CN113558246B - Symbiotic bifidobacterium composite microcapsule and preparation method thereof - Google Patents
Symbiotic bifidobacterium composite microcapsule and preparation method thereof Download PDFInfo
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- CN113558246B CN113558246B CN202110826168.8A CN202110826168A CN113558246B CN 113558246 B CN113558246 B CN 113558246B CN 202110826168 A CN202110826168 A CN 202110826168A CN 113558246 B CN113558246 B CN 113558246B
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- 238000002360 preparation method Methods 0.000 title abstract description 30
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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
-
- 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
-
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/51—Bifidobacterium
- A23V2400/515—Animalis
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/51—Bifidobacterium
- A23V2400/533—Longum
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Mycology (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Manufacturing Of Micro-Capsules (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention belongs to the technical field of biology, and particularly relates to a symbiotic bifidobacterium composite microcapsule and a preparation method thereof. The composite microcapsule takes bifidobacterium longum subspecies infancy and bifidobacterium animalis subspecies lactis and fructo-oligosaccharides and inulin as core materials, so that not only can intestinal flora be enriched, but also bacterial strain proliferation can be promoted, and intestinal microecological health can be further promoted; pectin and sodium alginate are used as wall materials, so that the coating has no toxic or side effect, has stronger gelatinization property and improves the embedding effect. The composite microcapsule prepared by the invention can improve the survival rate of strains, and the embedded probiotics can stably colonize in gastrointestinal tracts, so that physiological effects can be better exerted; the particle size of the prepared microcapsule is 2.0-2.5mm, the embedding rate is more than or equal to 80.07%, and the survival rate of the simulated gastrointestinal fluid strain is higher than 83.46%; the survival rate after heat treatment at 50 ℃ and 60 ℃ is higher than 63.52 percent, and the product has good gastrointestinal tolerance and heat stability.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a symbiotic bifidobacterium composite microcapsule and a preparation method thereof.
Background
Bifidobacteria are widely used as common intestinal probiotics in the industries of food processing, medicine, feed and the like. During processing, its viability is often affected by external conditions, resulting in reduced activity. How to adopt effective physical and chemical methods to improve the tolerance of probiotics in production, processing and storage and the survival rate of the probiotics in the upper digestive tract of a host has become the key point of the current domestic and foreign research, and the method of relatively mature research still forms a microecological preparation by utilizing the microcapsule embedding technology. In the process of optimizing the microecological preparation, the prebiotics are usually associated with probiotics, and microecology is taken as an important theoretical basis; the composite microcapsule combines polysaccharides such as pectin, sodium alginate, chitosan and the like, and the processing technology of the microcapsule protects the activity of the strain and simultaneously forms a composite microcapsule containing living bacteria and metabolites thereof, can effectively inhibit the growth and propagation of pathogenic bacteria and pathogenic microorganisms in intestinal tracts, improves the micro-ecological balance in a host body, plays a probiotic effect and provides the health level and immunity of the organism.
The prebiotics serving as a dietary supplement cannot be hydrolyzed and absorbed by the upper digestive tract of a human body, and can only selectively stimulate growth and reproduction or activate metabolic functions of beneficial bacteria in the intestinal tract. Common prebiotics mainly include functional oligosaccharides: fructo-oligosaccharides, xylo-oligosaccharides, galacto-oligosaccharides, isomalto-oligosaccharides, etc., and natural plant polysaccharide extracts such as inulin.
Pectin is a class of anionic heteropolysaccharides widely present in the primary wall and cell intermediate lamellae of plant cell walls, mainly consisting of D-galacturonic acid (D-Galacturonic Acids, D-Gal-A) linked by alpha-1, 4 glycosidic linkages, usually extracted from the pericarp of apples, citrus, etc., and capable of forming a gel with divalent cations as sodium alginate. At present, pectin has been widely used as a natural food additive for the fields of food, medicine, cosmetics and the like due to good safety, no toxic or harmful effect, low preparation cost and high availability, and mainly has the effects of gelation, thickening, improving texture, emulsifying and stabilizing.
