CN116949029A - Pectin with different esterification degrees as well as W/O/W type lactobacillus plantarum L3 microcapsule wall material, preparation method and application thereof - Google Patents
Pectin with different esterification degrees as well as W/O/W type lactobacillus plantarum L3 microcapsule wall material, preparation method and application thereof Download PDFInfo
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- 239000003094 microcapsule Substances 0.000 title claims abstract description 129
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- 235000010987 pectin Nutrition 0.000 title claims abstract description 99
- 229920001277 pectin Polymers 0.000 title claims abstract description 99
- 238000005886 esterification reaction Methods 0.000 title claims abstract description 54
- 230000032050 esterification Effects 0.000 title claims abstract description 48
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 239000000463 material Substances 0.000 title claims abstract description 15
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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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
- A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
- A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
-
- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5021—Organic macromolecular compounds
- A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
-
- 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
Abstract
Preparation of pectin with different esterification degrees and wall material embedded lactobacillus plantarum L 3 (Lactobacillus plantarum L 3 ) A method for obtaining W/O/W type microcapsules belongs to the technical field of food science. The invention aims to carry out enzymatic de-esterification on commercial high methoxyl pectin to obtain pectin with different esterification degrees, and then embedding the pectin with different esterification degrees by an ionic crosslinking method to produce bacteriocin L 3 Is L.plantarum L 3 The prepared microcapsule is inoculated into MRS culture medium (De Man Rogosa Sharpe), on the one hand, the L-caused L-plantarum L can be reduced 3 The fermentation is participated to cause the post acidification problem of MRS culture medium, and on the other hand, the L.plantarum L can be prolonged 3 The bacteriocin producing time can achieve the long-term bacteriostasis effect. The invention not only provides a new wall material for embedding probiotics, but also can reduce the post-acidification effect of the MRS culture medium, thereby achieving the long-term antibacterial effect. The invention obtains pectin with different esterification degrees, and the prepared microcapsule can effectively delay L.plantarum L under the condition of 4 DEG C 3 The acid production speed reduces the post acidification of the MRS culture medium, and can play a long-term antibacterial effect in the storage period 28 d.
Description
Technical Field
The invention relates to a preparation method of pectin with different esterification degrees and a W/O/W type L.plantarum L with the pectin as a wall material 3 A method for preparing microcapsules.
Background
Pectin is a dietary fiber which exists in the interstitial layer and primary wall of plants, can maintain the morphological structure of plant cells, and can be used as a prebiotic to promote intestinal peristalsis and improve the digestion capacity of human bodies. A part of galacturonic acid residues in the pectin main chain are methyl esterified at the C-6 position, the esterification degree is higher than 50% and the esterification degree is lower than 50%. Due to the structural characteristics of the low methoxy pectin, the low methoxy pectin can be crosslinked with divalent metal ions to form a gel structure, and the low methoxy pectin has wide application in drug and probiotic delivery. The low methoxy pectin has the properties of increasing the viscosity of the product, improving the flavor, improving the stability and the like, is a very good natural additive, and has very wide application in low-sugar and low-heat foods. Large in naturePectin in most plants is high methoxyl pectin, and low methoxyl pectin is prepared by reducing the esterification degree. Biological preservation, i.e., the use of microorganisms or their metabolites (typified by bacteriocins, organic acids), and mixtures of both, to help extend shelf life. Lactic acid bacteria are the main bacteriocin-producing microorganism and are most widely used in biological preservation. Most bacteriocins produced by lactic acid bacteria are extracellular proteins or polypeptides, and are attracting more and more attention because of their broad-spectrum antibacterial properties. However, the direct addition of lactobacillus to the lactobacillus culture medium (De Man Rogosa Sharpe, MRS) can further accelerate the post-acidification process in the MRS culture medium, and is influenced by the metabolic process of thallus growth, so that the goal of continuously producing bacteriocin in a long time cannot be achieved, and therefore, a novel preservative and a delivery system thereof are urgently needed to be searched, so that bacteriocin is slowly delivered in the MRS culture medium, and the sterilization effect is achieved. The subject group screens out a plant lactobacillus L capable of producing bacteriocin from pickled Chinese cabbage with longer shelf life 3 (Lactobacillus plantarum L 3 NCBI sequence No. MT 781360), the bacteriocin L3 produced by the strain has certain antibacterial activity on gram-positive bacteria and gram-negative bacteria and part of fungi. Based on the above research background, the purpose and meaning of the invention are as follows: preparation of low methoxyl pectin with different esterification degrees by using pectin methylesterase and embedding L 3 Obtaining the bacteriocin-producing W/O/W type L.plantarum L 3 The microcapsule can make bacteriocin L3 slowly delivered in lactic acid bacteria culture medium (De Man Rogosa Sharpe, MRS) to achieve the purpose of long-term sterilization and delay post acidification. The theoretical value of the invention is: the theoretical basis is provided for the development of a novel probiotic delivery system in the future by researching the delivery of a probiotic metabolite bacteriocin L3 through microcapsules; the universality is that W/O/W type L.plantarum L 3 Pectin microcapsules are directly put into an MRS culture medium for fermentation, the fermentation speed of the pectin microcapsules in the MRS culture medium and the speed of bacteriocin L3 production are delayed, and the delivery process and mechanism of micromolecular substances produced by metabolism of probiotics are clear.
Disclosure of Invention
The invention provides a preparation technology of pectin with different esterification degreesW/O/W type L. plantarum L using pectin as wall material 3 Method for preparing microcapsules, which method comprises the step of preparing L.plantarum L 3 Embedding to make it produce bacteriocin L3 and slowly deliver it in MRS culture medium for long-term sterilization.
