CN116570029A - Walnut oligopeptide probiotics microcapsule and preparation method thereof - Google Patents
Walnut oligopeptide probiotics microcapsule and preparation method thereof Download PDFInfo
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- CN116570029A CN116570029A CN202310235895.6A CN202310235895A CN116570029A CN 116570029 A CN116570029 A CN 116570029A CN 202310235895 A CN202310235895 A CN 202310235895A CN 116570029 A CN116570029 A CN 116570029A
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
- A23P10/00—Shaping or working of foodstuffs characterised by the products
- A23P10/30—Encapsulation of particles, e.g. foodstuff additives
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
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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
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Life Sciences & Earth Sciences (AREA)
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- Polymers & Plastics (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Nutrition Science (AREA)
- Mycology (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention discloses a walnut oligopeptide probiotic microcapsule and a preparation method thereof, and belongs to the technical field of probiotic products. The invention prepares walnut oligopeptide microcapsules by a instilling method and an endogenous emulsifying method respectively, wherein the instilling method is to take probiotics and walnut oligopeptide as core materials, take mixed solution of sodium alginate and whey protein isolate as wall materials, and drop the mixture of the core materials and the wall materials into calcium chloride solution to form the microcapsules; the endogenous emulsification method is to add the mixed solution of probiotics, walnut oligopeptide and sodium alginate into the soybean salad oil containing tween, and then add the calcium chloride solution after emulsification to form the microcapsule. The microcapsule is prepared from natural polymer materials, and the walnut oligopeptide and the lactobacillus plantarum are wrapped in the microcapsule, so that the intestinal tolerance and the storage stability of probiotics can be effectively improved, the survival rate of the probiotics in intestinal juice is improved, and theoretical basis and technical support are provided for promoting the proliferation of the probiotics and the development of probiotic products.
Description
Technical Field
The invention belongs to the technical field of probiotic products, and particularly relates to a walnut oligopeptide probiotic microcapsule and a preparation method thereof.
Background
Probiotics are a class of active microorganisms beneficial to a host by colonizing the human body and altering the flora composition of a part of the host. Has the physiological functions of immunoregulation, antibiosis, antivirus, antioxidation and the like, and particularly plays an important role in regulating the balance of intestinal flora, promoting nutrition absorption and keeping intestinal health. With the rapid development of medical science, broad-spectrum and powerful antibiotics are widely applied to treat various diseases, so that the normal flora balance in human intestinal tracts is destroyed to different degrees, and therefore, the aim of increasing the number of beneficial bacteria in the intestinal tracts is necessary. However, probiotics are easily inactivated by temperature, oxygen, gastric acid with low pH, bile, digestive enzymes, etc. during processing, marketing and passing through the digestive tract, and thus it is difficult to exert their physiological activities, so that it is necessary to select suitable prebiotics to promote their growth and reproduction.
The walnut is a long-term food and a medicine, is rich in various active ingredients such as unsaturated fatty acid, protein, vitamin, folic acid, polyphenol and the like, and has extremely high nutritive value. The walnut oligopeptide is a bioactive peptide and is obtained from walnut through a biological enzymolysis technology. It was found that walnut oligopeptides showed a wide variety of physiological activities. Such as anti-fatigue, improving learning and memory, regulating metabolic disorder diseases, anti-tumor, antibacterial, anti-hangover, etc. At present, no report about walnut oligopeptide as a prebiotic is found.
The microcapsule technology is used as a most effective and promising strategy for protecting probiotics and is widely applied in the fields of food, medicine and the like. The microcapsule is prepared by using natural or synthetic polymer material as wall material and physically or chemically packing active matter (core material) to form semipermeable or sealing capsule. The microcapsule can embed probiotics in the enteric-coated wall material, effectively strengthen the resistance of the thalli to the reverse environment of the digestive tract, enable the probiotics to be planted on the intestinal mucosa in a higher viable count, realize the accurate slow release in the intestinal tract and play a role. In addition, the probiotics are prepared into microcapsule products with good fluidity and dispersibility, and the probiotics are easy to be uniformly mixed with substances, so that the probiotics are convenient to transport, store and add for use; the microcapsule can also enable water-insoluble substances to be uniformly dispersed in a water-soluble medium, so that the stability of probiotics in the production, storage and consumption processes is greatly improved.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a walnut oligopeptide probiotics microcapsule and a preparation method thereof.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a microcapsule of walnut oligopeptide and probiotics is prepared by taking probiotics and walnut oligopeptide as core materials and adopting a instillation method or an endogenous emulsification method.
