High-activity natural dietary supplement for improving intestinal flora of children and preparation method of high-activity natural dietary supplement

Abstract

The invention discloses a high-activity natural dietary supplement for improving intestinal flora of children and a preparation method of the high-activity natural dietary supplement. Water is added into natural substance powder to prepare paste, and a calcium chloride solution and alpha-amylase are added for enzymolysis; adding a sodium chloride solution, adjusting the pH value, adding pepsase, and carrying out enzymolysis; adding a phosphate solution, adjusting the pH value, adding pancreatin, carrying out enzymolysis, inhibiting enzyme activity at low temperature, and freeze-drying to obtain enzymolysis natural substance powder; macromolecular substances in natural substances are decomposed through enzymolysis, products are more beneficial to digestion and absorption of children, meanwhile, substances obtained after enzymolysis contain more dietary fibers or resistant starch, experiments prove that the food has a good improvement effect on intestinal flora of the children, growth of beneficial bacteria can be promoted, and therefore the health level of the children is improved; the growth and development of children are facilitated.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional foods, and in particular relates to a highly active natural dietary supplement for improving children's intestinal flora and a preparation method thereof. Background Art

[0002] Intestinal microorganisms are inextricably linked to human health, and have become an important acquired "organ" in the long-term co-evolution process with the host. The symbiotic relationship between the human body and the intestinal microbiota is established from birth. At the same time, during the growth and development of children, changes in dietary structure, environmental exposure, and antibiotic use will affect their intestinal flora, thereby affecting their immune, metabolic, digestive system, and the development and maturation of neural and cognitive abilities. In addition, changes in the composition of the intestinal microbiota are also associated with short-term and long-term health disorders, such as malnutrition, obesity, and inflammatory diseases. Therefore, various dietary supplements for children have emerged to help protect children's gastrointestinal health and promote their growth and development.

[0003] The rich nutrients and active ingredients in natural substances are of great benefit to children's health. For example, polysaccharides, resistant starch, polypeptides, etc. can not only improve children's immunity but also prevent and alleviate some diseases. Enzymatic hydrolysis is suitable for the extraction of various active ingredients, but it is often used to extract a single substance. When extracting the substance, it will destroy or remove other nutrients or active ingredients. In addition, although the optimal action conditions of the enzyme used can achieve a relatively ideal effect in vitro, this is often very different from the digestive decomposition environment in the body. In order to make up for the gap between in vitro enzymatic hydrolysis and in vivo enzymatic hydrolysis, the bionic extraction method is used to enzymatically hydrolyze natural substances. After enzymatic hydrolysis under simulated gastrointestinal fluid acid-base conditions, a part of the macromolecules in the substance are hydrolyzed into relatively small molecules that are easy to absorb. At the same time, the effective ingredient group is also retained, and the mixed active substances in the original substance are retained to a greater extent, so that natural substances can play their maximum effect. Summary of the invention

[0004] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0005] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0006] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a highly active natural dietary supplement for improving children's intestinal flora.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions: the highly active natural dietary supplement is an enzymatic hydrolysis product of natural substances;

[0008] Among them, dietary fiber content>400mg / g, total flavonoids content>1mg / g, total polyphenols content>1.5mg / g, unsaturated fatty acid content>30mg / g

[0009] As a preferred embodiment of the highly active natural dietary supplement for improving children's intestinal flora of the present invention, the enzymatic hydrolysis product of the natural substance has an enrichment rate of 7.9-10.9% higher than that of inulin for beneficial bacteria in children's intestines, and an inhibition rate of 14.5-14.8% higher than that of inulin for harmful bacteria in children's intestines;

[0010] Among them, beneficial bacteria include Bifidobacterium and Weissella, and harmful bacteria include Klebsiella and some genera in the Enterobacteriaceae family.

[0011] Another object of the present invention is to provide a method for preparing a highly active natural dietary supplement for improving children's intestinal flora.

