CN116035217B - Composition with weight-losing and fat-reducing effects and application thereof - Google Patents

Composition with weight-losing and fat-reducing effects and application thereof Download PDF

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CN116035217B
CN116035217B CN202211672459.7A CN202211672459A CN116035217B CN 116035217 B CN116035217 B CN 116035217B CN 202211672459 A CN202211672459 A CN 202211672459A CN 116035217 B CN116035217 B CN 116035217B
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yam
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CN116035217A (en
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何蓉蓉
梁磊
李怡芳
闫昌誉
栗原博
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Jinan University
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Abstract

The invention discloses a composition with the effects of losing weight and reducing fat and application thereof, and belongs to the field of food and medicine. The invention relates to a composition with weight-losing and fat-reducing effects, which comprises the following components in parts by weight: 10-35 parts of yam peptide and 0.8-1.2 parts of N-lactoyl phenylalanine; the relative molecular mass of the yam peptide is less than or equal to 5kDa. The composition is matched with N-lactoyl phenylalanine by the yam peptide, the weight-losing and fat-reducing effects of the yam peptide are obviously improved by taking the N-lactoyl phenylalanine as an auxiliary, the obtained product can effectively inhibit the activity of pancreatic lipase in a human body, has good weight-losing and weight-reducing effects, is safe and free of toxic and side effects, and can be directly used as a dietary supplement to supplement other nutrition required by the human body.

Description

Composition with weight-losing and fat-reducing effects and application thereof
Technical Field
The invention relates to the field of food and medicine, in particular to a composition with the functions of losing weight and reducing fat and application thereof.
Background
Overweight and obesity are mainly manifested as excessive accumulation of adipose tissue in the human body. There are many causes of overweight and obesity, including genetic and environmental factors, alterations in metabolism and endocrine function, poor lifestyle and eating habits, and psycho-psychological factors. At present, besides diet control and exercise enhancement, overweight and obesity lack corresponding clinical weight-losing methods with definite efficacy and small side effects.
On the other hand, although the chemical weight-reducing medicines currently used mainly have a plurality of types, in order to inhibit the intake of heat of human bodies, the main chemical weight-reducing medicines adopt some neurohormonal substances for controlling the appetite of the human bodies as key components, and the substances can quickly help people to get rid of obesity and overweight by being matched with other weight-reducing medicines or fat-reducing operations, but the compounds have certain effects on similar receptors in the complex central nervous system, cannot avoid causing corresponding side effects, and are easy to addict to people with special physique or even cause bad consequences such as anorexia.
Aiming at the bottleneck problem of the current synthetic chemical weight-reducing medicine, people begin to seek weight-reducing medicines with homology of medicine and food and no toxic or side effect in the direction of traditional Chinese medicine. Mainly characterized in that the traditional Chinese medicine has relatively mild effect on human body, and simultaneously the method of 'medicine and food simultaneous therapy' can further reduce the side effect of the medicine on human body. The traditional Chinese medicine researches show that the main constitution types of obese people are phlegm-dampness and qi deficiency, so the spleen strengthening and spleen tonifying have very important significance for weight and lipid reduction of obese people according to the angle of traditional Chinese medicine.
Disclosure of Invention
Based on the defects existing in the prior art, the invention aims to provide a composition with the functions of losing weight and reducing fat, which is prepared by matching yam peptide with specific relative molecular mass with N-lactoyl phenylalanine, and taking the N-lactoyl phenylalanine as an auxiliary to remarkably improve the weight and fat reducing efficacy of the yam peptide, the obtained product can effectively inhibit the activity of pancreatic lipase in a human body, has good weight losing and weight reducing efficacy, is safe and has no toxic or side effect, and can be directly used as a dietary supplement to supplement other nutrition required by the human body.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a composition with the effects of losing weight and reducing fat comprises the following components in parts by weight:
10-35 parts of yam peptide and 0.8-1.2 parts of N-lactoyl phenylalanine;
the relative molecular mass of the yam peptide is less than or equal to 5kDa.