Based on the scheme, the instilling method is to take probiotics and walnut oligopeptide as core materials, mix the core materials with wall materials and then drop the mixture into calcium chloride solution to form microcapsules; the wall material is a mixture of sodium alginate and whey protein isolate or a mixture of sodium alginate and resistant starch;
the endogenous emulsification method comprises the steps of uniformly mixing probiotic bacteria liquid, walnut oligopeptide solution and sodium alginate solution, adding soybean salad oil containing tween for emulsification, then adding soybean salad oil containing glacial acetic acid, finally adding calcium chloride solution, uniformly stirring, standing, pouring soybean oil, and centrifugally flushing to obtain the walnut oligopeptide probiotic microcapsule.
In a specific embodiment, the probiotic is lactobacillus plantarum.
The number of viable bacteria in the walnut oligopeptide probiotic microcapsule is 10 9 ～10 10 CFU/g。
The preparation method of the walnut oligopeptide probiotic microcapsule is characterized in that probiotics and walnut oligopeptide are used as core materials, and the walnut oligopeptide microcapsule is prepared by adopting a instillation method or an endogenous emulsification method;
the instilling method is to take probiotics and walnut oligopeptide as core materials, mix the core materials with wall materials and then drop the mixture into calcium chloride solution to form microcapsules; the wall material is a mixture of sodium alginate and whey protein isolate or a mixture of sodium alginate and resistant starch;
the endogenous emulsification method comprises the steps of uniformly mixing probiotic bacteria liquid, walnut oligopeptide solution and sodium alginate solution, adding soybean salad oil containing tween for emulsification, then adding soybean salad oil containing glacial acetic acid, finally adding calcium chloride solution, uniformly stirring, standing, pouring soybean oil, and centrifugally flushing to obtain the walnut oligopeptide probiotic microcapsule.
Based on the scheme, the instillation method comprises the following steps:
(1) Uniformly mixing the probiotic bacteria liquid and the walnut oligopeptide solution to obtain a core material solution;
(2) Uniformly mixing the sodium alginate solution with the whey protein isolate solution or the resistant starch solution to obtain a wall material solution;
(3) Uniformly mixing the core material solution and the wall material solution in proportion, dropwise adding a calcium chloride solution to form a microcapsule, fixing for 20min at room temperature, washing with distilled water, and removing excessive calcium ions and unencapsulated probiotics to obtain the walnut oligopeptide probiotics microcapsule.
In a specific embodiment, the concentration of the walnut oligopeptide solution is 2% -5%; the concentration of the sodium alginate solution is 1% -1.5%, the concentration of the whey protein isolate solution is 1% -1.5%, and the concentration of the calcium chloride solution is 1% -2%.
In a specific embodiment, the mixing ratio of the probiotic bacteria liquid and the walnut oligopeptide solution is 1:5-1:10, the mixing ratio of the sodium alginate solution and the whey protein isolate solution is 1:1-1:3, the mixing ratio of the sodium alginate solution and the resistant starch solution is 1:1-1:5, and the mixing ratio of the core material solution and the wall material solution is 1:5-1:10.
Based on the scheme, the endogenous emulsification method comprises the following steps:
(1) Uniformly mixing the probiotic bacteria liquid with the walnut oligopeptide solution and the sodium alginate solution to obtain a mixed solution A;
(2) Adding soybean salad oil containing tween into the mixed solution A, stirring and mixing uniformly, emulsifying for 20min to obtain a mixed solution B, adding soybean salad oil containing glacial acetic acid, and stirring and mixing uniformly to obtain a mixed solution C;
(3) Adding the mixed solution C into a calcium chloride solution, stirring uniformly, standing, pouring soybean oil, centrifuging at 1000r/min for 5min, flushing with normal saline for three times, and removing excessive calcium ions and unencapsulated probiotics to obtain the walnut oligopeptide probiotics microcapsule.
