CN111480849A - Composite probiotic microcapsule powder for emulsification and preparation method and application thereof - Google Patents
Composite probiotic microcapsule powder for emulsification and preparation method and application thereof Download PDFInfo
- Publication number
- CN111480849A CN111480849A CN202010258804.7A CN202010258804A CN111480849A CN 111480849 A CN111480849 A CN 111480849A CN 202010258804 A CN202010258804 A CN 202010258804A CN 111480849 A CN111480849 A CN 111480849A
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- China
- Prior art keywords
- powder
- bacterial
- probiotic microcapsule
- microcapsule powder
- parts
- Prior art date
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- Granted
Links
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Images
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
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/19—Dairy proteins
-
- 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
- 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
- A23V2400/11—Lactobacillus
- A23V2400/165—Paracasei
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/21—Streptococcus, lactococcus
- A23V2400/249—Thermophilus
-
- 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
Abstract
The invention discloses a composite probiotic microcapsule powder for mother emulsification, which belongs to the field of food and comprises bifidobacterium animalis subsp lactis I797, lactobacillus paracasei N1115, lactobacillus plantarum N3117 and streptococcus thermophilus JMCC 0003; the raw materials for preparing the effective components of the milk powder comprise soybean oligosaccharide, galacto-oligosaccharide, 3' sialyllactose, maltodextrin, whey protein powder, sodium alginate, gelatin and glycerol; the invention also discloses a preparation method of the compound probiotic microcapsule powder, which comprises the steps of preparing the bacterial powder and the compound probiotic microcapsule powder; the invention also discloses the application of the composite probiotic microcapsule powder which is independently eaten or mixed with the infant formula milk powder to eat the breast emulsion. The product of the invention can promote the colonization of beneficial bacteria, enhance the stability of intestinal flora colonization of infants, solve the intestinal flora colonization fluctuation of infants caused by the difference of delivery modes, and is suitable for infants.
Description
Technical Field
The invention belongs to the field of food, and relates to composite probiotic microcapsule powder, in particular to composite probiotic microcapsule powder for emulsification and a preparation method and application thereof.
Background
Because the metabolic system of the infant is not completely developed, the infant has special requirements on nutrition intake, and breast milk is the most natural and most suitable food in the growth process of the infant. The World Health Organization (WHO) states that infants should be fed pure breast milk 6 months before life in order to achieve optimal growth and development levels and health levels. The breast milk contains abundant nutrient components including lactose, fat, protein, vitamins, minerals, oligosaccharide, immunoglobulin, etc. Moreover, the newborn is breastfed within 1 hour after birth, so that the newborn can be effectively protected from disease infection.
Moreover, breast milk is also able to transport bacteria from the mother, one of the main sources of gastrointestinal flora in infants. Researches show that colostrum contains abundant microorganisms, and the colostrum can promote the establishment of intestinal flora and the development and maturity of immune function of infants by mother-infant vertical transmission through breast feeding.
The infant's intestinal flora is established from the beginning of life and interacts with the development of the nervous system, continuing into adulthood. The intestinal flora development of infants is divided into 3 stages, the development period is less than or equal to 14 months, and bifidobacteria are dominant; the transition period is 15-30 months, and the diversity of the flora is increased; the stationary phase is more than or equal to 31 months, and the firmicutes are dominant.
However, in addition to breast feeding, gut flora establishment and stability in infants is also influenced by a number of factors, such as the mode of delivery, genetic factors, eating habits, etc. Studies have shown that the initial colonization of the intestinal flora of a child born by caesarean section is determined to a large extent by the abundance of microorganisms that the infant comes into contact with from the mother's vagina, skin, etc., whereas the intestinal flora of a child born by caesarean section is largely dependent on the external environment and the microorganisms on the mother's skin, and differs greatly from the intestinal flora of a child born by caesarean section.
However, in the existing technical scheme, no probiotic product capable of being added to infant formula milk powder in an auxiliary manner is provided, so that intestinal flora colonization fluctuation of infants caused by delivery mode difference is improved, and intestinal flora colonization stability of infants is enhanced.
Disclosure of Invention
The invention aims to provide a compound probiotic microcapsule powder for mother emulsification, which aims to solve the problem of intestinal flora colonization fluctuation of infants caused by delivery mode difference;
the invention also aims to provide a preparation method of the composite probiotic microcapsule powder for the emulsification;
still another object of the present invention is to provide the use of the above-mentioned composite probiotic microcapsule powder for emulsification.
In order to achieve the purpose, the invention adopts the technical scheme that:
the raw materials of the compound probiotic microcapsule powder prepared by the present invention comprise bifidobacterium animalis subsp lactis i797, lactobacillus paracasei N1115, lactobacillus plantarum N3117 and streptococcus thermophilus JMCC 0003.