The invention relates to a W/O/W type L.plantarum L with pectin with different esterification degrees as wall materials 3 The preparation method of the bacteriocin-producing microcapsule is realized by the following steps:
(1) Dissolving high methoxyl pectin in sterile water to prepare high methoxyl pectin solution;
(2) Adding pectin methylesterase into the high methoxy pectin solution obtained in the step (1), and respectively carrying out enzymolysis for 40-120 min under the conditions of controlling the pH value to be 4-5 and the temperature to be 40-50 ℃;
(3) Adding the pectin solution obtained in the step (2) into ethanol solution until pectin is completely precipitated, filtering and drying to obtain low-ester pectin samples with different esterification degrees, and placing the low-ester pectin samples in a shade and dry place for standby;
(4) Activated L.Plantarum L 3 Inoculating to MRS liquid culture medium, culturing at 37deg.C for 18-30 hr to obtain L.plantarum L 3 A bacterial suspension; then collecting the thalli and washing with sterile water for 2-3 times to obtain L.plantarum L 3 Is prepared from the bacterial mud;
(5) L.Plantarum L 3 The mass volume ratio of the bacterial mud to the normal saline is 1g: mixing 5mL to obtain a bacterial concentrated solution;
(6) Preparing pectin with different esterification degrees prepared in the step (3) into pectin water solution with mass fraction of 0.5% -4%;
(7) Taking 1-2 g of bacterial sludge prepared in the step (4), blowing and uniformly mixing the bacterial sludge with a small amount of normal saline, uniformly mixing the bacterial sludge with 1-5 mL of oil under a magnetic stirrer, and then adding the bacterial sludge into pectin solutions with different esterification degrees prepared in the step (6), and oscillating the pectin solutions on a vortex oscillator to fully mix the pectin solutions to obtain a mixed solution of bacteria, oil and pectin; dropping the mixed solution into sterile CaCl with the concentration of 100-500mmol/L 2 Obtaining wet microcapsules in the solution;
(8) Standing and solidifying the wet microcapsule at room temperature for 10-120 min, filtering out, and flushing the surface of the microcapsule with sterile water to obtain a finished microcapsule;
(9) The W/O/W type L.plantarum L prepared in the step (8) is processed 3 Culturing the microcapsule in MRS culture medium at 40-45 deg.C for 6-10 h, treating at 4 deg.C for 10-12 h, and storing at 4 deg.C, room temperature and 30 deg.C for 28d;
(10) Samples from the different treatment groups in step (9) were sampled every 7d to prepare cell-free fermentation supernatants, and the pH of each group was determined. Then selecting the optimal temperature condition for deferring acidification, treating for 6-10 h at 40-45 ℃ and 10-12 h at 4 ℃ in MRS culture medium, and then carrying out fermentation 7d experiment, and respectively measuring pH after treating for 6-10 h at 40-45 ℃ and 10-12 h at 4 ℃ and within 7d fermentation;
(11) Using three indicator bacteria to deliver bacteriocin L by using oxford cup method to obtain cell-free fermentation supernatant of different treatment group samples in step (10) 3 Is a study of antibacterial activity.
According to the preparation method, in the step (1), the high methoxyl pectin is citrus high methoxyl pectin with the esterification degree of 60-80%.
Further, the pectin solution concentration in step (1) is 1%.
Further, in the step (2), the pH of the solution was adjusted with a 1M aqueous NaOH solution, and the temperature was kept at 45 ℃.
Further, the pectin methylesterase in step (2) is of Aspergillus niger origin.
Further, in the step (2), the adding amount of pectin methylesterase is 300PEU/g, and the enzymolysis time is 40, 60, 80, 100 and 120min respectively.
Further, the ethanol solution in the step (3) is an absolute ethanol solution.
The invention also provides the low-ester pectin prepared by the preparation method.
The esterification degree of the low-ester pectin in the step (3) is 36.40%, 27.79%, 22.30%, 18.47% and 16.12% respectively.
Further, L.plantarum L as described in step (4) 3 (Lactobacillus plantarum L 3 NCBI serial number MT 781360) strain, and is selected from pickled Chinese cabbage with longer shelf life in the market for the subject group.
Further, L.plantarum L as described in step (4) 3 The activation method comprises collecting L.Plantarum L stored in glycerol at-20deg.C 3 After slowly heating up in an ultra-clean bench, taking 100 mu L of the ultra-clean bench, inoculating to 5mL of MRS liquid culture medium, and then placing the inoculated culture medium into a constant temperature incubator at 37 ℃ for culturing for 24 hours; culturing the activated bacterial liquid on a solid MRS culture medium for 48 hours at 37 ℃ through three dividing lines by a bacterial inoculating loop; single colony is selected and inoculated into 5mL of MRS liquid culture medium, and the culture is repeatedly passaged for a plurality of times to completely revive the thalli.
Further, the MRS liquid culture medium in the step (4) is prepared by adding 800mL of distilled water to 38.6g of MRS liquid culture medium and sterilizing at 121 ℃ for 20min.
Further, the bacterial concentrate in the step (5) is L.plantarum L 3 The mass volume ratio of the bacterial mud to the normal saline is 1g: dilution was carried out at a ratio of 5mL to give L.plantarum L 3 And (3) concentrating the liquid.
Further, the mass concentration of the pectin aqueous solution in the step (6) is 1%.
Further, the bacterial sludge added in the step (6) is 1g.
Further, the oil in the step (6) is peanut oil.
Further, the amount of the oil added in the step (6) is 3.8mL.
Further, caCl as described in step (6) 2 Is 300mmol/L.
Further, the microcapsule curing time in the step (7) is 1h.
Further, the culture temperature in the step (9) is 42 ℃.
Further, the culture time in the step (9) is 8 hours.
Further, the treatment is carried out for 12 hours at the temperature of 4 ℃ in the step (9).
Further, the optimal post-delay acidification storage temperature in the step (9) is 4 ℃.
Further, the fermentation supernatant in the step (10) is prepared by centrifuging the bacterial suspension at 5000rpm for 10min, filtering with a 0.22 μm filter membrane, and removing the bacterial cells to obtain a cell-free fermentation supernatant.
Further, the indicator bacteria in the step (11) are two gram-positive bacteria, namely staphylococcus aureus (Staphylococcus aureus) and listeria monocytogenes (Listeria monocytogenes), and gram-negative bacteria, namely Escherichia coli.
Further, the oxford cup method in the step (11) is to put the sterilized oxford cup into a sterile culture dish, and adjust the number of the indicator bacteria to 10 8 CFU/mL, inoculating to solid culture medium cooled to 46 ℃ according to 1% inoculum size, shaking and mixing uniformly, pouring into a plate, solidifying the culture medium, taking out oxford cups, adding 200 mu L of cell-free fermentation supernatant of each group of samples into the formed holes, standing for 1h in a refrigerator at 4 ℃, culturing at the corresponding temperature of the indicator bacteria for 12h, and observing the diameter of a bacteriostasis ring. The invention prepares the W/O/W type L.plantarum L with pectin with different esterification degrees as wall materials 3 Bacteriocin-producing microcapsules are applicable to a variety of related fields, such as biotechnology, food industry, and the like.
Bacteriocin L is produced by embedding pectin with different esterification degrees 3 Is L.plantarum L 3 The prepared microcapsule is put into MRS culture medium, on one hand, the L-caused L-plantarum L can be reduced 3 After participating in fermentation, the MRS culture medium is acidified, and on the other hand, L.plantarum L can be prolonged 3 The time for producing bacteriocin and the degradation of bacteriocin in intestinal tracts are reduced, and in a word, the invention can not only reduce the post-acidification effect of MRS culture medium, but also can inhibit bacteria for a long time.
By adopting the preparation method of the low-ester pectin and the microcapsule, the invention has the following advantages:
the invention adopts an enzyme method to prepare low-ester pectin with different esterification degrees and prepares the W/O/W type L.plantarum L 3 The bacteriocin microcapsule can reduce the occurrence of post acidification in the fermentation process of MRS culture medium, and can slowly deliver bacteriocin in intestinal tracts to exert broad-spectrum antibacterial activity, thereby achieving the effect of long-term bacteriostasis.