The Chinese yam is sweet and flat, has the effects of tonifying spleen and lung, nourishing yin and promoting the production of body fluid, and is good for tonifying spleen and yin, so that the Chinese yam is also a model of homology of medicine and food, the protein content in the Chinese yam is rich, and when the protein in the Chinese yam is degraded by utilizing an enzymolysis technology, the Chinese yam peptide with biological activity can be produced, and the substance has obvious effects of inhibiting pancreatic lipase in a human body and achieving the effects of losing weight and reducing fat. On the other hand, the pure yam peptide has a certain limit on the weight-losing and fat-reducing effects of the human body, and the main reason is that the yam peptide is a mixture, the content of specific active sequences in the yam peptide is not high, and good biological effects can be realized by taking enough yam peptide; however, excessive intake affects metabolism. In order to solve the practical problem of low overall action efficiency of the yam peptide, the inventor discovers through experiments that the addition of a certain content of N-lactoyl phenylalanine into the yam peptide can effectively and synergistically promote the inhibition degree of the yam peptide on pancreatic lipase activity, which is possibly related to the promotion of the combination of specific active sequences in the yam peptide and lipase. Among them, N-lactoyl phenylalanine is also called lac-phe molecule, which is An endogenous amino acid metabolite and can be detected in blood, and researches show that (such as An exhale-inducible metabolite that suppresses feeding and obesity) can suppress appetite of mice to a certain extent, but the effect in human body has not been confirmed yet. The inventor finds that when the Chinese yam peptide is used as an auxiliary ingredient to act together, the appetite inhibition effect is not obvious, which indicates that the Chinese yam peptide does not excessively influence the nerve center within a certain dosage range. Meanwhile, the inventor finds through experiments that the addition amount of the N-lactoyl phenylalanine is not as large as possible, and excessive lac-phe molecule intake can lead to low pancreatic lipase inhibition efficiency of the whole product, and the two key components of the composition need to be maintained in a specific proportion range to realize the optimal weight and fat reducing effect.
On the other hand, the Chinese yam is a natural plant part with rich nutrition, and after enzymolysis treatment, the Chinese yam contains various enzymolysis peptide substances with different sizes, and the larger Chinese yam peptide still needs digestion of the human digestive system, has higher absorption difficulty, and has less effective components with the weight-reducing and fat-reducing effects.
Preferably, the weight part of N-lactoyl phenylalanine in the composition is 1 part.
Preferably, the weight part of the yam peptide in the composition is 20-30 parts.
As described above, the single yam peptide cannot effectively achieve the effect of inhibiting pancreatic lipase in human body, and along with the introduction of N-lactoyl phenylalanine, the inhibiting effect of the whole composition on pancreatic lipase is gradually improved, so that the overall effect of losing weight and reducing fat (weight) is gradually improved, when the weight ratio of the two is (20-30): 1, the activity efficacy degree of the product is highest, and when the addition amount of the N-lactoyl phenylalanine is further increased, the performance of the product is reduced.
Preferably, the preparation method of the N-lactoyl phenylalanine comprises the following steps: mixing and dispersing L-phenylalanine, lactic acid and calcium oxide in a solvent, mixing and stirring at 80-100 ℃ for 2.5-3.5 h, filtering, concentrating and drying to obtain the N-lactoyl phenylalanine.
More preferably, the mass ratio of the L-phenylalanine, the lactic acid and the calcium oxide is L-phenylalanine: lactic acid: calcium oxide= (0.9 to 1.05): (4.3 to 4.5): (0.09-0.13).
More preferably, the preparation method of the N-lactoyl phenylalanine further comprises the following steps: crushing the obtained N-lactoyl phenylalanine by using a vibration crusher, sieving with a 20-25 mesh sieve, and then placing the sieved powder into a low-temperature wall breaking crusher to perform vibration crushing for 10-20 min at the temperature of-8 to-12 ℃.
Preferably, the resulting product may be structurally identified by HPLC-MS/MS system.
The N-lactoyl phenylalanine is produced by combining lactic acid and phenylalanine produced by human body as raw materials mainly by catalysis of cndp2 protein in human body, and in the invention, L-phenylalanine, lactic acid and calcium oxide are used as raw materials, and lac-phe molecules with higher purity can be prepared by proper heating and mixing conditions and applied to the composition.
The invention also aims to provide a preparation method of the yam peptide, which comprises the following steps:
(1) Pulverizing rhizoma Dioscoreae, adding water, mixing, and adding alkaline protease for enzymolysis to obtain enzymolysis solution A;
(2) And (3) inactivating enzyme of the enzymolysis liquid A, centrifuging, filtering the supernatant by using an ultrafiltration membrane, and freeze-drying to obtain the yam peptide.
Preferably, the ultrafiltration membrane is a 5kDa ultrafiltration membrane.
In the prior art, the yield of the yam peptide is generally lower, and as yam enzymolysis products are not all capable of playing roles in losing weight and reducing fat, when the yam enzymolysis products are applied to the composition provided by the invention, the more the target enzymolysis oligopeptide content is, the better the weight and fat reducing effect is, meanwhile, the production economic efficiency is also higher, and experimental researches show that the enzymolysis degrees of different enzymes on the yam are different, the products obtained by enzymolysis are naturally different, and when the yam is subjected to enzymolysis by using alkaline protease, compared with other common enzyme types, the yield of the target enzymolysis oligopeptide is higher, and the activity of losing weight and reducing fat is stronger.
Preferably, in the step (1), the mass ratio of the yam to the alkaline protease is 100: (0.2-2.5);
more preferably, the mass ratio of yam to alkaline protease in the step (1) is 100: (0.2-1).