The invention isBifidobacterium animalis subsp. lactis i797 (C.animalis) may be usedBifidobacterium animalis subsp. lactisi797) The culture has been preserved in the general microbiological culture collection center of China general microbiological culture collection management Committee (national patent office assigned patent microbiological Collection center) of Xilu No.1 Hospital No. 3, North Cheng, the area of the rising of Beijing City, 20 days 8.2019, with the preservation numbers: CGMCC No. 18403;
lactobacillus paracasei N1115 (Lactobacillus paracaseiN1115), which has been deposited at 17 days 3/2011 in beijing, at the general microorganism center of the china microorganism culture collection management committee (national patent microorganism collection center designated as patent microorganism collection center) located at north west road No.1 hospital 3, north township, japan, with the collection number: CGMCC No.4691, which is disclosed in Chinese invention patent with the patent number of 201110357058.8 for the first time;
lactobacillus plantarum N3117 (Lactobacillus plantarumN3117) which has been deposited at 5 days 12 months 2014 at the general microorganism center of the china microorganism culture collection management committee (national patent microorganism collection center designated as patent microorganism collection center) located at north west road No.1 hospital 3, north township, tokyo, and the deposit numbers: CGMCC No.10133, which is disclosed in the Chinese invention patent with the patent number of 201510936243.0 for the first time;
streptococcus thermophilus JMCC0003 (Streptococcus thermophilusJMCC 0003) which has been deposited at 15.10.2012 days in the general microbiological culture collection center of the china microbiological culture collection administration (national patent collection center for assigned patent microbiological collection) No. 3, west road No.1, north chen, township, beijing, with the deposit numbers: CGMCC No.6674, which was first disclosed in the Chinese invention patent with patent number 201510938581.8.
A compound probiotic microcapsule powder contains four kinds of probiotic bacteria, including Bifidobacterium animalis subsp lactis i797, Lactobacillus paracasei N1115, Lactobacillus plantarum N3117 and Streptococcus thermophilus JMCC 0003;
the raw materials for preparing the active ingredients of the composite probiotic microcapsule powder comprise, by weight, 1-5 parts of soybean oligosaccharide, 5-10 parts of galactooligosaccharide, 0.1-2 parts of 3' sialyllactose, 5-15 parts of maltodextrin, 5-10 parts of whey protein powder, 1-3 parts of sodium alginate, 1-5 parts of gelatin and 0.1-3 parts of glycerol.
As a limitation of the invention, the raw materials for preparing the effective components of the composite probiotic microcapsule powder for the breast emulsification further comprise 1-10 parts by weight of animal bifidobacterium lactis subsp.lactis i797 bacterial mud, 0.5-5 parts by weight of lactobacillus paracasei N1115 bacterial mud, 0.5-5 parts by weight of lactobacillus plantarum N3117 bacterial mud and 0.5-5 parts by weight of streptococcus thermophilus JMCC0003 bacterial mud, wherein the four bacterial mud respectively contains corresponding probiotics which is more than or equal to 1 × 1011CFU/g。
As another limitation of the invention, the composite probiotic microcapsule powder emulsified by the mother contains bifidobacterium animalis subsp lactis i797 ≥ 1 × 1010CFU/g, lactobacillus paracasei N1115 is more than or equal to 1 × 1010CFU/g, Lactobacillus plantarum N3117 is more than or equal to 1 × 1010CFU/g and Streptococcus thermophilus JMCC0003 ≥ 1 × 1010CFU/g。
The invention also provides a preparation method of the composite probiotic microcapsule powder for emulsification, which comprises the following steps in sequence:
1) preparation of fungal powder
The method comprises the following steps of preparing bifidobacterium animalis subsp lactis i797 bacterial powder, lactobacillus paracasei N1115 bacterial powder, lactobacillus plantarum N3117 bacterial powder and streptococcus thermophilus JMCC0003 bacterial powder, wherein the bifidobacterium animalis subsp lactis i797 bacterial powder, the lactobacillus paracasei N1115 bacterial powder and the streptococcus thermophilus JMCC0003 bacterial powder are respectively prepared by the following operations:
11) uniformly mixing maltodextrin, whey protein powder, sodium alginate, gelatin and glycerol to obtain a mixture A;
12) adding the mixture A into purified water, hydrating and dissolving, homogenizing, and sterilizing to obtain sterile protectant B;
13) respectively mixing the bacterial sludge of the corresponding probiotics with the sterile protective agent B, freeze-drying, crushing and sieving to prepare bacterial powder of the corresponding probiotics;
2) preparing a composite probiotic microcapsule powder which is emulsified by mother:
and (3) mixing the bacterial powder of the four probiotics, namely bifidobacterium animalis subsp lactis i797 bacterial powder, lactobacillus paracasei N1115 bacterial powder, lactobacillus plantarum N3117 bacterial powder and streptococcus thermophilus JMCC0003 bacterial powder, with soybean oligosaccharide, galactooligosaccharide and 3' sialyllactose uniformly to obtain the composite probiotic microcapsule powder emulsified by the mother.
As a limitation of the invention, each bacterial sludge is fermented by corresponding probiotics until the bacterial number is more than or equal to 1 × 1011After CFU/m L, the strain liquid is collected and centrifuged to obtain the strain.
As a further limitation of the present invention, each bacterial sludge is prepared, washed and centrifuged.
As a further limitation of the present invention, the washing process is washing 1-2 times with sterile physiological saline;
the fermentation temperature is 30-42 ℃, and the fermentation time is 36-48 h;
the rotation speed of the centrifugation is 4000-6000 rpm, and the centrifugation time is 8-15 min.
As another limitation of the present invention, in the step 1), the temperature of the hydration dissolution is 35-40 ℃;
the homogenizing temperature is 60-65 ℃;
the sterilization is carried out at the temperature of 110-121 ℃, and the sterilization time is 20-25 min;
in the step 2), the uniformly mixing is to stir and mix for 10-15 min.