Drawings
Table 1 shows the measurement of the properties of the low-ester pectins prepared in examples 1 to 5 according to the invention;
FIG. 1 shows the variation of time and degree of esterification of low ester pectin prepared in examples 1-5 of the present invention;
FIG. 2 is a low ester pectin prepared in examples 1-5 of the present invention;
FIG. 3 is an infrared spectrum of the low-ester pectin prepared in examples 1-5 of the present invention;
FIG. 4 shows the W/O/W type L.plantarum L prepared in examples 1 to 5 of the present invention 3 A microcapsule;
FIG. 5 shows the W/O/W type L.plantarum L prepared in examples 1 to 5 of the present invention 3 The swelling degree of the microcapsules varies;
FIG. 6 is a W/O/W type L.plantarum L prepared in example 5 of the present invention 3 Antibacterial activity of the microcapsule on staphylococcus aureus at 4 ℃ in an MRS culture medium fermentation experiment;
FIG. 7 is a W/O/W type L.plantarum L prepared in example 5 of the present invention 3 Antibacterial activity of the microcapsule on staphylococcus aureus at room temperature in an MRS culture medium fermentation experiment;
FIG. 8 is a W/O/W type L.plantarum L prepared in example 5 of the present invention 3 Antibacterial activity of the microcapsule on staphylococcus aureus at 30 ℃ in an MRS culture medium fermentation experiment;
FIG. 9 is a W/O/W type L.plantarum L prepared in example 5 of the present invention 3 Antibacterial activity of the microcapsule on listeria monocytogenes at 4 ℃ in MRS culture medium fermentation experiments;
FIG. 10 is a W/O/W-type L.plantarum L prepared in example 5 of the present invention 3 Antibacterial activity of the microcapsule on listeria monocytogenes at room temperature in MRS culture medium fermentation experiments;
FIG. 11 is a W/O/W type L.plantarum L prepared in example 5 of the present invention 3 Antibacterial activity of the microcapsule on listeria monocytogenes at 30 ℃ in MRS culture medium fermentation experiments;
FIG. 12 is a W/O/W type L.plantarum L prepared in example 5 of the present invention 3 Antibacterial activity of the microcapsule on escherichia coli at 4 ℃ in an MRS culture medium fermentation experiment;
FIG. 13 is a W/O/W type L.plantarum L prepared in example 5 of the present invention 3 Antibacterial activity of the microcapsule on escherichia coli at room temperature in an MRS culture medium fermentation experiment;
FIG. 14 shows the present inventionW/O/W type L.plantarum L prepared in example 5 3 Antibacterial activity of the microcapsule on escherichia coli at 30 ℃ in an MRS culture medium fermentation experiment;
FIG. 15 is a W/O/W-type L.plantarum L prepared in example 5 of the present invention 3 Bacteriocin L3 in MRS culture medium fermentation experiments is exemplified by bacteriostasis circle of staphylococcus aureus, listeria monocytogenes, escherichia coli and pichia pastoris;
FIG. 16 is a W/O/W type L.plantarum L prepared in example 5 of the present invention 3 The microcapsule has pH change under the storage period condition of 4 ℃ in an MRS culture medium fermentation experiment;
FIG. 17 is a W/O/W type L.plantarum L prepared in example 5 of the present invention 3 The microcapsule has pH change under the condition of room temperature storage period in MRS culture medium fermentation experiment;
FIG. 18 is a W/O/W type L.plantarum L prepared in example 5 of the present invention 3 The microcapsules were subjected to a pH change under storage conditions of 30℃in MRS medium fermentation experiments.
FIG. 19 is a W/O/W-type L.plantarum L prepared in example 5 of the present invention 3 The microcapsules were treated at 42℃for 8h, at 4℃for 12h and stored at 4℃for 7d pH change in MRS medium fermentation experiments.
Detailed Description
The following examples and figures illustrate the invention in further detail, but embodiments of the invention are not limited thereto.
The experimental methods in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The degree of esterification of the low ester pectin obtained in the examples below was determined according to the method in QB 2484-2000.
The solubility of the low ester pectin obtained in the examples below was determined according to the method in QB 2484-2000.
The ash content of the low ester pectin obtained in the examples below was determined according to the method in GB 25533-2010.
The acid insolubles of the low ester pectin obtained in the examples below were determined according to the method in GB 25533-2010.
The water content of the low ester pectin obtained in the examples below was determined according to the method in GB 5009.3-2010.
In the examples below, citrus high methoxy pectin is purchased from anderil pectin limited; the degree of esterification was measured in the laboratory to be 66.9%.
Example 1
Enzymatic preparation of low-ester pectin with different esterification degrees and preparation of W/O/W type L.plantarum L 3 The method for preparing the microcapsule specifically comprises the following steps:
the first step: modification of materials
(1) Mixing citrus high methoxyl pectin and distilled water according to the ratio of 1g to 50mL, regulating the pH value to 4.5 by using a 1M NaOH aqueous solution, and regulating the temperature to 45 ℃;
(2) Adding pectin methylesterase into the high methoxyl pectin solution, wherein the adding amount is 300PEU/g, the temperature is kept at 45 ℃, and the enzymolysis is carried out for 40min, so that the purpose of de-esterification is achieved;
(3) Slowly pouring the mixture treated by pectin methylesterase into boiling ethanol, continuously stirring, keeping the mixture boiling for 10min, and standing at room temperature for 30min to achieve complete precipitation. Filtering with gauze, oven drying the residue at 65deg.C, and pulverizing to 60 mesh to obtain low-ester pectin with esterification degree of 36.40%;
and a second step of: preparation of L.Plantarum L 3 Bacterial suspension
(1) Activated L.Plantarum L 3 Inoculating into MRS liquid culture medium, and culturing at 37deg.C for 24 hr to obtain L 3 A culture solution; centrifuging, removing supernatant to obtain L.plantarum L 3 Is prepared from the bacterial mud;
(2) L.Plantarum L 3 Washing the bacterial mud with sterilized normal saline, centrifuging, discarding supernatant, and diluting with 5mL normal saline per 1g bacterial mud precipitate at a ratio of 6 times to obtain L.plantarum L 3 The naked fungus concentrated solution is preserved for standby at the temperature of 4 ℃;
and a third step of: preparation of W/O/W type L.plantarum L 3 Microcapsule
(1) Preparing pectin aqueous solution with the mass fraction of 1% from the low-ester pectin prepared in the first step, taking 1.2g of bacterial mud, blowing and mixing uniformly with a small amount of physiological saline, uniformly mixing with 3.8mL of oil under a magnetic stirrer, adding the mixture into the pectin aqueous solution, and oscillating on a vortex oscillator to fully mix the mixture to obtain a mixture of bacteria, oil and pectin;
(2) Connecting the medical needle with a sterile hose, dripping the mixture into 300mmol/L sterile CaCl by using a peristaltic pump 2 Preparing wet microcapsules;
(3) And (3) placing the wet capsules at room temperature for curing for 1h, filtering out, discarding capsules with different sizes and irregular shapes, and flushing the surfaces of the microcapsules with sterile water to obtain finished microcapsules.