At present, the research and development of main probiotics microcapsules in domestic and foreign markets are mainly focused on the development and application of bacillus subtilis and lactobacillus, and bifidobacteria are more limited in daily processing due to the extremely strong anaerobic performance, so that the application difficulty is higher. Most of the strain is prepared from a plurality of single strains, and the strains form a single structure; or the optimization and selection of wall materials, and are mostly applied to probiotic preparations and animal husbandry related products. The invention patent CN108323571A discloses a preparation method of a wall material of a probiotic microcapsule, which comprises the steps of selecting sodium alginate, carrageenan and dietary fiber to be mixed as a wall material solution, utilizing good biocompatibility of natural biomacromolecules, no toxic or side effect and the like, improving the tolerance of probiotics to pepsin and the like, and increasing the gel strength, elasticity and the like of a system. The invention patent CN108888726A discloses a preparation method of a composite probiotic capsule, which is a composite probiotic capsule prepared by preparing beneficial metabolites rich in probiotics, traditional Chinese medicine active ingredients, and produced by metabolism of the probiotics. The invention patent CN110833193A discloses a preparation method for preparing bifidobacterium capsules by using banana peel insoluble fibers. However, the microcapsules disclosed in the prior patent have the defects of single structure, low cell survival rate, weak functionality and the like. Although the use of the composite wall material can improve the defects of low embedding rate, poor enteric solubility and the like of the common wall material to a certain extent. However, the wall material is difficult to select, and the development of a wall material which has enteric solubility and is harmless to human body is rare, so that the development of a novel wall material is still difficult; the existing probiotics are mostly mixed in the feed in the form of preparations and applied to the animal husbandry, and how to fully apply the prebiotics as a supplement to the products to exert the efficacy, the development of a microencapsulated product is in need of improvement.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to develop a novel multi-prebiotic and symbiotic bifidobacterium composite microcapsule which can stably improve the activity and survival rate of probiotics, better colonise the gastrointestinal tract and exert the efficacy of the probiotics as a supplement.
In order to achieve the above purpose, the present invention is realized by the following technical scheme:
in a first aspect, the invention provides a probiotic composite microbial inoculant for symbiotic bifidobacteria, wherein the composite microbial inoculant comprises probiotics and prebiotics in a mass ratio of 1-9:9-1; the prebiotics comprise one or more of fructo-oligosaccharide, galacto-oligosaccharide, isomalto-oligosaccharide, xylo-oligosaccharide and inulin; the probiotics comprise one or two of bifidobacterium longum subspecies infantis (Bifidobacterium longum ssp.infantis) and bifidobacterium animalis subspecies lactis (Bifidobacterium animalis ssp.lactis).
Preferably, the prebiotics consist of fructo-oligosaccharides and inulin in a mass ratio of 1-9:9-1; the probiotics are composed of bifidobacterium longum subspecies infancy and bifidobacterium animalis subspecies lactis with the mass ratio of 1-9:9-1.
Preferably, the prebiotic consists of fructo-oligosaccharides and inulin in a mass ratio of 3:7; the probiotics consist of bifidobacterium longum subspecies infancy and bifidobacterium animalis subspecies lactis in a mass ratio of 1:1.
Preferably, the mass ratio of the probiotics to the prebiotics is 1:1.
In a second aspect, the invention provides a symbiotic bifidobacterium probiotic composite microcapsule, wherein the composite microcapsule consists of a wall material and a core material in a mass ratio of 1-10:1, and the core material is the symbiotic bifidobacterium probiotic composite microbial agent in the first aspect; the wall material comprises pectin and sodium alginate with the mass ratio of 1-4:4-1.
Preferably, the mass ratio of the wall material to the core material is 8:1; the mass ratio of the core material to the wall material is 1: 1-10, preferably 1:8, and screening the optimal core material and wall material composite effect in a reasonable proportion range, wherein the specific expression is that when the proportion is too small, the release effect of the strain is affected, the content of colloid wall material is higher, the effective release of the strain is limited, and the colonization effect is poor; when the ratio is too large, the effect of embedding the probiotic strain is poor.
Preferably, the mass ratio of pectin to sodium alginate in the wall material is 2:2.5. The pectin and the sodium alginate are used as wall materials and have smaller mass percent, but in practical application, the content range with optimal embedding effect, higher activity and stability for the bifidobacterium probiotics is obtained through screening; so that the embedded strain is minimally affected by the simulated gastrointestinal environment and the heat treatment condition.
In a third aspect, the present invention provides a method for preparing the composite microcapsule according to the second aspect, the method comprising the steps of:
(1) Mixing the probiotic suspension and the prebiotic solution to form a core material solution;
(2) Mixing the core material solution obtained in the step (1) with the wall material solution to obtain a bacterial gel solution;
(3)dripping CaCl into the bacterial gel solution obtained in the step (2) by an extrusion method 2 Solidifying the solution to form wet microcapsules, and drying to obtain composite microcapsules;
wherein the mass percentage of sodium alginate and pectin in the wall material solution is 1-4% respectively; the concentration of each component of the prebiotics in the core material solution is 10-30 g/L respectively; the CaCl 2 The mass percentage of the solution is 1-4%. The CaCl 2 The concentration of the solution is selected to ensure the embedding efficiency of the strain to the maximum extent and avoid the bacterial loss caused by the incomplete embedding with too low concentration and too high concentration.
Preferably, the method for preparing the bacterial suspension comprises the following steps: inoculating bifidobacteria preserved at-80 ℃ into MRS liquid culture medium respectively, performing anaerobic culture at 37 ℃ for 24-48 hours, and continuously transferring for 2-3 times; and (3) centrifuging the obtained final activated bacterial liquid, discarding the supernatant, repeatedly washing bacterial mud twice by using sterile physiological saline, re-suspending by using the physiological saline, and collecting the probiotic bacterial body.