In the enzymolysis process, the adding amount of the substrate yam and the inoculation amount of the enzyme need to be maintained in a specific ratio, and not so much is better, mainly because the yam remains a plurality of substances after enzymolysis, including the yam which is not subjected to enzymolysis (saturation phenomenon exists in enzymolysis), metabolites of the enzyme, the enzyme and other enzymolysis structural products except oligopeptide after enzymolysis, and because the key substances needed by the product are only yam peptide, the optimal concentration and yield need to be maintained. Through screening, the yam peptide prepared by the substrate/enzyme adding ratio has higher yield, higher purity and better efficacy.
Preferably, the temperature of the enzymolysis treatment in the step (1) is 35-65 ℃;
more preferably, the temperature of the enzymolysis treatment in the step (1) is 45-50 ℃.
In the present invention, the enzymatic oligopeptide needs to be maintained in a low relative molecular weight range, so that the enzyme needs to be ensured to have high activity in the enzymatic hydrolysis process, and experiments show that the enzymatic hydrolysis temperature has a great influence on the activity of the enzyme, and the activity of the enzyme is reduced no matter the enzymatic hydrolysis temperature is too low or too high, and meanwhile, the enzymatic activity of the enzyme metabolite is rapidly increased, so that the enzymatic hydrolysis efficiency of the enzyme is inhibited, and therefore, the enzymatic hydrolysis process is best performed at the temperature of 35-65 ℃, especially 45-50 ℃.
Preferably, the enzymolysis treatment time in the step (1) is 6-24 hours;
more preferably, the enzymolysis treatment in the step (1) is performed for 10 to 12 hours.
As described above, as the progress of the enzymolysis treatment advances, the enzymatic hydrolysis products and the metabolites of the enzyme rather decrease the enzymatic activity, so that the longer the time of the enzymolysis is, the better the time is, the more preferably 6 to 24 hours is, through experimental screening.
Preferably, the temperature of the centrifugation in the step (2) is 4-8 ℃, the time is 10-25 min, and the centrifugation speed is 8000-12000 rpm.
It is still another object of the present invention to provide a method for preparing the composition, comprising the steps of: and uniformly mixing the components to obtain the composition with the weight and fat reducing effects.
The preparation method of the composition with the weight and fat reducing effects is simple, and industrial mass production can be easily realized without depending on equipment.
It is a further object of the present invention to provide the use of said composition for the preparation of a product having a weight-reducing effect.
Preferably, the product is a pharmaceutical product, a food product or a health product.
More preferably, the dosage form of the medicament is a clinically pharmaceutically acceptable dosage form.
More preferably, the polarity is a tablet, pill, granule or liquid.
The composition with the weight and fat reducing effect has good efficacy, can effectively inhibit the activity of pancreatic lipase in a human body, reduces the fat content of the human body, finally achieves the functions of reducing fat and weight, and also has rich dietary fibers, and can synchronously supplement nutrition required by the human body. The product is mainly a human body containing substances and natural biological enzymolysis products, is safe and free of toxic and side effects, and can be used for a long time. The product has good stability, can be prepared into various products or dosage forms, and has high application value.
Meanwhile, it should be noted that the composition of the present invention has stable properties, and when the product is applied to the preparation of commercial products according to actual needs, a plurality of processing aids or additives such as sweeteners, preservatives, stabilizers, etc. can be added by those skilled in the art, so long as the efficacy of the composition of the present invention is not affected.
The invention has the beneficial effects that the composition is provided, the yam peptide with specific relative molecular mass is matched with the N-lactoyl phenylalanine, the N-lactoyl phenylalanine is used as an auxiliary for remarkably improving the weight-losing and fat-reducing effects of the yam peptide, the obtained product can effectively inhibit the activity of pancreatic lipase in a human body, has good weight-losing and weight-reducing effects, is safe and free from toxic and side effects, and can be directly used as a dietary supplement to supplement other nutrition required by the human body. The invention also provides a preparation method of the yam peptide, which aims at the requirement of the specific activity low molecular weight oligopeptide of the composition, and realizes high resource utilization rate and high yield of yam enzymolysis through specific enzyme types. The invention also provides application of the composition in preparation of products with weight-losing efficacy. The product is safe and nontoxic, has strong action and effect, is suitable for being applied to a plurality of product forms, and has high commercial value.
Drawings
FIG. 1 is a HPLC-MS/MS structure standard chart of a product prepared by the preparation method of the N-lactoyl phenylalanine and an N-lactoyl phenylalanine standard reference substance.
Detailed Description
The present invention will be further described with reference to specific examples and comparative examples for better illustrating the objects, technical solutions and advantages of the present invention, and the object of the present invention is to be understood in detail, not to limit the present invention. All other embodiments, which can be made by those skilled in the art without the inventive effort, are intended to be within the scope of the present invention. The experimental reagents, raw materials and instruments designed in the practice and comparative examples of the present invention are common reagents, raw materials and instruments unless otherwise specified.