As a third limitation of the present invention, in step 1), the weight ratio of each bacterial sludge to the sterility protectant B is 1:1 to 20;
the grain diameter of each bacterial powder is less than or equal to 40 meshes;
in the step 2), the number of live bacteria of each probiotic is kept to be more than or equal to 1 × 10 in the process of uniformly mixing10CFU/g。
The invention also provides an application of the compound probiotics microcapsule powder, and the compound probiotics microcapsule powder can be eaten independently or mixed with infant formula milk powder.
Bifidobacterium animalis subsp lactis i797, which is separated and screened from feces of breast-fed infants or infants, has good effect of regulating intestinal tract, and can regulate intestinal tract flora balance and improve feces characteristics;
the 16SrRNA sequence of the bifidobacterium animalis subsp lactis strain i797 is as follows:
ACGGCTCCCCCACAAGGGTCGGGCCACCGGCTTCGGGTGCTACCCACTTTCATGACTTGACGGGCGGTGTGTACAAGGCCCGGGAACGCATTCACCGCGGCGTTGCTGATCCGCGATTACTAGCGACTCCGCCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGACCGGTTTTCAGCGATCCGCCCCACGTCACCGTGTCGCACCGCGTTGTACCGGCCATTGTAGCATGCGTGAAGCCCTGGACGTAAGGGGCATGATGATCTGACGTCATCCCCACCTTCCTCCGAGTTGACCCCGGCGGTCCCACATGAGTTCCCGGCATCACCCGCTGGCAACATGCGGCGAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTGAACCGGCCCCGAAGGGAAACCGTGTCTCCACGGCGATCCGGCACATGTCAAGCCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCGCATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTTCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGATGCTTAACGCGTTGGCTCCGACACGGGACCCGTGGAAAGGGCCCCACATCCAGCATCCACCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTGACGGCCCAGAGACCTGCCTTCGCCATTGGTGTTCTTCCCGATATCTACACATTCCACCGTTACACCGGGAATTCCAGTCTCCCCTACCGCACTCCAGCCCGCCCGTACCCGGCGCAGATCCACCGTTAGGCGATGGACTTTCACACCGGACGCGACGAACCGCCTACGAGCCCTTTACGCCCAATAAATCCGGATAACGCTCGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTATTCGAACAATCCACTCAACACGGCCGAAACCGTGCCTTGCCCTTGAACAAAAGCGGTTTACAACCCGAAGGCCTCCATCCCGCACGCGGCGTCGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTATCTCAGTCCCAATGTGGCCGGTCACCCTCTCAGGCCGGCTACCCGTCAACGCCTTGGTGGGCCATCACCCCGCCAACAAGCTGATAGGACGCGACCCCATCCCATGCCGCAAAAGCATTTCCCACCCCACCATGCGATGGAGCGGAGCATCCGGTATTACCACCCGTTTCCAGGAGCTATTCCGGTGCACAGGGCAGGTTGGTCACGCATTACTCACCCGTTCGCCACTCTCACCCGACAGCAAGCTGCCAGGGATCCCGTTCGACTGCATGTGTAAG。
the tuf gene sequence is as follows:
GGATCTCGATGAGAGCAGCGTGGTATCACCATCAACATTGCCCACATCGAGTACCAGACGGCCAAGCGTCACTACGCCCACGTCGACTGCCCGGGCCACGCCGACTTCGTGAAGAACATGATCACCGGCGCTGCCCAGATGGATGGCGCCATCCTCGTTGTGGCCGCCACCGACGGCCCGATGGCCCAGACCCGCGAGCACGTGCTGCTCGCCCGTCAGGTCGGCGTCCCGAAGATCCTCGTCGCTCTGAACAAGTGCGATATGGTCGATGACGAAGAGCTCATCGAGCTCGTCGAAGAAGAGGTCCGCGACCTCCTCGACGAGAACGGCTTCGACCGCGACTGCCCGGTCGTGCACACCTCCGCTTACGGCGCTCTGCATGACGACGCTCCCGGATCACGACAAGTGGGTTGCCACCATCAAGGAGCTCATGGACGACGTCGACGAGTACATCCCGACCCCGGTCCACGACCTCGACAAGCCGTTCCTGATGCCGATCGAGGACGTCTTCACCATCTCCGGCCGTGGCACCGTCGTCACCGGTCGTGTCGAGCGCGGCAAGCTGCCGATCAACACGAACGTCGAGATCGTCGGCATCCGCCCGACCCAGACCACCACCGTCACCTCCATCGAGACCTTCCACAAGCAGATGGATGAGTGCGAGGCCGGCGACAACACCGGTCTGCTGCTCCGCGGCATCAACCGCACCGACGTCGAGCGTGGCCAGGTCGTGGCTGCTCCGGGTTCGGTCACCCCGCACACCAAGTTCGAAGGCGAAGTCTACGTCCTTACCAAGGATGAGGGCGGCCGTCACTCGCCGTTCTTCTCGAACTACCGTCCGCAGTTCTACTTCCGCACCACCGACGTCACCGGCGTCATCACGCTGCCGGAAGGCGTCGAGATGGTTCAGCCTGGCGATCACGCGACCTTCACGGTTGAGCTGATCCAGCCGATCGCTATGGAAGAGGGCTTCACCTTCCCAGTGCTTGAAGGC。
compared with the prior art, the invention has the following progress:
the compound probiotic microcapsule powder for the breast emulsification prepared according to the microbial diversity change in the breast milk contains various probiotics, wherein animal bifidobacterium lactis I797 is taken as a main material, lactobacillus paracasei N1115, lactobacillus plantarum N3117 and streptococcus thermophilus JMCC0003 are added, and the beneficial bacterium field planting is promoted by optimizing the proportion, so that the intestinal flora field planting fluctuation of infants caused by the difference of delivery modes is improved, and the intestinal flora field planting stability of the infants is enhanced;
the soybean oligosaccharide is the general term of soluble sugar in soybean, accounts for about 10% of the content of mature soybean, and comprises sucrose, raffinose and stachyose. The soybean oligosaccharide cannot be digested and utilized by human body and some harmful bacteria, but can be utilized by bifidobacterium. The soybean oligosaccharide is added to help bifidobacterium to proliferate in human intestinal tracts;
the 3 'sialyllactose is a sialylated oligosaccharide in breast milk, has a content of about 0.1-0.3 g/L in breast milk, and is relatively stable throughout lactation, and the 3' sialyllactose can support the growth of probiotics such as bifidobacteria in the intestinal tract, has anti-inflammatory properties, can reduce the attachment of conditional pathogenic bacteria in the intestinal tract, and further can promote the development of the nervous system of infants through the gut-brain axis.
