CN110897166B - Edible composition containing probiotics and casein phosphopeptide with digestion promoting effect - Google Patents
Edible composition containing probiotics and casein phosphopeptide with digestion promoting effect Download PDFInfo
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- CN110897166B CN110897166B CN201910942027.5A CN201910942027A CN110897166B CN 110897166 B CN110897166 B CN 110897166B CN 201910942027 A CN201910942027 A CN 201910942027A CN 110897166 B CN110897166 B CN 110897166B
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- lactobacillus paracasei
- bifidobacterium lactis
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- composition
- probiotics
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Images
Classifications
-
- 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
- A23L33/18—Peptides; Protein hydrolysates
-
- 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
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention provides an edible composition with digestion promoting effect, which contains probiotics and casein phosphopeptide. In the edible composition containing probiotics and casein phosphopeptide, the probiotics comprise bifidobacterium lactis (Bifidobacterium lactis) and lactobacillus paracasei (Lactobacillus paracasei), the bifidobacterium lactis comprises bifidobacterium lactis BL-99, and the lactobacillus paracasei comprises lactobacillus paracasei K56 and lactobacillus paracasei ET-22. The composition of the present invention can be added to various health foods and health foods such as liquid beverages, solid beverages, oral liquids, milk products, tablets or capsules, etc., and can be used for effectively promoting digestion.
Description
Technical Field
The invention mainly relates to an edible composition with digestion promoting effect, in particular to an edible composition containing probiotics and casein phosphopeptide, which can be added into various health foods and health foods such as liquid beverage, solid beverage, oral liquid, dairy products (liquid milk, yoghourt, cheese, milk slices and the like), tablets, capsules and the like.
Background
According to the 2010-2013 Chinese resident nutrition and health monitoring data, the growth retardation rate of children aged 0-5 in the whole country is about 8%, and the growth retardation rate in rural areas reaches 11%; the anemia rate of children aged 0-5 years nationwide is 11%. Another representative national survey shows that children 3-12 years old in China have a variety of micronutrient intake deficiency, for example, dietary calcium intake is over 90%, and vitamin B1\B2\A\C intake is over half. Approximately half of children have a picky eating phenomenon. The children cannot concentrate on complete meals due to specific physiological characteristics such as strong curiosity, poor tolerance, and good mind, and unbalanced nutrient intake and insufficient micronutrient intake in the children caused by the above reasons become one of the main problems affecting the growth and development of the children. For children whose digestive system is still in development stage, digestive dysfunction problems such as dyspepsia, constipation, diarrhea and the like are more frequent occurrence.
At present, dietary intervention and nutrition propaganda are mostly adopted for the measures for overcoming the deficiency of dietary nutrient intake of children, but the measures are required to be put into higher manpower and material resources, are only suitable for local test areas, and have slow effect and high cost for large-scale popularization. Although the nutrient supplement is a measure capable of improving the current state of nutrient intake in children, there are problems of poor adherence and high cost.
Probiotics have effects of regulating intestinal flora of human body and improving constipation, but have few reports on the effects of improving intestinal motility, promoting digestive enzyme activity and improving intestinal tissue development.
Casein phosphopeptide (CPP) is a bioactive peptide derived from milk, and can be combined with divalent mineral ions such as calcium, iron, zinc, selenium and the like in the small intestine environment of animals, so that precipitation is prevented, the concentration of soluble mineral in the intestines is enhanced, and the absorption and utilization of calcium, iron, zinc, selenium, especially calcium, by intestinal mucosa are promoted. In the prior art, the CPP is applied to calcium-supplementing functional foods or health-care products, but the related report of the CPP on digestion promotion function is not found after search.
Disclosure of Invention
It is an object of the present invention to provide an edible composition having digestion promoting effect to prepare a food for consumer's convenience and at the same time having digestion promoting effect.
Another object of the present invention is to provide the use of said edible composition for the preparation of a food product having digestion promoting effects.
Another object of the present invention is to provide a food product having digestion promoting effects.
In one aspect, the invention provides an edible composition comprising a probiotic and a casein phosphopeptide, wherein the probiotic comprises bifidobacterium lactis (Bifidobacterium lactis) and lactobacillus paracasei (Lactobacillus paracasei).