Example 2
The first step: modification of materials
(1) Mixing citrus high methoxyl pectin and distilled water according to the ratio of 1g to 50mL, regulating the pH value to 4.5 by using a 1M NaOH aqueous solution, and regulating the temperature to 45 ℃;
(2) Adding pectin methylesterase into the high methoxy pectin solution, wherein the adding amount is 300PEU/g, the temperature is kept at 45 ℃, and the enzymolysis is carried out for 60 minutes, so that the purpose of de-esterification is achieved;
(3) Slowly pouring the mixture treated by pectin methylesterase into boiling ethanol, continuously stirring, keeping the mixture boiling for 10min, and standing at room temperature for 30min to achieve complete precipitation. Filtering with gauze, oven drying the residue at 65deg.C, and pulverizing to 60 mesh to obtain low-ester pectin with esterification degree of 27.79%;
and a second step of: preparation of L.Plantarum L 3 Bacterial suspension
(1) Activated L.Plantarum L 3 Inoculating into liquid culture medium, and culturing at 37deg.C for 24 hr to obtain L 3 A bacterial suspension; then the cell phone is homogenized and washed for 2 to 3 times by sterile water to obtain L.plantarum L 3 Is prepared from the bacterial mud;
(2) L.Plantarum L 3 The mass volume ratio of the bacterial mud to the normal saline is 1g: mixing at a ratio of 5mL physiological saline to obtain L.Plantarum L 3 The concentrated solution of the bacteria is preserved for standby at the temperature of 4 ℃;
and a third step of: preparation of W/O/W type L.plantarum L 3 Microcapsule
(1) Preparing pectin water solution with the mass fraction of 1% from the low-ester pectin prepared in the first step, taking 1.2g of bacterial mud, blowing and mixing the bacterial mud with a small amount of physiological saline, uniformly mixing the bacterial mud with 3.8mL of oil under a magnetic stirrer, adding the bacterial mud into the pectin water solution, and oscillating the bacterial mud on a vortex oscillator to fully mix the bacterial mud, the oil and the pectin to obtain a mixture of bacteria, the pectin;
(2) Connecting the medical needle with a sterile hose, dripping the mixture into 300mmol/L sterile CaCl by using a peristaltic pump 2 Preparing wet microcapsules;
(3) Placing the wet capsules at room temperature for curing for 1h, filtering out, discarding capsules with different sizes and irregular shapes, and flushing the surfaces of the microcapsules with sterile water to obtain finished microcapsules;
example 3
The first step: modification of materials
(1) Mixing citrus high methoxyl pectin and distilled water according to the ratio of 1g to 50mL, regulating the pH value to 4.5 by using a 1M NaOH aqueous solution, and regulating the temperature to 45 ℃;
(2) Adding pectin methylesterase into the high methoxy pectin solution, wherein the adding amount is 300PEU/g, the temperature is kept at 45 ℃, and the enzymolysis is carried out for 80 minutes, so that the purpose of de-esterification is achieved;
(3) Slowly pouring the mixture treated by pectin methylesterase into boiling ethanol, continuously stirring, keeping the mixture boiling for 10min, and standing at room temperature for 30min to achieve complete precipitation. Filtering with gauze, oven drying the residue at 65deg.C, and pulverizing to 60 mesh to obtain low-ester pectin with esterification degree of 22.30%;
and a second step of: preparation of L.Plantarum L 3 Bacterial suspension
(1) Activated L.Plantarum L 3 Inoculating into liquid culture medium, and culturing at 37deg.C for 24 hr to obtain L 3 A bacterial suspension; then the cell phone is homogenized and washed for 2 to 3 times by sterile water to obtain L.plantarum L 3 Is prepared from the bacterial mud;
(2) L.Plantarum L 3 The mass volume ratio of the bacterial mud to the normal saline is 1g: mixing at a ratio of 5mL physiological saline to obtain L.Plantarum L 3 The concentrated solution of the bacteria is preserved for standby at the temperature of 4 ℃;
and a third step of: preparation of W/O/W type L.plantarum L 3 Microcapsule
(1) Preparing pectin water solution with the mass fraction of 1% from the low-ester pectin prepared in the first step, taking 1.2g of bacterial mud, blowing and mixing the bacterial mud with a small amount of physiological saline, uniformly mixing the bacterial mud with 3.8mL of oil under a magnetic stirrer, adding the bacterial mud into the pectin water solution, and oscillating the bacterial mud on a vortex oscillator to fully mix the bacterial mud, the oil and the pectin to obtain a mixture of bacteria, the pectin;
(2) Connecting the medical needle with a sterile hose, dripping the mixture into 300mmol/L sterile CaCl by using a peristaltic pump 2 Preparing wet microcapsules;
(3) And (3) placing the wet capsules at room temperature for curing for 1h, filtering out, discarding capsules with different sizes and irregular shapes, and flushing the surfaces of the microcapsules with sterile water to obtain finished microcapsules.
Example 4
The first step: modification of materials
(1) Mixing citrus high methoxyl pectin and distilled water according to the ratio of 1g to 50mL, regulating the pH value to 4.5 by using a 1M NaOH aqueous solution, and regulating the temperature to 45 ℃;
(2) Adding pectin methylesterase into the high methoxy pectin solution, wherein the adding amount is 300PEU/g, and the temperature is kept at 45 ℃ for enzymolysis for 100min, so that the purpose of de-esterification is achieved;
(3) Slowly pouring the mixture treated by pectin methylesterase into boiling ethanol, continuously stirring, keeping the mixture boiling for 10min, and standing at room temperature for 30min to achieve complete precipitation. Filtering with gauze, oven drying the residue at 65deg.C, and pulverizing to 60 mesh to obtain low-ester pectin with esterification degree of 18.47%;
and a second step of: preparation of L.Plantarum L 3 Bacterial suspension
(1) Activated L.Plantarum L 3 Inoculating into MRS liquid culture medium, and culturing at 37deg.C for 24 hr to obtain L 3 A culture solution; centrifuging, removing supernatant to obtain L.plantarum L 3 Is prepared from the bacterial mud;
(2) L.Plantarum L 3 Washing the bacterial mud with sterilized normal saline, centrifuging, discarding supernatant, and diluting with 5mL normal saline per 1g bacterial mud precipitate at a ratio of 6 times to obtain L.plantarum L 3 The naked fungus concentrated solution is preserved for standby at the temperature of 4 ℃;
and a third step of: preparation of W/O/W type L.plantarum L 3 Microcapsule
(1) Preparing pectin water solution with the mass fraction of 1% from the low-ester pectin prepared in the first step, taking 1.2g of bacterial mud, blowing and mixing the bacterial mud with a small amount of physiological saline, uniformly mixing the bacterial mud with 3.8mL of oil under a magnetic stirrer, adding the bacterial mud into the pectin water solution, and oscillating the bacterial mud on a vortex oscillator to fully mix the bacterial mud, the oil and the pectin to obtain a mixture of bacteria, the pectin;
(2) Connecting the medical needle with a sterile hose, dripping the mixture into 300mmol/L sterile CaCl by using a peristaltic pump 2 Preparing wet microcapsules;
(3) And (3) placing the wet capsules at room temperature for curing for 1h, filtering out, discarding capsules with different sizes and irregular shapes, and flushing the surfaces of the microcapsules with sterile water to obtain finished microcapsules.