The compound probiotic microcapsule powder emulsified by the mother is suitable for infants, and can improve the intestinal flora colonization fluctuation of the infants caused by the difference of delivery modes.
Description of the drawings:
FIG. 1 is a graph showing the effect of bacterial suspension of a complex probiotic mixture on the barrier function of the intestine according to example 8 of the present invention;
FIG. 2 is a graph showing the effect of oligosaccharide mixtures on the proliferation of bifidobacteria enterobacteria in example 9 of the present invention;
FIG. 3 is a graph showing the change in body mass of a newborn mouse in example 10 of the present invention;
FIG. 4 is a graph showing the changes in Bifidobacterium enterobacter in neonatal mouse in example 10 of the present invention.
Detailed Description
The present invention is further illustrated in detail by the following specific examples, which are to be construed as merely illustrative, and not limitative of the remainder of the disclosure.
EXAMPLES 1-6 composite probiotic microcapsule powders for emulsification and preparation thereof
Examples 1 to 6 are composite probiotic microcapsule powders, which are emulsified in a mother, and the active ingredients of which are prepared from the same raw materials, except that the amounts of the raw materials in different examples are different, and the details are shown in table 1:
table 1 raw material ratio summary table
The following is a preparation method of the composite probiotic microcapsule powder emulsified by mother in example 1, which comprises the following steps performed in sequence according to the raw material dosage in example 1:
1) preparing probiotic powder:
the method comprises the following steps of preparing bifidobacterium animalis subsp lactis i797 bacterial powder, lactobacillus paracasei N1115 bacterial powder, lactobacillus plantarum N3117 bacterial powder and streptococcus thermophilus JMCC0003 bacterial powder, wherein the bifidobacterium animalis subsp lactis i797 bacterial powder, the lactobacillus paracasei N1115 bacterial powder and the streptococcus thermophilus JMCC0003 bacterial powder are respectively prepared by the following operations:
11) weighing 5kg of maltodextrin, 10kg of whey protein powder, 3kg of sodium alginate, 2kg of gelatin and 1kg of glycerin, and stirring for 10min at room temperature until the mixture is fully and uniformly mixed to obtain a mixture A;
adding mixture A into 29L purified water, hydrating and dissolving at 35 deg.C, heating to 60 deg.C, homogenizing at 60 deg.C, and sterilizing at 110 deg.C for 20min to obtain 50kg sterile protectant B;
weighing 1kg of bifidobacterium animalis subsp lactis i797 bacterial mud and 20kg of sterile protective agent B, stirring at room temperature for 20min, uniformly mixing, freeze-drying, crushing, and sieving with a 40-mesh sieve to obtain 1kg of bifidobacterium animalis subsp lactis i797 bacterial powder, wherein the particle size of the bacterial powder is less than or equal to 40 meshes;
weighing 0.5kg of lactobacillus paracasei N1115 bacterial mud and 10kg of aseptic protective agent B, stirring for 20min at room temperature, uniformly mixing, freeze-drying, crushing, and sieving with a 40-mesh sieve to obtain 0.5kg of lactobacillus paracasei N1115 bacterial powder, wherein the particle size of the bacterial powder is less than or equal to 40 meshes;
weighing 0.5kg of lactobacillus plantarum N3117 bacterial sludge and 10kg of sterile protective agent B, stirring for 20min at room temperature, uniformly mixing, freeze-drying, crushing, and sieving with a 40-mesh sieve to obtain 0.5kg of lactobacillus plantarum N3117 bacterial powder, wherein the particle size of the bacterial powder is less than or equal to 40 meshes;
weighing 0.5kg of streptococcus thermophilus JMCC0003 bacterial mud and 10kg of aseptic protective agent B, stirring for 20min at room temperature, uniformly mixing, freeze-drying, crushing, and sieving with a 40-mesh sieve to obtain 0.5kg of streptococcus thermophilus JMCC0003 bacterial powder, wherein the particle size of the bacterial powder is less than or equal to 40 meshes.