Preferably, the temperature of the centrifugation is 0-10 ℃; the rotational speed of the centrifugation is 4000-8000 r/min; the centrifugation time is 5-15 min. The centrifugal temperature, the rotational speed and the time for preparing the bacterial suspension are selected to ensure the centrifugal efficiency and the bacterial collection integrity as much as possible.
Preferably, the mixed solution in the steps (1) and (2) is required to be stirred at the temperature of 25-40 ℃; the stirring speed is 100-200 r/min; the stirring time is 10-30 min. The selection range of the stirring temperature, the stirring speed and the stirring time aims to ensure that the bacterial strain is more uniformly dispersed in the wall material in the mixing and preparation process, and simultaneously improve the embedding rate of the bacterial strain and the viable count.
Preferably, the method further comprises the step (3) of drying by vacuum freeze-drying, wherein the vacuum freeze-drying method is as follows: pre-freezing the prepared wet microcapsule for 8-16 h at-80 to-75 ℃, and then freeze-drying for 24-36h in a vacuum freeze dryer at-50 to-45 ℃ and a vacuum degree of 0 Pa. The multi-prebiotic and symbiotic bifidobacterium composite microcapsule is subjected to freeze-drying treatment under the freeze-drying condition, so that survival and stability of the strain are ensured to the greatest extent.
Preferably, the mass percentage of sodium alginate in the wall material solution is 2.5%, and the mass percentage of pectin is 2.0%; the concentration of prebiotics in the core material solution is 25g/L of fructo-oligosaccharide and 30g/L of inulin.
Preferably, the total book of bifidobacterium longum subspecies infantis and bifidobacterium animalis subspecies lactis in the probiotic bacterial suspension is 1 x 10 9 ～1×10 10 CFU/mL。
The beneficial effects of the invention are as follows:
(1) In the composite microbial agent provided by the invention, the mixed probiotics of the bifidobacterium longum subspecies infantis and the bifidobacterium animalis subspecies lactis have a good symbiotic effect; the bacterial colony diversity is enriched, and the guarantee is provided for further maintaining the micro-ecological balance in the host body while the number of viable bacteria reaching the intestinal tract is increased, the activity is maintained and the probiotic characteristics are guaranteed;
(2) The embedding technology provided by the invention forms a composite microcapsule which is easy to store, convenient to carry and use, protects active substances, simultaneously slows down the damage of strains in the stress environment such as gastric acid, bile salt and heat treatment in the digestive tract of a human body, and improves the stability of products;
(3) The particle size of the microcapsule prepared by the invention is 2.0-2.5mm, the embedding rate is more than or equal to 80.07%, and the survival rate of simulated gastrointestinal fluid strain is higher than 83.46%; the survival rate after heat treatment at 50 ℃ and 60 ℃ is higher than 63.52 percent.
(4) The invention adds a plurality of probiotics which are easy for the strain to cooperatively utilize and proliferate into the embedding system, further protects sensitive components, improves the taste and provides a new path for implementing the concept of promoting nutrition and health benefit and the product of promoting aging;
(5) The invention selects the bifidobacterium strain with excellent performance and can be utilized in the food production field as the core material of the microcapsule so as to enrich the intestinal flora structure; natural apple pectin is selected as part of wall material substances, and the natural apple pectin has no toxic or side effect and strong gelation property, and is combined with common wall materials such as sodium alginate, so that the embedding effect is further improved; adding a plurality of prebiotics capable of promoting strain proliferation as a supplement thereof to further promote intestinal microecological health; finally, a composite microcapsule of the multi-prebiotics and the bifidobacteria, which can improve the survival rate of the strain and fully exert the functions of the strain, is developed, so that the embedded probiotics can stably colonize in the gastrointestinal tract, and better physiological effects are exerted.