Example 5
The first step: modification of materials
(1) Mixing citrus high methoxyl pectin and distilled water according to the ratio of 1g to 50mL, regulating the pH value to 4.5 by using a 1M NaOH aqueous solution, and regulating the temperature to 45 ℃;
(2) Adding pectin methylesterase into the high methoxy pectin solution with the addition amount of 300PEU/g, keeping the temperature at 45 ℃ and carrying out enzymolysis for 120min so as to achieve the purpose of de-esterification;
(3) Slowly pouring the mixture treated by pectin methylesterase into boiling ethanol, continuously stirring, keeping the mixture boiling for 10min, and standing at room temperature for 30min to achieve complete precipitation. Filtering with gauze, oven drying the residue at 65deg.C, and pulverizing to 60 mesh to obtain low-ester pectin with esterification degree of 16.12%;
and a second step of: preparation of L.Plantarum L 3 Bacterial suspension
(1) Activated L.Plantarum L 3 Inoculating into MRS liquid culture medium, and culturing at 37deg.C for 24 hr to obtain L 3 A culture solution; centrifuging, removing supernatant to obtain L.plantarum L 3 Is prepared from the bacterial mud;
(2) L.Plantarum L 3 Sterilized physiological salt for bacterial mudWashing with water, centrifuging, discarding supernatant, and diluting with 5mL physiological saline per 1g bacterial sludge precipitate at a ratio of 6 times to obtain L.Plantarum L 3 The naked fungus concentrated solution is preserved for standby at the temperature of 4 ℃;
and a third step of: preparation of W/O/W type L.plantarum L 3 Microcapsule
(1) Preparing pectin water solution with the mass fraction of 1% from the low-ester pectin prepared in the first step, taking 1.2g of bacterial mud, blowing and mixing the bacterial mud with a small amount of physiological saline, uniformly mixing the bacterial mud with 3.8mL of oil under a magnetic stirrer, adding the bacterial mud into the pectin water solution, and oscillating the bacterial mud on a vortex oscillator to fully mix the bacterial mud, the oil and the pectin to obtain a mixture of bacteria, the pectin;
(2) Connecting the medical needle with a sterile hose, dripping the mixture into 300mmol/L sterile CaCl by using a peristaltic pump 2 Preparing wet microcapsules;
(3) And (3) placing the wet capsules at room temperature for curing for 1h, filtering out, discarding capsules with different sizes and irregular shapes, and flushing the surfaces of the microcapsules with sterile water to obtain finished microcapsules.
Examples 1 to 5 L.Plantarum L in the second step (1) 3 The inoculum size of (2) was 5%.
Examples 1 to 5 the preparation of MRS medium in the second step (1) was as follows: 38.64g of finished MRS medium, 800mL of distilled water is added, and the mixture is sterilized by high-pressure steam at 121 ℃ for 20min.
The low-ester pectins prepared in examples 1 to 5 were compared in terms of degree of esterification, ash, acid insolubles, solubility, galacturonic acid content, and the like, and the results are shown in Table 1.
The method for measuring the esterification degree comprises the following steps:
determining the esterification degree of pectin by adopting a titration method:
accurately weighing 0.5g of dried pectin, transferring into a 250mL conical flask, and wetting with 2mL absolute ethanol. 100mL of distilled water is added, the bottle stopper is added, the sample is rotated from time to time until the sample is completely dissolved, 5 drops of phenolphthalein indicator are added, the titration is performed by using a 0.1mol/L sodium hydroxide standard titration solution until pink color is not discolored for 30 seconds, and the consumed 0.1mol/L sodium hydroxide volume V1 (initial titer) is recorded. Then, 20.0mL of 0.5mol/L sodium hydroxide solution is added to the solution, a bottle stopper is added, the solution is shaken vigorously and then is kept stand for 15min, 20.0mL of 0.5mol/L hydrochloric acid standard titration solution is added, shaking is performed until pink color disappears, then 0.1mol/L sodium hydroxide standard titration solution is used for titration, and shaking is performed vigorously until weak pink color is not faded for 30s as an end point. The volume V2 of the 0.1mol/L sodium hydroxide standard titration solution consumed is recorded. The calculation of the degree of esterification of pectin is shown in formula (1).
As can be seen from Table 1 and FIG. 1, the pectin having five esterification degrees prepared by the present invention was compared, and the pectin has the fastest reduction in esterification degree for the first 60 minutes, because the esterified galacturonic acid content in pectin is higher in the initial stage of the reaction, and is easily continuously de-esterified. The lower the degree of esterification, the lower the pectin galacturonic acid content and solubility. The ash content, acid insoluble matter and solubility of each low-ester pectin all meet the corresponding standards.
The low-ester pectins obtained in examples 1 to 5 were analyzed by infrared spectroscopy, and the results are shown in FIG. 3. 1741.21cm -1 The absorption peak at the position is the stretching vibration of C=O bond in ester group (-COOR), and represents the absorption peak of ester bond in pectin galacturonic acid; 1622.31cm -1 The absorption peak at the position is asymmetric stretching vibration of carboxylate (-COO-) so that the change of the esterification degree of a pectin sample can be intuitively reflected by comparing the changes of the intensities of the two absorption peaks, and the result of the titration measurement of the esterification degree is also confirmed from the side surface.
For the W/O/W type L.plantarum L prepared in examples 1 to 5 3 The microcapsules were validated by the following experiments:
(1) For W/O/W type L.plantarum L 3 The microcapsules were subjected to embedding rate measurement.
The method comprises the following specific steps: 1g of the microcapsule is weighed and added into 8mL of EDTA-2Na solution with pH of 8.0, and the microcapsule is oscillated in a shaking table for 180r/min at 37 ℃ until the microcapsule disintegrates, and then a proper dilution coating plate is taken for calculating the number of viable bacteria. Taking 1mL of bacteria concentrate 3790 Xg, centrifuging for 15min, discarding the supernatant, washing the precipitate with sterile physiological saline for 2-3 times, clarifying and transparentizing the supernatant, carrying out gradient dilution with the sterile physiological saline, and plating to count the number of viable bacteria in the bacteria concentrate.
L.plantarum L 3 Eubacterium and W/O/W type L.plantarum L 3 The number of viable bacteria and the embedding rate in the microcapsules are shown in Table 2, and the W/O/W type L.plantarum L prepared in examples 1 to 5 3 The embedding rate of the microcapsules is 90.45%, 91.67%, 93.66%, 95.14% and 96.79%, respectively, which indicates that the embedding rate of the microcapsules is related to the esterification degree of pectin, and the lower the esterification degree is, the better the embedding effect is.