In a specific embodiment, the tween content in the tween-containing soybean salad oil is 1% -4% (V/V); the content of glacial acetic acid in the soybean salad oil containing glacial acetic acid is 0.1% -0.5% (V/V); the mass fraction of the calcium chloride solution is 1% -2%.
In a specific embodiment, the volume ratio of the mixed solution A to the soybean salad oil containing tween in the step (2) is 1:1-1:4; the adding proportion of the soybean salad oil containing glacial acetic acid is 50-80% of the volume of the mixed solution B.
In a specific embodiment, the bacterial content of the probiotic bacterial liquid is more than or equal to 10 10 CFU/mL。
In a specific embodiment, the preparation method of the lactobacillus plantarum bacterial liquid comprises the following steps:
inoculating Lactobacillus plantarum into MRS broth, anaerobic culturing at 37deg.C for 24 hr, activating for 2 passages continuously according to 1% inoculum size, collecting logarithmic phase bacterial liquid, centrifuging at 4000r/min for 10min, removing supernatant, collecting thallus, washing thallus with sterile physiological saline for 2-3 times, and resuspending at 10 concentration 10 CFU/mL to obtain lactobacillus plantarum bacterial liquid.
In a specific embodiment, for convenience of transportation and storage, the method further comprises the step of placing the prepared microcapsule in a vacuum freeze dryer for drying for 48 hours to prepare the freeze-dried walnut oligopeptide probiotic microcapsule.
In a specific embodiment, the walnut oligopeptide solution, the sodium alginate solution, the whey protein isolate solution and the resistant starch solution are subjected to filtration sterilization by a 0.22 μm filter membrane before use; the calcium chloride solution is sterilized at 121 ℃ for 15min before use.
In order to better protect the activity of the probiotics and prevent the oxygen from invading the probiotics, the mixed liquid of the wall material and the core material is continuously filled with sterile nitrogen in the preparation process so as to block the oxygen.
Advantages of the technical proposal of the invention
According to the invention, the walnut oligopeptide is used as a prebiotic to be embedded with probiotics in a double layer, and is prepared into the microcapsule by using different processes together with materials such as sodium alginate, whey protein, resistant starch and the like, the preparation process is simple, the cost is low, the raw materials are easy to obtain, the prepared microcapsule has good morphology, and the added walnut oligopeptide can not only ensure the activity of the probiotics, but also is rich in nutrition, and has the effects of antioxidation and the like. Sodium alginate, whey protein and resistant starch are respectively used as wall materials to wrap probiotics and prebiotics, and the sodium alginate and the whey protein have better protection effect on the probiotics, probably because the sodium alginate and the beta-lactoglobulin in the whey protein have cross-linking and condensation reaction to form a composite microcapsule with stronger acid resistance, and the walnut oligopeptide and the lactobacillus plantarum are embedded in the composite microcapsule, so that the killing effect of the lactobacillus plantarum on gastric acid resistance is improved, the probiotics can successfully reach the intestinal tract, and the probiotics with higher activity can be released in the intestinal tract to play a role. The protection effect of the microcapsule prepared by the instillation method on probiotics is better than that of the microcapsule prepared by the endogenous emulsification method. The invention provides a reference for exploring the walnut oligopeptide as a potential prebiotic and the application of the probiotic microcapsule in the food industry.
