CN112167341A - Preparation method of dairy product containing lactobacillus plantarum JLAU103 exopolysaccharide - Google Patents
Preparation method of dairy product containing lactobacillus plantarum JLAU103 exopolysaccharide Download PDFInfo
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- CN112167341A CN112167341A CN202011034332.3A CN202011034332A CN112167341A CN 112167341 A CN112167341 A CN 112167341A CN 202011034332 A CN202011034332 A CN 202011034332A CN 112167341 A CN112167341 A CN 112167341A
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
- A23C9/1307—Milk products or derivatives; Fruit or vegetable juices; Sugars, sugar alcohols, sweeteners; Oligosaccharides; Organic acids or salts thereof or acidifying agents; Flavours, dyes or pigments; Inert or aerosol gases; Carbonation methods
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
- A23C9/1315—Non-milk proteins or fats; Seeds, pulses, cereals or soja; Fatty acids, phospholipids, mono- or diglycerides or derivatives therefrom; Egg products
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/123—Bulgaricus
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/21—Streptococcus, lactococcus
- A23V2400/249—Thermophilus
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Abstract
The invention discloses a preparation method of a dairy product containing lactobacillus plantarum JLAU103 exopolysaccharide, which comprises the following operation steps: (1) adding trichloroacetic acid aqueous solution into the inactivated lactobacillus plantarum JLAU103 fermentation liquor, and centrifuging to obtain supernatant; (2) adding absolute ethyl alcohol into the supernatant, and centrifuging to obtain a precipitate; (3) dissolving the precipitate with distilled water, dialyzing, concentrating, and freeze drying to obtain crude polysaccharide; (4) dissolving the crude polysaccharide with distilled water, performing ion exchange column chromatography and deionized water dialysis, and freeze drying to obtain Lactobacillus plantarum JLAU103 extracellular polysaccharide; (5) mixing whole milk, white granulated sugar, chlorella powder, egg powder, lactobacillus plantarum JLAU103 exopolysaccharide and sodium citrate, adding water, fermenting, and making into milk product containing lactobacillus plantarum JLAU103 exopolysaccharide. The dairy product prepared by the invention has high nutrition content and immunoregulation function.
Description
Technical Field
The invention belongs to the technical field of functional dairy product preparation, and particularly relates to a preparation method of a dairy product containing lactobacillus plantarum JLAU103 exopolysaccharide.
Background
The intestinal mucosa is the largest contact surface between the animal body and the external environment, plays an important role in digestion and absorption of nutrients, and has a defense barrier effect on invasion of most of external pathogenic microorganisms and toxins. The intestinal mucosa barrier mainly comprises an intestinal epithelial cell barrier, an immune barrier, a biological barrier and a chemical barrier. Recent studies have shown that intestinal barrier dysfunction is closely related to the pathogenesis of various intestinal diseases (diarrhea, inflammatory bowel disease, crohn's disease, ulcerative colitis, food allergy). Therefore, the natural active substance for regulating the barrier effect of the intestinal mucosa is developed, and the practical significance is provided for the treatment and prevention of intestinal diseases.
Lactobacillus plantarum Exopolysaccharides (EPS) is a generic term for mucopolysaccharides or capsular polysaccharides secreted by lactobacillus plantarum outside the cell wall during the growth metabolism. As a green and safe natural active high molecular polymer, the natural active high molecular polymer has proved to have various physiological activities, including oxidation resistance, blood pressure reduction, tumor resistance, intestinal micro-ecological environment improvement, human immunity enhancement and the like. At present, no dairy product is added with lactobacillus plantarum exopolysaccharide.
Disclosure of Invention
The invention aims to provide a preparation method of a dairy product added with lactobacillus plantarum exopolysaccharide.
The invention is realized by the following technical scheme.