According to a specific embodiment of the invention, in the edible composition comprising probiotics and casein phosphopeptide of the invention, the bifidobacterium lactis comprises bifidobacterium lactis BL-99, and the lactobacillus paracasei comprises lactobacillus paracasei K56 and lactobacillus paracasei ET-22.
According to a specific embodiment of the present invention, in the edible composition of the present invention, bifidobacterium lactis BL-99 is a strain having the accession number CGMCC No.15650 (which is also referred to as BL-99 in the present invention). The strain has gastric acid resistance, the survival rate of viable bacteria is more than 62% when the strain is treated in gastric acid liquid with pH of 2.5 for 30min, and the survival rate of viable bacteria is more than 61% when the strain is treated for 2 hours. The bifidobacterium lactis BL-99 provided by the invention also has intestinal juice resistance, and the survival rate of viable bacteria in small intestinal juice with pH of 6.8 for 2 hours is more than 70%. The mouse experiment shows that the strain has no acute toxicity for oral administration, no antibiotic tolerance and safety and can be used for food processing. The strain is preserved in China general microbiological culture collection center (CGMCC) (address: north West Lu No.1, north West Lu No. 3, china academy of sciences microbiological study) in the Korean area of Beijing) at 2018, and is named after classification: bifidobacterium lactis (Bifidobacterium lactis); the preservation number is CGMCC No.15650.
According to a specific embodiment of the present invention, lactobacillus paracasei K56 is a strain with the accession number CGMCC 15139 or DSM27447 in the edible composition according to the present invention. Lactobacillus paracasei (Lactobacillus paracasei subsp. Paracasei) strain K56 was stored in german collection for microorganisms and cell cultures (German Collection of Microorganisms and Cell Cultures) at month 6 and 27 of 2013 under accession number DSM27447; in addition, lactobacillus paracasei (Lactobacillus paracasei subsp.paracasei) strain K56 was also stored in China general microbiological culture Collection center (CGMCC) (address: north Chen West Lu 1, university of China academy of sciences of China) at 12 and 29 days in 2017: lactobacillus paracasei (Lactobacillus paracasei subsp.paramasasei); the preservation number is CGMCC 15139. In addition, lactobacillus paracasei K56 was deposited in the institute of food industry development for mass legal people (Food Industry Research and Development Institute, FIRDI) in taiwan area at 4/8 of 2014, and deposited under the accession number BCRC 910621.
According to a specific embodiment of the invention, lactobacillus paracasei ET-22 is a strain with the preservation number of CGMCC No.15077 in the edible composition of the invention. The strain is preserved in China general microbiological culture Collection center (CGMCC) (address: national institute of microbiology, national academy of sciences of China, including national academy of sciences of China) at 12 months 18 of 2017: lactobacillus paracasei (Lactobacillus paracasei); the preservation number is CGMCC No.15077.
According to a specific embodiment of the invention, the edible composition of the invention has a weight ratio of probiotics to casein phosphopeptide of 240-700: 0.03 to 50, preferably 293 to 476:0.3 to 30.
Casein phosphopeptide (CPP) is prepared by refining and purifying casein hydrolyzed by trypsin. The molecular structure consists of twenty to thirty amino acid residues, including 4 to 7 phosphate silk acyl groups in clusters. At present, the purity of CPP in the commercially available CPP product is 12% -80%. In the edible composition of the invention, the parts by weight of CPP are calculated by the amount of CPP in the CPP product.
In some embodiments of the invention, the edible composition of the invention has a viable count ratio of bifidobacterium lactis BL-99, lactobacillus paracasei K56, lactobacillus paracasei ET-22 of from 0.01 to 10:0.01 to 20:0.01 to 20, preferably 0.1 to 10:0.1 to 10:0.1 to 10.
In other embodiments of the invention, the edible composition of the invention comprises bifidobacterium lactis BL-99, lactobacillus paracasei K56 and lactobacillus paracasei ET-22 in a weight ratio (based on the weight of the bacterial powder of each bacterium) of 0.01 to 10:0.01 to 20:0.01 to 20, preferably 0.1 to 10:0.1 to 10:0.1 to 10.
According to a specific embodiment of the present invention, in the edible composition of the present invention, each of bifidobacterium lactis BL-99, lactobacillus paracasei K56 and lactobacillus paracasei ET-22 is independently a viable bacterium.