[0012] In order to solve the above technical problems, the present invention provides the following technical solutions: comprising:

[0013] Powdered natural substances are mixed with pure water to obtain a paste-like liquid, wherein the powdered natural substances include one or more of green banana powder, galactomannan powder, and wheat germ powder;

[0014] The paste liquid is mixed with a calcium chloride solution, heated in a water bath and stirred, and then α-amylase is added to perform enzymolysis to obtain an enzymolysis solution A;

[0015] The enzymatic solution A is mixed with a sodium chloride solution, the pH is adjusted to be acidic, pepsin is added, and the mixture is heated in a water bath and stirred, and enzymatically treated to obtain an enzymatic solution B;

[0016] The enzymatic solution B is mixed with a dipotassium hydrogen phosphate solution, and the pH is adjusted to be neutral, and then pancreatin is added, and the mixture is heated in a water bath and stirred, and enzymatically treated to obtain an enzymatic solution C;

[0017] The enzymatic hydrolysate C is placed at low temperature to inhibit enzyme activity, and then freeze-dried and crushed to obtain a highly active natural dietary supplement.

[0018] As a preferred embodiment of the highly active natural dietary supplement for improving children's intestinal flora according to the present invention, the mass volume ratio of the powdered natural substance to pure water is 1:3-5.

[0019] As a preferred embodiment of the highly active natural dietary supplement for improving children's intestinal flora according to the present invention, the concentration of the calcium chloride solution is 1-3 mM, and the volume ratio of the calcium chloride solution to the pasty liquid is 1:1.

[0020] As a preferred embodiment of the highly active natural dietary supplement for improving children's intestinal flora described in the present invention, after the pasty liquid is mixed with the calcium chloride solution, the temperature of water bath heating and stirring is 35-40°C, the speed is 200-300 rpm, and the time is 10-30 min. α-amylase is added so that the enzyme activity of the enzymatic hydrolyzate A is 75-100 U / ml. The enzymatic hydrolysis conditions are temperature 30-45°C, pH 5-8, speed 200-300 rpm, and time 5-15 min.

[0021] As a preferred embodiment of the highly active natural dietary supplement for improving children's intestinal flora according to the present invention, the concentration of the sodium chloride solution is 30-40 mM, and the volume ratio of the sodium chloride solution to the enzymatic hydrolysate A is 1-3:1.

[0022] As a preferred embodiment of the highly active natural dietary supplement for improving children's intestinal flora described in the present invention, after the enzymatic hydrolyzate A is mixed with the sodium chloride solution, pepsin is added so that the enzyme activity of the enzymatic hydrolyzate B is 50-70 U / ml, and the enzymatic hydrolysis conditions are temperature 30-45°C, pH 1-4, rotation speed 200-300 rpm, and time 1-3 h.

[0023] As a preferred embodiment of the highly active natural dietary supplement for improving children's intestinal flora according to the present invention, the concentration of the dipotassium hydrogen phosphate solution is 45-55 mM, and the volume ratio of the enzymatic hydrolyzate B is 1-3:1.

[0024] As a preferred embodiment of the highly active natural dietary supplement for improving children's intestinal flora according to the present invention, after the enzymatic hydrolysate B is mixed with the dipotassium hydrogen phosphate solution, pancreatic enzyme is added so that the enzyme activity of the enzymatic hydrolysate C is 90-110 U / ml, and the enzymatic hydrolysis conditions are temperature 30-45°C, pH 5-8, rotation speed 200-300 rpm, and time 1-3h.

[0025] Beneficial effects of the present invention:

[0026] The present invention performs bionic extraction on natural substances, decomposing macromolecular nutrients into relatively small molecules that are easily absorbed, while also retaining the effective component group and retaining the mixed active substances in the original substances to a greater extent, thereby reducing the gastrointestinal burden on children and promoting digestion and absorption of nutrients;

[0027] The present invention determines the improvement effect of natural substances after bionic extraction on children's intestinal flora, explores their prebiotic potential, and promotes the development of related children's dietary supplements. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0029] Figure 1 This is the effect of different α-amylase enzymatic hydrolysis conditions on the growth effect of a single probiotic strain as determined in Example 21.