Drawings
FIG. 1 shows a graph of the utilization of walnut oligopeptide by Lactobacillus plantarum;
FIG. 2 is a graph showing the effect of walnut oligopeptide on a biological membrane of Lactobacillus plantarum (wherein, a: lactobacillus plantarum without walnut oligopeptide added; b: lactobacillus plantarum with walnut oligopeptide added);
FIG. 3 is a photograph of a wet walnut oligopeptide probiotic microcapsule prepared by a instillation method (wherein a: a walnut oligopeptide probiotic microcapsule with sodium alginate/whey protein isolate as a wall material; b: a walnut oligopeptide probiotic microcapsule with sodium alginate/resistant starch as a wall material);
FIG. 4 electron microscope observation of the walnut oligopeptide probiotics microcapsule prepared by an endogenous emulsification method;
FIG. 5 is an infrared spectrum of a walnut oligopeptide probiotic microcapsule prepared by a instillation method of sodium alginate/whey protein isolate (wherein a: the probiotic microcapsule without walnut oligopeptide added; b: the probiotic microcapsule with walnut oligopeptide added);
FIG. 6X-ray image of a sodium alginate/whey protein isolate walnut oligopeptide probiotic microcapsule prepared by instillation (wherein a: probiotic microcapsule without walnut oligopeptide added; b: probiotic microcapsule with walnut oligopeptide added);
figure 7 is a graph showing the protective effect of walnut oligopeptide probiotic microcapsules on probiotics.
Detailed Description
The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art unless otherwise indicated.
The invention will be described in further detail below in connection with specific embodiments and with reference to the data. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
In the following examples, the experimental methods used, unless otherwise specified, were all conventional; the materials, reagents and the like used, unless otherwise specified, are all commercially available.
The walnut oligopeptide used in the following examples is light yellow solid powder, and the main component is small molecular oligopeptide with molecular weight less than 1000, wherein the content of glutamic acid, aspartic acid, arginine and leucine is more, and the small molecular oligopeptide is provided by Beijing-Tian peptide biotechnology limited company. Lactobacillus plantarum is preserved in a refrigerator of-80 ℃ in Qingdao university laboratory for special food.
Use of lactobacillus plantarum for walnut oligopeptide
8.6mg of fluorescein isothiocyanate is weighed and dissolved in 0.2mL of 0.1M potassium hydroxide solution to be evenly vibrated, and 1.6mL of 0.1M carbonate buffer solution (Na) with pH of 8.3 is added 2 CO 3 /NaHCO 3 ) 1mL of peptide solution containing 0.5% of walnut oligopeptide is dripped, and fluorescein isothiocyanate is marked on the walnut oligopeptide after light-shielding reaction is carried out for 4 hours. The labeled peptide solution was added to MRS medium, followed by inoculation with 1% Lactobacillus plantarum, and after incubation at 37℃for 24 hours, 2.5. Mu.L of the bacterial solution was aspirated onto a clean slide glass and observed with a laser confocal microscope (Confocal Laser Scanning Microscope, CLSM) (TCS-SP 8, leica company, germany). Maximum emission wavelength: 520-530 nm, maximum absorption wavelength: and (5) measuring the utilization condition of the lactobacillus plantarum on the walnut oligopeptide at 490-495 nm.
The walnut oligopeptide dyed by the fluorescein isothiocyanate is added into a liquid culture medium of the lactobacillus plantarum, and an isothiocyanate group in the fluorescein isothiocyanate can be covalently bonded with amino acid in the peptide, so that the utilization condition of the lactobacillus plantarum on the walnut oligopeptide can be observed under a laser confocal microscope. As can be seen from FIG. 1, lactobacillus plantarum can well utilize walnut oligopeptides, and fluorescent-labeled walnut oligopeptides are found in most bacteria. The fluorescent marked walnut oligopeptide is mostly distributed in cytoplasm of bacteria, which indicates that the walnut oligopeptide can freely penetrate cell membrane of bacteria to enter the bacteria for various metabolic activities of the bacteria and provide required energy.
Influence of walnut oligopeptide on lactobacillus plantarum biofilm
Lactobacillus plantarum strains were inoculated in a 1% (V/V) ratio in MRS broth for shaking culture for 24h (37 ℃,160 rpm). 100 mu L of bacterial liquid is added into a 24-hole plate after dilution according to the ratio of 1:100 (V/V), and 2.0% (m/V) of walnut oligopeptide and MRS broth (control) are respectively added. Placing a sterile cover glass into a 24-well plate, standing at 37 ℃ for culturing for 48 hours, rinsing the cover glass with ultrapure water for 3 times, dyeing with 0.4% crystal violet for 20 minutes, and observing the influence condition of walnut oligopeptide on lactobacillus plantarum biofilm under a microscope.