The inventor surprisingly found in the research that the combination of bifidobacterium lactis (Bifidobacterium lactis) BL-99, lactobacillus paracasei (Lactobacillus paracasei) K56 and lactobacillus paracasei (Lactobacillus paracasei) ET-22 with CPP has remarkable digestion promoting effect, and the composition has synergistic effect on the aspects of promoting trypsin activity, promoting intestinal motility and the like. The composition of the invention is an edible composition with digestion promoting effect.
Therefore, on the other hand, the invention also provides application of the probiotic edible composition in preparing food with digestion promoting effect. In particular, the digestion promotion includes promotion of trypsin activity and/or promotion of intestinal motility, etc.
The invention also provides a food with digestion promoting effect, and the food comprises the edible composition.
According to a specific embodiment of the present invention, the food product comprising the edible composition of the present invention may be a liquid beverage, a solid beverage, an oral liquid, a dairy product, a tablet or a capsule.
According to a specific embodiment of the present invention, in the food product comprising the edible composition of the present invention, the edible composition may be used in an amount of 10 in terms of viable bacteria therein 3 CFU～10 11 CFU/day.
The food comprising the edible composition of the present invention has a digestion promoting function due to the inclusion of the edible composition.
In conclusion, the edible composition provided by the invention has the digestion promoting effect, is applied to children foods, and can improve the digestion and absorption functions of children on foods, improve the digestion capacity of children on foods and the absorption and utilization of children on nutrients in meals, so that the situation of insufficient nutrient intake is improved.
Drawings
Figure 1 shows the effect of the edible composition of the present invention on the motility of the small intestine of experimental animals.
Microbial preservation of the patent procedure:
bifidobacterium lactis BL-99 of the present invention:
preservation date: 26 days of 2018, 04 months;
preservation unit: china general microbiological culture Collection center (CGMCC);
deposit unit address: beijing city, the region of Chaoyang, north Chen Xili, no.1, 3, china academy of sciences microbiological institute
Preservation number: CGMCC No.15650;
classification naming: bifidobacterium lactis (Bifidobacterium lactis).
(II) Lactobacillus paracasei ET-22 of the invention:
preservation date: 12 months 18 days 2017;
preservation unit: china general microbiological culture Collection center (CGMCC);
deposit unit address: beijing city, the region of Chaoyang, north Chen Xili, no.1, 3, china academy of sciences microbiological institute
Preservation number: CGMCC No.15077;
classification naming: lactobacillus paracasei (Lactobacillus paracasei).
Detailed Description
In order to more clearly understand the technical features, objects and advantages of the present invention, the technical solutions of the present invention will now be described in detail with reference to specific examples, which should be understood to be only illustrative of the present invention and not limiting the scope of the present invention.
Example 1
1. Bifidobacterium lactis BL-99
The bifidobacterium lactis BL-99 is isolated from the intestinal tract of infants. The strain is preserved in China general microbiological culture collection center (CGMCC) (address: north West Lu No.1, north West Lu No. 3, china academy of sciences microbiological study) in the Korean area of Beijing) at 2018, and is named after classification: bifidobacterium lactis (Bifidobacterium lactis); the preservation number is CGMCC No.15650.
1. Taxonomic characterization of bifidobacterium lactis BL-99
Physical and chemical test results:
2. artificial gastric juice and intestinal juice tolerance of bifidobacterium lactis BL-99
Bifidobacteria are generally non-acid-fast bacteria. In this example, the artificial gastric juice and intestinal juice tolerance of bifidobacterium lactis BL-99 of the present invention was tested, and bifidobacterium lactis BB-12, which can survive through the gastrointestinal tract, was recognized in the current art as excellent in acid resistance
As a comparison.
The testing method comprises the following steps: after culturing bifidobacterium lactis BL-99 strain in MRS liquid culture medium at 37 ℃ for 16 hours, centrifuging at 4 ℃ and 2500rpm for 10min, and collecting thalli.
The strain to be tested is respectively cultured in artificial gastric juice and artificial intestinal juice, and is subjected to viable count analysis after being treated for 0min, 30min and 2h at 37 ℃ so as to evaluate the acid resistance and intestinal juice resistance of the strain according to the survival rate. Survival rate= (number of viable bacteria after treatment/number of viable bacteria at time 0) ×100%.