(2) For W/O/W type L.plantarum L 3 The microcapsules were subjected to a swelling degree measurement.
The method comprises the following specific steps: 0.5g of the lyophilized microcapsules were weighed on an analytical balance and then hydrated in water for 15min and in simulated gastric and intestinal fluids for 15min. Then, it was placed on cellulose paper, dried at 20 ℃ for 5min, and then weighed again. The swelling degree was measured according to the following formula.
The swelling performance has important influence on the protection function and the controlled release function of the microcapsule, and the swelling rule is mainly characterized by the water absorption swelling degree, L.plantarum L 3 Eubacterium and W/O/W type L.plantarum L 3 The swelling degree of the microcapsules is shown in fig. 5, and the swelling degree of the microcapsules prepared from the pectin with the shortest enzymolysis time is the lowest in the swelling degree of the microcapsules in examples 1 to 5, and may be that the outer layer of the microcapsules is swelled to form a thin sol, and the thin sol is dispersed in a liquid, while the swelling state of the microcapsules in example 5 is still kept in a spherical swelling state. Therefore, the microcapsule prepared from pectin with long enzymolysis time has better swelling effect.
The low-ester pectin prepared in example 5 was finally selected to prepare W/O/W-type L.plantarum L by combining the measurement results of the above indexes of the low-ester pectin and the microcapsules prepared in examples 1 to 5 3 The microcapsules were validated by the following experiments:
fermentation experiments in MRS Medium
The method comprises the following specific steps: W/O/W type L.plantarum L in MRS Medium 3 Culturing the microcapsule at 42deg.C for 8 hr, treating at 4deg.C for 12 hr, storing at 4deg.C and 30deg.C for 28d, sampling every 7d, centrifuging at 5000rpm for 10min, filtering with 0.22 μm filter membrane, removing thallus to obtain cell-free fermentation supernatant, and storing at-20deg.C for use; and measuring the pH of the fermentation broth at various times to give L.plantarum L 3 Nude bacteria were used as controls, three samples per group were in parallel. And then selecting the optimal temperature condition for deferring acidification, carrying out 42 ℃ treatment for 8 hours and 4 ℃ treatment for 12 hours in an MRS culture medium, and carrying out fermentation 7d experiments to respectively determine the pH value in 42 ℃ treatment for 8 hours, 4 ℃ treatment for 12 hours and fermentation 7 d. Determination of bacteriocin L in fermentation broths during different storage periods 3 The antibacterial activity of the composition is achieved by an oxford cup method. Antibacterial studies were performed with two gram-positive bacteria, namely staphylococcus aureus and listeria monocytogenes, and two gram-negative bacteria, namely escherichia coli, as indicator bacteria. Placing sterilized oxford cup into sterile culture dish, and adjusting the number of indicator bacteria to 10 8 CFU/mL, inoculating to solid culture medium cooled to 46 deg.C according to 1% inoculum size, shaking, mixing, pouring into a plate, solidifying the culture medium, taking out oxford cup, adding 200 μl of cell-free fermentation supernatant of each group of samples into the formed holes, standing in refrigerator at 4deg.C for 1 hr, culturing at corresponding temperature for 12 hr, observing diameter of antibacterial zone, and collecting L.plantarum L 3 Nude bacteria were used as controls, three samples per group were in parallel.
L.plantarum L 3 Eubacterium and W/O/W type L.plantarum L 3 The microcapsule uses staphylococcus aureus as indicator bacteria, and the measurement results of bacteriocin L3 antibacterial activity in fermentation broth under 4 ℃ storage conditions in different time periods are shown in figure 6, and in the fermentation and storage period in MRS culture medium, two groups of antibacterial activity reach peak value at 7d and W/O/W type L.plantarum L 3 The antibacterial activity of the microcapsule group is obviously greater than that of L.plantarum L 3 A naked fungus group; and L.plantarum L between 14d and 28d 3 The antibacterial activity of the naked bacteria is slightly smaller than that of the W/O/W type L.plantarum L 3 A group of microcapsules. Description of W/O/W type L.plantarum L 3 The microcapsule group has better and lasting antibacterial effect.
L.plantarum L 3 Eubacterium and W/O/W type L.plantarum L 3 The microcapsule uses staphylococcus aureus as indicator bacteria, and has good therapeutic effectThe results of bacteriocin L3 antibacterial activity measurement in fermentation broth under room temperature storage conditions at different time periods are shown in FIG. 7, and in the fermentation and storage period in MRS culture medium, two groups of antibacterial activities reach peak values at 7d and W/O/W type L. plantarum L between 7d and 21d 3 The antibacterial activity of the microcapsule group is larger than that of L.plantarum L 3 There were significant differences in nude bacteria groups, 14d and 21 d. Description of W/O/W type L.plantarum L 3 The microcapsule group has better and lasting antibacterial effect.
L.plantarum L 3 Eubacterium and W/O/W type L.plantarum L 3 The microcapsule uses staphylococcus aureus as indicator bacteria, and the measurement results of bacteriocin L3 antibacterial activity in fermentation liquor under different time periods under 30 ℃ and storage conditions are shown in figure 8, and in the fermentation and storage period in MRS culture medium, two groups of antibacterial activities reach peak value at 7d and W/O/W type L. plantarum L at 14d 3 The antibacterial activity of the microcapsule group is obviously greater than that of L.plantarum L 3 A naked fungus group; the antibacterial activity during the rest of storage period has no significant difference. Description of W/O/W type L.plantarum L 3 The microcapsule group has better and lasting antibacterial effect.
L.plantarum L 3 Eubacterium and W/O/W type L.plantarum L 3 The results of measuring bacteriocin L3 bacteriostatic activity in fermentation broth of different time periods under the storage condition of 4 ℃ are shown in figure 9, and the results of measuring the bacteriocin L3 bacteriostatic activity in fermentation broth of different time periods under the storage condition of 4 ℃ are shown in the figure, wherein the two groups of bacteriocin L3 bacteriostatic activity reach peak values at 7d and reach W/O/W type L.plantarum L between 14d and 28d in the fermentation and storage period of MRS culture medium 3 The antibacterial activity of the microcapsule group is slightly larger than that of L.plantarum L 3 Bare bacteria group. Description of W/O/W type L.Plantarum L 3 The microcapsule group has better and lasting antibacterial effect.
L.plantarum L 3 Eubacterium and W/O/W type L.plantarum L 3 The results of measuring bacteriocin L3 bacteriostatic activity in fermentation broth of different time periods under room temperature storage condition by using Listeria monocytogenes as indicator bacteria of the microcapsule are shown in figure 10, and the two groups of bacteriocin L3 bacteriostatic activity reach peak value at 7d and reach W/O/W type L.plantarum L between 14d and 28d in the fermentation and storage period of MRS culture medium 3 The antibacterial activity of the microcapsule group is obviously larger than that of the naked bacterium group. Description of W/O/W type L.plantarum L 3 MicrocapsuleThe group antibacterial effect is better and more durable.