The biological envelope is an aggregate membrane sample formed by the way that thalli are suitable for the environment, adhere to the surface of an object or human tissue and wrap themselves in the extracellular matrix such as polysaccharide, protein, nucleic acid and the like by secreting a large amount of extracellular matrix. It has been demonstrated that probiotics in the biofilm state have a more pronounced resistance to the external adverse environment than probiotics in the planktonic state. As shown in fig. 2, after the lactobacillus plantarum biofilm is dyed by crystal violet under an optical microscope, the bacterial distribution without walnut oligopeptide is dispersed, and after the walnut oligopeptide is added, cells are aggregated to form a large-piece compact biofilm, and bacteria are wrapped in the biofilm to form a complex three-dimensional structure. The walnut oligopeptide has the effects of promoting the generation of lactobacillus plantarum biofilm and enhancing the tolerance of the lactobacillus plantarum biofilm.
Example 1
The walnut oligopeptide probiotics microcapsule is prepared by the following instillation method:
(1) And uniformly mixing lactobacillus plantarum bacterial liquid and a walnut oligopeptide solution with the mass fraction of 5% according to the volume ratio of 1:10 to obtain a core material solution.
(2) Uniformly mixing 1.5% (m/V) sodium alginate solution and 1.5% (m/V) whey protein isolate solution according to a ratio (V/V) of 1:1 to obtain sodium alginate and whey protein isolate wall material solution; uniformly mixing 1.5% (m/V) sodium alginate solution and 2% (m/V) resistant starch solution according to a ratio of 1:1 (V/V), and obtaining sodium alginate and resistant starch wall material solution;
(3) And (3) uniformly mixing the core material solution with the sodium alginate and whey separated protein wall material solution, the sodium alginate and the resistant starch wall material solution according to the volume ratio of 1:10, dropwise adding the mixture into the calcium chloride solution with the mass concentration of 2% by using a 10mL syringe at a height of 15cm from the calcium chloride liquid level to form microcapsules, fixing the microcapsules at room temperature for 20min, flushing the microcapsules with distilled water for 3 times, and removing excessive calcium ions and unencapsulated probiotics to obtain the wet microcapsules.
(4) And (3) after pre-freezing for 24 hours at the temperature of minus 20 ℃ in a vacuum freeze dryer, placing the wet microcapsule prepared in the step (3) in the vacuum freeze dryer for drying for 48 hours to obtain the freeze-dried walnut oligopeptide probiotic microcapsule with two different wall materials.
The lactobacillus plantarum bacterial liquid is prepared by the following method:
inoculating Lactobacillus plantarum into MRS broth, anaerobic culturing at 37deg.C for 24 hr, activating for 2 passages continuously according to 1% inoculum size, collecting logarithmic phase bacterial liquid, centrifuging at 4000r/min for 10min, removing supernatant, collecting thallus, washing thallus with sterile physiological saline for 2-3 times, and resuspending at 10 concentration 10 CFU/mL, the lactobacillus plantarum bacterial liquid is prepared.
The walnut oligopeptide solution with the mass fraction of 5% is prepared and filtered and sterilized by a 0.22 mu m filter membrane; preparing 1.5% sodium alginate solution and 1.5% whey protein isolate solution, magnetically stirring to completely dissolve, preparing 1.5% sodium alginate solution and 2% resistant starch solution, magnetically stirring to completely dissolve, and filtering and sterilizing with 0.22 μm filter membrane; the calcium chloride solution with the mass concentration of 2% is sterilized for 15min at 121 ℃.
The walnut oligopeptide probiotic micro-capsules prepared by the method are shown in figure 3, and are light brown round pellets, smooth in surface and uniform in size, and are prepared by taking sodium alginate/whey protein isolate (a in figure 3) and sodium alginate/resistant starch (b in figure 3) as wall materials respectively.