The detection result of the survival rate of the strain in artificial gastric acid (pH 2.5) is shown in table 1, the survival rate of BB-12 in artificial gastric acid (pH 2.5) is 7.04% when treated for 30min, and the survival rate of BB-12 in artificial gastric acid (pH 2.5) is only 1.64% when treated for 2 hours; whereas the viable bacteria survival rate of the bifidobacterium lactis BL-99 of the invention is 62.60% when being treated in artificial gastric acid (pH 2.5) for 30min, and 61.83% when being treated for 2 hours. The bifidobacterium lactis BL-99 has excellent gastric acid resistance and can smoothly pass through the stomach to reach intestinal tracts to play a probiotic role.
Table 1 survival of the strains in Artificial gastric acid (pH 2.5)
The results of the survival rate detection of the strain in artificial intestinal juice (pH 6.8) are shown in Table 2. The data show that the survival rate of the live bacteria of BB-12 treated in artificial intestinal juice (pH 6.8) for 2 hours is only 28.95 percent; whereas the viable bacteria survival rate of the bifidobacterium lactis BL-99 of the invention is 70.23% after being treated in artificial intestinal juice (pH 6.8) for 2 hours. The bifidobacterium lactis BL-99 has excellent intestinal fluid resistance and can survive and colonise in intestinal tracts.
TABLE 2 survival of strains in Artificial intestinal juice (pH 6.8)
3. Toxicity experiment and safety detection of bifidobacterium lactis BL-99
Inoculating the bifidobacterium lactis BL-99 of the invention into BBL liquid culture medium, anaerobic culturing at 36+/-1 ℃ for 48+/-2 hours, and counting the viable count of the bifidobacterium lactis BL-99 in the culture solution to be 3.7X10 8 cfu/mL, stock solution of the culture and 5-fold concentrated solution were continuously perfused orally at 20.0mL/kg BW for 3 days, and observed for 7 days. The test was performed with medium stock and 5-fold concentrate control. The test results show that: the BBL culture stock and 5-fold concentrate of Bifidobacterium lactis BL-99 had no statistical significance (p > 0.05) on the effect of weight gain in mice compared to the respective control group, while no toxic reaction or death of the tested mice was observed.
The antibiotic susceptibility performance of bifidobacterium lactis BL-99 was evaluated by the method of SN/T1944-2007 determination of bacterial resistance in animals and products thereof. The evaluation results show that the bifidobacterium lactis BL-99 is sensitive to Ampicillin, penicillin G Penicilling, erythromycin, chloramphenicol chlorhenicol, clindamycin, vancomycin, tetracycline and the like. Meets the requirements of European food safety Commission (European Food Safety Authority) on edible bacteria drug resistance evaluation standards. The bifidobacterium lactis BL-99 does not contain exogenous antibiotic resistance genes, and is safe to eat.
2. Lactobacillus paracasei ET-22
The lactobacillus paracasei ET-22 of the invention is preserved in China general microbiological culture Collection center (CGMCC) (address: national institute of microbiology, national academy of sciences of China, including North Chen West Lu No.1 and No. 3) of the Korean area of Beijing) in 12 months and is named after classification: lactobacillus paracasei (Lactobacillus paracasei); the preservation number is CGMCC No.15077.
The taxonomic characteristics of the ET-22 strain were confirmed based on the 16S rDNA sequence analysis and the API bacteria identification system analysis results. The method is characterized by comprising the following steps:
morphological features: 1. when the MRS culture solution is used for culturing, the bacterial cells are in a middle-short rod shape, have round two ends, are usually chain-shaped, and occasionally appear in pairs. 2. Gram-positive bacillus does not generate spores, does not have thixotropic enzyme, oxidase and motility, can grow in aerobic and anaerobic environments, has an optimal growth temperature of 37+/-1 ℃, belongs to facultative heterogeneous fermentation strains, and does not generate gas during glucose metabolism.
The fermentation conditions of the strain are as follows: MRS liquid medium: peptone, 10.0g; beef extract, 10.0g; yeast extract powder, 5.0g; glucose, 20.0g; dipotassium hydrogen phosphate, 5.0g; 2.0g of diammonium hydrogen citrate; sodium acetate, 5.0g; magnesium sulfate heptahydrate, 0.5g; manganese sulfate tetrahydrate, 0.2g; tween 80,1.0g; 15.0g of agar; distilled water 1000mL. Adjusting the pH value to between 6.2 and 6.4, and sterilizing for 15 minutes at the temperature of 121 ℃.
