CN110897166A - Edible composition containing probiotics and casein phosphopeptides and having digestion promoting effect - Google Patents
Edible composition containing probiotics and casein phosphopeptides and having digestion promoting effect Download PDFInfo
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- CN110897166A CN110897166A CN201910942027.5A CN201910942027A CN110897166A CN 110897166 A CN110897166 A CN 110897166A CN 201910942027 A CN201910942027 A CN 201910942027A CN 110897166 A CN110897166 A CN 110897166A
- Authority
- CN
- China
- Prior art keywords
- lactobacillus paracasei
- bifidobacterium lactis
- probiotic
- edible composition
- cpp
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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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 containing probiotics and casein phosphopeptides, which has the digestion promoting effect. In the edible composition containing probiotics and casein phosphopeptides, 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 into 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 phosphopeptides, which can effectively promote digestion, and can be added into various health foods and health foods such as liquid beverages, solid beverages, oral liquids, milk products (liquid milk, yoghourt, cheese, milk slices and the like), tablets, capsules and the like.
Background
According to 2010-2013 Chinese resident nutrition and health monitoring data, the growth retardation rate of children in 0-5 years in China is about 8%, and the growth retardation rate in rural areas reaches 11%; the anemia rate of children aged 0-5 years in China is 11%. Another representative national survey shows that children aged 3-12 years in China have inadequate intake of various micronutrients, such as dietary calcium intake of more than 90%, and inadequate intake rates of more than half of all vitamins B1\ B2\ A \ C. Approximately half of children have the phenomenon of food pickings. In the infant stage, people cannot eat fully with concentrated energy due to specific physiological characteristics such as strong curiosity, insufficient patience, hyperactivity and the like, and unbalanced nutrient intake and insufficient micronutrient intake in the child stage are one of the main problems influencing the growth and development of children due to the various reasons. In children whose digestive systems are still in development, problems of digestive disorders such as dyspepsia, constipation, and diarrhea are more frequent.
At present, dietary intervention and nutrition propaganda and education are mostly adopted for making up measures for insufficient intake of dietary nutrients of children, but the measures all need to invest higher manpower and material resources, are only suitable for local trial areas, and are slow in effect and high in cost for large-scale popularization. Although the enriched nutrient supplement is a measure capable of improving the current status of nutrient intake of children, it has problems of poor adherence and high cost.
The probiotics have the effects of regulating intestinal flora of human bodies and improving constipation, but the effects of improving the intestinal motility and promoting the activity of digestive enzymes and improving the development of intestinal tissues are rarely reported.
Casein phosphopeptide (CPP) is a milk-derived bioactive peptide, can be combined with divalent mineral ions such as calcium, iron, zinc, selenium and the like in the small intestine environment of animals, prevents the generation of precipitates, and enhances the concentration of soluble minerals in the intestines, thereby promoting the absorption and utilization of calcium, iron, zinc and selenium, particularly calcium, by intestinal mucosa. In the prior art, the CPP is applied to functional food or health care products for calcium supplement, but no report about the digestion promoting function of the CPP is found through retrieval.
Disclosure of Invention
The invention aims to provide an edible composition with digestion promoting effect, so that the edible composition can be prepared into food for consumers to conveniently eat, and has the digestion promoting effect.
The invention also aims to provide application of the edible composition in preparing food with digestion promoting effect.
Another object of the present invention is to provide a food product having digestion promoting effect.
In one aspect, the present invention provides an edible composition comprising a probiotic and 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 containing probiotics and casein phosphopeptides, the bifidobacterium lactis comprises bifidobacterium lactis BL-99, 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 a preservation number of CGMCC No.15650 (also referred to as BL-99 herein). The strain has gastric acid resistance, and 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 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 is more than 70% after being treated in small intestinal juice with pH of 6.8 for 2 hours. Mouse experiments show that the strain has no oral acute toxicity, no antibiotic tolerance and safety and can be used for food processing. The strain has been preserved in China general microbiological culture Collection center (CGMCC) (address: No. 3 Xilu-Beijing province No.1, Beijing Korean district, Ministry of China microbiology institute) 26.04.2018, and is named after classification: bifidobacterium lactis (Bifidobacterium lactis); the preservation number is CGMCC number 15650.
