CN115153027A - Supercritical mixed pectin coating process for improving stress resistance of probiotics - Google Patents
Supercritical mixed pectin coating process for improving stress resistance of probiotics Download PDFInfo
- Publication number
- CN115153027A CN115153027A CN202210541287.3A CN202210541287A CN115153027A CN 115153027 A CN115153027 A CN 115153027A CN 202210541287 A CN202210541287 A CN 202210541287A CN 115153027 A CN115153027 A CN 115153027A
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- Prior art keywords
- pectin
- carbon dioxide
- supercritical
- coating process
- probiotics
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- 229910002092 carbon dioxide Inorganic materials 0.000 claims abstract description 48
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- 238000009650 gentamicin protection assay Methods 0.000 description 1
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- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- CDUFCUKTJFSWPL-UHFFFAOYSA-L manganese(II) sulfate tetrahydrate Chemical compound O.O.O.O.[Mn+2].[O-]S([O-])(=O)=O CDUFCUKTJFSWPL-UHFFFAOYSA-L 0.000 description 1
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- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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Abstract
The invention relates to the technical field of microbial preparations, in particular to a supercritical mixed pectin coating process for improving stress resistance of probiotics. The supercritical mixed pectin coating process is characterized in that a probiotic product and a pectin coating agent are added into a supercritical carbon dioxide device for supercritical carbon dioxide dissolving and mixing, so that pectin and the probiotic product are fully mixed, and the pectin-coated probiotic product is finally obtained. The invention adopts the supercritical carbon dioxide dissolving and mixing technology, effectively avoids the negative effects of uneven coating and easy agglomeration of the probiotic products, does not harm the probiotics due to the operation in the low-temperature environment, can ensure the activity of the probiotics and improve the stress resistance of the probiotics.
Description
Technical Field
The invention relates to the technical field of microbial preparations, in particular to a supercritical mixed pectin coating process for improving stress resistance of probiotics.
Background
Probiotics refer to a group of active microorganisms beneficial to a host by changing the composition of flora at a certain part of the host through colonization in a human body. In order to prolong the survival time of the probiotic bacteria and increase their survival in the intestine, the skilled person chooses to coat the probiotic bacteria in order to improve their stability.
The existing probiotic bacteria coating methods mainly comprise a conventional coating method, a semi-dry coating method and a microcapsule coating method.
The conventional coating method comprises the following steps: generally, artificially synthesized chemical additive packages are adopted. Chinese patent application CN109464425A uses enteric material, plasticizer, anticaking agent, however, the natural product coating material is becoming the main stream of the market in pursuit of green healthy diet.
The pectin is extracted from plants, belongs to natural high molecular compounds, and accords with the green and healthy idea. At present, the pectin coating process is still in the preliminary research stage, and the main reason is that pectin belongs to one kind of plant-derived macromolecules and has low solubility in an aqueous solution, but the current technology is still traditional mixed coating, so that the defects of uneven coating and unstable quality still exist in the actual coating process.
The semi-dry coating method comprises the following steps: the technology mechanically mixes and embeds the probiotic bacteria mud and the solid powder coating agent. But the mechanical mixing embedding is easy to cause the phenomena of uneven mixing and caking, so that the full embedding of the probiotics is difficult to realize.
The microcapsule coating method comprises the following steps: the microcapsule coating method still belongs to the solid coating method. For example, patent CN105595359 "a positioning slow release microcapsule probiotics and its preparation method" introduces a preparation method by mixing coating materials using galacto-oligosaccharide, xylo-oligosaccharide, fructo-oligosaccharide, and isomalto-oligosaccharide as coating agents. Because the coating method still belongs to the solid mixed coating category, the phenomena of uneven mixed coating, caking and the like still occur in the coating process.
Disclosure of Invention
Aiming at the technical problems of nonuniform coating and unstable quality of pectin coated probiotics, the invention provides a supercritical mixed pectin coating process for improving stress resistance of probiotics, a supercritical carbon dioxide dissolving and mixing technology is adopted, negative effects of nonuniform coating and easy agglomeration of probiotic products are effectively avoided, and due to operation in a low-temperature environment, the probiotics cannot be damaged, the activity of the probiotics can be ensured, and the stress resistance of the probiotics is improved.