Drawings
FIG. 1 color change of a bifidobacterium primary screening solution through a prebiotic solution;
FIG. 2 is a graph of the viable count results of a re-screening of Bifidobacterium longum subspecies infantis and Bifidobacterium animalis subspecies lactis, wherein the left graph is Bifidobacterium longum subspecies infantis and the right graph is Bifidobacterium animalis subspecies lactis;
FIG. 3 results of screening of bifidobacterium longum subspecies infantis and bifidobacterium animalis subspecies lactis for optimal prebiotic solution concentrations, wherein the left panel shows bifidobacterium longum subspecies infantis and the right panel shows bifidobacterium animalis subspecies lactis;
FIG. 4 results of screening of Bifidobacterium longum subspecies infantis and Bifidobacterium animalis subspecies lactis, respectively, in a mixed prebiotic system, wherein each group A-I represents 25g/L inulin: the volume ratio of 30g/L fructooligosaccharides is 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1;
FIG. 5 growth curve and pH change of composite strain in mixed prebiotic system;
FIG. 6 is a process flow diagram for preparing microcapsules;
FIG. 7 is a graph simulating the change of viable count and survival rate of a microcapsule composite strain in a gastric juice environment, wherein B represents a microcapsule embedded strain, and F represents a free strain;
FIG. 8 is a graph of the viable count and survival rate of the microcapsule complex strain in a simulated intestinal fluid environment, wherein B represents the microcapsule embedded strain and F represents the free strain;
FIG. 9 is a graph simulating the change of viable count and survival rate of a microcapsule complex strain in a gastrointestinal fluid environment, wherein B represents a microcapsule embedded strain, and F represents a free strain; 0-120 min represents the change condition of the simulated gastric fluid treatment strain, and 120-240 min represents the change condition of the simulated intestinal fluid treatment strain;
FIG. 10 sodium alginate-pectin wet microcapsules and morphology under an optical microscope, wherein the left panel is the wet microcapsules; the right figure is a morphological image of wet microcapsules under a normal light microscope (x 4);
FIG. 11 is a representation of the morphology of the lyophilized sodium alginate-pectin microcapsules under a common optical microscope, wherein the left panel is the lyophilized microcapsules (. Times.4); the right panel is a morphological image under a freeze-dried normal light microscope (. Times.4).
Detailed Description
The present invention will be described in further detail by way of examples, which are only for illustration of the present invention and are not limited to the scope of the examples.
The bifidobacterium strains used in this example were respectively bifidobacterium longum subspecies infantis (Bifidobacterium longum ssp.) CGMCC 1.2202; bifidobacterium animalis subspecies Lactis (Bifidobacterium animalis ssp.) lacts, CGMCC 1.2226; all purchased from China general microbiological culture Collection center.
EXAMPLE 1 Co-cultivation of probiotics and prebiotics
1. Strain activation and preparation of bacterial suspension
Inoculating 6 bifidobacterium strains (54 strains in total) preserved at-80 ℃ into MRS liquid culture medium (0.5 g/L L-cysteine hydrochloride is added) respectively, performing anaerobic culture at 37 ℃ for 24-48h, and continuously transferring for 2-3 times; centrifuging the culture solution of the last time at 4 ℃ for 10min at 8000r/min to obtain bacterial precipitate, washing the bacterial precipitate twice with sterile physiological saline, and re-suspending with sterile physiological saline with the same volume to obtain bacterial suspension for later use;
TABLE 1 bifidobacterium strains
2. Effect of prebiotics and additive amount thereof on bifidobacterium growth
(1) And (5) primary screening of prebiotics:
preparing a Basal-MRS culture medium, adding 10g/L of five probiotics (fructo-oligosaccharide FOS, xylo-oligosaccharide XOS, isomaltooligosaccharide IMO, galacto-oligosaccharide GOS and Inulin Inulin respectively), 10g/L of glucose as a positive control group and a Basal-MRS culture medium without sugar as a negative control group, inoculating the activated bacterial suspension into the culture medium according to an inoculum size of 2% v/v for preliminary screening, standing and culturing at 37 ℃ for 36-48h, and observing the color change of the solution (wherein the color change of the solution represents the growth results of the strain in the negative control group and part of the strain which cannot utilize the prebiotic solution, the yellow solution represents the growth results of the strain in the positive control group and the strain which can better utilize the prebiotic solution, and the color change of the solution represents the strain which can better utilize the prebiotic solution).
The statistical results are shown in figure 1, and the results show that all strains in the bifidobacterium adolescentis, the bifidobacterium bifidum and the bifidobacterium breve have poor utilization conditions of the prebiotics in each strain, and the solution is purple orange in color; the strain in the bifidobacterium longum subspecies infantis and bifidobacterium animalis subspecies lactis can better utilize the prebiotics, and the solution is yellow.
(2) And (3) re-screening the prebiotics:
activating the infant subspecies of bifidobacterium longum, the milk subspecies of bifidobacterium animalis and the long subspecies of bifidobacterium longum obtained through preliminary screening, respectively inoculating the infant subspecies of bifidobacterium longum, the milk subspecies of bifidobacterium longum and the long subspecies of bifidobacterium longum into a culture medium containing 10g/L of each prebiotic solution according to the inoculum size of 2 percent v/v, standing and culturing at 37 ℃ for 36-48 hours, and then diluting the plate to count the number of viable bacteria, and comparing the growth effects of different bifidobacteria.
The results are shown in FIG. 2, wherein the strains corresponding to Bifidobacterium longum subspecies infantis and Bifidobacterium animalis subspecies lactis grew well in prebiotics containing fructooligosaccharides and inulin, and the average colony count was greater than 9.04LogCFU/mL and 9.01LogCFU/mL, respectively (as shown in FIG. 2); and the average colony count of the strain corresponding to the long subspecies of bifidobacterium longum is lower than 8.64LogCFU/mL. The results show that only bifidobacterium longum subspecies infancy and bifidobacterium animalis subspecies lactis are better able to utilize the prebiotics and grow well in the prebiotic containing medium.