A method for preparing a dairy product containing lactobacillus plantarum JLAU103 exopolysaccharide comprises the following operation steps:
(1) adding trichloroacetic acid aqueous solution into the inactivated lactobacillus plantarum JLAU103 fermentation liquor until the trichloroacetic acid final concentration mass fraction is 4-6%, stirring at room temperature for 1.5-2.5 hours, and centrifuging to obtain supernatant;
(2) adding 2-3 times of anhydrous ethanol into the supernatant, refrigerating, standing for 10-12 hr, and centrifuging to obtain precipitate;
(3) dissolving the precipitate with distilled water, putting into a dialysis bag with molecular cut-off of 8000-14000 Da, dialyzing for 45-50h, changing distilled water every 8h, concentrating, and freeze-drying to obtain crude polysaccharide;
(4) dissolving crude polysaccharide with distilled water, performing DEAE-Sepharose Fast Flow ion exchange column chromatography, loading sample amount of 90-110mg, sequentially performing linear gradient elution with distilled water, 0.2mol/L NaCl and 0.5mol/L NaCl at an elution speed of 0.8-1.2mL/min, collecting 4-6mL in each tube, detecting exopolysaccharide content tube by adopting a phenol-sulfuric acid method, respectively combining and collecting single peak components according to the exopolysaccharide content detection value, performing deionized water dialysis, and freeze-drying to obtain Lactobacillus plantarum JLAU103 exopolysaccharide;
(5) according to the weight parts, 70-80 parts of whole milk, 10-14 parts of white granulated sugar, 7-10 parts of chlorella powder, 12-16 parts of egg powder, 0.2-0.6 part of lactobacillus plantarum JLAU103 exopolysaccharides and 0.05-0.09 part of sodium citrate are mixed, water is added into the mixture according to the ratio of the material to the water of 1:20-25g/ml, and after stirring treatment, homogenization, sterilization, cooling, inoculation, constant-temperature fermentation, cooling, filling and after-ripening refrigeration are sequentially carried out, so as to prepare the dairy product containing lactobacillus plantarum JLAU103 exopolysaccharides.
Specifically, in the step (1), the lactobacillus plantarum JLAU103 fermentation broth is prepared by inoculating lactobacillus plantarum JLAU103 into a liquid SDM medium at an inoculum size of 3% by volume, and culturing at 37 ℃ for 24 h.
Specifically, in the step (1), the trichloroacetic acid aqueous solution has an initial mass fraction of 75-85%.
Specifically, in the step (1) and the step (2), the centrifugal force is 10000g during the centrifugal treatment, the temperature is 4 ℃ during the centrifugal treatment, and the centrifugal time is 40-50 min.
Specifically, in the step (2), the temperature during the cold storage and standing treatment is 3 to 5 ℃.
Specifically, in the step (4), the homogenization temperature is 58-62 ℃ and the homogenization pressure is 18-20MPa during the homogenization treatment.
Specifically, in the step (4), the strains used for inoculation are lactobacillus bulgaricus and streptococcus thermophilus, and the inoculation amount is 1-3%.
Specifically, in the step (4), the temperature of the constant-temperature fermentation is 39-41 ℃, and the time of the constant-temperature fermentation treatment is 6-7 hours.
According to the technical scheme, the beneficial effects of the invention are as follows:
1. the dairy product containing the extracellular polysaccharide of the lactobacillus plantarum JLAU103 provided by the invention has the advantages of good glossiness, harmonious milk fragrance, strong fragrance and high nutrient content, and has the function of immunoregulation;
2. according to the method for preparing the extracellular polysaccharide of lactobacillus plantarum JLAU103 provided in the steps (1) to (4), the product yield is high, and the obtained product has high purity;
3. the dairy product prepared by the invention contains active substances fermented by chlorella and egg powder, can act together with lactobacillus plantarum JLAU103 extracellular polysaccharide, and can effectively improve the immunoregulation function of immunosuppressive mice.
Drawings
FIG. 1 is a thymus index (A) and spleen index (B) of immunosuppressed mice with different letters indicating a significant p <0.05 difference between groups.
FIG. 2 is a graph of the ratio of villus length V to crypt depth C in intestinal tissue of immunosuppressed mice, wherein different letters indicate that the difference between groups is significant p < 0.05.
FIG. 3 is a graph of the serum cytokines IL-2(A) and IL-6(B) in immunosuppressed mice with different letters indicating a significant p <0.05 difference between groups.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. 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.