[0030] Figure 2 The effect of different pepsin enzymatic hydrolysis conditions on the growth effect of a single probiotic strain measured in Example 21.

[0031] Figure 3 The effects of different pancreatic enzyme hydrolysis conditions on the growth effect of a single probiotic strain as measured in Example 21 are shown.

[0032] Figure 4 The effects of different enzymatic hydrolysis products on the phylum and genus levels of bacterial community composition measured in Example 22.

[0033] Figure 5 This is a comparison chart of the relative abundance of Bifidobacterium, Weissella, Klebsiella and some bacterial genera in the Enterobacteriaceae family between the blank group and the in vitro fermentation group using different enzymatic hydrolysates as substrates as determined in Example 22. DETAILED DESCRIPTION

[0034] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0035] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0036] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0037] Unless otherwise specified, the raw materials used in the present invention are all commercially available in the art. Among them, green banana powder (Philippines) and wheat germ powder (COFCO Lijin Grain and Oil) were purchased from Miying Food Technology (Suzhou) Co., Ltd.

[0038] The content of the relevant active substances in the product of the present invention is determined by the following method:

[0039] Resistant starch content

[0040] The resistant starch content was determined according to the agricultural industry standard “Spectrophotometric method for determination of resistant starch in plant-derived foods”.

[0041] Determination of total polyphenol content

[0042] The polyphenol content was detected using a plant polyphenol content detection kit, which was purchased from Shanghai Myrrel Biochemical Technology Co., Ltd.

[0043] Determination of total flavonoids content

[0044] The sodium nitrite-aluminum nitrate method is used for determination, specifically:

[0045] Preparation of extract: Take 0.5g of green banana powder and add 5ml of 80% ethanol, ultrasonicate at 40℃ for 2h, centrifuge at 6000r / min for 10min, and the supernatant is the flavonoid extract. Add 0.15ml of 5% sodium nitrite solution to 0.5ml sample solution, let stand for 6min, add 0.15ml of 10% aluminum nitrate solution. Let stand for 6min, add 2ml of 4% sodium hydroxide solution, add distilled water to 5ml, shake and mix well, let stand for 3min, and measure the liquid absorbance at 510nm.

[0046] Taking rutin as the standard, 10.4 mg was dissolved in 25 ml 80% ethanol. 0.0, 0.2, 0.4, 0.6, 0.8, and 1.0 ml of rutin standard solution were measured to replace 0.5 ml of sample solution in the above process for liquid absorbance measurement. The concentration and absorbance standard curve was plotted to obtain the linear regression equation.

[0047] The flavonoid content in green banana powder was calculated by linear regression equation, with rutin as equivalent.

[0048] Peptide content determination

[0049] The BCA protein concentration was determined using a kit purchased from Shanghai Myrrel Biochemical Technology Co., Ltd.

[0050] Determination of polyunsaturated fatty acid content

[0051] The polyunsaturated fatty acid content was determined by referring to the national standard "GB 5009.168-2016 National Food Safety Standard Determination of Fatty Acids in Food".

[0052] Example 1

[0053] This embodiment provides a method for preparing a highly active natural dietary supplement (enzymatically hydrolyzed green banana powder) for improving children's intestinal flora, specifically:

[0054] 1) adding pure water to green banana powder in a ratio of 1:2 (m:v) to obtain a paste-like liquid;

[0055] 2) adding a 1.5 mM calcium chloride solution with a volume of 1 times that of the paste liquid, incubating at 35°C in a water bath at 200 rpm for 30 min to adjust the pH to 6.0, adding α-amylase (so that the enzyme activity in the hydrolyzate reaches 8 U / mg), and performing enzymolysis at 35°C for 10 min to obtain hydrolyzate A;

[0056] 3) Add 34 mM sodium chloride solution with a volume of 1 times that of enzymatic solution A, adjust the pH to 2.0, add pepsin to make the enzyme activity in the enzymatic solution reach 60 U / ml, stir and enzymolyze in a 40°C water bath at 200 rpm for 2 h to obtain enzymatic solution B;