The L.Paracasei ET-22 is microaerobic bacteria, the facultative anaerobic environment grows better, lactic acid is produced, the acid resistance is realized, the acid environment with the pH value of 2.5 and the bile salt environment with the concentration of 0.4% can be resisted for 4 hours, the mesophilic bacteria grow at the temperature ranging from 15 ℃ to 45 ℃, and the optimal growth temperature is about 37 ℃.
Example 2
The components are compounded into a composition according to the following weight: bifidobacterium lactis BL-99 kg (viable count 10) 9 CFU/g); lactobacillus paracasei K56 1kg (viable count 10) 9 CFU/g); lactobacillus paracasei ET22 kg (viable count 10) 9 CFU/g); CPP 40kg (purity about 40%).
The following experiment was performed using the composition prepared in example 2 as a sample:
1. purpose of experiment
The effect of 3 probiotics (BL-99, K56, ET-22) and CPP composition on the digestion and absorption effects of feed nutrients by weaned rats is evaluated by taking weaned rats as model animals, and the effect of composition intervention on food intake, digestive enzyme activity and digestive tract tissue structure is evaluated with great importance.
2. Experimental method
2.1 animal feeding and grouping
40 healthy SPF grade ICR male weaned rats with weight of 83.2-101.5g are fed into animal house of barrier system by Beijing Veitz Lihua laboratory animal technology Co., maintaining indoor temperature 22 deg.C and humidity 10-60%, and are illuminated alternately for 12 hr to eat and drink water freely. The experimental animals were randomly divided into 4 groups of 10 animals each according to body weight. After 1 week of adaptive feeding with large mouse standard feed (feed formula supplied by the company of the synergetics of beijing), the experiment was carried out.
2.2 preparation of gastric lavage samples and intervention
Before daily gastric lavage, respectively dissolving the bacterial powder which is packaged in advance according to the types and the contents of probiotics of each experimental group in 10mL of physiological saline for standby. The standard feed was administered to the blank and probiotic groups, the ad hoc feed (standard feed + casein phosphopeptide) was administered to the CPP and "CPP + probiotic" groups, and the probiotic test samples were administered simultaneously to the probiotic groups and "CPP + probiotic" groups in a gastric lavage manner. Each experimental animal was perfused with 0.5mL, once daily in the morning for 35 consecutive days. The control group was perfused with 0.5mL of physiological saline.
TABLE 3 grouping of experiments
2.3 measurement of weight gain, food utilization, apparent digestibility of protein, and protein utilization
The initial body weight of the rats, the fasting body weight at the end of the experiment, and the body weight 1 time per week during the intervention period (5 times total during the experiment period) were measured. Feed intake was recorded and rat status was observed. And calculating the weight gain, the food utilization rate and the protein utilization rate.
2.4 intestinal digestive enzyme Activity assay
After the experiment is finished, the rats are fasted for 24 hours and are free to drink water, gastric juice discharged in 2 hours is collected by adopting an ether anesthetized rat pylorus ligation method, and the pepsin amount and activity are detected. Small intestine tissues were taken after slaughter and intestinal trypsin and intestinal amylase activities were determined. The activity of pepsin, trypsin and amylase are detected by using a kit produced by Nanjing's established bioengineering research institute, and the operation is performed according to the instruction of the kit.