Example 1
A method for preparing a dairy product containing lactobacillus plantarum JLAU103 exopolysaccharide comprises the following operation steps:
(1) adding trichloroacetic acid aqueous solution with the mass fraction of 75% into inactivated lactobacillus plantarum JLAU103 fermentation liquor until the final concentration mass fraction of trichloroacetic acid is 4%, stirring at room temperature for 1.5 hours, centrifuging to obtain supernatant, centrifuging at 4 ℃ for 40min under the condition that the centrifugal force is 10000g, wherein the lactobacillus plantarum JLAU103 fermentation liquor is prepared by inoculating lactobacillus plantarum JLAU103 into a liquid SDM culture medium according to the inoculation amount with the volume fraction of 3%, culturing at 37 ℃ for 24 hours,
(2) adding 2 times of anhydrous ethanol into the supernatant, refrigerating at 3 deg.C, standing for 10 hr, centrifuging at 4 deg.C for 40min to obtain precipitate, wherein the centrifugal force is 10000 g;
(3) dissolving the precipitate with distilled water, dialyzing in dialysis bag with molecular cut-off of 8000Da for 45 hr, changing distilled water every 8 hr, concentrating, and freeze drying to obtain crude polysaccharide;
(4) dissolving crude polysaccharide with distilled water, performing DEAE-Sepharose Fast Flow ion exchange column chromatography, loading sample amount of 90mg, sequentially performing linear gradient elution with distilled water, 0.2mol/L NaCl and 0.5mol/L NaCl, wherein the elution speed is 0.8mL/min, collecting 4mL per tube, detecting exopolysaccharide content tube by adopting a phenol-sulfuric acid method, respectively combining and collecting single peak components according to the exopolysaccharide content detection values, performing deionized water dialysis, and freeze-drying to obtain the exopolysaccharide of lactobacillus plantarum JLAU 103;
(5) according to the weight parts, 70 parts of whole milk, 10 parts of white granulated sugar, 7 parts of chlorella powder, 12 parts of egg powder, 0.2 part of lactobacillus plantarum JLAU103 exopolysaccharide and 0.05 part of sodium citrate are mixed, water is added into the mixture according to the material-water ratio of 1:20g/ml, after stirring treatment, homogenization, sterilization, cooling, inoculation, constant-temperature fermentation, cooling, filling and after-ripening and refrigeration are sequentially carried out, and the dairy product containing the lactobacillus plantarum JLAU103 exopolysaccharide is prepared, wherein during homogenization treatment, the homogenization temperature is 58 ℃, the homogenization pressure is 18MPa, the strains used for inoculation are lactobacillus bulgaricus and streptococcus thermophilus, the inoculation amount is 1%, the constant-temperature fermentation temperature is 39 ℃, and the constant-temperature fermentation treatment time is 6 hours.
Example 2
A method for preparing a dairy product containing lactobacillus plantarum JLAU103 exopolysaccharide comprises the following operation steps:
(1) adding 80% trichloroacetic acid aqueous solution into inactivated lactobacillus plantarum JLAU103 fermentation liquor until the final trichloroacetic acid concentration mass fraction is 5%, stirring at room temperature for 2.0 hours, centrifuging to obtain supernatant, centrifuging at 4 deg.C for 45min under centrifugal force of 10000g, wherein the lactobacillus plantarum JLAU103 fermentation liquor is prepared by inoculating lactobacillus plantarum JLAU103 into liquid SDM culture medium according to 3% inoculation amount, and culturing at 37 deg.C for 24 hours,
(2) adding 2.5 times volume of anhydrous ethanol into the supernatant, refrigerating at 4 deg.C, standing for 11 hr, centrifuging at 4 deg.C for 45min to obtain precipitate, wherein the centrifugal force is 10000 g;
(3) dissolving the precipitate with distilled water, dialyzing in dialysis bag with molecular cut-off of 10000Da for 48 hr, changing distilled water every 8 hr, concentrating, and freeze drying to obtain crude polysaccharide;