According to a particular embodiment of the invention, the edible composition according to the invention, the lactobacillus paracasei K56 is a strain with a preservation number of CGMCC 15139 or DSM 27447. Lactobacillus paracasei (Lactobacillus paracasei) K56 strain has been deposited at the German Collection of Microorganisms and Cell Cultures (German Collection of Microorganisms and Cell Cultures) at 27 days 6 months in 2013 under accession No. DSM 27447; in addition, the strain Lactobacillus paracasei (Lactobacillus paracasei subsp. paracasei) K56 has also been stored in the china microbiological culture collection center CGMCC (address: north american society No.1, 3, institute of microbiology, china academy of sciences) at 29.12.2017, under the classification name: lactobacillus paracasei (Lactobacillus paracasei subsp. paracasei); the preservation number is CGMCC 15139. In addition, lactobacillus paracasei K56 was deposited in the center of the Food industry Development research Institute (Food industry research and Development Institute, FIRDI) in 2014, 4, 8, with accession number BCRC 910621.
According to a particular embodiment of the invention, in the edible composition according to the invention, Lactobacillus paracasei ET-22 is a strain with a accession number CGMCC No. 15077. The strain has been preserved in China general microbiological culture Collection center (CGMCC) for 12 months and 18 days in 2017 (address: Xilu No.1 Hospital No. 3, Ministry of China academy of sciences, North Cheng, south China), and is named after classification: lactobacillus paracasei (Lactobacillus paracasei); the preservation number is CGMCC No. 15077.
According to a specific embodiment of the invention, in the edible composition, the weight ratio of the probiotics to the casein phosphopeptides is 240-700: 0.03 to 50, preferably 293 to 476: 0.3 to 30.
Casein phosphopeptide (CPP) is prepared by hydrolyzing casein with trypsin, refining, and purifying. The molecular structure is composed of twenty to thirty amino acid residues, wherein the molecular structure comprises 4-7 phosphoric acid serine acyl groups which exist in clusters. At present, the purity of CPP in a commercial CPP product is 12-80%. In the edible composition of the invention, the weight parts of the CPP are calculated by the amount of the CPP in the CPP product.
In some embodiments of the present invention, the ratio of viable count of bifidobacterium lactis BL-99, lactobacillus paracasei K56 and lactobacillus paracasei ET-22 in the edible composition of the present invention is 0.01-10: 0.01-20: 0.01 to 20, preferably 0.1 to 10: 0.1-10: 0.1 to 10.
In other embodiments of the present invention, the edible composition of the present invention comprises bifidobacterium lactis BL-99, lactobacillus paracasei K56 and lactobacillus paracasei ET-22 in a ratio of 0.01 to 10 by weight (based on the weight of the bacterial powder of each bacterium): 0.01-20: 0.01 to 20, preferably 0.1 to 10: 0.1-10: 0.1 to 10.
According to a specific embodiment of the invention, in the edible composition of the invention, bifidobacterium lactis BL-99, lactobacillus paracasei K56 and lactobacillus paracasei ET-22 are each independently live bacteria.
The inventor of the present invention surprisingly found in research that by combining Bifidobacterium lactis (BL-99), Lactobacillus paracasei (Lactobacillus paracasei) K56 and Lactobacillus paracasei (ET-22) with CPP, the obtained composition has a significant digestion promoting effect, and the composition has a synergistic effect on the aspects of promoting trypsin activity, promoting intestinal motility and the like. The composition is an edible composition with digestion promoting efficacy.
Therefore, in another aspect, the invention also provides the application of the probiotic edible composition in preparing food with digestion promoting efficacy. In particular, said digestion promoting includes promoting trypsin activity and/or promoting intestinal motility, etc.