The technical scheme of the invention is as follows:
a supercritical mixed pectin coating process for improving stress resistance of probiotics is characterized in that a probiotic product and a pectin coating agent are added into a supercritical carbon dioxide device for supercritical carbon dioxide dissolving and mixing, so that pectin and the probiotic product are fully mixed, and finally the pectin-coated probiotic product is obtained;
wherein, the supercritical carbon dioxide dissolving and mixing comprises the following steps:
s1, filling carbon dioxide gas into a device, enabling the pressure of a supercritical carbon dioxide device to reach 11 to 15atm, reducing the temperature of the supercritical carbon dioxide device to-50 to-55 ℃, and keeping the reaction condition for 1.5 to 2.5 hours;
s2, sequentially adjusting the temperature and the pressure to enable the temperature to be-55 ℃, the pressure to be 15atm, and controlling the cooling speed to be 1 ℃/20 to 30 minutes;
s3, maintaining the reaction condition of-55 ℃ and 15atm for 2 to 3 hours;
s4, continuously cooling to-60 to-64 ℃, controlling the cooling speed to be 1 ℃/15 to 25 minutes, reducing the pressure to 3 to 7atm after the set temperature is reached, and maintaining the reaction condition for 5 to 7 hours;
and S5, releasing the pressure, and then heating to room temperature.
Further, the mass ratio of the probiotic product to the pectin coating agent is 1: (3 to 5).
Furthermore, the probiotic product is streptococcus thermophilus powder, bifidobacterium lactis powder, lactobacillus casei powder or lactobacillus plantarum powder.
Furthermore, the probiotic product can be a commercially available product or a self-made product, and the preparation method of the probiotic product comprises the following steps:
(1) Activating probiotics, inoculating the activated probiotics into an MRS liquid culture medium, and culturing to obtain a bacterial liquid;
(2) Centrifuging the bacterial liquid, collecting thalli, and suspending in recovered skim milk to obtain a suspension;
(3) Adjusting the concentration of the suspension to 1.0 to 2.0 × 10 10 cfu/mL to obtain a bacterial suspension, and freeze-drying the bacterial suspension to obtain the probiotic product.
Further, the pectin coating agent is citrus pectin, lemon pectin, banana pectin or pumpkin pectin, preferably lemon pectin.
Further, the pectin coating agent is formed by mixing low-ester pectin and high-ester pectin, wherein the mass ratio of the low-ester pectin to the high-ester pectin is (4-6): 1. the low ester pectin and the high ester pectin have different water-soluble viscosity and dispersibility, and the proportion is selected for mixing in order to ensure that the mixture is uniformly mixed to reach the optimal production state.
The invention has the beneficial effects that:
the invention adopts a supercritical carbon dioxide dissolving and mixing technology, completely dissolves pectin in liquid carbon dioxide and fully mixes the pectin with probiotic products, then strictly controls technological parameters to convert the carbon dioxide into a solid state, regulates the parameters again after freezing, converts the carbon dioxide from the solid phase into a gaseous phase, and discharges the gaseous phase out of a reactor, thereby obtaining the evenly mixed pectin-coated probiotic products.
Supercritical II used in the inventionCarbon oxide means CO 2 At its critical temperature (T) c = 304.1K) and above a critical pressure (pc =7.38 MPa), which has a dissolution capacity similar to that of a liquid, and good mass transfer properties, and which, because of its absence of surface tension, is also easily penetrated into the probiotic product. At the same time, CO 2 Non-toxic, non-polluting, non-flammable, inexpensive and readily available, and, relative to other supercritical fluids, CO 2 The supercritical condition is easy to realize, the energy consumption is low, and the equipment requirement is low.
Drawings
In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.
FIG. 1 is a first SEM photograph of a pectin-coated Streptococcus thermophilus product prepared in example 1 of the invention.
FIG. 2 is a second SEM photograph of a pectin-coated Streptococcus thermophilus product prepared in example 1 of the invention.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The probiotic powder (streptococcus thermophilus powder, bifidobacterium lactis powder, lactobacillus casei powder and lactobacillus plantarum powder) used in the specific embodiment of the invention is self-made, and the preparation method is as follows:
(1) Inoculating probiotics to a plate culture medium for activation, then inoculating the probiotics to an MRS liquid culture medium according to the inoculation amount of 1%, and culturing for 24h at 37 ℃ to obtain a bacterial liquid;
wherein, the raw materials of the MRS liquid culture medium comprise 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of dipotassium phosphate heptahydrate, 80 mL of tween, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate and 1000mL of distilled water; the preparation method comprises mixing the above materials, adjusting pH to 6.8, stirring, and sterilizing at 121 deg.C and 0.1MPa for 20 min;
(2) Centrifuging the bacterial liquid, collecting thalli, washing the thalli with sterile normal saline, and then suspending the thalli in 15% (w/w) recovered skim milk to obtain suspension;
(3) Adjusting the concentration of the suspension to 1.0 to 2.0 × 10 10 cfu/mL to obtain a bacterial suspension, and freeze-drying the bacterial suspension to obtain the probiotic powder.
