feed additive.
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| CN104711247A (en) * | 2015-03-10 | 2015-06-17 | 南京农业大学 | Preparation method of high-activity microencapsulated lactic acid bacteria starter |
| CN105360600A (en) * | 2015-11-30 | 2016-03-02 | 曲靖市千村农牧科技有限公司 | Preparation method of feed lactic acid bacterium microcapsule |
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| CN104711247A (en) * | 2015-03-10 | 2015-06-17 | 南京农业大学 | Preparation method of high-activity microencapsulated lactic acid bacteria starter |
| CN105360600A (en) * | 2015-11-30 | 2016-03-02 | 曲靖市千村农牧科技有限公司 | Preparation method of feed lactic acid bacterium microcapsule |
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