The invention also provides a food with digestion promoting effect, and the raw material composition of 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 particular embodiment of the invention, the food product comprising the edible composition according to the invention comprises the edible composition applied to the food product in an amount calculated as the number of viable bacteria thereinIs 103CFU～1011CFU/day.
The food comprising the edible composition has digestion promoting function due to the edible composition.
In conclusion, the edible composition has the digestion promoting effect, and can be applied to children food, so that the digestion and absorption functions of the children can be promoted, the digestion capability of the children on food can be improved, the absorption and utilization of nutrients in diet of the children can be improved, and the current situation of insufficient nutrient intake can be further improved.
Drawings
Figure 1 shows the effect of the edible composition of the invention on small intestine motility in experimental animals.
Microbial preservation of the patent procedure:
(one) Bifidobacterium lactis BL-99 of the present invention:
the preservation date is as follows: 26/04/2018;
the preservation unit: china general microbiological culture Collection center (CGMCC);
the address of the depository: xilu No.1 Hospital No. 3, the institute of microbiology, China academy of sciences, Beijing, Chaoyang
The preservation number is: CGMCC No. 15650;
and (3) classification and naming: bifidobacterium lactis (Bifidobacterium lactis).
(II) Lactobacillus paracasei ET-22 of the present invention:
the preservation date is as follows: 12 months and 18 days 2017;
the preservation unit: china general microbiological culture Collection center (CGMCC);
the address of the depository: xilu No.1 Hospital No. 3, the institute of microbiology, China academy of sciences, Beijing, Chaoyang
The preservation number is: CGMCC No. 15077;
and (3) classification and naming: lactobacillus paracasei (Lactobacillus paracasei).
Detailed Description
For a more clear understanding of the technical features, objects and advantages of the present invention, reference is now made to the following detailed description of the technical aspects of the present invention with reference to specific examples, which are intended to illustrate the present invention and not to limit the scope of the present invention.
Example 1
Bifidobacterium lactis BL-99
The bifidobacterium lactis BL-99 is separated from the intestinal tract of the infant. The strain has been preserved in China general microbiological culture Collection center (CGMCC) (address: No. 3 Xilu-Beijing province No.1, Beijing Korean district, Ministry of China microbiology institute) 26.04.2018, and is named after classification: bifidobacterium lactis (Bifidobacterium lactis); the preservation number is CGMCC No. 15650.
1. Taxonomical characterization of Bifidobacterium lactis BL-99
The results of the physical and chemical tests are as follows:
2. tolerance of bifidobacterium lactis BL-99 to artificial gastric juice and intestinal juice
Bifidobacteria are genera that are generally not acid-fast. In this example, the tolerance of the bifidobacterium lactis BL-99 of the present invention to artificial gastric juice and intestinal juice was tested, and bifidobacterium lactis which is known in the art to have excellent acid resistance and can survive through the gastrointestinal tract
For comparison.
The test method comprises the following steps: culturing Bifidobacterium lactis BL-99 strain in MRS liquid culture medium at 37 deg.C for 16 hr, centrifuging at 4 deg.C and 2500rpm for 10min, and collecting thallus.
Respectively culturing the strains to be tested in artificial gastric juice and artificial small intestinal juice, processing at 37 ℃ for 0, 30min and 2h, and then performing viable count analysis to evaluate the acid resistance and intestinal juice resistance of the strains according to the survival rate. Survival rate (viable cell count after treatment/viable cell count at time 0) × 100%.
The survival rate detection result of the bacterial strain in artificial gastric acid (pH2.5) is shown in Table 1, the survival rate of the viable bacteria is 7.04% when BB-12 is treated in the artificial gastric acid (pH2.5) for 30min, and the survival rate of the viable bacteria is only 1.64% after 2 hours of treatment; the survival rate of the live bacteria of the bifidobacterium lactis BL-99 is 62.60 percent when the bifidobacterium lactis BL-99 is treated in artificial gastric acid (pH2.5) for 30min, and the survival rate of the live bacteria is 61.83 percent when the bifidobacterium lactis BL-99 is treated for 2 hours. The bifidobacterium lactis BL-99 disclosed by the invention has excellent gastric acid resistance and can smoothly pass through the stomach to reach the intestinal tract to play a probiotic role.