[0057] 4) Add 50 mM potassium dihydrogen phosphate solution with a volume that is 1 times that of enzymatic solution B, adjust the pH to 8.0, add pancreatic enzyme to make the enzyme activity in the enzymatic solution reach 100 U / ml, stir and enzymolyze in a 40°C water bath at 200 rpm for 2 h to obtain enzymatic solution C;

[0058] 5) The enzymolysis solution C is placed at low temperature to inhibit enzyme activity, freeze-dried and crushed to obtain the highly active natural dietary supplement of this embodiment, enzymolysis green banana powder.

[0059] Example 2

[0060] The difference between this embodiment and embodiment 1 is that the raw material green banana powder is replaced with wheat germ powder, and the remaining steps and processes are all referred to embodiment 1 to obtain the enzymatically hydrolyzed wheat germ powder of this embodiment.

[0061] Example 3

[0062] The difference between this embodiment and embodiment 1 is that the enzymatic hydrolysis time of step 2) is adjusted to 5, 10, and 15 minutes respectively, and the remaining steps and processes are all referred to embodiment 1, to obtain enzymatically hydrolyzed green banana powder with different α-amylase hydrolysis times in this embodiment, and the content of active substances therein is measured, and the results are shown in Table 1.

[0063] Table 1

[0064]

[0065] Example 4

[0066] The difference between this embodiment and embodiment 2 is that the enzymatic hydrolysis time of step 2) is adjusted to 5, 10, and 15 minutes respectively, and the remaining steps and processes are all referred to embodiment 2, to obtain enzymatically hydrolyzed wheat germ powder with different α-amylase hydrolysis times in this embodiment, and the content of active substances therein is measured, and the results are shown in Table 2.

[0067] Table 2

[0068]

[0069] It can be seen from the results in Table 1 and Table 2 that when the enzymatic hydrolysis time of α-amylase in the scheme of the present invention is 5 min, the active ingredient content in the enzymatic hydrolysis products obtained from green banana powder and wheat germ powder is the highest.

[0070] Example 5

[0071] The difference between this embodiment and embodiment 1 is that the enzymatic hydrolysis temperature of step 2) is adjusted to 30, 35, 40, and 45° C., respectively, and the remaining steps and processes are all referred to embodiment 1, to obtain enzymatic hydrolysis green banana powder at different α-amylase hydrolysis temperatures of this embodiment, and the active substance content thereof is measured, and the results are shown in Table 3.

[0072] Table 3

[0073]

[0074] Example 6

[0075] The difference between this embodiment and embodiment 2 is that the enzymatic hydrolysis temperature of step 2) is adjusted to 30, 35, 40, and 45° C., respectively, and the remaining steps and processes are all referred to embodiment 2, to obtain enzymatically hydrolyzed wheat germ powder at different α-amylase hydrolysis temperatures of this embodiment, and the active substance content thereof is measured, and the results are shown in Table 4.

[0076] Table 4

[0077]

[0078] It can be seen from the results in Table 3 and Table 4 that when the enzymatic hydrolysis temperature of α-amylase in the scheme of the present invention is 35-40°C, the active ingredient content in the enzymatic hydrolysis products obtained from green banana powder and wheat germ powder is the highest.

[0079] Example 7

[0080] The difference between this embodiment and embodiment 1 is that the enzymatic hydrolysis pH of step 2) is adjusted to 5, 6, 7, and 8 respectively, and the remaining steps and processes are referred to embodiment 1, to obtain enzymatic hydrolysis green banana powder with different α-amylase enzymatic hydrolysis pH of this embodiment, and the active substance content thereof is measured, and the results are shown in Table 5.

[0081] Table 5

[0082]

[0083]

[0084] Example 8

[0085] The difference between this embodiment and embodiment 2 is that the enzymatic hydrolysis pH of step 2) is adjusted to 5, 6, 7, and 8 respectively, and the remaining steps and processes are referred to embodiment 2, to obtain enzymatically hydrolyzed wheat germ powder with different α-amylase enzymatic hydrolysis pH of this embodiment, and the active substance content thereof is measured, and the results are shown in Table 6.