Example 1
In one embodiment of the composition with the weight and fat reducing effect, the composition comprises 5 parts of yam peptide and 1 part of N-lactoyl phenylalanine, and the two are directly mixed to obtain a product.
The preparation method of the yam peptide comprises the following steps:
(1) Crushing peeled and cleaned fresh Chinese yam to 100 meshes, and then mixing the Chinese yam according to the mass ratio: after water is added into water according to a mass ratio of 1:25 and uniformly mixed, the pH value of the mixed solution is adjusted to 9.5, and then alkaline protease is added according to the mass ratio: adding alkaline protease into the yam with the inoculation amount of 0.2:100, stirring for 6 hours at the temperature of 45 ℃ for enzymolysis treatment to obtain enzymolysis liquid A;
(2) And (3) carrying out enzyme deactivation treatment on the enzymolysis liquid A at 90 ℃ for 30min, centrifuging at 4 ℃ and 10000rpm for 15min until the solution is layered, filtering the supernatant with an ultrafiltration membrane to obtain oligopeptide with the molecular weight of less than 5kDa, and freeze-drying to obtain the yam peptide.
The preparation method of the N-lactoyl phenylalanine comprises the following steps:
165g of edible L-phenylalanine, 720g of edible lactic acid and 17g of edible calcium oxide are mixed and dispersed in 2000mL of water, then are mixed and stirred for 3 hours at 100 ℃, are filtered, are dried by rotary evaporation, are crushed into coarse powder by a vibration crusher, are filtered by a 24-mesh screen, and are transferred into an ultralow temperature ultrafine wall-breaking crusher to be crushed by vibration at-10 ℃ for 15 minutes, so that the N-lactoyl phenylalanine is obtained. The obtained product and a commercially available N-lactoyl phenylalanine standard reference substance are subjected to structural identification through an HPLC-MS/MS system to obtain the N-lactoyl phenylalanine.
Example 2
In one embodiment of the composition with the weight and fat reducing effect, the composition comprises 40 parts of yam peptide and 1 part of N-lactoyl phenylalanine, and the two components are directly mixed to obtain a product.
The preparation method of the yam peptide comprises the following steps:
(1) Crushing peeled and cleaned fresh Chinese yam to 100 meshes, and then mixing the Chinese yam according to the mass ratio: after water is added into water according to a mass ratio of 1:25 and uniformly mixed, the pH value of the mixed solution is adjusted to 9.5, and then alkaline protease is added according to the mass ratio: adding alkaline protease into the yam with the inoculation amount of 1.5:100, stirring for 12 hours at the temperature of 55 ℃ for enzymolysis treatment to obtain enzymolysis liquid A;
(2) And (3) carrying out enzyme deactivation treatment on the enzymolysis liquid A at 90 ℃ for 30min, centrifuging at 4 ℃ and 10000rpm for 15min until the solution is layered, filtering the supernatant with an ultrafiltration membrane to obtain oligopeptide with the molecular weight of less than 5kDa, and freeze-drying to obtain the yam peptide.
The preparation method of the N-lactoyl phenylalanine is the same as that of the example 1.
Example 3
In one embodiment of the composition with the weight and fat reducing effect, the composition comprises 15 parts of yam peptide and 1 part of N-lactoyl phenylalanine, and the two components are directly mixed to obtain a product.
The preparation method of the yam peptide comprises the following steps:
(1) Crushing peeled and cleaned fresh Chinese yam to 100 meshes, and then mixing the Chinese yam according to the mass ratio: after water is added into water according to a mass ratio of 1:25 and uniformly mixed, the pH value of the mixed solution is adjusted to 9.5, and then alkaline protease is added according to the mass ratio: inoculating alkaline protease with the inoculum size of 2.5:100 of the yam, stirring for 18 hours at 65 ℃ for enzymolysis treatment to obtain enzymolysis liquid A;
(2) And (3) carrying out enzyme deactivation treatment on the enzymolysis liquid A at 90 ℃ for 30min, centrifuging at 4 ℃ and 10000rpm for 15min until the solution is layered, filtering the supernatant with an ultrafiltration membrane to obtain oligopeptide with the molecular weight of less than 5kDa, and freeze-drying to obtain the yam peptide.
The preparation method of the N-lactoyl phenylalanine is the same as that of the example 1.
Example 4
The difference between this example and example 1 is only that in the step (1) of the preparation method of the yam peptide, the mass ratio of alkaline protease: the yam is inoculated with alkaline protease at an inoculum size of 0.5:100.
Example 5
The difference between this example and example 1 is only that in the step (1) of the preparation method of the yam peptide, the mass ratio of alkaline protease: the Chinese yam is inoculated with alkaline protease at an inoculum size of 1:100.