TABLE 1 survival rate of the strains in artificial gastric acid (pH2.5)
The survival rate of the strain in the artificial small intestine solution (pH6.8) is tested and shown in Table 2. The data show that the viable bacteria survival rate of BB-12 in artificial small intestine solution (pH6.8) for 2 hours is only 28.95%; the viable bacteria survival rate of the bifidobacterium lactis BL-99 is 70.23 percent when the bifidobacterium lactis BL-99 is treated in artificial gastric acid (pH2.5) for 2 hours. The bifidobacterium lactis BL-99 disclosed by the invention has excellent intestinal juice resistance and can survive and colonize in intestinal tracts.
TABLE 2 survival rate of the strains in artificial intestinal juice (pH6.8)
3. Toxicity experiment and safety detection of bifidobacterium lactis BL-99
Inoculating the bifidobacterium lactis BL-99 of the invention into a BBL liquid culture medium, carrying out anaerobic culture for 48 +/-2 hours at 36 +/-1 ℃, and counting the viable count of the bifidobacterium lactis BL-99 in the culture solution to be 3.7 multiplied by 108cfu/mL, stock and 5-fold concentrate of the culture were continuously gavaged to the test mice at 20.0mL/kg BW for 3 days and observed for 7 days. The experiment was performed with a control group of 5-fold concentrated solution and a stock solution of the medium. The test result shows that: the BBL culture stock solution and 5-fold concentrated solution of Bifidobacterium lactis BL-99 had no statistical effect on the weight gain of mice (p > 0.05) compared with the respective control group, and no toxic reaction or death of the tested mice was observed.
The antibiotic sensitivity of the bifidobacterium lactis BL-99 is evaluated by adopting an SN/T1944-2007 method of determination of bacterial resistance in animals and products thereof. The evaluation results show that the bifidobacterium lactis BL-99 is sensitive to Ampicillin Ampicillin, penicillin G Penicillin G, Erythromycin Erythromycin, Chloramphenicol Chloramphenicol, Clindamycin Clindamycin, Vancomycin Vancomycin, Tetracycline and the like. Meets the requirements of European Food Safety Authority (European Food Safety Authority) on the evaluation specification of the resistance of the edible bacteria. Bifidobacterium lactis BL-99 does not contain exogenous antibiotic drug resistance gene, and is safe to eat.
Lactobacillus paracasei ET-22
The lactobacillus paracasei ET-22 has been preserved in China general microbiological culture Collection center (CGMCC) (the address: No. 3 Xilu No.1 of Kyoho, Beijing City, Chaoyang, the institute of microbiology, Chinese academy of sciences) for 18 days at 12 months and 18 days in 2017, and is named after classification: lactobacillus paracasei (Lactobacillus paracasei); the preservation number is CGMCC No. 15077.
The characteristics of the ET-22 strain in taxonomy were confirmed based on the results of 16S rDNA sequence analysis and analysis by the API bacterial identification system. The method comprises the following specific steps:
morphological characteristics: 1. when the strain is cultured in an MRS culture solution, the strain is in a medium-short rod shape, two ends of the strain are in a circular shape, and the strain is usually in a chain shape and occasionally appears in pairs. 2. Gram-positive bacillus can grow in aerobic and anaerobic environment without producing spore, catalase, oxidase and motility, has optimum growth temperature of 37 +/-1 ℃, belongs to facultative heterofermentation strain and does not produce gas during glucose metabolism.