The preparation process of the bacterial sludge comprises the following steps:
placing Bifidobacterium animalis subsp lactis i797 in a fermenter, fermenting at 35 deg.C for 40 hr until the number of bacteria is not less than 1 × 1011CFU/m L, collecting bacterial liquid, centrifuging at room temperature and 5000rpm for 10min to obtain thallus, washing with sterile normal saline solution at equal amount of thallus for 2 times, centrifuging at room temperature and 5000rpm for 10min to obtain Bifidobacterium animalis subspecies lactis i797 bacterial mud with viable count of 1 × 1011CFU/g；
Placing Lactobacillus paracasei N1115 into a fermentation tank, fermenting and culturing at 35 deg.C for 40h until the number of bacteria is not less than 1 × 1011CFU/m L, collecting bacterial liquid, centrifuging at room temperature and 5000rpm for 10min to obtain thallus, washing with sterile normal saline solution at equal amount of thallus for 2 times, centrifuging at room temperature and 5000rpm for 10min to obtain Lactobacillus paracasei N1115 bacterial mud with viable count of 1 × 1011CFU/g；
Placing Lactobacillus plantarum N3117 in a fermentation tank, fermenting at 35 deg.C for 40 hr until the number of bacteria is not less than 1 × 1011CFU/m L, collecting bacterial liquid, centrifuging at room temperature and 5000rpm for 10min to obtain thallus, washing with sterile normal saline solution for 2 times, centrifuging at room temperature and 5000rpm for 10min to obtain Lactobacillus plantarum N3117 bacterial mud with viable count of 1 × 1011CFU/g；
Placing Streptococcus thermophilus JMCC0003 in a fermentation tank, fermenting and culturing at 35 ℃ for 40h until the bacterial count is more than or equal to 1 × 1011CFU/m L, collecting bacterial liquid, centrifuging at room temperature and 5000rpm for 10min to obtain thallus, washing with sterile normal saline solution at equal amount of thallus for 2 times, centrifuging at room temperature and 5000rpm for 10min to obtain JMCC0003 sludge of Streptococcus thermophilus with viable count of 1 × 1011CFU/g。
2) Preparing a composite probiotic microcapsule powder which is emulsified by mother:
weighing 1kg of bifidobacterium animalis subsp lactis i797 powder, 0.5kg of lactobacillus paracasei N1115 powder, 0.5kg of lactobacillus plantarum N3117 powder, 0.5kg of streptococcus thermophilus JMCC0003 powder, 1kg of soybean oligosaccharide, 8kg of galactooligosaccharide and 0.5kg of 3' sialyllactose, putting into a mixing tank, stirring for 10min at room temperature, uniformly mixing, and keeping the viable count of each probiotic to be not less than 1 × 1010CFU/g, and preparing the compound probiotic microcapsule powder for the mother emulsion.
The following preparation methods of the composite probiotic microcapsule powder emulsified in mother according to examples 2 to 6 respectively are the same as the preparation method of example 1 in terms of the amount of raw materials used in examples 2 to 6 respectively, and are different in the process parameters in the steps, and the specific different process parameters are as follows:
TABLE 2 summary of the different process parameters of examples 2 to 6
The indexes of the composite probiotic microcapsule powder emulsified by the mother prepared in the examples 1 to 6 are shown in the table 3:
TABLE 3 indices of the products
Example 7 acid and bile salt resistance of the composite probiotic microcapsule powder
7 parts of low-pH culture solution (pH = 2.0) and 7 parts of high-bile-salt culture solution (containing 3 per thousand of pig bile salt) which are approximate to the environment of the gastrointestinal tract of a human body are prepared respectively.
1g of the composite probiotic microcapsule powder prepared in the examples 1 to 6 and 1g of the composite probiotic thallus are respectively added to 7 parts of low-pH culture solution which is preheated at 37 ℃ and is similar to the gastrointestinal tract environment of a human body, 9m L is added, and the mixture is incubated for 2 hours;
similarly, 1g of the composite probiotic microcapsule powder prepared in examples 1-6 and 1g of the composite probiotic bacteria were added to 7 parts of 9m L of the high-bile-salt culture solution preheated at 37 ℃ and incubated for 6 hours;
after completion of the incubation, colonies of the 14 mixtures were counted.
After the counting, 14 parts of the mixed solution were dispersed for 60 seconds by a homogenizer, and after shaking slowly for 15min, the 14 parts of the mixed solution were counted again for colonies.
TABLE 4 evaluation results of acid and bile salt resistance
As can be seen from table 4, compared with the non-embedded probiotics, the embedding treatment of the composite probiotic microcapsule powder prepared in examples 1-6 improves the acid and alkali resistance of the probiotics, so that the composite probiotic microcapsule powder prepared by the present invention has a certain acid and alkali resistance, and can better adapt to the gastrointestinal environment of human body.
Example 8 Effect of the composite probiotic microcapsule powder in Breast emulsification on intestinal Barrier function
The intestinal development is very important in the growth process of infants, and provides guarantee for the infants to absorb necessary nutrient substances. Some probiotics can be attached to the intestinal development process of infants, and can maintain the activity of intestinal cells by adjusting the intestinal permeability, so that the tightness among the intestinal cells is maintainedThe purpose of the connecting structure. Based on this, this example employs H2O2An in-vitro Caco-2 cell research model for oxidizing damaged intestinal cells evaluates the influence of the composite probiotics on the barrier function of the intestinal tracts of infants.