(4) dissolving crude polysaccharide with distilled water, performing DEAE-Sepharose Fast Flow ion exchange column chromatography, loading sample amount of 100mg, sequentially performing linear gradient elution with distilled water, 0.2mol/L NaCl and 0.5mol/L NaCl, wherein the elution speed is 1.0mL/min, collecting 5mL in each tube, detecting exopolysaccharide content tube by adopting a phenol-sulfuric acid method, respectively combining and collecting single peak components according to the exopolysaccharide content detection values, performing deionized water dialysis, and freeze-drying to obtain the exopolysaccharide of lactobacillus plantarum JLAU 103;
(5) according to the weight parts, 75 parts of whole milk, 12 parts of white granulated sugar, 8 parts of chlorella powder, 14 parts of egg powder, 0.4 part of lactobacillus plantarum JLAU103 exopolysaccharide and 0.07 part of sodium citrate are mixed, water is added into the mixture according to the material-water ratio of 1:23g/ml, after stirring treatment, homogenization, sterilization, cooling, inoculation, constant-temperature fermentation, cooling, filling and after-ripening and refrigeration are sequentially carried out, and the dairy product containing the lactobacillus plantarum JLAU103 exopolysaccharide is prepared, wherein during homogenization treatment, the homogenization temperature is 60 ℃, the homogenization pressure is 19MPa, the strains used for inoculation are lactobacillus bulgaricus and streptococcus thermophilus, the inoculation amount is 2%, the constant-temperature fermentation temperature is 40 ℃, and the constant-temperature fermentation treatment time is 6.5 hours.
Example 3
A method for preparing a dairy product containing lactobacillus plantarum JLAU103 exopolysaccharide comprises the following operation steps:
(1) adding trichloroacetic acid aqueous solution with the mass fraction of 85% into inactivated lactobacillus plantarum JLAU103 fermentation liquor until the final concentration mass fraction of trichloroacetic acid is 6%, stirring at room temperature for 2.5 hours, centrifuging to obtain supernatant, centrifuging at 4 ℃ for 50min under the condition that the centrifugal force is 10000g, wherein the lactobacillus plantarum JLAU103 fermentation liquor is prepared by inoculating lactobacillus plantarum JLAU103 into a liquid SDM culture medium according to the inoculation amount with the volume fraction of 3%, culturing at 37 ℃ for 24 hours,
(2) adding 3 times of anhydrous ethanol into the supernatant, refrigerating at 5 deg.C, standing for 12 hr, centrifuging at 4 deg.C for 50min to obtain precipitate, wherein the centrifugal force is 10000 g;
(3) dissolving the precipitate with distilled water, dialyzing in dialysis bag with molecular cut-off of 14000Da for 50 hr, changing distilled water every 8 hr, concentrating, and freeze drying to obtain crude polysaccharide;
(4) dissolving crude polysaccharide with distilled water, performing DEAE-Sepharose Fast Flow ion exchange column chromatography, loading a sample amount of 110mg, sequentially performing linear gradient elution with distilled water, 0.2mol/L NaCl and 0.5mol/L NaCl, wherein the elution speed is 1.2mL/min, collecting 6mL in each tube, detecting the content of extracellular polysaccharide tube by adopting a phenol-sulfuric acid method, respectively combining and collecting single peak components according to the detection value of the content of extracellular polysaccharide, performing deionized water dialysis, and performing freeze drying to obtain the extracellular polysaccharide of lactobacillus plantarum JLAU 103;
(5) according to the weight parts, 80 parts of whole milk, 14 parts of white granulated sugar, 10 parts of chlorella powder, 16 parts of egg powder, 0.6 part of lactobacillus plantarum JLAU103 exopolysaccharide and 0.09 part of sodium citrate are mixed, water is added according to the ratio of material to water of 1:25g/ml, after stirring treatment, homogenization, sterilization, cooling, inoculation, constant-temperature fermentation, cooling, filling and after-ripening and refrigeration are sequentially carried out, and the dairy product containing the lactobacillus plantarum JLAU103 exopolysaccharide is prepared, wherein during homogenization treatment, the homogenization temperature is 62 ℃, the homogenization pressure is 20MPa, the strains used for inoculation are lactobacillus bulgaricus and streptococcus thermophilus, the inoculation amount is 3%, the constant-temperature fermentation temperature is 41 ℃, and the constant-temperature fermentation treatment time is 7 hours.