The fermentation conditions of the strain are as follows: MRS liquid medium: peptone, 10.0 g; 10.0g of beef extract; 5.0g of yeast extract powder; glucose, 20.0 g; dipotassium hydrogen phosphate, 5.0 g; diammonium hydrogen citrate, 2.0 g; sodium acetate, 5.0 g; magnesium sulfate heptahydrate, 0.5 g; 0.2g of manganese sulfate tetrahydrate; tween 80, 1.0 g; 15.0g of agar; 1000mL of distilled water. Adjusting the pH value to 6.2-6.4, and sterilizing for 15 minutes at 121 ℃.
L. paracasei ET-22 is a microaerophilic bacterium, grows well in a facultative anaerobic environment, produces lactic acid, has acid resistance, can resist an acid environment with a pH value of 2.5 and a 0.4% bile salt environment for 4 hours, is mesophilic, has a growth temperature range of 15-45 ℃, and has an optimal growth temperature of about 37 ℃.
Example 2
The components are compounded into a composition according to the following weight percentage: bifidobacterium lactis BL-991 kg (viable count 10)9CFU/g); lactobacillus paracasei K561 kg (viable count 10)9CFU/g); lactobacillus paracasei ET 221 kg (viable count 10)9CFU/g); CPP 40kg (purity about 40%).
The following experiments were carried out using the composition prepared in example 2 as a sample:
1. purpose of experiment
The influence of 3 probiotics (BL-99, K56 and ET-22) and CPP compositions on the digestion and absorption efficacy of feed nutrients of weaned rats is evaluated by taking weaned rats as model animals, and the influence of composition intervention on food intake, digestive enzyme activity and digestive tract tissue structure is mainly evaluated.
2. Experimental methods
2.1 animal feeding and grouping
40 healthy SPF-grade ICR males, weaned at 21 weeks, weighed 83.2-101.5g, were housed in a barrier animal house maintained at 22 deg.C and 10-60% humidity in the house, and were exposed to 12 hours of alternating light and dark illumination, and were allowed to freely eat and drink water, provided by Beijing Wintolite laboratory animal technology, Inc. The experimental animals were randomly divided into 4 groups of 10 animals each according to body weight. After the rats are fed with standard feed (the feed formula is provided by Olympic feed Co., Ltd. of Beijing Ke) for 1 week, formal experiments are carried out.
2.2 intragastric sample preparation and intervention
The preparation method comprises the steps of preparing the probiotic powder before daily gavage, and 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 normal saline for later use. The blank control group and the probiotic group are given standard feed, the CPP group and the CPP + probiotic group are given special feed (standard feed + casein phosphopeptide), and the probiotic group and the CPP + probiotic group are simultaneously given a probiotic test sample in a gastric perfusion mode. Each experimental animal was gavaged with 0.5mL once a day in the morning for 35 consecutive days. The control group was gavaged with 0.5mL of physiological saline.
TABLE 3 Experimental groups
2.3 body weight gain, food utilization, apparent protein digestibility, protein utilization measurements
Initial body weights were measured in rats, fasting body weights at the end of the experiment and 1 body weight per week for the dry expectation (5 total measurements during the experimental period). The feed intake was recorded and the rat status was observed. Calculating weight gain, food utilization rate and protein utilization rate.
2.4 detection of intestinal digestive enzyme Activity
After the experiment is finished, the patient is fasted for 24 hours, water is freely drunk, gastric juice discharged within 2 hours is collected by adopting a pyloric ligation method of ether-anesthetized rats, and the amount and the activity of pepsin are detected. After slaughter, intestinal tissue was taken and intestinal trypsin and intestinal amylase activities were measured. The kit produced by Nanjing institute of bioengineering is adopted to detect the activity of pepsin, trypsin and amylase respectively, and the operation is carried out according to the instruction of the kit.