Example 6
The difference between this example and example 1 is only that in the step (1) of the preparation method of the yam peptide, the mass ratio of alkaline protease: the yam is inoculated with alkaline protease at an inoculum size of 1.5:100.
Example 7
The difference between this example and example 1 is only that in the step (1) of the preparation method of the yam peptide, the mass ratio of alkaline protease: the yam is inoculated with alkaline protease at an inoculum size of 2:100.
Example 8
The difference between this example and example 1 is only that in the step (1) of the preparation method of the yam peptide, the mass ratio of alkaline protease: the yam is inoculated with alkaline protease at an inoculum size of 2.5:100.
Example 9
The difference between this example and example 1 is that the temperature during the enzymolysis treatment in the step (1) of the preparation method of the yam peptide is 35 ℃.
Example 10
The difference between this example and example 1 is that the temperature during the enzymolysis treatment in the step (1) of the preparation method of the yam peptide is 40 ℃.
Example 11
The difference between this example and example 1 is that the temperature during the enzymolysis treatment in the step (1) of the preparation method of the yam peptide is 50 ℃.
Example 12
The difference between this example and example 1 is that the temperature during the enzymolysis treatment in the step (1) of the preparation method of the yam peptide is 55 ℃.
Example 13
The difference between this example and example 1 is that the temperature during the enzymolysis treatment in the step (1) of the preparation method of the yam peptide is 60 ℃.
Example 14
The difference between this example and example 1 is that the temperature during the enzymolysis treatment in the step (1) of the preparation method of the yam peptide is 65 ℃.
Example 15
The difference between this example and example 1 is that the time for the enzymolysis treatment is 8 hours in the step (1) of the preparation method of the yam peptide.
Example 16
The difference between this example and example 1 is that the time for the enzymolysis treatment is 10 hours in the step (1) of the preparation method of the yam peptide.
Example 17
The difference between this example and example 1 is that the time for the enzymolysis treatment in the step (1) of the preparation method of the yam peptide is 12 hours.
Example 18
The difference between this example and example 1 is that the time for the enzymolysis treatment in the step (1) of the preparation method of the yam peptide is 16 hours.
Example 19
The difference between this example and example 1 is that the time for the enzymolysis treatment is 20 hours in the step (1) of the preparation method of the yam peptide.
Example 20
The difference between this example and example 1 is that the time for the enzymolysis treatment is 24 hours in the step (1) of the preparation method of the yam peptide.
Effect example 1
In order to verify the fat-reducing efficacy of the composition with fat-reducing and fat-reducing effects, the product obtained in the embodiment 1 of the invention is subjected to the measurement of the effect of inhibiting pancreatic lipase activity by adopting an olive oil substrate emulsification method, and the specific test method is as follows:
a 2% polyvinyl alcohol (Polyvinyl alcohol, PVA) solution was prepared with distilled water and filtered with gauze. Adding 1/3 volume of olive oil into 2% of PVA solution, and shearing at high speed of 10000rpm for 9min by adopting a shearing emulsifying machine to prepare PVA emulsion, namely substrate solution. To a 25mL cuvette, 2mL olive oil substrate emulsion and 2.5mL PBS were added, vortexed and mixed well, and in a 40℃water bath for 5min, 1mL of the diluted example 1 product (2 mg/mL) and 0.5mL pancreatic lipase solution (3 mg/mL) were addedAfter thoroughly mixing, the mixture was heated in a water bath for 20 minutes, and 6mL of 95% ethanol and 1mL of HCl (6 mol/L) were added to terminate the reaction. Adding 3mL of isooctane, vibrating for 90s, standing in a water bath at 60 ℃ for layering, taking 1mL of upper layer solution, adding 4mL of isooctane and 1mL of copper acetate color developing agent, vibrating for 90s in a vortex, standing for layering, taking the upper layer solution, and measuring absorbance at 710 nm. The control group (control) was not added with the composition solution with weight-reducing and fat-reducing effects according to the present invention. All absorbance values were calibrated through a complete blank well (blank, without addition of sample solution and enzyme). The calculation is carried out according to the following formula: pancreatic lipase inhibition (%) = (1-a) sample /A control )×100。
Meanwhile, similar experimental sample groups were prepared by the same method as in example 1, and each experimental sample group was not different except for the weight parts of the yam peptide and the N-lactoyl phenylalanine, and had the comparison shown in table 1, and the same test experiment was performed on each control group, and the results are shown in table 1.