L.plantarum L 3 Eubacterium and W/O/W type L.plantarum L 3 The results of measuring bacteriocin L3 bacteriostatic activity in fermentation broth of different time periods under 30 ℃ storage condition by using Listeria monocytogenes as indicator bacteria are shown in figure 11, and the W/O/W type L.plantarum L at 14d in the fermentation and storage period of MRS culture medium 3 The antibacterial activity of the microcapsule group is slightly larger than that of L.plantarum L 3 W/O/W type L.plantarum L between 21d and 28d of naked bacteria group 3 The antibacterial activity of the microcapsule group is obviously greater than that of L.plantarum L 3 Antibacterial activity of naked bacteria. Description of W/O/W type L.plantarum L 3 The microcapsule group has better and lasting antibacterial effect.
L.plantarum L 3 Eubacterium and W/O/W type L.plantarum L 3 The results of measuring bacteriocin L3 bacteriostatic activity of the microcapsule, which uses Escherichia coli as indicator bacteria, in fermentation broth at 4 ℃ under different time periods are shown in FIG. 12, and W/O/W type L.plantarum L is fermented in MRS culture medium and stored for a period of time 3 The antibacterial activity of the microcapsule group is continuously increased between 0d and 28d while the antibacterial activity of the microcapsule group is continuously increased to L.plantarum L 3 The naked bacteria reach a peak value at 14 d; and W/O/W type L.plantarum L between 21d and 28d 3 The antibacterial activity of the microcapsule group is also larger than that of L.plantarum L 3 There was a significant difference at 28d for the nude bacteria group. Description of W/O/W type L.plantarum L 3 The microcapsule group has better and lasting antibacterial effect.
L.plantarum L 3 Eubacterium and W/O/W type L.plantarum L 3 The results of measuring bacteriocin L3 bacteriostatic activity in fermentation broth of different time periods under room temperature storage condition by using Escherichia coli as indicator bacteria of the microcapsule are shown in FIG. 13, and W/O/W type L.plantarum L is fermented in MRS culture medium and stored for a period of time 3 The antibacterial activity of the microcapsule group is continuously increased between 0d and 28d while the antibacterial activity of the microcapsule group is continuously increased to L.plantarum L 3 The bare bacteria group reaches a peak value at 14 d; and L.plantarum L between 21d and 28d 3 The bacteriostatic activity of the naked bacteria group is reduced, and the W/O/W type L.plantarum L is carried out at 28 days 3 The microcapsule group is significantly larger than L.plantarum L 3 Bare bacteria group. Description of W/O/W type L.plantarum L 3 The microcapsule group has better and lasting antibacterial effect.
L.plantarum L 3 Eubacterium and W/O/W type L.plantarum L 3 The results of measuring bacteriocin L3 bacteriostatic activity in fermentation broth of different time periods under 30 ℃ storage condition by using Escherichia coli as indicator bacteria are shown in FIG. 14, and the W/O/W type L.plantarum L is between 7d and 28d in the fermentation and storage period of MRS culture medium 3 The antibacterial activity of the microcapsule group is slightly greater than that of L 3 Bare bacteria group. Description of W/O/W type L.plantarum L 3 The microcapsule group has better and lasting antibacterial effect.
The bacteriocin L3 has certain bacteriostasis activity on staphylococcus aureus, listeria monocytogenes, escherichia coli and pichia pastoris as shown in figure 15, and the bacteriocin L3 produced by the strain has certain bacteriostasis activity on gram-positive bacteria, gram-negative bacteria and partial fungi, has broad-spectrum bacteriostasis performance, and has a greater gram-positive bacteria inhibition effect than gram-negative bacteria.
L.plantarum L 3 Eubacterium and W/O/W type L.plantarum L 3 The results of pH measurements in the fermentation broth at 4deg.C for different time periods are shown in FIG. 16, with W/O/W type L.plantarum L during fermentation and storage in MRS medium 3 The pH of the microcapsule group is higher than that of L.plantarum L 3 Nude bacteria group pH. Description of W/O/W type L.plantarum L 3 The microcapsule group can delay post acidification in MRS culture medium.
L.plantarum L 3 Eubacterium and W/O/W type L.plantarum L 3 The results of pH measurements in the fermentation broth at room temperature under storage conditions for different periods of time are shown in FIG. 17, and W/O/W type L.plantarum L during fermentation and storage in MRS medium 3 The pH of the microcapsule group is slightly higher than that of L.plantarum L 3 Nude bacteria group pH. Description of W/O/W type L.plantarum L 3 The microcapsule group can delay post acidification in MRS culture medium, but W/O/W type L.plantarum L 3 The acidification effect of the microcapsule group after delay is slightly lower than that under the condition of 4 ℃.
L.plantarum L 3 Eubacterium and W/O/W type L.plantarum L 3 The results of pH measurements in the fermentation broth at 30℃for different time periods are shown in FIG. 18, and during fermentation and storage in MRS medium,W/O/W type L.plantarum L between 0 and 14d 3 The pH of the microcapsule group is slightly higher than that of L.plantarum L 3 Naked bacteria group pH, W/O/W type L.plantarum L between 21d and 28d 3 Microcapsule group and L.plantarum L 3 The pH of the nude bacteria group was not different. Description of W/O/W type L.Plantarum L 3 The microcapsule group can delay post acidification in MRS culture medium, but W/O/W type L.plantarum L 3 The acidification effect of the microcapsule group after delay is slightly lower than that under the condition of 4 ℃.
L.plantarum L 3 Eubacterium and W/O/W type L.plantarum L 3 The pH measurement results of the microcapsule in the fermentation broth at the optimal delayed acidification temperature, namely 7 days under 4 ℃ storage condition are shown in FIG. 19, and the microcapsule is treated for 8 hours at 42 ℃ in MRS culture medium, 12 hours at 4 ℃ and 7 days in storage period, and the W/O/W type L.plantarum L 3 The pH of the microcapsule group is higher than that of L.plantarum L 3 Nude bacteria group pH. Description of W/O/W type L.plantarum L 3 The microcapsule group can effectively delay post acidification in MRS culture medium under the storage condition of 4 ℃.