Preparation of a bacterial suspension of a complex probiotic mixture of four strains of Bifidobacterium animalis, Lactobacillus paracasei, Lactobacillus plantarum, N3117 and Streptococcus thermophilus, JMCC0003 (final total concentration about 1 × 10)8CFU/well), incubated with the injured in vitro Caco-2 cell study model for 6h, and the transmembrane resistance values were determined at 2h, 4h, and 6h, respectively, and the relative transmembrane resistance value was calculated according to the following formula:
the intercellular tight junction structure is the basis of intestinal mechanical barrier, and in a research model of the Caco-2 cells of Transwell, if the tight junction structure is broken, the resistance to ion flow is reduced, and the transmembrane resistance value is reduced.
The above experimental results, referring to fig. 1, prove that the bacterial suspension of the composite probiotic mixture has a certain maintenance effect on the intercellular tight junction structure, and can alleviate H2O2The intestinal cell is oxidized and damaged, the tight connection structure of the intestinal cell can be maintained and protected, and the healthy development of the intestinal tract of the infant is promoted.
Example 9 oligosaccharides promote intestinal Bifidobacterium proliferation
Selecting 50 Kunming male mice, randomly dividing into 5 groups, performing intragastric gavage on 0.2 m L normal saline every morning in a control group, performing intragastric gavage on 0.2 m L normal saline solution containing 2% oligosaccharide mixture every morning in an intervention group 1-4 groups respectively every morning, continuously performing intragastric gavage culture for 2 weeks, tracking for 1 week, and detecting the amount of bifidobacterium in the feces of the mice on 0 th, 3 th, 5 th, 7 th, 9 th, 11 th, 13 th, 15 th and 21 th days.
Wherein, the oligosaccharide mixture for intragastric administration of the intervention groups 1-4 is added with oligosaccharide with different proportions: the proportion of the intervention group 1 is soybean oligosaccharide: galacto-oligosaccharide: 3' sialyllactose = 1: 1: 1. the proportion of the intervention group 2 is soybean oligosaccharide: galacto-oligosaccharide: 3' sialyllactose = 1: 1: 2. the proportion of the dried group 3 is soybean oligosaccharide: galacto-oligosaccharide: 3' sialyllactose = 2: 1: 2. the proportion of the dried group 4 is soybean oligosaccharide: galacto-oligosaccharide: 3' sialyllactose = 2: 2: 1.
after 2 weeks of intragastric gavage culture and 1 week of tracking detection, the results are shown in fig. 2, and compared with the control group, the oligosaccharide mixture in the results of the intervention group 1-4 groups can promote the proliferation of the intestinal bifidobacteria of the mice. Wherein, the intervention group 3 has the best result, and the addition ratio of the oligosaccharide mixture is soybean oligosaccharide: galacto-oligosaccharide: 3' sialyllactose = 2: 1: 2.
it was thus demonstrated that the addition of galactooligosaccharides and 3' sialyllactose helps to promote the proliferation of bifidobacteria in the intestinal tract.
Example 10 evaluation of the Pre-growth and Bifidobacterium colonization by the composite probiotic microcapsule powder in accordance with the parent emulsion
30 newborn C57B L/6 mice were selected and randomly divided into 2 groups, the control group was gavaged with 20 μ L of physiological saline every morning, and the intervention group was gavaged with 20 μ L of physiological saline solution containing 10 μ g of the composite probiotic microcapsule powder prepared in example 1 every morning, and the gavaged culture was continued for 2 weeks and followed for 1 week, and the body mass was recorded every 2 days.
The initial body mass was set to 1, and then the body mass measured was a multiple of the initial body mass, and the amount of bifidobacteria in the mouse feces was measured on days 0, 3, 5, 7, 9, 11, 13, 15, and 21.
After 2 weeks of intragastric gavage culture and 1 week of tracking detection, the results are shown in fig. 3 and fig. 4, the body mass of the intervention group and the body mass of the control group are both continuously increased, the intervention group is increased faster than the control group, and the content of the bifidobacterium longum in the intestinal tract of the mice in the intervention group is higher than that in the control group.
Therefore, the compound probiotic microcapsule powder emulsified by the mother can promote the growth of newborn mice and the growth and the colonization of intestinal beneficial bacteria, and has beneficial effects on early growth.