(1) The method for measuring pepsin comprises the following steps: taking out gastric juice of the mice, carrying out moderate dilution, and temporarily storing in a refrigerator at 4 ℃ for later use. The pepsin assay was performed according to the kit procedure. Firstly, carrying out enzymatic reaction, marking two 1.5mL centrifuge tubes as an empty control tube and a measuring tube, firstly, respectively adding 0.04mL diluted gastric juice into the two centrifuge tubes, preheating for 2min at 37 ℃, then adding 0.4mL of first reagent and 0.2mL of second reagent into the control tube, adding 0.2mL of second reagent into the measuring tube, fully mixing, then incubating for 10min at 37 ℃, adding 0.4mL of first reagent into the measuring tube, fully mixing, incubating for 10min at 37 ℃, centrifuging for 10min at 3500r/min, and taking 0.3mL of supernatant for color reaction. Taking 4 centrifuge tubes of 5mL, respectively marked as a control tube, a measuring tube, a standard tube and a blank tube, adding 0.3mL of supernatant into the control tube and the measuring tube, adding 0.3mL of standard substance application liquid (50 mug/mL) into the standard tube, adding 0.3mL of standard substance diluent into the blank tube, then adding 1.5mL of reagent III and 0.3mL of reagent IV into the four tubes respectively, fully and uniformly mixing, incubating at 37 ℃ for 20min, and measuring the absorbance value (zeroing of distilled water) of each tube at 660nm by using a visible light spectrophotometer. The calculation formula is as follows:
(2) The method for measuring trypsin comprises the following steps: taking out the intestinal juice of the mice, and temporarily storing the intestinal juice in a refrigerator at the temperature of 4 ℃ for standby. The trypsin assay was performed according to the kit procedure. Marking two 5mL centrifuge tubes as a blank tube and a measuring tube, adding 1.5mL trypsin substrate application liquid into the two centrifuge tubes respectively, preheating for 5min at 37 ℃, adding 50 mu L sample homogenate medium into the blank tube, adding 50 mu L intestinal juice into the measuring tube, respectively and rapidly mixing, timing, rapidly pouring into a quartz cuvette, and measuring absorbance value (zeroing by double distilled water) at 253nm by using an ultraviolet spectrophotometer at 30s, and marking as A1; the solution was poured into an original tube and incubated at 37℃for 20min, and then rapidly poured into a quartz cuvette, and its absorbance was measured at 253nm using an ultraviolet spectrophotometer at 20.5min, and was designated as A2. The calculation formula is as follows:
(3) Method for measuring alpha-amylase: taking out the intestinal juice of the mice, carrying out moderate dilution, and temporarily storing in a refrigerator at 4 ℃ for later use. The determination of alpha-amylase was performed according to the kit procedure. Two 5mL centrifuge tubes were labeled as a blank tube and a measurement tube, 0.5mL substrate buffer was added to the two centrifuge tubes, 0.1mL diluted intestinal fluid was added to the measurement tube, the mixture was homogenized, after 7.5 minutes of incubation at 37℃the blank tube and the measurement tube were each added with 0.5mL iodine-applying fluid, 3.1mL double distilled water was added to the blank tube, 3mL double distilled water was added to the measurement tube, the mixture was homogenized again, and the absorbance values of the two tubes were measured at 660nm with a visible light spectrophotometer (double distilled water zeroing). The calculation formula is as follows:
2.5 rats intestinal tissue morphology index
After the experiment is finished, the animals are fasted for 24 hours and are free to drink water, and small intestine (jejunum and ileum), anterior large intestine and posterior large intestine tissues are taken for slaughtering all the animals. Large and small intestine sections were stained and morphologically observed using software Image-Pro Plus 6.0, including intestinal villus height and intestinal villus surface smoothness.
2.6 statistical analysis method
Unless otherwise noted, experimental results were expressed as mean ± standard deviation
To represent. Statistical analysis of experimental data using SPSS16.0 software, homogeneous test of data using single-factor analysis of variance, and comparison between groups of data using Duncan's method in single-factor analysis of variance (one-way ANOVA), with significance level set at P<0.05。
3. Experimental results
3.1 Effect of the composition on weight gain, food intake, food utilization and protein utilization in laboratory animals
During the experimental period, the animals are characterized as normal, have good mental and activity conditions, and do not have any adverse reaction after daily administration of the test substance. Compared with the control group, each intervention group has no obvious difference in weight gain, food intake, food utilization, apparent digestibility of protein and protein utilization.
3.2 Effect of compositions on intestinal digestive enzyme Activity
As shown in table 4, trypsin activity was significantly higher in the "CPP + probiotic" group than in the control group, as well as significantly higher in the CPP and probiotic groups (p < 0.05). The trypsin activity of the CPP alone and the probiotic alone was not significantly higher than that of the control group, indicating that the combination of CPP and probiotic has a synergistic effect, synergistically promoting trypsin activity. The pepsin and amylopsin activities of each experimental group were not significantly different (p > 0.05) from those of the control group.
The results show that the CPP+probiotics group has the effect of promoting the intestinal digestive enzyme activity of mice, so that the intestinal digestive function is promoted.