(1) Determination method of pepsin: the gastric juice of the mouse is taken out, diluted properly and stored temporarily in a refrigerator at 4 ℃ for later use. The pepsin assay was performed according to the kit procedures. 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 reagent I and 0.2mL reagent II into the control tube, adding 0.2mL reagent II into the measuring tube, fully and uniformly mixing, incubating for 10min at 37 ℃, then adding 0.4mL reagent I into the measuring tube, fully and uniformly mixing, incubating for 10min at 37 ℃, centrifuging for 10min at 3500r/min, and taking 0.3mL supernatant for color reaction. Taking 45 mL centrifuge tubes, respectively marking as a reference tube, a measuring tube, a standard tube and a blank tube, adding 0.3mL supernatant into the reference tube and the measuring tube, adding 0.3mL standard substance application liquid (50 mu g/mL) into the standard tube, adding 0.3mL standard substance diluent into the blank tube, then respectively adding 1.5mL reagent III and 0.3mL reagent IV into the four tubes, fully mixing, incubating for 20min at 37 ℃, and measuring the absorbance value of each tube (zero adjustment of distilled water) at 660nm by using a visible spectrophotometer. The calculation formula is as follows:
(2) determination method of trypsin: after taking out intestinal juice of the mouse, temporarily storing the intestinal juice in a refrigerator at 4 ℃ for later use. The trypsin assay was performed according to the kit procedures. Marking two 5mL centrifuge tubes as a blank tube and a measuring tube, respectively adding 1.5mL trypsin substrate application liquid into the two centrifuge tubes, preheating for 5min at 37 ℃, then 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 uniformly, timing, rapidly pouring into a quartz cuvette, and measuring the absorbance value (zero-adjusted by double distilled water) at 253nm by using an ultraviolet spectrophotometer at 30s, and marking as A1; pouring the solution into a primary test tube, incubating at 37 ℃ for 20min, quickly pouring the solution into a quartz cuvette, and measuring the absorbance value of the solution at 253nm by using an ultraviolet spectrophotometer at the time of 20.5min, wherein the absorbance value is recorded as A2. The calculation formula is as follows:
(3) α -determination method of amylase, taking out intestinal juice of mouse, diluting properly, storing in 4 deg.C refrigerator for standby, α -determination of amylase is carried out according to the steps of kit, marking two 5mL centrifugal tubes as blank tube and determination tube, adding 0.5mL substrate buffer solution into two centrifugal tubes, adding 0.1mL diluted intestinal juice into determination tube, mixing, incubating at 37 deg.C for 7.5 min, adding 0.5mL iodine application solution into blank tube and determination tube, adding 3.1mL double distilled water into blank tube, adding 3mL double distilled water into determination tube, mixing again, determining absorbance value (double distilled water zero adjustment) of two tubes at 660nm with visible light spectrophotometer, calculating formula as follows:
2.5 morphological index of rat intestinal tract tissue
After the experiment, the animal was fasted for 24h, water was freely fed, all animals were slaughtered, and small intestine (jejunum, ileum), anterior large intestine and posterior large intestine tissues were taken. Sections of large and small intestine were stained and morphologically observed using the software Image-Pro Plus 6.0, including intestinal villus height and intestinal villus surface smoothness.
2.6 statistical analysis method
The results are expressed as mean. + -. standard deviation, unless otherwise indicated
To perform the presentation. SPSS16.0 software is adopted to carry out statistical analysis on experimental data, firstly, single-factor analysis of variance is adopted to carry out homogeneous detection on the data, then, the comparison among all groups of data is analyzed by a Duncan's method in the single-factor analysis of variance (one-way ANOVA), and the significance level is set as P<0.05。
3. Results of the experiment
3.1 Effect of the composition on weight gain, feed intake, food and protein availability in laboratory animals
During the experimental period, the animals are normal in characteristic, good in mental and activity conditions, and free of any adverse reaction after the test substances are given every day. Compared with the control group, the intervention groups have no significant difference in weight gain, food intake, food utilization rate, apparent protein digestibility and protein utilization rate.
3.2 Effect of the composition on the Activity of intestinal digestive enzymes
As shown in Table 4, the trypsin activity of the "CPP + probiotic" group was significantly higher than that of the control group, and also significantly higher than that of the CPP group and the probiotic group (p < 0.05). The trypsin activity of the CPP group and the probiotic bacteria combination group is not obviously higher than that of the control group, which shows that the combination of the CPP and the probiotics has a synergistic effect and synergistically promotes the trypsin activity. The pepsin activity and the amylopsin activity of each experimental group have no significant difference compared with the control group (p is more than 0.05).