Example 11A method for isolation and purification of Bifidobacterium animalis subsp lactis i797
This example provides a method for the isolation and purification of bifidobacterium animalis subsp lactis i797, which comprises the following steps:
firstly, collecting a sample
Taking 1g of intestinal excrement of infants or young children, adding the intestinal excrement into 9ml of physiological saline, and fully and uniformly mixing to obtain a sample A;
second, sample enrichment
Adding 2ml of the sample A into 100m L modified MRS liquid culture medium, and carrying out anaerobic culture at 37 ℃ for 72h to obtain a culture solution B;
thirdly, separating and screening strains
Taking 1ml of culture solution B1, diluting with sterile physiological saline with concentration of 0.9% by gradient multiplication by 10 times, sequentially diluting with gradient 10-1、10-2、10-3、10-4、10-5Doubling, corresponding to obtaining bacterial suspension C11～C15；
Taking the improved MRS solid culture medium, melting, pouring into the first to fifth culture dishes respectively, and obtaining a culture medium D after cooling and complete solidification1～ D5Respectively sucking the bacterial suspension C1～C50.1m L each and applied to the medium D in one-to-one correspondence1～D5Then, inverting the flat plate, placing the flat plate in an environment at 37 ℃ for anaerobic culture for 72 hours, and observing the growth condition of bacterial colonies;
after the plate has the typical bacteria, selecting a corresponding single bacterial colony E according to the bacterial colony characteristics of the standard bifidobacterium and the reference of related literature pictures;
fourthly, strain purification
Selecting a selected single colony E, streaking and inoculating a single colony E culture on an improved MRS solid culture medium, and culturing for 72 hours in an anaerobic environment at 37 ℃ to obtain a single colony F;
continuously streaking and inoculating the single colony F to an improved MRS solid culture medium, and culturing for 72h in an anaerobic environment at 37 ℃ to obtain a single colony G;
then, continuously carrying out streak inoculation on the single colony G on an improved MRS solid culture medium, and carrying out anaerobic environment culture for 72H at the temperature of 37 ℃ to obtain a pure culture H, namely the strain of the bifidobacterium animalis subsp lactis i 797;
fifthly, storing
Mixing the pure culture H and sterile glycerol with the mass portion of 50% according to the proportion of 1:1, placing the mixture into a strain storage tube, uniformly mixing the mixture, storing the mixture at the temperature of 70 ℃ below zero, and simultaneously inoculating a test tube inclined plane of the modified MRS solid culture medium for temporary storage.
In this embodiment, the raw materials of the improved MRS liquid medium include: casein peptone, beef extract, yeast extract, glucose, sodium acetate, diamine citrate, Tween-80, and K2HPO4、MgSO4·7H2O、MnSO4·7H2O, cysteine and distilled water; wherein casein peptone, beef extract, yeast extract, glucose, sodium acetate, citric acid diamine, tween-80, and K2HPO4、MgSO4·7H2O、MnSO4·7H2The dosage proportion relation of O, cysteine and distilled water is 10g, 20g, 5g, 2g, 1g, 2g, 0.2g, 0.05g, 0.5g, 1000m L, and the modified MRS solid culture medium is formed by adding 15 mass percent of agar into each 1000m L modified MRS liquid culture medium.
Example 12 basic bacteriological characteristics of Bifidobacterium animalis strain Lactobacillus subsp.lactis I797
The basic bacteriological characteristics of Bifidobacterium animalis, Lactobacillus subspecies i797, are shown in Table 5:
TABLE 5 basic characteristics of Bifidobacterium animalis lactococcus i797
Example 13 sugar fermentation characteristics of Bifidobacterium animalis subsp
This example shows the sugar fermentation characteristics of Bifidobacterium animalis strain Lactobacillus 797 of example 1. The experimental method for the sugar fermentation characteristics comprises the following steps: selecting a single colony of the bifidobacterium animalis subsp lactis i797 strain, inoculating the single colony into a sterilized improved MRS liquid culture medium, culturing for 24 hours at 37 ℃, inoculating the bacterial suspension into a sugar fermentation tube, performing anaerobic culture for 48 hours at 37 ℃, and observing color change. The results of the identification of the sugar fermentation characteristics are shown in Table 6:
TABLE 6 identification of Bifidobacterium animalis lactococcus i797 sugar fermentation characteristics
Note: "+" indicates fermentation utilization; "-" indicates no fermentative utilization.
The modified MRS liquid medium used in this example has the same composition as the modified MRS liquid medium of example 11.
Example 14 gastric acid and intestinal fluid tolerance Properties of Bifidobacterium animalis strain lactococcus i797
Activating 3 generations of bifidobacterium lactis i797 strain to be tested, taking 1m L, respectively placing in artificial gastric juice with pH value of 3.0 and subjected to filtration sterilization treatment of 9m L, shaking uniformly, carrying out anaerobic culture at 37 ℃, respectively sampling at the beginning of culture and 2h of culture, and measuring the viable count, then taking 1m L of culture solution digested for 2h in the artificial gastric juice with pH value of 3.0, respectively inoculating in artificial intestinal juice with pH value of 8.0 and subjected to filtration sterilization of 9m L, continuously culturing at 37 ℃, and respectively measuring the viable count at 0h, 4h and 6 h.
The control experiment is carried out by taking bifidobacterium BB-12 as a standard strain, and the experimental parameters are the same as those of the bifidobacterium animalis subsp.
Survival (%) = (cfu N1/cfu N0) × 100%
In the formula, N1 represents the viable count of 6 hours after being treated by the artificial digestive juice, and N0 represents the viable count of 0 hour after being treated by the artificial digestive juice.
b. Intestinal juice, Bile Salts (Difco) 0.9 g/100 m L, adjusting pH to 8.0, and filtering and sterilizing for later use.
TABLE 7 Bifidobacterium animalis subsp lactis i797 results in simulated gastric fluid tolerance
Experiments show that BB-12 has strong gastric acid tolerance and poor intestinal juice tolerance, and the bifidobacterium animalis subsp lactis i797 has poor gastric acid tolerance and strong intestinal juice tolerance. Compared with animal bifidobacterium lactis I797, the simulated digestive juice survival rate is better, and 7.4 percent is better than BB-12.