TABLE 4 influence of probiotics on the digestive enzyme Activity of rats
| Group of | Trypsin (U/ml) |
| Control | 652.72±145.02abc |
| CPP | 724.81±138.18bcd |
| BL-99 | 840.44±139.81d |
| K56 | 607.94±56.76ab |
| ET-22 | 776.72±56.00cd |
| Composite probiotics | 628.86±67.87ab |
| CPP+probiotic combinations | 1126.30±150.46e |
Note that: different lower case letters in the same column represent significant differences (P < 0.05)
3.3 Effect of the composition on the morphology of intestinal tissue of laboratory animals
As shown in table 5, the ileal villi height of the cpp+ probiotic group was significantly higher than the control group, and was also highest in each treatment group. The jejunum villus height of the composite probiotic group is significantly higher than any single probiotic group. The jejunum and ileum villus surface smoothness of the CPP+ probiotic group is higher than that of the control group, which shows that the CPP+ probiotic group has the strongest effect of improving the small intestine tissue morphology structure, and the higher the villus height and the higher the villus surface smoothness, the stronger the small intestine absorption capacity.
TABLE 5 influence of the compositions on the morphology of the intestinal tissues of rats
| Jejunum villus height | Ileum villus height | |
| Control | 359.67±32.85ab | 255.18±34.99a |
| Composite probiotics | 421.17±34.22c | 276.13±9.78abc |
| CPP | 349.23±43.35a | 287.17±18.28bc |
| CPP+probiotic | 369.08±31.34ab | 294.12±26.32c |
The difference between different lower case letters in the same column is significant (p < 0.05).
The results of example 2 are combined, and the trypsin activity of the "probiotic+CPP" composition intervention group is obviously higher than that of the control group, and is also obviously higher than that of the CPP group and the probiotic group (p is less than 0.05), so that the combination of CPP and probiotics has a synergistic effect, and the activity of trypsin is synergistically promoted. The ileal villus height of the CPP+ probiotic group was significantly higher than that of the control group, and was highest in each treatment group. The more powerful the CPP+ probiotic group is to improve small intestine absorption.
Example 3
1. Purpose of experiment
The effect of 3 probiotic (BL-99, K56, ET-22) and CPP compositions on the intestinal motility of weaned mice was evaluated using weaned mice as model animals.
2. Experimental method
2.1 animal feeding and grouping
50 healthy SPF grade ICR male weaned mice with weight of 11.6-19.5g are fed into animal house of barrier system, maintaining indoor temperature 22 deg.C and humidity 10-60%, and lighting alternately for 12 hr to eat and drink water freely. The experimental animals were randomly divided into 5 groups of 10 animals each according to body weight. After 1 week of feeding with large and small mouse standard feeds (feed formula is provided by Beijing ao Kogyo feed Co., ltd.), the final experiment was entered.
2.2 preparation of gastric lavage samples and intervention
Before daily gastric lavage, respectively dissolving the bacterial powder which is packaged in advance according to the types and the contents of probiotics of each experimental group in 10mL of physiological saline for standby. The standard feed, the CPP group and the "cpp+probiotic" group were administered to the blank control group, the model control group and the probiotic group, the special feed (standard feed+casein phosphopeptide) was administered to the "cpp+probiotic" group, and the probiotic test sample was administered simultaneously in a gastric lavage manner to the "cpp+probiotic" group. Each experimental animal was perfused with 0.5mL, once daily in the morning for 35 consecutive days. The control group was perfused with 0.5mL of physiological saline.
TABLE 6 grouping of experiments
2.3 small intestine movement test
After the 30 th day is finished, the small intestine exercise experiment is carried out, and the small intestine exercise experiment is fasted for 16 hours before the experiment, and the small intestine exercise experiment is free to drink water. The test sample was administered on the day of the assay, and after 30min the model control and each experimental group were filled with the compound diphenoxylate (0.025%), and the blank control was filled with the same volume of distilled water. After 30min of compound diphenoxylate, each group was filled with stomach ink (5% active carbon powder, 10% gum arabic). After 25min of ink supply, the mice were sacrificed by cervical dislocation, the abdominal cavity was opened, and the upper end was cut to the pylorus and the lower end to the intestinal canal of the ileocecum. The small intestine was pulled in a straight line, the length of the intestine was measured as "total length of small intestine", and the distance from the pylorus to the leading edge of the carbon powder was measured as "distance of advance of the carbon powder in the intestine", and the percentage of advance of the carbon powder was calculated as follows.