[0086] Table 6

[0087]

[0088] It can be seen from the results in Table 5 and Table 6 that when the enzymatic hydrolysis pH of α-amylase in the scheme of the present invention is 6, the active ingredient content in the enzymatic hydrolysis products obtained from green banana powder and wheat germ powder is the highest.

[0089] Example 9

[0090] The difference between this embodiment and embodiment 1 is that the enzymatic hydrolysis time of step 3) is adjusted to 1, 1.5, 2, 2.5, and 3 hours respectively, and the processes of the remaining steps are referred to embodiment 1, to obtain enzymatically hydrolyzed green banana powder with different pepsin hydrolysis times in this embodiment, and the content of active substances therein is determined, and the results are shown in Table 7.

[0091] Table 7

[0092]

[0093] Example 10

[0094] The difference between this embodiment and embodiment 2 is that the enzymatic hydrolysis time of step 3) is adjusted to 1, 1.5, 2, 2.5, and 3 h, respectively, and the processes of the remaining steps are referred to embodiment 2, to obtain enzymatically hydrolyzed wheat germ powder with different pepsin hydrolysis times in this embodiment, and the content of active substances therein is determined, and the results are shown in Table 8.

[0095] Table 8

[0096]

[0097] It can be seen from the results in Tables 7 and 8 that when the enzymatic hydrolysis time of pepsin in the scheme of the present invention is 2 h, the active ingredient content in the enzymatic hydrolysis products obtained from green banana powder and wheat germ powder is the highest.

[0098] Embodiment 11

[0099] The difference between this embodiment and embodiment 1 is that the enzymatic hydrolysis temperature of step 3) is adjusted to 30, 35, 40, and 45° C., respectively, and the processes of the remaining steps are all referred to embodiment 1, to obtain enzymatic hydrolysis green banana powder at different pepsin hydrolysis temperatures of this embodiment, and the content of active substances therein is measured, and the results are shown in Table 9.

[0100] Table 9

[0101]

[0102] Example 12

[0103] The difference between this embodiment and embodiment 2 is that the enzymatic hydrolysis temperature of step 3) is adjusted to 30, 35, 40, and 45° C., respectively, and the processes of the remaining steps are all referred to embodiment 2, to obtain enzymatically hydrolyzed wheat germ powder at different pepsin hydrolysis temperatures of this embodiment, and the content of active substances therein is measured, and the results are shown in Table 10.

[0104] Table 10

[0105]

[0106] It can be seen from the results in Tables 9 and 10 that when the enzymatic hydrolysis temperature of pepsin in the scheme of the present invention is 40° C., the active ingredient content in the enzymatic hydrolysis products obtained from green banana powder and wheat germ powder is the highest.

[0107] Embodiment 13

[0108] The difference between this embodiment and embodiment 1 is that the enzymatic hydrolysis pH of step 3) is adjusted to 1, 2, 3, and 4 respectively, and the processes of the remaining steps are referred to embodiment 1, to obtain enzymatic hydrolysis green banana powder at different pepsin enzymatic hydrolysis pH of this embodiment, and the active substance content thereof is measured, and the results are shown in Table 11.

[0109] Table 11

[0110]

[0111] Embodiment 14

[0112] The difference between this embodiment and embodiment 2 is that the enzymatic hydrolysis pH of step 3) is adjusted to 1, 2, 3, and 4 respectively, and the processes of the remaining steps are referred to embodiment 2, to obtain enzymatically hydrolyzed wheat germ powder with different pepsin hydrolysis pH of this embodiment, and the content of active substances therein is measured, and the results are shown in Table 12.

[0113] Table 12

[0114]

[0115]

[0116] It can be seen from the results in Table 11 and Table 12 that when the enzymatic hydrolysis pH of pepsin in the scheme of the present invention is 2, the active ingredient content in the enzymatic hydrolysis products obtained from green banana powder and wheat germ powder is the highest.