(3) Utilization of prebiotics with different contents:
and adding prebiotics with different contents into the culture medium to ensure that the final concentration reaches 10g/L, 15 g/L, 20 g/L, 25g/L and 30g/L respectively, and sterilizing the prepared culture medium for later use. Inoculating activated Bifidobacterium longum subspecies infantis and Bifidobacterium animalis subspecies lactis bacterial suspensions into the culture medium according to the inoculum size of 2% v/v respectively, standing at 37 ℃ for culturing for 36-48 hours, then diluting the culture medium to count the number of viable bacteria, and comparing the influence of the addition amounts of the prebiotics with different concentrations on the growth effect of the Bifidobacterium.
The results are shown in FIG. 3: the comparison of the plate counting results shows that the bifidobacterium longum subspecies infantis and the bifidobacterium animalis subspecies lactis have the best utilization effect on fructo-oligosaccharides and inulin, and the viable count of the final strain is higher than 8.97LogCFU/mL when the prebiotic concentration is 25g/L fructo-oligosaccharides and 30g/L inulin respectively; the average colony count in the culture medium containing xylo-oligosaccharide, isomalto-oligosaccharide and galacto-oligosaccharide is respectively lower than 8.51LogCFU/mL, 8.35LogCFU/mL and 7.94LogCFU/mL.
On the basis, the bifidobacterium longum subspecies infancy (Bifidobacterium longum ssp. Infantis) and the bifidobacterium animalis subspecies lactis are selected, and the subsequent experiments are carried out on the CGMCC 1.2202 and the bifidobacterium animalis subspecies lactis (Bifidobacterium animalis ssp. Lactis).
3. Co-culture of Bifidobacterium longum subspecies infantis and Bifidobacterium animalis subspecies lactis and prebiotics
(1) The prebiotics have the combined action effect:
respectively preparing 25g/L fructo-oligosaccharide and 30g/L inulin solution according to the ratio of 1:9, 2:8, 3:7, 4:6 and 5:5; the mixed solution of the two is prepared according to the ratio of 6:4, 7:3, 8:2 and 9:1, a liquid culture medium is prepared according to the corresponding ratio, sugar-free and glucose culture media are used as negative and positive controls respectively, the optimal combined concentration is determined through viable bacteria counting after anaerobic constant temperature culture for 24 hours at 37 ℃, the result is shown in fig. 4, and the optimal volume ratio of the combined concentration of the prebiotics is found to be optimal when 25g/L fructo-oligosaccharide is 30g/L inulin=3:7 through further screening by a plate counting method.
(2) Bifidobacterium co-culture effect:
preparing 25g/L fructo-oligosaccharide and 30g/L inulin solution respectively, and preparing mixed solution of the fructo-oligosaccharide and the inulin solution according to the ratio of 3:7, and mixing the above bifidobacterium longum with the infantThe method comprises the steps of respectively adding suspension of the Lactobacillus subspecies of the Bifidobacterium subspecies of the animals into the prepared mixed solution of the prebiotics according to the proportion of 1:0, 2:8, 4:6, 8:2 and 0:1, standing and culturing for 24-36 hours at 37 ℃, measuring the total number of bacteria and the pH value change by using a turbidimetry method, finally screening to obtain the optimal co-culture combination, and adding the two strains of bacteria into the optimal concentration of the prebiotic combination according to different proportions, wherein the two strains of bacteria are found to have a certain symbiotic effect when the two bacterial suspensions are added according to the proportion of 1:1 by measuring the growth curve and the pH value change of each combination, and the specific appearance is OD (optical density) 600 The increase in measured value and the decrease in pH value.
The results show that the bifidobacterium longum subspecies infantis and the bifidobacterium animalis subspecies lactis have good prebiotic utilization conditions, and when the ratio of the subspecies infantis to the bifidobacterium animalis subspecies lactis is 1:1, 2.5g/L fructo-oligosaccharides and 3.0g/L inulin with the volume ratio of 3:7 are added, so that the bifidobacterium longum subspecies infantis and the bifidobacterium animalis subspecies lactis have good symbiotic proliferation effect.