Ink advance = ink advance length (cm)/small intestine total length (cm) ×100%
2.4 statistical analysis method
Unless otherwise noted, experimental results were expressed as mean ± standard deviation
To represent. Statistical analysis of experimental data using SPSS16.0 software, homogeneous test of data using single-factor analysis of variance, and comparison between groups of data using Duncan's method in single-factor analysis of variance (one-way ANOVA), with significance level set at P<0.05。
3. Experimental results
As a result, referring to fig. 1, the ink propulsion rate of the mice with the probiotics K56 and the probiotics ET-22, the compound probiotics group and the cpp+ probiotics group is significantly increased (p < 0.05), which indicates that the probiotics K56, the probiotics ET-22, the compound probiotics group and the "cpp+ probiotics" group can promote intestinal tract movement of the mice, and the compound probiotics group has the intestinal tract movement promotion ability which is higher than that of the probiotics group alone, and the cpp+ probiotics group has the intestinal tract movement promotion ability which is higher than that of the probiotics group alone and the CPP group alone.
By combining the results of example 2 and example 3, the probiotic and CPP compositions exhibit synergistic effects in significantly improving digestive enzyme activity, enhancing intestinal motility, and enhancing absorption capacity by improving intestinal tissue morphology in experimental animals.
Claims (19)
1. An edible composition comprising a probiotic and casein phosphopeptide, wherein the probiotic comprises bifidobacterium lactisBifidobacterium lactis) Lactobacillus paracasei @Lactobacillus paracasei）；
Wherein the Lactobacillus bifidus comprises Lactobacillus bifidus BL-99, and the Lactobacillus paracasei comprises Lactobacillus paracasei K56 and Lactobacillus paracasei ET-22; the bifidobacterium lactis BL-99 is a strain with a preservation number of CGMCC No.15650, the lactobacillus paracasei K56 is a strain with a preservation number of CGMCC 15139 or DSM27447, and the lactobacillus paracasei ET-22 is a strain with a preservation number of CGMCC No.15077.
2. The composition of claim 1, wherein the weight ratio of probiotics to casein phosphopeptide is 240-700: 0.03-50.
3. The composition of claim 2, wherein the weight ratio of probiotics to casein phosphopeptide is 293-476: 0.3 to 30.
4. A composition according to claim 1, 2 or 3, wherein the ratio of viable count of bifidobacterium lactis BL-99, lactobacillus paracasei K56, lactobacillus paracasei ET-22 is 0.01 to 10: 0.01-20: 0.01-20.
5. The composition according to claim 4, wherein the ratio of viable count of bifidobacterium lactis BL-99, lactobacillus paracasei K56 and lactobacillus paracasei ET-22 is 0.1 to 10: 0.1-10: 0.1 to 10.
6. A composition according to claim 1, 2 or 3, wherein the weight ratio of bifidobacterium lactis BL-99, lactobacillus paracasei K56, lactobacillus paracasei ET-22 is 0.01-10: 0.01-20: 0.01-20.
7. The composition of claim 6, wherein the weight ratio of bifidobacterium lactis BL-99, lactobacillus paracasei K56 and lactobacillus paracasei ET-22 is 0.1-10: 0.1-10: 0.1 to 10.
8. The composition of claim 1, wherein bifidobacterium lactis BL-99, lactobacillus paracasei K56, lactobacillus paracasei ET-22 are each independently viable bacteria.
9. Use of the composition according to any one of claims 1 to 8 for preparing a food product having digestion promoting effect.
10. The use according to claim 9, wherein the digestion promotion comprises promotion of trypsin activity and/or promotion of intestinal motility.
11. Use according to claim 9, wherein the food product is a liquid beverage or a solid beverage.
12. The use according to claim 9, wherein the food product is an oral liquid.
13. Use according to claim 9, wherein the food product is a dairy product.
14. The use according to claim 9, wherein the food product is a tablet or capsule.
15. A food product having digestion promoting effect, the food product comprising the probiotic composition of any one of claims 1 to 8 in its raw material composition.
16. The food product of claim 15, wherein the food product is a liquid beverage or a solid beverage.
17. The food product of claim 15, wherein the food product is an oral liquid.
18. The food product of claim 15, wherein the food product is a dairy product.
19. The food product of claim 15, wherein the food product is a tablet or capsule.
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