[0117] Embodiment 15

[0118] The difference between this embodiment and embodiment 1 is that the enzymatic hydrolysis time of step 4) is adjusted to 1, 1.5, 2, 2.5, and 3 hours respectively, and the processes of the remaining steps are referred to embodiment 1, to obtain enzymatically hydrolyzed green banana powder with different pancreatic enzymatic hydrolysis times in this embodiment, and the content of active substances therein is measured, and the results are shown in Table 13.

[0119] Table 13

[0120]

[0121] Example 16

[0122] The difference between this embodiment and embodiment 2 is that the enzymatic hydrolysis time of step 4) is adjusted to 1, 1.5, 2, 2.5, and 3 hours respectively, and the remaining steps and processes are referred to embodiment 2, to obtain enzymatically hydrolyzed wheat germ powder with different pancreatic enzymatic hydrolysis times in this embodiment, and the content of active substances therein is measured, and the results are shown in Table 14.

[0123] Table 14

[0124]

[0125]

[0126] It can be seen from the results in Table 13 and Table 14 that when the enzymatic hydrolysis time of pancreatic enzyme in the scheme of the present invention is 2 hours, the active ingredient content in the enzymatic hydrolysis products obtained from green banana powder and wheat germ powder is the highest.

[0127] Embodiment 17

[0128] The difference between this embodiment and embodiment 1 is that the enzymatic hydrolysis temperature of step 4) is adjusted to 30, 35, 40, and 45° C., respectively, and the remaining steps and processes are all referred to embodiment 1, to obtain enzymatically hydrolyzed green banana powder at different pancreatic enzymatic hydrolysis temperatures of this embodiment, and the active substance content thereof is measured, and the results are shown in Table 15.

[0129] Table 15

[0130]

[0131] Embodiment 18

[0132] The difference between this embodiment and embodiment 2 is that the enzymatic hydrolysis temperature of step 4) is adjusted to 30, 35, 40, and 45° C., respectively, and the processes of the remaining steps are all referred to embodiment 2, to obtain enzymatically hydrolyzed wheat germ powder at different pancreatic enzymatic hydrolysis temperatures of this embodiment, and the active substance content thereof is measured. The results are shown in Table 16.

[0133] Table 16

[0134]

[0135]

[0136] It can be seen from the results in Table 15 and Table 16 that when the enzymatic hydrolysis temperature of pancreatic enzyme in the scheme of the present invention is 40°C, the active ingredient content in the enzymatic hydrolysis products obtained from green banana powder and wheat germ powder is the highest.

[0137] Embodiment 19

[0138] The difference between this embodiment and embodiment 1 is that the enzymatic hydrolysis pH of step 4) is adjusted to 5, 6, 7, and 8 respectively, and the remaining steps and processes are referred to embodiment 1, to obtain enzymatically hydrolyzed green banana powder with different pancreatic enzymatic hydrolysis pH of this embodiment, and the active substance content thereof is measured. The results are shown in Table 17.

[0139] Table 17

[0140]

[0141] Embodiment 20

[0142] The difference between this embodiment and embodiment 2 is that the enzymatic hydrolysis pH of step 4) is adjusted to 5, 6, 7, and 8 respectively, and the remaining steps and processes are referred to embodiment 2, to obtain enzymatically hydrolyzed wheat germ powder with different pancreatic enzymatic hydrolysis pH of this embodiment, and the active substance content thereof is measured. The results are shown in Table 18.

[0143] Table 18

[0144]

[0145] It can be seen from the results in Tables 17 and 18 that when the enzymatic hydrolysis pH of pancreatic enzyme in the scheme of the present invention is 8, the active ingredient content in the enzymatic hydrolysis products obtained from green banana powder and wheat germ powder is the highest.