Example 2 preparation of composite prebiotic microcapsules 1
The embodiment provides a bifidobacterium and composite prebiotic microcapsule, the composite microcapsule comprises a core material and a wall material, the core material is a composite prebiotic of bifidobacterium longum subspecies infancy thallus, fructo-oligosaccharide and inulin combination, the wall material is sodium alginate-pectin, the preparation flow is shown in figure 6, and the specific preparation method is as follows:
(1) Inoculating the bifidobacterium longum subspecies infancy strain into a liquid MRS culture medium for activating proliferation, wherein the culture temperature is 37 ℃, the culture time is 36 hours, centrifuging the culture medium at 4 ℃ for 10 minutes at 8000r/min after the period of maturation and stabilization, collecting, washing and re-suspending to obtain bifidobacterium longum subspecies infancy suspension; the mass percentage of the sodium alginate and the pectin in the wall material solution is 2.5 percent and 2.0 percent respectively, and the ratio of the sodium alginate to the pectin is 1:1; the ratio of the bacterial suspension in the core material to the composite prebiotic solution is 1:2, and the volume composition of the composite prebiotic is 25g/L fructo-oligosaccharide and 30g/L inulin=3:7; the mass ratio of the wall material to the core material solution is 1:8;
(2) Stirring and mixing the wall material and the core material solution obtained in the step (1) at the temperature of 37 ℃ for 20min at the speed of 120r/min to form a fungus gel mixed solution;
(3) Extruding the mixed solution of the bacterial gel obtained in the step (2) into 2% CaCl through a nozzle injector with the diameter of 450 mu m 2 Solidifying for 30min under stirring at a speed of 100r/min at room temperature in the solution to obtain probiotics and composite prebiotics wet microcapsules;
(4) Pre-freezing the obtained wet microcapsule at-80 ℃ for 10 hours, and then freeze-drying the wet microcapsule in a vacuum freeze dryer at the temperature of-45 ℃ and the vacuum degree of 0Pa for 24-36 hours to obtain the dry microcapsule.
Example 3 preparation of composite prebiotic microcapsules 2
The embodiment provides a bifidobacterium and composite prebiotic microcapsule, which comprises a core material and a wall material, wherein the core material is a composite prebiotic formed by combining bifidobacterium lactis subspecies of animals, fructo-oligosaccharides and inulin, and the wall material is sodium alginate-pectin. The preparation method comprises the following steps:
(1) Inoculating the bifidobacterium animalis subspecies lactis into a liquid MRS culture medium for activating proliferation, wherein the culture temperature is 37 ℃, the culture time is 36 hours, centrifuging the culture medium at 4 ℃ for 10 minutes at 8000r/min after the culture medium is mature and stable, collecting, washing and re-suspending to obtain bifidobacterium animalis subspecies lactis suspension; the mass percentage of the sodium alginate and the pectin in the wall material solution is 2.5 percent and 2.0 percent respectively, and the ratio of the sodium alginate to the pectin is 1:1; the ratio of the bacterial suspension in the core material to the composite prebiotic solution is 1:2, and the composite prebiotic composition is 25g/L fructo-oligosaccharide, 30g/L inulin=3:7; the mass ratio of the wall material to the core material solution is 1:8;
(2) Stirring and mixing the wall material and the core material solution obtained in the step (1) at the temperature of 37 ℃ for 20min at the speed of 120r/min to form a fungus gel mixed solution;
(3) Extruding the mixed solution of the bacterial gel obtained in the step (2) into 2% CaCl through a nozzle injector with the diameter of 450 mu m 2 Solidifying for 30min under stirring at a speed of 100r/min at room temperature in the solution to obtain a bifidobacterium and composite prebiotic wet microcapsule;
(4) Pre-freezing the obtained wet microcapsule at-80 ℃ for 10 hours, and then freeze-drying the wet microcapsule in a vacuum freeze dryer at the temperature of-45 ℃ and the vacuum degree of 0Pa for 24-36 hours to obtain the dry microcapsule.
Example 4 preparation of composite prebiotic microcapsules 3
The embodiment provides a composite prebiotic and symbiotic bifidobacterium microcapsule, which comprises a core material and a wall material, wherein the core material is a composite prebiotic formed by combining bifidobacterium longum subspecies infancy, bifidobacterium animalis subspecies lactis thalli, fructo-oligosaccharide and inulin, and the wall material is pectin and sodium alginate. The preparation method comprises the following steps:
(1) Inoculating Bifidobacterium longum subspecies infantis and Bifidobacterium animalis subspecies lactis into a liquid MRS culture medium for activating proliferation, wherein the culture temperature is 37 ℃, the culture time is 36h, centrifuging the culture medium at 4 ℃ for 10min at 8000r/min after the maturation and stabilization period, respectively collecting, washing and re-suspending to obtain bacterial suspensions, and mixing the bacterial suspensions according to the ratio of 1:1; the mass percentage of pectin and sodium alginate in the wall material solution is 2.0% and 2.5%, respectively, and the ratio of the pectin to the sodium alginate is 1:1; the ratio of the mixed bacterial suspension to the composite prebiotic solution in the core material is 1:2, and the composition of the composite prebiotic is 25g/L fructo-oligosaccharide and 30g/L inulin=3:7; the mass ratio of the wall material to the core material solution is 1:8;
(2) Stirring and mixing the wall material and the core material solution obtained in the step (1) at the temperature of 37 ℃ for 20min at the speed of 100r/min to form a fungus gel mixed solution;
(3) Extruding the mixed solution of the bacterial gel obtained in the step (2) into 2% CaCl through a nozzle injector with the diameter of 450 mu m 2 Solidifying the solution for 30min under stirring at a speed of 100r/min at room temperature, and filtering to obtain wet microcapsules;
(4) Pre-freezing the obtained wet microcapsule at-80 ℃ for 10 hours, and then freeze-drying the wet microcapsule in a vacuum freeze dryer at the temperature of-45 ℃ and the vacuum degree of 0Pa for 24-36 hours to obtain the dry microcapsule.