The milk product containing the exopolysaccharide of lactobacillus plantarum JLAU103 prepared in example 1 was tested for immunomodulatory effects on immunosuppressed mice:
after SPF-grade BALB/c mice were fully acclimated, they were labeled with picric acid, and 60 mice were randomly divided into six groups: normal control group (NC), model group (MC), positive control group (PC), dairy low dose group (LD), dairy medium dose group (MD) and dairy high dose group (HD). The experiment adopts a mode of intraperitoneal injection for 3 continuous days to build an immunosuppressive model, and a mode of intragastric administration for 10 continuous days. During the molding period: the normal control group was injected with sterile physiological saline, and the remaining mice were injected with cyclophosphamide at a corresponding volume concentration of 80mg/kg per day. During the period of gavage: the positive control group is perfused with 40mg/kg levamisole hydrochloride water solution every day, the high, medium and low dosage groups are separately perfused with 200, 100, 50mg/(kg multiplied by d) dairy products, and the normal control group and the model group are perfused with normal saline with corresponding volume. After gavage, all test animals were sacrificed without water intake.
Immune organ index determination
24h after the completion of the gavage cycle, the experimental mice were weighed and sacrificed, the intact thymus and spleen were isolated, washed in physiological saline, and the remaining physiological saline was aspirated by filter paper, and the ratio of the weight (mg) of the thymus and spleen to the weight (g) of the mice was measured to represent the index of the thymus and spleen. The formula is as follows:
histological morphology observation
The method comprises the following steps of rapidly performing laparotomy after a mouse is sacrificed, taking out small intestine tissues, fixing the small intestine tissues in 10% paraformaldehyde for 48 hours, embedding the small intestine tissues in normal paraffin, slicing the small intestine tissues, performing HE staining, observing tissue morphology under an optical microscope, accurately measuring the length of intestinal villi and the depth of crypts by using an image data analysis system, taking the average value of the intestinal villi and the depth of crypts, and calculating the ratio of the intestinal villi and.
Determination of cytokines in serum
Blood is obtained by adopting a mode of taking blood from eyeballs, standing for 2h at room temperature, 3000 Xg at 4 ℃, centrifuging for 10min, taking the serum of a book, and measuring the concentration of IL-2 and IL-6 in the serum by utilizing an ELISA kit according to the requirements of kit specifications.
Data analysis
Data between groups were compared using ANOVA using Tukey 'and Dunnett' multiple comparison tests. Statistical significance of significant results was set at P < 0.05. All data are expressed as mean ± standard deviation (n ≧ 3).
Results and analysis
Example 1 Effect of the milk product obtained in the example 1 on the thymus index and spleen index of immunosuppressed mice
As shown in fig. 1A and 1B, both thymus index and spleen index of model group Mice (MC) were significantly decreased (p <0.05) compared to the blank control group (NC), indicating that cyclophosphamide caused immunosuppression in the mice. Compared with a model control group, the low, medium and high dose dairy product treatment groups can remarkably improve the thymus index (p is less than 0.05) of immunosuppressive mice and show higher concentration dependence. Meanwhile, spleen index of immunosuppressed mice can be obviously improved by medium and high dose dairy product treatment groups (p is less than 0.05).
Protective effect of dairy products on small intestine damage of immunosuppressed mice
The integrity and functionality of the small intestine are kept on the premise of playing a normal barrier function, when the small intestine is damaged, the villus surface area of the intestinal cavity is reduced, the intestinal permeability is increased, and the crypt is continuously differentiated to generate new epithelial cell renewal injury. As shown in fig. 2, the ratio of intestinal villus length (V) to crypt depth (C) was significantly decreased in the model group mice compared to the blank control group (p < 0.05). The ratio of the intestinal villus length (V) to crypt depth (C) was significantly increased in the dairy treated mice compared to the model control group (p < 0.05). The lactobacillus plantarum JLAU103 exopolysaccharide is shown to play a certain protection role in the damage of small intestine of immunosuppressive mice, and is helpful for recovering the integrity of small intestine epithelial cells of the immunosuppressive mice.