TABLE 1
Grouping Composition of the product components Pancreatic lipase inhibition rate (%)
Example 1 5 parts of yam peptide plus 1 part of N-lactoyl phenylalanine 30.6±3.18
Experiment group 1 5 parts of yam peptide plus 0 part of N-lactoyl phenylalanine 25.5±2.61
Experiment group 2 10 parts of yam peptide plus 1 part of N-lactoyl phenylalanine 32.6±2.86
Experiment group 3 15 parts of yam peptide plus 1 part of N-lactoyl phenylalanine 37.6±1.91
Experiment group 4 20 parts of yam peptide plus 1 part of N-lactoyl phenylalanine 40.6±3.74
Experiment group 5 25 parts of yam peptide and 1 part of N-lactoyl phenylalanine 45.6±2.68
Experiment group 6 30 parts of yam peptide plus 1 part of N-lactoyl phenylalanine 42.6±2.34
Experiment group 7 35 parts of yam peptide plus 1 part of N-lactoyl phenylalanine 35.6±3.76
Experiment group 8 40 parts of yam peptide plus 1 part of N-lactoyl phenylalanine 30.2±3.25
From table 1, it can be seen that experiment group 1 only containing yam peptide shows a certain pancreatic lipase inhibition activity, but the inhibition degree is not high, and the pancreatic lipase inhibition rate of the product of example 1 after matching N-lactoyl phenylalanine with yam peptide is improved by about 5%, which indicates that the introduction of lac-phe molecule can effectively assist yam peptide to realize better inhibition effect. Along with the increase of the specific gravity of the yam peptide, the pancreatic lipase activity inhibition effect shown by the whole product is gradually enhanced, which shows that the yam peptide is still used as a main functional substance, the lac-phe molecule is used as an auxiliary substance to a certain extent, and the pancreatic lipase inhibition rate of the obtained composition is highest and reaches about 45.6% when the weight ratio of the yam peptide to the lac-phe molecule is 25:1. However, as the specific gravity of the yam peptide is continuously increased to be more than 25:1, the effect of the product is reduced, and when the weight ratio of the yam peptide to the product is 40:1, the inhibition rate of the product is reduced to 30.2%, which means that the yam peptide and the product are required to be maintained in a special proportion range to realize a good synergistic effect, wherein the weight ratio of the yam peptide to the product is (5-40): 1, the pancreatic lipase inhibition rate of the product can reach more than 30 percent, and the pancreatic lipase inhibition rate reaches (20-30): in the case of 1, the inhibition rate can reach more than 40 percent.
Effect example 2
Further, in order to explore the actual weight-losing and weight-losing effects of the composition in the living organism application, SPF-grade male C57 mice are fed with high-fat feed for 10 weeks to construct an obese mouse model. Obese mice were randomly divided into 5 groups, model control group and 4 dosing group, and subjects of the 4 dosing group were experimental group 1 product, experimental group 2 product, experimental group 5 product and experimental group 8 product in effect example 1, respectively, with 12 mice per group. The administration group was filled with 0.5g/kg of the corresponding product per day, and the model control group was given an equal volume of distilled water, and the mice were still free to ingest the high-fat feed during the administration period. The weight and feeding amount of the mice were recorded daily from the start of self-administration, the mice were anesthetized by the time of week 6, their abdominal fat was weighed, the average weight values at different time periods were calculated, and the statistical weight gain rate and feeding amount inhibition rate after the end of the experiment were recorded, and the test results are shown in tables 2 to 4.
TABLE 2
TABLE 3 Table 3
TABLE 4 Table 4
Grouping Abdomen fat weight (mg, mean±sd) of sixth week mice
Model control group 364.7±40.8
Experiment group 1 305.1±25.7
Experiment group 2 289.5±32.1
Experiment group 5 228.4±21.3
Experiment group 8 271.5±27.9
As can be seen from tables 2 to 4, the weight gain rate of the model group mice during the experiment is as high as 27.5% due to high caloric intake, and the experimental model is successfully constructed, whereas compared with the model group, the weight gain rate of the mice can be effectively inhibited by using only the product of the experimental group 1 of the yam peptide, so that the weight gain rate is reduced to 20.4%, on the other hand, the normal intake appetite of the mice can be reduced by using the yam peptide, mainly because the yam peptide has a certain fiber satiety, a certain appetite inhibition effect can exist under the normal intake, and the accumulated fat amount in the mice is reduced to a certain extent under the two effects. The products of experimental groups 2, 5 and 8 have more remarkable effects when applied than the product of experimental group 1, wherein the product of experimental group 5 can lead the weight gain rate of mice to be less than 10%, the fat mass after the experiment is only about 230mg, and almost only half of the model group, which indicates that the composition of the invention is beneficial to reducing the absorption and accumulation of fat in food in the digestive system, and can inhibit the conversion of external lipid substances into fat in vivo. In contrast, the effect of the product of experiment set 5 is better than that of the products of experiment sets 2 and 8, and the experiment result is similar to that of effect example 1, which shows that the weight-losing and weight-reducing function of the composition in biological application can be related to pancreatic lipase activity in organisms, and the ratio of two key substances in the composition needs to be maintained within a certain range to realize ideal weight-losing and weight-reducing effects in organisms. In addition, as can be seen from table 3, the feeding inhibition rate of each experimental group is almost the same for mice, which indicates that the composition of the invention does not realize the weight-losing effect by directly inhibiting the appetite of organisms like the action mechanism of lac-phe molecules in organisms, the lac-phe molecules only bring weak (less than 10%) feeding inhibition effect as auxiliary substances in combination with satiety brought by yam peptide, and the main function mechanism of the product is also to inhibit the activity of pancreatic lipase.