The probiotic powder may be replaced by commercially available products.
Example 1 preparation of pectin-coated Streptococcus thermophilus product
10g of streptococcus thermophilus powder and 50g of lemon pectin powder (42 g of low-ester lemon pectin powder and 8g of high-ester lemon pectin powder are mixed) are placed in a supercritical carbon dioxide device, the carbon dioxide is used for replacing the device for 3 times, and then supercritical carbon dioxide dissolving and mixing are carried out according to the following steps:
s1, filling carbon dioxide gas into a device to enable the pressure of a supercritical carbon dioxide device to reach 15atm, reducing the temperature of the supercritical carbon dioxide device to-50 ℃, paying attention to pressure compensation in the temperature reduction process to maintain the pressure in a container unchanged, and keeping the reaction condition for 2 hours;
s2, after S1, reducing the temperature to-55 ℃ at a cooling rate of 1 ℃/30 minutes, and keeping the pressure in the container unchanged by pressure supplement in the cooling process;
s3, maintaining the reaction condition of-55 ℃ and 15atm for 2 hours;
s4, after 2 hours, continuously cooling to-60 ℃ at a cooling rate of 1 ℃/15 minutes, slowly reducing the pressure to 3atm after the set temperature is reached, and maintaining the reaction condition for 7 hours;
and S5, after the completion of the reaction, releasing the pressure to normal pressure for 30 minutes, then slowly heating to room temperature to complete the coating process to obtain a pectin-coated streptococcus thermophilus product, wherein each gram of the pectin-coated streptococcus thermophilus product contains 150 hundred million cfu streptococcus thermophilus through detection.
The shape of the pectin-coated streptococcus thermophilus product is shown in figures 1 and 2, and as can be seen from figure 1, the product coated by the method of the invention shows a perfect spherical state and has a round and smooth surface. The pectin is uniformly coated on the surface of the thallus, so that the defects that the conventional coating is not uniformly mixed and the coated product is thick and thin are overcome; the surface of the thallus is enlarged (figure 2), the uniform coverage condition of the pectin can be clearly seen, and the method can excellently realize the uniform coating of the pectin on the thallus under the supercritical carbon dioxide technology.
Example 2 preparation of pectin-coated Bifidobacterium lactis product
Taking 10g of bifidobacterium lactis powder and 40g of citrus pectin powder (mixing 33g of low-ester citrus pectin powder and 7g of high-ester citrus pectin powder), placing in a supercritical carbon dioxide device, replacing for 3 times by using a carbon dioxide replacement device, and then carrying out supercritical carbon dioxide dissolving and mixing according to the following steps:
s1, filling carbon dioxide gas into a device to enable the pressure of a supercritical carbon dioxide device to reach 12atm, reducing the temperature of the supercritical carbon dioxide device to-55 ℃, paying attention to pressure compensation in the process of temperature reduction to keep the pressure in a container unchanged, and keeping the reaction condition for 2.5 hours;
after S2 and S1 are finished, supplementing carbon dioxide gas, and increasing the pressure to 15atm;
s3, maintaining the reaction condition of-55 ℃ and 15atm for 2.5 hours;
s4, after 2.5 hours, continuously cooling to-62 ℃ at a cooling rate of 1 ℃/20 minutes, slowly reducing the pressure to 4atm after the set temperature is reached, and maintaining the reaction condition for 6 hours;
and S5, after the pressure is released to normal pressure for 30 minutes, and then the temperature is slowly increased to room temperature, so that the coating process is completed, and the pectin-coated bifidobacterium lactis product is obtained.
EXAMPLE 3 preparation of pectin-coated Lactobacillus casei product
Taking 10g of lactobacillus casei powder and 30g of banana pectin powder (24 g of low-ester banana pectin powder and 6g of high-ester banana pectin powder are mixed), placing the mixture in a supercritical carbon dioxide device, replacing the mixture for 3 times by using a carbon dioxide replacement device, and then carrying out supercritical carbon dioxide dissolving and mixing according to the following steps:
s1, filling carbon dioxide gas into a device to enable the pressure of a supercritical carbon dioxide device to reach 14atm, reducing the temperature of the supercritical carbon dioxide device to-54 ℃, paying attention to pressure compensation in the temperature reduction process to maintain the pressure in a container unchanged, and keeping the reaction condition for 2 hours;
s2, reducing the temperature to-55 ℃ according to the cooling speed of 1 ℃/20 minutes; then supplementing carbon dioxide gas, and increasing the pressure to 15atm;
s3, maintaining the reaction condition of-55 ℃ and 15atm for 2 hours;
s4, after 2 hours, continuously cooling to-64 ℃ according to the cooling speed of 1 ℃/25 minutes, slowly reducing the pressure to 6atm after the set temperature is reached, and maintaining the reaction condition for 5 hours;
and S5, after the pressure is released to normal pressure for 30 minutes, and then the temperature is slowly increased to room temperature, so that the coating process is completed, and the pectin-coated lactobacillus casei product is obtained.