Effect of Dairy products on immunosuppressive mouse serum cytokines
As shown in FIGS. 3A and 3B, both IL-2 and IL-6 levels in the serum of the model mice were significantly reduced (p <0.05) compared to the blank control group. Compared with a model control group, the low-dose, medium-dose and high-dose dairy product treatment groups can improve the IL-2 level in the serum of the immunosuppressed mice to different degrees, wherein the improvement effect of the high-dose dairy product treatment group is obvious (p is less than 0.05); meanwhile, the low, medium and high dose dairy product treatment groups can obviously improve the level of IL-6 in the serum of an immunosuppressed mouse.
In the experiment, cyclophosphamide is used for constructing an immunosuppressive mouse model, and the research shows that the dairy product prepared in the example 1 has an immunoregulation effect on the immunosuppressive mouse. The result shows that the dairy product prepared in the example 1 can improve thymus index and spleen index of cyclophosphamide induced immunosuppression mice, improve the ratio of the length of villi in jejunum of the mice to the depth of crypts, and increase the secretion of cytokines IL-6 and IL-2 in serum of the mice. In conclusion, the dairy product prepared in example 1 has the effect of improving immunosuppression.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. 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.
Claims (8)
1. A preparation method of a dairy product containing lactobacillus plantarum JLAU103 exopolysaccharide is characterized by comprising the following operation steps:
(1) adding trichloroacetic acid aqueous solution into the inactivated lactobacillus plantarum JLAU103 fermentation liquor until the trichloroacetic acid final concentration mass fraction is 4-6%, stirring at room temperature for 1.5-2.5 hours, and centrifuging to obtain supernatant;
(2) adding 2-3 times of anhydrous ethanol into the supernatant, refrigerating, standing for 10-12 hr, and centrifuging to obtain precipitate;
(3) dissolving the precipitate with distilled water, putting into a dialysis bag with molecular cut-off of 8000-14000 Da, dialyzing for 45-50h, changing distilled water every 8h, concentrating, and freeze-drying to obtain crude polysaccharide;
(4) dissolving crude polysaccharide with distilled water, performing DEAE-Sepharose Fast Flow ion exchange column chromatography, loading sample amount of 90-110mg, sequentially performing linear gradient elution with distilled water, 0.2mol/L NaCl and 0.5mol/L NaCl at an elution speed of 0.8-1.2mL/min, collecting 4-6mL in each tube, detecting exopolysaccharide content tube by adopting a phenol-sulfuric acid method, respectively combining and collecting single peak components according to the exopolysaccharide content detection value, performing deionized water dialysis, and freeze-drying to obtain Lactobacillus plantarum JLAU103 exopolysaccharide;
(5) according to the weight parts, 70-80 parts of whole milk, 10-14 parts of white granulated sugar, 7-10 parts of chlorella powder, 12-16 parts of egg powder, 0.2-0.6 part of lactobacillus plantarum JLAU103 exopolysaccharides and 0.05-0.09 part of sodium citrate are mixed, water is added into the mixture according to the ratio of the material to the water of 1:20-25g/ml, and after stirring treatment, homogenization, sterilization, cooling, inoculation, constant-temperature fermentation, cooling, filling and after-ripening refrigeration are sequentially carried out, so as to prepare the dairy product containing lactobacillus plantarum JLAU103 exopolysaccharides.
2. The method for preparing a dairy product containing extracellular polysaccharide of Lactobacillus plantarum JLAU103 according to claim 1, wherein in step (1), the fermentation broth of Lactobacillus plantarum JLAU103 is prepared by inoculating Lactobacillus plantarum JLAU103 into liquid SDM medium at a volume fraction of 3%, and culturing at 37 ℃ for 24 h.
3. The method for preparing a dairy product containing extracellular polysaccharide of lactobacillus plantarum JLAU103 according to claim 1, wherein the initial mass fraction of the trichloroacetic acid aqueous solution in step (1) is 75-85%.
4. The method for preparing a dairy product containing extracellular polysaccharide of lactobacillus plantarum JLAU103 according to claim 1, wherein the centrifugation in step (1) and step (2) is carried out at 10000g for 4 ℃ for 40-50 min.