Comparative example 1 Bifidobacterium Single culture
Placing Bifidobacterium longum subspecies infantis and Bifidobacterium animalis subspecies lactis strains into a prepared prebiotic system of 25g/L fructo-oligosaccharide and 30g/L inulin solution mixed according to the ratio of 3:7 respectively, standing and culturing at 37 ℃ for 24-36h, and measuring the total bacterial count and pH value change by using a turbidimetry method.
The results are shown in figure 5 of the drawings,after a single strain is cultured for 36 hours in a mixed prebiotic system, the strain is cultured by OD 600 The total bacterial count is found when the bacterial count is measured, the measurement result is obviously lower than the result when the bacterial strain is mixed and cultured (P is less than 0.05), and indirectly indicates that the bacterial strain mixed and cultured can better promote the growth of the bacterial strain and the bacterial strain compared with the bacterial strain mixed and cultured singly, and the bacterial strain mixed and cultured bacterial strain has certain symbiotic application value.
Comparative example 2 preparation of Single microcapsules 1
This comparative example provides the preparation of a bifidobacterium longum subspecies infancy microcapsule comprising a core material and a wall material, the difference from example 2 is that the core material is only bifidobacterium subspecies infancy cells and the wall material is sodium alginate and pectin, the rest of the preparation method is the same as example 2.
Comparative example 3 preparation of Single microcapsules 2
This comparative example provides the preparation of a bifidobacterium animalis subspecies lactis microcapsule comprising a core material and a wall material, the difference from example 3 being that the core material is only bifidobacterium animalis subspecies cells and the wall material is sodium alginate and pectin, the remainder of the preparation process being the same as example 3.
Comparative example 4 preparation of Single microcapsules 3
This comparative example provides the preparation of a mixed microcapsule of bifidobacterium longum subspecies infantis and bifidobacterium animalis subspecies lactis comprising a core material and a wall material, the remainder of the preparation being the same as in example 4, except that the core material is solely a mixed cell of bifidobacterium longum subspecies infantis and bifidobacterium animalis subspecies lactis, and the wall material is sodium alginate and pectin.
The results of comparative examples 2-4 and examples 2-4 are analyzed to find that the mixed addition of the prebiotic fructo-oligosaccharide and inulin for preparing the microcapsule can effectively improve the embedding rate and the viable count of the strain, so that the microencapsulation can more effectively protect the bifidobacterium strain in the core material, further improve the stability and the survival activity of the bifidobacterium strain, effectively avoid the adverse stimulation of the gastrointestinal tract environment, and reach the colonisation and the action of the intestinal tract in a sufficient quantity.
Example 5 evaluation of the Effect of composite prebiotic microcapsules
1. Determination of embedding Rate and viable count
The specific method comprises the following steps: 1.0g of the microcapsules prepared in examples 2 to 4 and comparative examples 2 to 4 was weighed and added to 9mL of a solution for decolonizing (mass fraction: 0.1mol/L phosphate buffer, pH=7.0, sterilization at 121 ℃ C. For 15 min), and the mixture was shaken in a shaker at 37 ℃ for 90min at a shaking table rotation speed of 150rpm to completely release the cells, and then subjected to sampling and gradient dilution, and viable count was performed by a plate count method, by the formula: the embedding rate = (number of viable bacteria in embedded microcapsule/number of viable bacteria of embedded free cells) ×100%. The results are shown in Table 3, the embedding rate of the microcapsules prepared in examples 2-4 is higher than 80%, and the viable count reaches more than 9 LogCFU/g; the microcapsule prepared by taking bifidobacterium longum subspecies infantis and bifidobacterium animalis subspecies lactis as mixed probiotics has higher embedding rate and viable count content, and the viable count reaches more than 10 LogCFU/g.
TABLE 3 microcapsule entrapment and viable count statistics
2. Simulated gastrointestinal fluid tolerance analysis
The specific operation method comprises the following steps: weighing 1.0g of the microcapsule in example 4, adding the microcapsule into 9.0mL of simulated gastric fluid (10 g of pepsin is added into 800mL of deionized water, the pH is regulated to 2.5 by 0.1mol/L of hydrochloric acid), and shake culturing for 2 hours at 37 ℃; then placing the mixture in simulated intestinal juice (adding 6.8g of dipotassium hydrogen phosphate into 500mL of deionized water, adding 10g of trypsin into the deionized water for dissolution, adjusting the pH to 6.8 with 0.4% sodium hydroxide solution, and adding water to a constant volume of 1000 mL) for 2 hours; or transferring the capsule treated by the simulated gastric fluid for 2 hours into the simulated intestinal fluid for 2 hours, and performing viable count through plate coating, wherein the result is shown in fig. 7-9, the microcapsule prepared in the embodiment 4 of the invention shows better tolerance in the simulated gastric intestinal fluid, and the survival rate of the strain is higher than 83.46%.