Effect example 3
In order to verify the preference of the enzyme species used in the preparation method of the yam peptide of the present invention, an enzymolysis control experiment was performed according to the preparation method of the yam peptide of example 1, and the control experiment was performed by using pepsin, trypsin, papain and neutral protease respectively, except for alkaline protease, using the same preparation method, and the yields of the product of example 1 and each control group (i.e., fresh yam x 100% after peeling and cleaning of yam peptide product/raw material) were counted, and the yields of the yam peptides obtained in example 2 and example 3 were counted. The pancrelipase inhibitory activity of the above yam peptides was also measured by the method of effect example 1, and the test results are shown in table 5.
TABLE 5
Enzyme species Yield of yam peptide product (%) Pancreatic lipase inhibition rate (%)
Alkaline protease (example 1) 1.37 25.5
Pepsin 0.79 13.1
Trypsin, trypsin and its preparation method 0.81 15.6
Papain 1.06 18.6
Neutral protease 0.96 17.3
Alkaline protease (example 2) 1.51 26.3
Alkaline protease (example 3) 1.62 22.8
As can be seen from Table 5, not all enzymes can well carry out enzymolysis on yam and obtain small-molecule oligopeptide, but alkaline protease has better hydrolysis effect on yam compared with other types of protease, and the yield of the yam peptide obtained by the preparation method in each embodiment is as high as 1.37-1.62%. Correspondingly, the type of the enzyme also determines the pancrelipase inhibition efficiency of the obtained yam peptide, and compared with other proteases, the yam peptide pancrelipase prepared by alkaline protease enzymolysis has better inhibition activity.
Further, in order to investigate the optimal addition ratio of enzyme/substrate in the enzymolysis process of the yam peptide of the present invention, yield statistics and pancrelipase inhibition activity evaluation were performed on the yam peptides obtained in examples 4 to 8 except for example 1, and the results are shown in table 6.
TABLE 6
Examples Yield of yam peptide product (%) Pancreatic lipase inhibition rate (%)
Example 1 1.37 25.5
Example 4 1.43 26.1
Example 5 1.58 26.6
Example 6 1.51 26.3
Example 7 1.55 26.5
Example 8 1.60 26.8
As can be seen from Table 6, as the ratio of enzyme to substrate was gradually increased from 0.2:100 to (0.5-2.5) 100 described in examples 4-8, the yield of the obtained yam peptide was increased, but the increase was not significant, and the yield of example 5, which was increased by 5 times, was increased by about 15% as compared with example 1, but the yield of the product was not increased significantly, even decreased, as the enzyme addition was further increased, indicating that too much enzyme addition could inhibit the activity of the enzyme and the efficiency of the enzymolysis, and the product having a molecular weight of less than 5kDa could not be hydrolyzed. On the other hand, when the enzyme usage amount reaches (0.5-2.5): 100, the pancrelipase inhibition activity of the yam peptide is changed in a small range of 26.1-26.8%, and the increase of the enzyme amount does not greatly improve the pancrelipase activity of the yam peptide prepared. Therefore, based on the consideration of process cost and effect, the mass ratio of enzyme to substrate is (0.2-1): 100 is most suitable.
Further, in order to investigate the optimal enzymolysis temperature of the yam peptide in the enzymolysis process, yield statistics and pancrelipase inhibition activity evaluation were carried out on the yam peptides obtained in examples 9 to 14 except for example 1, and the results are shown in table 7.
TABLE 7
Examples Yield of yam peptide product (%) Pancreatic lipase inhibition rate (%)
Example 1 1.37 25.5
Example 9 1.28 22.5
Example 10 1.32 23.6
Example 11 1.45 26.5
Example 12 1.43 26.2
Example 13 1.38 24.6
Example 14 1.32 23.1
As can be seen from Table 7, the yield of yam peptide was lower when the enzymatic hydrolysis temperature was in the range of 35-45℃and increased gradually with increasing temperature, indicating that the temperature range was not optimal for alkaline protease. When the enzymolysis temperature is 45-60 ℃, the yield of the product is higher, but when the temperature is higher than 50 ℃, the yield of the product begins to drop, which indicates that the optimal enzymolysis temperature of the alkaline protease is about 45-50 ℃, and when the optimal enzymolysis temperature exceeds the range, the enzymolysis activity of the enzyme is reduced, and the process energy consumption is increased. In addition, the pancrelipase inhibition activity and the yield of the yam peptide prepared at different enzymolysis temperatures show the same trend, and it can be seen that the optimal activity of the enzyme determines the effective release of the active sequence. Therefore, the optimal enzymolysis temperature of the yam peptide in the preparation process is 45-50 ℃.