5. The method for preparing a dairy product containing extracellular polysaccharide of lactobacillus plantarum JLAU103 according to claim 1, wherein the temperature for cold storage and standing treatment in step (2) is 3-5 ℃.
6. The method of claim 1, wherein in the step (4), the homogenization temperature is 58-62 ℃, and the homogenization pressure is 18-20 MPa.
7. The method for preparing a dairy product containing exopolysaccharides of lactobacillus plantarum JLAU103 according to claim 1, wherein the species used for inoculation in step (4) are lactobacillus bulgaricus and streptococcus thermophilus in an amount of 1-3%.
8. The method for preparing a dairy product containing extracellular polysaccharide of lactobacillus plantarum JLAU103 according to claim 7, wherein the fermentation temperature in the constant temperature fermentation in the step (4) is 39-41 ℃ and the fermentation time in the constant temperature fermentation is 6-7 hours.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112126664A (en) * | 2020-09-27 | 2020-12-25 | 吉林农业大学 | Method for producing extracellular polysaccharide by fermenting lactobacillus plantarum JLAU103 |
| CN114561436A (en) * | 2022-02-28 | 2022-05-31 | 西北农林科技大学 | Preparation method and application of lactobacillus plantarum exopolysaccharide |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250984A (en) * | 2011-07-11 | 2011-11-23 | 宁波大学 | Method for preparing functional extracellular polysaccharide of lactic acid bacteria |
| CN105028639A (en) * | 2015-07-13 | 2015-11-11 | 杨淑珍 | Immunity enhancing yoghourt and preparation method thereof |
| CN105441357A (en) * | 2015-12-16 | 2016-03-30 | 北京工商大学 | Lactobacillus plantarum for producing antitumor activity exopolysaccharides |
| CN109295126A (en) * | 2018-08-31 | 2019-02-01 | 四川农业大学 | A kind of lactobacillus plantarum exocellular polysaccharide and preparation method with immunoregulatory activity |
-
2020
- 2020-09-27 CN CN202011034332.3A patent/CN112167341A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250984A (en) * | 2011-07-11 | 2011-11-23 | 宁波大学 | Method for preparing functional extracellular polysaccharide of lactic acid bacteria |
| CN105028639A (en) * | 2015-07-13 | 2015-11-11 | 杨淑珍 | Immunity enhancing yoghourt and preparation method thereof |
| CN105441357A (en) * | 2015-12-16 | 2016-03-30 | 北京工商大学 | Lactobacillus plantarum for producing antitumor activity exopolysaccharides |
| CN109295126A (en) * | 2018-08-31 | 2019-02-01 | 四川农业大学 | A kind of lactobacillus plantarum exocellular polysaccharide and preparation method with immunoregulatory activity |
Non-Patent Citations (4)
| Title |
|---|
| JI WANG等: "In vitro immunomodulatory effects of acidic exopolysaccharide produced by Lactobacillus planetarium JLAU103 on RAW264.7 macrophages", 《INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES》 * |
| WEI-HONG MIN等: "Characterization and antioxidant activity of an acidic exopolysaccharide from Lactobacillus plantarum JLAU103", 《JOURNAL OF BIOSCIENCE AND BIOENGINEERING》 * |
| 宋茂清: "《益生剂及其功效》", 30 November 2018, 科学技术文献出版社 * |
| 房晓彬: "植物乳杆菌JLAU103胞外多糖的理化特性及生物活性研究", 《中国优秀博硕士学位论文全文数据库（硕士） 基础科学辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112126664A (en) * | 2020-09-27 | 2020-12-25 | 吉林农业大学 | Method for producing extracellular polysaccharide by fermenting lactobacillus plantarum JLAU103 |
| CN112126664B (en) * | 2020-09-27 | 2022-03-11 | 吉林农业大学 | Method for producing extracellular polysaccharide by fermenting lactobacillus plantarum JLAU103 |
| CN114561436A (en) * | 2022-02-28 | 2022-05-31 | 西北农林科技大学 | Preparation method and application of lactobacillus plantarum exopolysaccharide |
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