Example 4 preparation of pectin coated Lactobacillus plantarum products
10g of lactobacillus plantarum powder and 35g of pumpkin pectin powder (28 g of low-ester pumpkin pectin powder and 7g of high-ester pumpkin pectin powder are mixed) are placed in a supercritical carbon dioxide device, the carbon dioxide replacement device is used for 3 times, and then the supercritical carbon dioxide is dissolved and mixed according to the following steps:
s1, filling carbon dioxide gas into a device to enable the pressure of a supercritical carbon dioxide device to reach 11atm, reducing the temperature of the supercritical carbon dioxide device to-50 ℃, paying attention to pressure compensation in the temperature reduction process to maintain the pressure in a container unchanged, and keeping the reaction condition for 1.5 hours;
s2, reducing the temperature to-55 ℃ according to the cooling speed of 1 ℃/20 minutes; then supplementing carbon dioxide gas, and increasing the pressure to 15atm;
s3, maintaining the reaction condition of-55 ℃ and 15atm for 3 hours;
s4, after 3 hours, continuously cooling to-64 ℃ according to the cooling speed of 1 ℃/15 minutes, slowly reducing the pressure to 7atm after the set temperature is reached, and maintaining the reaction condition for 5 hours;
and S5, after the pressure is released to normal pressure for 30 minutes, and then the temperature is slowly increased to room temperature, so that the coating process is completed, and the pectin-coated lactobacillus plantarum product is obtained.
Example 5 in vitro survival assays in simulated gastrointestinal environments
The pectin-coated S.thermophilus product prepared in example 1 was tested by simulating gastrointestinal digestive juices.
1. By simulating gastric juice
Several parts of the pectin-coated S.thermophilus product prepared in example 1, each part weighing 1g, were transferred to a preheated test tube containing 9mL of simulated gastric fluid and treated at 37 ℃ and 80r/min for 1h, 2h, 3h and 4h, 3 replicates each.
The simulated gastric juice is prepared according to the following method: diluting with 9.5% hydrochloric acid and distilled water to pH of 1.5, adding 1.0g pepsin per 100mL, mixing, filtering with 0.22 μm sterile filter membrane, and using.
And (4) carrying out gradient dilution on the treated sample solution, counting viable bacteria by using a pouring method, and then calculating the survival rate.
Meanwhile, a streptococcus thermophilus product prepared by a conventional non-pectin coating method is used as a reference sample 1 for testing the gastric juice survival rate, and the specific preparation method is as follows:
dissolving fructo-oligosaccharide, isomaltooligosaccharide and skimmed milk powder with water, adding into the centrifuged probiotic bacteria paste, stirring, mixing, and freezing in a low-temperature vacuum freezer.
Because some coating agents have low solubility in water, the stirring and mixing often generate a lump phenomenon which can be observed by naked eyes in a mixer, so that the final coating effect is not uniform, the efficiency is low during freeze-drying, and even the freeze-drying effect is not ideal.
A streptococcus thermophilus product prepared by a conventional pectin coating method is used as a reference sample 2 for testing the gastric juice survival rate, and the specific preparation method is as follows:
the conventional pectin coating method is different from the conventional non-pectin coating method only in that the coating agent also comprises pectin powder.
The pectin powder is relatively good in free-running property because the water soluble in the pectin powder is colloid, and the product is free from large agglomeration compared with a product obtained by a conventional non-pectin coating method. However, in the production process, the discharge port is often blocked when the mixer discharges materials, and the problem is repeated and difficult to solve.
2. By simulating intestinal juice experiment
Several parts of the pectin-coated S.thermophilus product prepared in example 1, weighing 1g of each sample, were transferred to a tube containing 9mL of simulated intestinal fluid and treated at 37 ℃ and 80r/min for 1h, 2h, 3h and 4h, 3 repeats each treatment.