[0146] In summary, in the enzymatic hydrolysis scheme for natural substances in the present invention, the enzymatic hydrolysis conditions of α-amylase are pH = 6.0, 35°C for 10 min, the enzymatic hydrolysis conditions of pepsin are pH = 2.0, 40°C for 2 h, and the enzymatic hydrolysis conditions of pancreatin are pH = 8.0, 40°C for 2 h. The contents of each active substance in green banana powder and wheat germ powder are relatively high.

[0147] Embodiment 21

[0148] This example is used to verify the in vitro proliferation effect of the natural enzymatic hydrolysates prepared in Examples 3 to 20 on probiotics, specifically:

[0149] Prepare a carbon source-free fermentation medium, the medium formula is shown in Table 19;

[0150] Table 19

[0151]

[0152] 1% of the enzymatic hydrolysate of the natural substances in each example was added to the fermentation medium without carbon source to supplement the carbon source, and then 6% of Bifidobacterium adolescentis was inoculated. After anaerobic fermentation at 37°C for 24 hours, OD600 was measured. The blank control group was the fermentation medium without carbon source. The results are as follows: Figures 1 to 3 shown.

[0153] Figure 1 The effects of different α-amylase hydrolysis conditions (corresponding to Examples 3 to 8) on the growth effect of a single probiotic strain;

[0154] Figure 2 The effects of different pepsin enzymatic hydrolysis conditions (corresponding to Examples 9 to 14) on the growth effect of a single probiotic strain;

[0155] Figure 3 The figure shows the effect of different pancreatic enzyme hydrolysis conditions (corresponding to Examples 15 to 20) on the growth effect of a single probiotic strain. As can be seen from the figure, the hydrolysis conditions of α-amylase are pH = 6.0, 35°C for 10 min, the hydrolysis conditions of pepsin are pH = 2.0, 40°C for 2 h, and the hydrolysis conditions of pancreatic enzyme are pH = 8.0, 40°C for 2 h. The OD600 of the probiotic solution using green banana powder and wheat germ powder as substrates is the highest, indicating that the concentration of the probiotic solution is the highest, which is consistent with the result of the effect of the aforementioned hydrolysis conditions on the content of active substances.

[0156] Comparative Example 1

[0157] This comparative example adopts conventional method to enzymatically hydrolyze green banana powder, specifically:

[0158] 1) adding pure water to green banana powder in a ratio of 1:3 (m:v) to obtain a paste-like liquid;

[0159] 2) adjusting the pH of the paste to 5.0, adding pectinase and cellulase in a ratio of 1:2 (m:m), with an addition amount of 0.22%, stirring at a speed of 200 rpm for 35 minutes in a water bath at 45°C to obtain enzymatic solution A;

[0160] 3) adjusting the pH of the enzymatic hydrolysate A to 6.3, adding medium-temperature α-amylase in an amount of 0.35%, and stirring for 3.5 hours in a 52° C. water bath to obtain enzymatic hydrolysate B;

[0161] 4) The enzymolysis solution B was placed at low temperature to inhibit enzyme activity, freeze-dried and crushed to obtain the enzymolysis green banana powder of this example.

[0162] The content of active substances was measured and compared with that in Example 1. The results are shown in Table 20.

[0163] Table 20

[0164]

[0165] As can be seen from Table 20, the total content of active substances in the green banana powder obtained by enzymatic hydrolysis using the method of the embodiment of the present invention is significantly higher.

[0166] Comparative Example 2

[0167] This comparative example adopts conventional enzymatic hydrolysis method. This comparative example adopts conventional enzymatic hydrolysis method for green banana powder, specifically:

[0168] 1) adding phosphate buffer to wheat germ powder in a ratio of 1:4 (m:v) to obtain a paste-like liquid;

[0169] 2) adding composite protease to the paste liquid to make the enzyme activity in the liquid 17600U / ml, and performing enzymolysis at 50°C for 2.5h to obtain an enzymolysis solution;

[0170] 3) The enzymatic hydrolyzate was placed at low temperature to inhibit enzyme activity, freeze-dried and pulverized to obtain the enzymatic hydrolyzed wheat germ powder of this embodiment.

[0171] The content of active substances was measured and compared with Example 2. The results are shown in Table 21.