Further, in order to investigate the optimal enzymolysis time of the yam peptide in the enzymolysis process, yield statistics and pancrelipase inhibition activity evaluation were performed on the yam peptides obtained in examples 15 to 20 except for example 1, and the results are shown in table 8.
TABLE 8
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As can be seen from Table 8, as the enzymolysis time increases gradually from 6 hours of example 1, the yield of the yam peptide increases, the yield reaches more than 1.5% when the enzymolysis time is 10 hours, and the yield reaches 1.63% after the enzymolysis time is doubled by 12 hours, and the increase rate reaches 19%. With further lapse of the enzymolysis time, the yield of the product is not further improved, which indicates that the enzymolysis is saturated, and even the enzymolysis reaction is inhibited due to accumulation of the enzymolysis product and the enzyme metabolite. Likewise, the pancrelipase inhibitory activity of the yam peptide prepared within the enzymolysis time of 6-12 h is gradually increased, but the pancrelipase inhibitory activity of the yam peptide obtained after the enzymolysis time of 12h starts to decrease, and excessive hydrolysis may cause the destruction of the active sequence thereof. Therefore, the enzymolysis time of the yam peptide is optimally 10-12 hours.
Effect example 4
In order to verify the preference of the filtered product in the preparation method of the yam peptide according to the present invention, except that the enzymolysis control experiment was performed according to the preparation method of the yam peptide according to example 1, a 10kDa ultrafiltration membrane (only capable of filtering substances having a relative molecular mass of 10kDa or more) and a 5kDa ultrafiltration membrane (only capable of filtering substances having a relative molecular mass of 5kDa or more) were used, and the enzymolysis product was filtered to obtain fragments having molecular weights of < 5kDa, 5-10kDa and > 10kDa, respectively. Pancreatic lipase inhibitory activity was measured by the method of effect example 1, and the test results are shown in table 9.
TABLE 9
Component fragments Pancreatic lipase inhibition rate (%)
＜5kDa 25.5±2.61
5～10kDa 8.9±2.82
＞10kDa 4.36±1.47
The results are shown in Table 9, wherein the pancrelipase inhibition rate of the yam peptide is related to the molecular weight, and the inhibition activity is higher when the molecular weight is smaller. Compared with the components with the molecular weight of more than 10kDa and 5-10kDa, the pancreatic lipase inhibition activity of the component with the molecular weight of less than 5kDa is better, which indicates that not all the components in the product prepared after enzymolysis have the pancreatic lipase inhibition activity, and the product needs to be specifically screened to obtain the active yam peptide in order to achieve the expected effect.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Claims (8)

1. The composition with the weight and fat reducing effects is characterized by comprising the following components in parts by weight:
20-30 parts of yam peptide and 1 part of N-lactoyl phenylalanine;
the relative molecular mass of the yam peptide is less than or equal to 5 kDa;
the preparation method of the yam peptide comprises the following steps:
(1) Pulverizing rhizoma Dioscoreae, adding water, mixing, and adding alkaline protease for enzymolysis to obtain enzymolysis solution A; the mass ratio of the yam to the alkaline protease is 100: (0.2-2.5); the enzymolysis treatment is carried out at the temperature of 35-65 ℃ for 6-24 hours;
(2) And (3) inactivating enzyme of the enzymolysis liquid A, centrifuging, filtering the supernatant by using an ultrafiltration membrane, and freeze-drying to obtain the yam peptide.
2. The composition of claim 1, wherein the process for preparing N-lactoyl phenylalanine comprises the steps of: and mixing and dispersing the L-phenylalanine, the lactic acid and the calcium oxide in a solvent, mixing and stirring for 2.5-3.5 hours at the temperature of 80-100 ℃, and filtering, concentrating and drying to obtain the N-lactoyl phenylalanine.
3. The composition of claim 1, wherein the ultrafiltration membrane is a 5kDa ultrafiltration membrane.
4. The composition of claim 1, wherein the mass ratio of yam to alkaline protease in step (1) is 100: (0.2-1).
5. The composition of claim 1, wherein the temperature of the enzymolysis treatment in step (1) is 45-50 ℃; the enzymolysis treatment time is 10-12 hours.
6. The composition of claim 1, wherein the centrifugation in step (2) is performed at a temperature of 4 to 8 ℃ for a period of 10 to 25 minutes and at a centrifugation rate of 8000 to 12000rpm.
7. The use of the composition with weight and fat reducing effects according to any one of claims 1 to 6 for preparing a product with weight reducing effect, wherein the product is a medicine or a health product.
8. The use according to claim 7, wherein the preparation has an inhibitory effect on the activity of human pancreatic lipase.
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