The results show that the CPP and probiotic group has the function of promoting the digestive enzyme activity of the intestinal tract of the mice, thereby having the function of promoting the digestive function of the intestinal tract.
TABLE 4 Effect of probiotics on digestive enzyme Activity in 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 combination | 1126.30±150.46e |
Note: the difference of different lower case letters in the same column is significant (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 villus height was significantly higher in the CPP + probiotic group than in the control group, which was also the highest in each treatment group. The jejunal villus height of the complex probiotic group was significantly higher than any of the individual probiotic groups. The surface smoothness of the villi of the jejunum and the ileum 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 morphological structure of the small intestine tissue, and the higher the height of the villi is, the higher the surface smoothness of the villi is, and the stronger the absorption capacity of the small intestine is.
TABLE 5 Effect of compositions on rat intestinal histomorphology
| Height of jejunum villi | Height of villi of ileum | |
| 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 + probiotics | 369.08±31.34ab | 294.12±26.32c |
The difference between different lower case letters in the same column is significant (p < 0.05).
Taken together with the results of example 2, the trypsin activity of the "probiotic + CPP" composition in the intervention group was significantly higher than that of the control group, and also significantly higher than that of the CPP group and the probiotic group (p <0.05), indicating that the combination of CPP and probiotic bacteria has a synergistic effect, and synergistically promotes the trypsin activity. The ileal villus height of the CPP + probiotic group was significantly higher than the control group, which was also the highest in each treatment group. Indicating that the stronger the CPP + probiotic group is for improving the small intestine absorption capacity.
Example 3
1. Purpose of experiment
The influence of 3 probiotics (BL-99, K56 and ET-22) and CPP composition on the small intestine power of weaned mice is evaluated by taking the weaned mice as model animals.
2. Experimental methods
2.1 animal feeding and grouping
50 healthy SPF-grade ICR males, weaned at 21 weeks, weighed 11.6-19.5g, were housed in a barrier system animal house maintained at an indoor temperature of 22 deg.C and a humidity of 10-60% under 12-hour alternating light and dark conditions, and were allowed to freely eat and drink water, supplied by Beijing Wintonli Hua laboratory animal technology Co., Ltd. The experimental animals were randomly divided into 5 groups of 10 animals each according to body weight. After the mice and rats are fed with standard feed (the feed formula is provided by Olympic feed Co., Ltd. of Beijing Ke) for 1 week, formal experiments are carried out.
2.2 intragastric sample preparation and intervention
The preparation method comprises the steps of preparing the probiotic powder before daily gavage, and 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 normal saline for later use. The blank control group, the model control group and the probiotic group are given standard feed, the CPP group and the CPP + probiotic group are given special feed (standard feed + casein phosphopeptide), and the probiotic group and the CPP + probiotic group are simultaneously given a probiotic test sample in a gastric perfusion mode. Each experimental animal was gavaged with 0.5mL once a day in the morning for 35 consecutive days. The control group was gavaged with 0.5mL of physiological saline.
TABLE 6 Experimental groups
2.3 intestinal motility test
After the 30 th day of intervention, small intestine exercise experiments were carried out, and fasting was carried out for 16h before the experiments, and water was freely drunk. The test sample is given on the current day of measurement, and after 30min, the model control group and each experimental group are perfused with compound diphenoxylate (0.025%) and the blank control group is perfused with distilled water with the same volume. After administering compound diphenoxylate for 30min, each group of gavage ink (containing 5% of activated carbon powder and 10% of acacia gum) was administered. The mice were sacrificed by cervical dislocation 25min after administration of ink, the abdominal cavity was opened, and the intestinal canal from the upper end to the pylorus and from the lower end to the ileocecal part was cut off. The small intestine was pulled in a straight line, the length of the intestinal canal was measured as "total length of small intestine", the distance from the pylorus to the front edge of the charcoal dust was measured as "advancing distance of charcoal dust in intestine", and the percentage of charcoal dust advancing was calculated by the following formula.