Example 2
A walnut oligopeptide probiotics microcapsule is prepared by the following endogenous emulsification method:
(1) Uniformly mixing lactobacillus plantarum bacterial liquid, a 5% walnut oligopeptide solution and a 1.5% sodium alginate solution according to the volume ratio of 1:2:10 to obtain a mixed solution A;
(2) Adding soybean salad oil containing 4% (V/V) Tween into the mixed solution A according to the volume ratio of 1:4, uniformly mixing by using a magnetic stirrer, emulsifying for 20min to obtain a mixed solution B, adding soybean salad oil containing 0.5% (V/V) glacial acetic acid, wherein the adding proportion of the soybean salad oil containing glacial acetic acid is 80% of the volume of the mixed solution B, and stirring for 30min to obtain a mixed solution C;
(3) Adding the mixed solution C into 2% calcium chloride solution, wherein the mixing ratio of the mixed solution C and the calcium chloride solution is 1:4 (V/V), standing for 30min after stirring uniformly, centrifuging for 5min at 1000r/min after soybean oil is removed, flushing with normal saline for three times, and removing excessive calcium ions and unencapsulated probiotics to obtain the walnut oligopeptide probiotics microcapsule.
(4) And (3) after pre-freezing for 24 hours at the temperature of minus 20 ℃ in a vacuum freeze dryer, placing the microcapsule prepared in the step (3) into the vacuum freeze dryer for drying for 48 hours, and obtaining the freeze-dried walnut oligopeptide probiotics microcapsule.
The walnut oligopeptide solution with the mass fraction of 5% is prepared and filtered and sterilized by a 0.22 mu m filter membrane; preparing 1.5% (m/V) sodium alginate solution, magnetically stirring until the sodium alginate solution is completely dissolved, and filtering and sterilizing with a 0.22 mu m filter membrane; the calcium chloride solution with the mass concentration of 2% is sterilized for 15min at 121 ℃.
The lactobacillus plantarum bacterial liquid is prepared by the following method:
inoculating Lactobacillus plantarum into MRS broth, anaerobic culturing at 37deg.C for 24 hr, activating for 2 passages continuously according to 1% inoculum size, collecting logarithmic phase bacterial liquid, centrifuging at 4000r/min for 10min, removing supernatant, collecting thallus, washing thallus with sterile physiological saline for 2-3 times, and resuspending at 10 concentration 10 CFU/mL, the lactobacillus plantarum bacterial liquid is prepared.
An electron microscope observation diagram of the walnut oligopeptide probiotic microcapsule prepared by the method is shown in fig. 4, and it can be seen from the diagram that the walnut oligopeptide probiotic microcapsule prepared by an endogenous emulsification method is round pellets, has small particle size and smooth outer wall.
Example 3
Characterization of walnut oligopeptide probiotic microcapsules
Fully grinding the walnut oligopeptide probiotics microcapsule prepared in the embodiment 1 and KBr, tabletting by a tabletting machine, fixing on a sample frame after the tabletting, and fixing 400-4000cm -1 The wavelength was measured by fourier infrared spectroscopy. X-ray diffraction test was performed on walnut oligopeptide probiotic micro-freeze-dried microcapsules using an X-ray powder diffractometer, cu target, K alpha line (40 kV,40 mA). Wavelength λ=0.15418 nm. The detection scanning speed is 6 degrees/min, and the 2 theta scanning range is 5-90 degrees.
From the infrared spectrum of fig. 5 (fig. 5 a shows the sodium alginate/whey protein isolate microcapsule without walnut oligopeptide added; b shows the sodium alginate/whey protein isolate microcapsule with walnut oligopeptide added), the walnut oligopeptide probiotic microcapsule contains characteristic peaks of walnut oligopeptide, sodium alginate and whey protein. The sodium alginate and the whey protein are used as wall materials to wrap the core material solution well, and the structure and chemical properties of the wrapped core layer solution are not affected. Fig. 6 shows an X-ray diffractometer analysis of sodium alginate/whey protein isolate walnut oligopeptide probiotic microcapsules (a in fig. 6 is sodium alginate/whey protein isolate without walnut oligopeptide microcapsules; b is sodium alginate/whey protein isolate with walnut oligopeptide microcapsules), and the X-ray diffractogram is found to comprise characteristic diffraction peaks of core materials and wall materials, and after walnut oligopeptide is added, the diffraction peaks show that the crystallinity of the microcapsules is changed.