Sequence listing
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Claims (10)
1. A compound probiotics microcapsule powder of emulsification, characterized by that:
the compound probiotic microcapsule powder emulsified by the mother contains four probiotics, namely bifidobacterium animalis subsp lactis i797, lactobacillus paracasei N1115, lactobacillus plantarum N3117 and streptococcus thermophilus JMCC 0003;
the raw materials for preparing the active ingredients of the composite probiotic microcapsule powder comprise, by weight, 1-5 parts of soybean oligosaccharide, 5-10 parts of galactooligosaccharide, 0.1-2 parts of 3' sialyllactose, 5-15 parts of maltodextrin, 5-10 parts of whey protein powder, 1-3 parts of sodium alginate, 1-5 parts of gelatin and 0.1-3 parts of glycerol.
2. The compound probiotic microcapsule powder according to claim 1, wherein the raw materials for preparing the effective components of the compound probiotic microcapsule powder further comprise, by weight, 1-10 parts of bifidobacterium animalis subsp lactis i797 bacterial mud, 0.5-5 parts of lactobacillus paracasei N bacterial mud, 0.5-5 parts of lactobacillus plantarum N3117 bacterial mud and 0.5-5 parts of streptococcus thermophilus JMCC0003 bacterial mud, wherein the four bacterial mud respectively contain corresponding probiotics which are not less than 1 × 1011CFU/g。
3. The composite probiotic microcapsule powder according to claim 1, wherein the bifidobacterium animalis subsp lactis i797 ≥ 1 × 10 is contained in the composite probiotic microcapsule powder10CFU/g, lactobacillus paracasei N1115 is more than or equal to 1 × 1010CFU/g, Lactobacillus plantarum N3117 is more than or equal to 1 × 1010CFU/g and Streptococcus thermophilus JMCC0003 ≥ 1 × 1010CFU/g。
4. A process for the preparation of a composite, emulsified probiotic microcapsule powder according to any one of claims 1 to 3, characterized in that it comprises the following steps carried out in succession:
1) preparation of fungal powder
The method comprises the following steps of preparing bifidobacterium animalis subsp lactis i797 bacterial powder, lactobacillus paracasei N1115 bacterial powder, lactobacillus plantarum N3117 bacterial powder and streptococcus thermophilus JMCC0003 bacterial powder, wherein the bifidobacterium animalis subsp lactis i797 bacterial powder, the lactobacillus paracasei N1115 bacterial powder and the streptococcus thermophilus JMCC0003 bacterial powder are respectively prepared by the following operations:
11) uniformly mixing maltodextrin, whey protein powder, sodium alginate, gelatin and glycerol to obtain a mixture A;
12) adding the mixture A into purified water, hydrating and dissolving, homogenizing, and sterilizing to obtain sterile protectant B;
13) respectively mixing the bacterial sludge of the corresponding probiotics with the sterile protective agent B, freeze-drying, crushing and sieving to prepare bacterial powder of the corresponding probiotics;
2) preparing a composite probiotic microcapsule powder which is emulsified by mother:
and (3) mixing the bacterial powder of the four probiotics, namely bifidobacterium animalis subsp lactis i797 bacterial powder, lactobacillus paracasei N1115 bacterial powder, lactobacillus plantarum N3117 bacterial powder and streptococcus thermophilus JMCC0003 bacterial powder, with soybean oligosaccharide, galactooligosaccharide and 3' sialyllactose uniformly to obtain the composite probiotic microcapsule powder emulsified by the mother.
5. The method for preparing the composite probiotic microcapsule powder according to claim 4, wherein each bacterial sludge is fermented by the corresponding probiotic bacteria to a bacterial count of not less than 1 × 1011After CFU/m L, the strain liquid is collected and centrifuged to obtain the strain.
6. The method for preparing a composite probiotic microcapsule powder according to claim 5, wherein each bacterial sludge is prepared, washed and centrifuged.
7. The method for preparing the composite probiotic microcapsule powder according to claim 6, wherein the washing process is performed 1-2 times with sterile physiological saline;
the fermentation temperature is 30-42 ℃, and the fermentation time is 36-48 h;
the rotation speed of the centrifugation is 4000-6000 rpm, and the centrifugation time is 8-15 min.
8. The method for preparing the composite probiotic microcapsule powder according to claim 4, wherein the probiotic microcapsule powder is prepared by mixing the probiotic micro-capsules,
in the step 1), the temperature of the hydration dissolution is 35-40 ℃;
the homogenizing temperature is 60-65 ℃;
the sterilization temperature is 110-121 ℃, and the sterilization time is 20-25 min;
in the step 2), the uniformly mixing is to stir and mix for 10-15 min.
9. The method for preparing the composite probiotic microcapsule powder according to any one of claims 4 to 8, wherein the weight ratio of each bacterial sludge to the sterile protective agent B in the step 1) is 1:1 to 20;
the grain diameter of each bacterial powder is less than or equal to 40 meshes;
in the step 2), the number of live bacteria of each probiotic is kept to be more than or equal to 1 × 10 in the process of uniformly mixing10CFU/g。
10. Use of the bus-emulsified composite probiotic microcapsule powder according to any one of claims 1 to 3, characterized in that it is intended to be consumed alone or in admixture with infant formula.
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| US20240000868A1 (en) * | 2020-11-16 | 2024-01-04 | Inner Mongolia Yili Industrial Group Co., Ltd. | Composition Containing Bifidobacterium Lactis and Human Milk Oligosaccharides and Application Thereof |
| CN115251393B (en) * | 2022-07-05 | 2024-02-27 | 上海菌小宝健康科技有限公司 | Probiotic microcapsule for improving intestinal flora and preparation method thereof |
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