TABLE 1
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Claims (24)
1. Preparation of pectin microcapsules with different esterification degrees, and W/O/W type microcapsule embedded lactobacillus plantarum L prepared by taking pectin microcapsules as wall materials 3 (L.plantarum L 3 ) And its use in a simulated fermentation system comprising:
1. dissolving high methoxyl pectin in sterile water to prepare high methoxyl pectin solution;
2. adding pectin methylesterase into the high methoxy pectin solution obtained in the step one, and respectively carrying out enzymolysis for 40-120 min under the conditions of controlling the pH value to be 4-5 and the temperature to be 40-50 ℃;
3. adding the pectin solution obtained in the second step into ethanol solution until pectin is completely precipitated, filtering and drying to obtain low methoxy pectin samples with different esterification degrees, and placing the low methoxy pectin samples in a cool and dry place for standby;
4. activated L.Plantarum L 3 Inoculating to MRS (De M)an Rogosa Sharpe) is cultured at 37℃for 18 to 30 hours to give L.plantarum L 3 A bacterial suspension; then collecting the thalli and washing with sterile water for 2-3 times to obtain L.plantarum L 3 Is prepared from the bacterial mud;
5. L.Plantarum L 3 The mass volume ratio of the bacterial mud to the normal saline is 1g: mixing 5mL to obtain a bacterial concentrated solution;
6. preparing pectin with different esterification degrees prepared in the third step into pectin water solution with mass fraction of 0.5% -4%;
7. adding the bacterial concentrate prepared in the fifth step into the pectin aqueous solution with different esterification degrees prepared in the sixth step, and fully mixing by oscillating on a vortex oscillator to obtain a mixed solution of bacteria, oil and pectin; dropping the mixed solution into sterile CaCl with the concentration of 100-500mmol/L 2 Obtaining wet microcapsules in the solution;
8. standing the wet microcapsule at room temperature for crosslinking for 10-120 min, filtering with gauze, and washing the surface of the microcapsule with sterile water to obtain the finished microcapsule;
9. the W/O/W type L.plantarum.L prepared in the step eight 3 Culturing the microcapsule in MRS culture medium at 40-45 deg.C for 6-10 h, treating at 4 deg.C for 10-12 h, and then respectively treating at 4 deg.C, room temperature and 30 deg.C;
10. sampling samples of different treatment groups in the step nine every 7d, preparing cell-free fermentation supernatant, and measuring the pH of each group of samples;
11. then selecting the optimal temperature condition for deferring acidification, treating for 6-10 h at 40-45 ℃ and 10-12 h at 4 ℃ in MRS culture medium, and then carrying out fermentation 7d experiment, and respectively measuring pH after treating for 6-10 h at 40-45 ℃ and 10-12 h at 4 ℃ and within 7d fermentation;
12. the bacteriostasis activity study of the bacteriocin L3 delivered by the cell-free fermentation supernatant prepared by the samples of the different treatment groups in the step ten is carried out by using three indicator bacteria and adopting an oxford cup method.
2. The method of manufacturing according to claim 1, characterized in that: the high methoxyl pectin in the first step is orange high methoxyl pectin with the esterification degree of 60-80%.
3. The method of manufacturing according to claim 1, characterized in that: the concentration of the high methoxyl pectin solution in the step one is 1% -3%.
4. The method of manufacturing according to claim 1, characterized in that: in the second step, the pH of the solution is adjusted by using 1M NaOH aqueous solution.
5. The method of manufacturing according to claim 1, characterized in that: and step two, pectin methylesterase is an aspergillus niger source.
6. The method of manufacturing according to claim 1, characterized in that: in the second step, the adding amount of the pectin methylesterase is 100-500PEU/g, and the enzymolysis time is 40-120 min.
7. The method of manufacturing according to claim 1, characterized in that: the ethanol solution in the third step is an absolute ethanol solution.
8. The method of manufacturing according to claim 1, characterized in that: the esterification degree of the low methoxy pectin with different esterification degrees in the step three is 16.12-36.40 percent.
9. The method of manufacturing according to claim 1, characterized in that: L.Plantarum L as described in step four 3 (NCBI serial number is MT 781360) strain, and is selected from pickled Chinese cabbage with long shelf life in the market for the subject group.
10. The method of manufacturing according to claim 1, characterized in that: L.Plantarum L as described in step four 3 The activation method comprises collecting L.Plantarum.L stored in glycerol at-20deg.C 3 After the super clean bench is slowly warmed up, 100 mu L of the super clean bench is inoculated to 5mL of MRS liquid culture medium, and the inoculated culture medium is placed in a constant temperature incubator at 37 ℃ for culture for 24 hours; the activated bacterial liquid is put in a fungus inoculating loopThree dividing lines are arranged on a solid MRS culture medium, and the culture is carried out for 48 hours at 37 ℃; single colony is selected and inoculated into 5mL of MRS liquid culture medium, and the culture is repeatedly passaged for a plurality of times to completely revive the thalli.
11. The method of manufacturing according to claim 1, characterized in that: the preparation method of the MRS liquid culture medium in the fourth step comprises the steps of adding 38.6g of MRS liquid culture medium into 800mL of distilled water, and sterilizing at 121 ℃ for 20min.
12. The method of manufacturing according to claim 1, characterized in that: the bacterial concentrate in the fifth step is L.plantarum L 3 The mass volume ratio of the mud to the normal saline is 1g: dilution was carried out at a ratio of 5mL to give L.plantarum L 3 And (3) concentrating the liquid.
13. The method of manufacturing according to claim 1, characterized in that: and step six, the mass concentration of the pectin solution is 1%.
14. The method of manufacturing according to claim 1, characterized in that: the oil in the step seven is peanut oil.
15. The method of manufacturing according to claim 1, characterized in that: the addition amount of the oil in the step seven is 0.5-5.5mL.
16. The method of manufacturing according to claim 1, characterized in that: caCl as described in step seven 2 The concentration is 100-500mmol/L.
17. The method of manufacturing according to claim 1, characterized in that: and step eight, curing the microcapsule for 30-60 min.
18. The method of manufacturing according to claim 1, characterized in that: the culture temperature in step nine was 42 ℃.
19. The method of manufacturing according to claim 1, characterized in that: the culture time in the step nine is 8 hours.
20. The method of manufacturing according to claim 1, characterized in that: and step nine, treating at 4 ℃ for 12 hours.
21. The method of manufacturing according to claim 1, characterized in that: the optimal post-delay acidification storage temperature in the step nine is 4 ℃.
22. The method of manufacturing according to claim 1, characterized in that: the fermentation supernatant in the step ten is prepared by centrifuging the bacterial suspension at 5000rpm for 10min, filtering with 0.22 μm filter membrane, and removing thallus to obtain cell-free fermentation supernatant.
23. The method of manufacturing according to claim 1, characterized in that: the indicator bacteria in step eleven are two gram positive bacteria, namely staphylococcus aureus (Staphylococcus aureus) and listeria monocytogenes (Listeria monocytogenes), and gram negative bacteria, namely Escherichia coli.
24. The method of manufacturing according to claim 1, characterized in that: the oxford cup method in step eleven is to put the sterilized oxford cup into a sterile culture dish, and adjust the number of the indicator bacteria to 10 8 CFU/mL, inoculating to solid culture medium cooled to 46 ℃ according to 1% inoculum size, shaking and mixing uniformly, pouring into a plate, solidifying the culture medium, taking out oxford cups, adding 200 mu L of cell-free fermentation supernatant of each group of samples into the formed holes, standing for 1h in a refrigerator at 4 ℃, culturing at the corresponding temperature of the indicator bacteria for 12h, and observing the diameter of a bacteriostasis ring.
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| CN117264854B (en) * | 2023-11-17 | 2024-01-26 | 云南农业大学 | Lactobacillus plantarum and application thereof |
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