Example 4
Protection effect of walnut oligopeptide probiotics microcapsule on probiotics
The artificial gastric juice and the artificial intestinal juice are prepared according to the pharmacopoeia of the people's republic of China (2015 edition IV). Taking 0.5g of the walnut oligopeptide probiotics microcapsule prepared by the method of the examples 1 and 2, placing the walnut oligopeptide probiotics microcapsule in simulated gastric fluid, oscillating for 2 hours at 100r/min under anaerobic conditions of 37 ℃, sampling at 0, 30, 60, 90 and 120min, coating a flat plate method to measure the number of viable bacteria for gastric acid resistance experiments, calculating the survival rate of lactobacillus plantarum, and evaluating the protection effect of the walnut oligopeptide microcapsule embedded with probiotics on the probiotics.
The calculation formula of the survival rate of probiotics is as follows: survival rate/% = a/b×100%
Wherein A: simulating the number of viable bacteria in the gastric juice treated sample, wherein CFU/mL; b: the number of viable bacteria in the sample before gastric juice treatment was simulated, CFU/mL.
And (3) placing 0.5g of the walnut oligopeptide probiotic microcapsule prepared by the methods of examples 1 and 2 in artificial intestinal juice, and performing anaerobic condition at 37 ℃ for 100r/min for shaking for 3 hours, and then measuring the viable count to perform an intestinal juice tolerance experiment.
The protective effect of the walnut oligopeptide probiotic microcapsules on the probiotics is shown in figure 7. FIG. 7 a shows walnut oligopeptide benefitThe tolerance of the probiotic microcapsules to gastric juice can be seen that the survival rate of the lactobacillus plantarum without embedding is rapidly reduced in the artificial simulated gastric juice, which indicates that the simulated gastric juice has strong killing capability to the lactobacillus plantarum without embedding, and the probiotics can not exert physiological functions of the lactobacillus plantarum without protecting the stomach. The microcapsule of the walnut oligopeptide prepared by the instillation method and the endogenous emulsification method embeds the lactobacillus plantarum in the microcapsule, and the survival rate of the microcapsule is far higher than that of the non-embedded lactobacillus plantarum, which indicates that the walnut oligopeptide probiotic microcapsule plays a good role in protecting the lactobacillus plantarum. The sodium alginate/whey protein isolate microcapsule and the sodium alginate/resistant starch microcapsule prepared by the instillation method have better protection effect on probiotics than the microcapsule prepared by the endogenous emulsification method, and the sodium alginate/whey protein isolate microcapsule has the best protection effect on probiotics. In fig. 7 b, the tolerance of the walnut oligopeptide probiotic microcapsule to intestinal juice can be seen, and in simulated intestinal juice, the walnut oligopeptide probiotic microcapsule prepared by the instillation method and the endogenous emulsification method can continuously release lactobacillus plantarum, so that the walnut oligopeptide probiotic microcapsule has good enteric solubility, and can protect lactobacillus plantarum from being damaged by gastric juice to reach the intestinal tract, and release and play a role in the intestinal tract. The sodium alginate/whey protein isolate microcapsule has best effect of protecting probiotics in intestinal juice, and the viable count is about 10 9 CFU/mL。
The above description is only a preferred embodiment of the present invention, and is not intended to limit the invention in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to the equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present invention still fall within the protection scope of the technical solution of the present invention.
Claims (10)
1. The walnut oligopeptide probiotics microcapsule is characterized in that probiotics and walnut oligopeptide are used as core materials, and a instillation method or an endogenous emulsification method is adopted to prepare the microcapsule.
2. The walnut oligopeptide probiotics microcapsule according to claim 1, wherein the instilling method is a microcapsule formed by taking probiotics and walnut oligopeptides as core materials, mixing the core materials with wall materials, and then dripping the mixture into a calcium chloride solution; the wall material is a mixture of sodium alginate and whey protein isolate or a mixture of sodium alginate and resistant starch;
the endogenous emulsification method comprises the steps of uniformly mixing probiotic bacteria liquid, walnut oligopeptide solution and sodium alginate solution, adding soybean salad oil containing tween for emulsification, then adding soybean salad oil containing glacial acetic acid, finally adding calcium chloride solution, uniformly stirring, standing, pouring soybean oil, and centrifugally flushing to obtain the walnut oligopeptide probiotic microcapsule.