The simulated intestinal juice is prepared according to the following method: mixing 6.8g KH 2 PO 4 Dissolving in 500mL of distilled water, adding 3g of bile salt and 10g of trypsin, adjusting the pH value of the solution to 6.8 by using a NaOH solution with the concentration of 4g/L, diluting to 1L by using distilled water, mixing uniformly, filtering by using a sterile filter membrane with the diameter of 0.22 mu m, and preparing the solution for use.
And (4) carrying out gradient dilution on the treated sample solution, counting viable bacteria by using a pouring method, and then calculating the survival rate.
Meanwhile, a streptococcus thermophilus product prepared by a conventional non-pectin coating method and a streptococcus thermophilus product prepared by a conventional pectin coating method are respectively used as a control sample 1 and a control sample 2 for intestinal fluid survival rate test, and the preparation method of the control sample 1 and the control sample 2 is the same as that of the control sample 1 and is recorded by a simulated gastric fluid experiment.
3. Results and analysis of the experiments
As shown in Table 1, the survival rate of the pectin-coated Streptococcus thermophilus product prepared by the method is very high in an in-vitro simulated gastrointestinal environment, the survival rate of the product treated in gastric juice and intestinal juice for 4 hours is still over 90%, and the survival rate of the product treated in comparative sample 1 and comparative sample 2 is reduced to below 90% after the product is treated for 4 hours. The pectin-coated probiotic product prepared by the method has better effects of gastric acid resistance and cholate resistance in a human body, can effectively improve the stress resistance of the probiotic, and can lay a foundation for subsequent bacterial colonization on intestinal tracts and function exertion.
TABLE 1 Streptococcus thermophilus survival Rate in vitro simulated gastrointestinal Environment
Although the present invention has been described in detail by referring to the drawings in connection with the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions should be within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present disclosure and the scope of the present invention.
Claims (7)
1. A supercritical mixed pectin coating process for improving stress resistance of probiotics is characterized in that a probiotic product and a pectin coating agent are added into a supercritical carbon dioxide device for supercritical carbon dioxide dissolving and mixing, so that the pectin and the probiotic product are fully mixed, and the pectin coated probiotic product is finally obtained;
wherein, the supercritical carbon dioxide dissolving and mixing comprises the following steps:
s1, filling carbon dioxide gas into a device, enabling the pressure of a supercritical carbon dioxide device to reach 11 to 15atm, reducing the temperature of the supercritical carbon dioxide device to-50 to-55 ℃, and keeping the reaction condition for 1.5 to 2.5 hours;
s2, sequentially adjusting the temperature and the pressure to enable the temperature to be-55 ℃, the pressure to be 15atm, and controlling the cooling speed to be 1 ℃/20 to 30 minutes;
s3, maintaining the reaction condition of-55 ℃ and 15atm for 2 to 3 hours;
s4, continuously cooling to minus 60 to minus 64 ℃, controlling the cooling speed to be 1 ℃/15 to 25 minutes, reducing the pressure to be 3 to 7atm after the set temperature is reached, and maintaining the reaction condition for 5 to 7 hours;
and S5, releasing the pressure, and then heating to room temperature.
2. The supercritical mixed pectin coating process according to claim 1, wherein the mass ratio of the probiotic product to the pectin coating agent is 1: (3 to 5).
3. The supercritical mixed pectin coating process of claim 1, wherein the probiotic product is streptococcus thermophilus powder, bifidobacterium lactis powder, lactobacillus casei powder or lactobacillus plantarum powder.
4. The supercritical mixed pectin coating process of claim 3, wherein the probiotic product is prepared by a process comprising the steps of:
(1) Activating probiotics, inoculating the activated probiotics into an MRS liquid culture medium, and culturing to obtain a bacterial liquid;
(2) Centrifuging the bacterial liquid, collecting thalli, and suspending in recovered skim milk to obtain a suspension;
(3) Adjusting the concentration of the suspension to 1.0 to 2.0 × 10 10 cfu/mL to obtain a bacterial suspension, and freeze-drying the bacterial suspension to obtain the probiotic product.
5. The supercritical mixed pectin coating process according to claim 1, wherein the pectin coating agent is citrus pectin, lemon pectin, banana pectin or pumpkin pectin.
6. The supercritical mixed pectin coating process of claim 1, wherein the pectin coating agent consists of a mixture of low ester pectin and high ester pectin.
7. The supercritical mixed pectin coating process according to claim 6, wherein the mass ratio of the low-ester pectin to the high-ester pectin is (4 to 6): 1.
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