The ink propelling rate is equal to the propelling length (cm) of the ink/the total length (cm) of the small intestine multiplied by 100 percent
2.4 statistical analysis method
The results are expressed as mean. + -. standard deviation, unless otherwise indicated
To perform the presentation. SPSS16.0 software is adopted to carry out statistical analysis on experimental data, firstly, single-factor analysis of variance is adopted to carry out homogeneous detection on the data, then, the comparison among all groups of data is analyzed by a Duncan's method in the single-factor analysis of variance (one-way ANOVA), and the significance level is set as P<0.05。
3. Results of the experiment
The results are shown in figure 1, the ink propulsion rate of the mice with the probiotics K56 and the probiotics ET-22, the composite probiotics group and the CPP + probiotics group is obviously increased (p is less than 0.05), and the results show that the probiotics K56, the probiotics ET-22, the composite probiotics group and the CPP + probiotics group can promote the intestinal movement of the mice, the intestinal movement promoting capability of the composite probiotics group is higher than that of the probiotics group alone, and the intestinal movement promoting capability of the CPP + probiotics group is higher than that of the probiotics group and the CPP group alone.
Combining the results of example 2 and example 3, the probiotic bacteria and CPP composition can significantly improve the digestive enzyme activity of experimental animals, promote the intestinal motility, improve the absorption capacity by improving the tissue morphology of small intestine, and show a synergistic effect in promoting the digestive absorption.
Claims (10)
1. An edible composition comprising a probiotic and casein phosphopeptide, wherein the probiotic comprises Bifidobacterium lactis and Lactobacillus paracasei.
2. The composition of claim 1, wherein the bifidobacterium lactis comprises bifidobacterium lactis BL-99, the lactobacillus paracasei comprises lactobacillus paracasei K56, and lactobacillus paracasei ET-22; bifidobacterium lactis BL-99 is a strain with a preservation number of CGMCC No.15650, Lactobacillus paracasei K56 is a strain with a preservation number of CGMCC 15139 or DSM27447, and Lactobacillus paracasei ET-22 is a strain with a preservation number of CGMCC No. 15077.
3. The composition according to claim 1 or 2, wherein the weight ratio of the probiotics to the casein phosphopeptides is 240-700: 0.03 to 50, preferably 293 to 476: 0.3 to 30.
4. The composition according to claim 1, 2 or 3, wherein the ratio of viable count of Bifidobacterium lactis BL-99, Lactobacillus paracasei K56 and Lactobacillus paracasei ET-22 is 0.01-10: 0.01-20: 0.01 to 20, preferably 0.1 to 10: 0.1-10: 0.1 to 10.
5. The composition according to claim 1, 2 or 3, wherein the weight ratio of bifidobacterium lactis BL-99, lactobacillus paracasei K56 and lactobacillus paracasei ET-22 is 0.01-10: 0.01-20: 0.01 to 20, preferably 0.1 to 10: 0.1-10: 0.1 to 10.
6. The composition according to claim 1 or 2, wherein bifidobacterium lactis BL-99, lactobacillus paracasei K56, lactobacillus paracasei ET-22 are each independently live bacteria.
7. Use of a composition according to any one of claims 1 to 6 for the preparation of a food product having a digestive effect.
8. Use according to claim 7, wherein said promoting digestion comprises promoting trypsin activity and/or promoting intestinal motility.
9. Use according to claim 7, wherein the food product is a liquid beverage, a solid beverage, an oral liquid, a dairy product, a tablet or a capsule.
10. A food with digestion promoting effect comprises the probiotic edible composition of any one of claims 1 to 6 as raw materials; preferably, the food product is a liquid beverage, a solid beverage, an oral liquid, a dairy product, a tablet or a capsule.
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