3. The walnut oligopeptide probiotic microcapsule according to claim 1 or 2, wherein the probiotic is lactobacillus plantarum.
4. The preparation method of the walnut oligopeptide probiotics microcapsule is characterized in that probiotics and walnut oligopeptide are used as core materials, and the walnut oligopeptide probiotics microcapsule is prepared by adopting a instillation method or an endogenous emulsification method;
the instilling method is to take probiotics and walnut oligopeptide as core materials, mix the core materials with wall materials and then drop the mixture into calcium chloride solution to form microcapsules; the wall material is a mixture of sodium alginate and whey protein isolate or a mixture of sodium alginate and resistant starch;
uniformly mixing a probiotic bacterial liquid, a walnut oligopeptide solution and a sodium alginate solution, adding soybean salad oil containing tween for emulsification, then adding soybean salad oil containing glacial acetic acid, finally adding a calcium chloride solution, uniformly stirring, standing, pouring out soybean oil, and centrifugally flushing to obtain the walnut oligopeptide probiotic microcapsule;
the probiotics are lactobacillus plantarum.
5. The method for preparing the walnut oligopeptide probiotic microcapsules according to claim 4, wherein the instillation method comprises the following steps:
(1) Uniformly mixing the probiotic bacteria liquid and the walnut oligopeptide solution to obtain a core material solution;
(2) Uniformly mixing the sodium alginate solution with the whey protein isolate solution or the resistant starch solution to obtain a wall material solution;
(3) Uniformly mixing the core material solution and the wall material solution in proportion, dropwise adding a calcium chloride solution to form a microcapsule, fixing for 20min at room temperature, washing with distilled water, and removing excessive calcium ions and unencapsulated probiotics to obtain the walnut oligopeptide probiotics microcapsule.
6. The method for preparing the walnut oligopeptide probiotics microcapsule according to claim 5, characterized in that the concentration of the walnut oligopeptide solution is 2% -5%; the concentration of the sodium alginate solution is 1% -1.5%, the concentration of the whey protein isolate solution is 1% -1.5%, and the concentration of the calcium chloride solution is 1% -2%.
7. The method for preparing the walnut oligopeptide probiotics microcapsule according to claim 5, characterized in that the mixing ratio of the probiotics bacterial liquid and the walnut oligopeptide solution is 1:5-1:10, the mixing ratio of the sodium alginate solution and the whey protein isolate solution is 1:1-1:3, the mixing ratio of the sodium alginate solution and the resistant starch solution is 1:1-1:5, and the mixing ratio of the core material solution and the wall material solution is 1:5-1:10.
8. The method for preparing the walnut oligopeptide probiotic microcapsules according to claim 4, wherein the endogenous emulsification method comprises the following steps:
(1) Uniformly mixing the probiotic bacteria liquid with the walnut oligopeptide solution and the sodium alginate solution to obtain a mixed solution A;
(2) Adding soybean salad oil containing tween into the mixed solution A, stirring and mixing uniformly, emulsifying for 20min to obtain a mixed solution B, adding soybean salad oil containing glacial acetic acid, and stirring and mixing uniformly to obtain a mixed solution C;
(3) Adding the mixed solution C into a calcium chloride solution, stirring uniformly, standing, pouring soybean oil, centrifuging at 1000r/min for 5min, flushing with normal saline for three times, and removing excessive calcium ions and unencapsulated probiotics to obtain the walnut oligopeptide probiotics microcapsule.
9. The method for preparing the walnut oligopeptide probiotics microcapsule according to claim 8, characterized in that tween content in the tween-containing soybean salad oil is 1% -4%; the content of glacial acetic acid in the soybean salad oil containing glacial acetic acid is 0.1% -0.5%; the mass fraction of the calcium chloride solution is 1% -2%.
10. The method for preparing the walnut oligopeptide probiotic microcapsules according to claim 8, wherein the volume ratio of the mixed solution A to the soybean salad oil containing tween in the step (2) is 1:1-1:4; the adding proportion of the soybean salad oil containing the glacial acetic acid is 50-80% of the volume of the mixed solution B.
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