3. Observation of microcapsule morphology and structure
Placing a proper amount of microcapsules on a glass slide, observing the surface morphology of the microcapsules under an optical microscope, and photographing and comparing the surface morphology of the microcapsules, wherein the results are shown in fig. 10 and 11, and fig. 10 shows the wet microcapsules and the morphology under the optical microscope; fig. 11 shows the dry microcapsules and the morphology under an optical microscope.
As can be seen from fig. 10, the wet microcapsules prepared under visual observation were slightly milky colloidal spheres with smooth surface and uniform morphology and particle size; the microcapsules are further observed to be uniformly distributed in a spherical shape and smooth in surface after being amplified by four times under an optical microscope, and further show that the prepared microcapsules are more successful. As can be seen from fig. 11, compared with the wet microcapsule, the particle size of the microcapsule after freeze-drying is obviously reduced, the microcapsule is changed from transparent colloid to white solid, the surface structure presents wrinkles, but the size and the shape of the microcapsule after freeze-drying have good consistency as a whole, which indicates that the microcapsule is successfully treated by the vacuum freeze-drying method, and has better value in actual production for subsequent better application.
4. Microcapsule particle size determination
The particle size of the microcapsules is measured by a vernier caliper, the particle size of 20 microcapsules is measured, the average value is calculated, the result is shown in table 4, and the average particle size of the microcapsules prepared by the invention is about 2.3mm and meets the standard.
TABLE 4 particle size (mm) of microcapsules
5. Analysis of Heat resistance of microcapsules
1g of the microcapsules of example 4 were each taken and placed in 9mL of sterile phosphate buffer preheated to 50℃and 60℃for heat resistance analysis, and placed in a water bath at the corresponding temperature, measured once every 5min for 0min, 5min, 10min and 15min, respectively, and counted on a plate. In addition, 1mL of the free bacterial suspension was subjected to the same conditions for viable count in the control experiment. As shown in Table 5, after the microcapsule is subjected to heat treatment at 50 ℃ and 60 ℃, the survival rate of each strain is higher than 63.52%, and the microcapsule has good adaptability to stress environment, thereby creating conditions for better physiological action.
TABLE 5 Heat resistance of microcapsules
The results show that the multi-prebiotics and bifidobacterium microcapsules can obviously improve the viable count of the strain to be more than 9.12LogCFU/g under the premise of ensuring higher embedding rate (more than or equal to 80.07 percent), and the results of simulating gastrointestinal fluids are shown as figures 7-9, and also show better tolerance and the survival rate of the strain to be higher than 83.46 percent; the survival rate of each strain is higher than 63.52% after heat treatment at 50 ℃ and 60 ℃, has good adaptability to stress environment, and creates conditions for better physiological function.
Claims (4)
1. The symbiotic bifidobacterium probiotic composite microbial agent is characterized by comprising probiotics and prebiotics in a mass ratio of 1:2; the probiotics consist of bifidobacterium longum subspecies infantis (Bifidobacterium longum ssp. Inffantis) CGMCC 1.2202 and bifidobacterium animalis subspecies lactis (Bifidobacterium animalis ssp. Lactis) CGMCC 1.2226 with the mass ratio of 1:1; the prebiotics consist of fructo-oligosaccharides and inulin in a mass ratio of 3:7; the concentration of the fructo-oligosaccharide is 25g/L, and the concentration of the inulin is 30g/L.
2. The symbiotic bifidobacterium probiotic composite microcapsule is characterized by comprising wall materials and core materials in a mass ratio of 1:8, wherein the core materials are the symbiotic bifidobacterium probiotic composite microbial agent in claim 1; the wall material is pectin and sodium alginate, and the mass percentage of the pectin and the sodium alginate in the wall material solution is 2.0% and 2.5% respectively.
3. A method of preparing the composite microcapsule of claim 2, comprising the steps of:
(1) Mixing the probiotic suspension and the prebiotic solution to form a core material solution;
(2) Mixing the core material solution obtained in the step (1) with the wall material solution to obtain a bacterial gel solution;
(3) Dripping CaCl with the mass percent of 2% into the bacterial gel solution obtained in the step (2) by an extrusion method 2 Solidifying the solution to form wet microcapsules.
4. A process according to claim 3, wherein the probiotic bacterial suspension comprises a total bacterial count of 1 x 10 for the bifidobacterium longum subspecies infantis and bifidobacterium animalis subspecies lactis 9 ～1×10 10 CFU/mL。
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