[0172] Table 21

[0173]

[0174]

[0175] It can be seen from Table 22 that the content of active substances in the wheat germ powder obtained by enzymatic hydrolysis using the method of the embodiment of the present invention is significantly higher.

[0176] Embodiment 22

[0177] This example measures the improvement effect of enzymatic hydrolysis products under different enzymatic hydrolysis conditions on children's intestinal flora, and conducts in vitro fermentation of children's fecal bacteria. Specifically:

[0178] 1. Experimental population

[0179] The experiment selected 10 children aged 2 to 6 years old, five boys and five girls, all of whom had no metabolic or intestinal diseases in the past three months and no history of taking antibiotics or probiotics.

[0180] 2. Experimental Methods

[0181] Collect fresh fecal samples, mix them in equal weight, add sterile PBS buffer at a ratio of 1:9 (m:v), filter through four layers of sterile gauze to remove impurities, collect the filtrate and complete the inoculation within 2 hours. Prepare the fermentation medium, adjust the pH to 7.0 with 0.5 mol / L HCl, sterilize by high pressure at 121℃ for 15 minutes, and set aside after cooling.

[0182] The fermentation medium (composition as shown in Table 20) and the fecal suspension were mixed at a ratio of 4:1, and the enzymatic natural dietary supplement freeze-dried powder (i.e., the enzymatic hydrolysis product of Example 1, Example 2, Comparative Example 1, and Comparative Example 2) was added at a 1% addition amount as the fermentation substrate, the blank control group was the intestinal anaerobic basic nutrient medium (without carbon source), and the positive control group was the addition of 1% inulin as the fermentation substrate. Fermentation was carried out under anaerobic conditions at 37°C for 24 hours, and the precipitate was obtained by centrifugation at 8000r / min for 15 minutes, which was stored at -80°C and tested for 16SrDNA.

[0183] Table 20

[0184]

[0185]

[0186] 3. Experimental results

[0187] Figure 4 The effect of in vitro fermentation of children's feces on the phylum and genus levels of the flora composition is shown in the figure using natural substances after enzymolysis (the abscissa Blank in the figure represents blank control, Inulin represents inulin, GBF-S represents the enzymolysis product green banana powder in Example 1, WGF-S represents the wheat germ powder after enzymolysis in Example 2, GBF-D represents the enzymolysis product green banana powder in Comparative Example 1, and WGF-D represents the wheat germ powder after enzymolysis in Comparative Example 2) as the substrate.

[0188] Figure 5 The abundance comparison diagram of Bifidobacterium, Weissella, Klebsiella and some genera in Enterobacteriaceae between the fermented substances and the blank group is shown. It can be seen that the enzymatic hydrolyzate of the natural substances in Examples 1 and 2 of the present invention has an enrichment rate of 7.9-10.9% for beneficial bacteria in the intestines of children compared with inulin, and an inhibition rate of 14.5-14.8% for harmful bacteria in the intestines of children compared with inulin. Among them, the beneficial bacteria include Bifidobacterium and Weissella, and the harmful bacteria include Klebsiella and some genera in Enterobacteriaceae.

[0189] The enzymatic hydrolysates of Comparative Examples 1-2 have lower enrichment effects on Bifidobacterium and Weissella and lower inhibitory effects on Klebsiella and some genera in the Enterobacteriaceae than those in the Examples.

[0190] In summary, the present invention performs bionic extraction on natural substances, decomposing macromolecular nutrients into relatively small molecules that are easily absorbed, while also retaining the effective component group and retaining the mixed active substances in the original substances to a greater extent, thereby reducing the gastrointestinal burden on children and promoting digestion and absorption of nutrients;

[0191] The present invention determines the improvement effect of natural substances after bionic extraction on children's intestinal flora, explores its prebiotic potential, and promotes the development of related children's dietary supplements. The natural substances after enzymatic hydrolysis can enrich some beneficial bacteria in the flora and inhibit harmful bacteria, and have a certain improvement effect on children's intestinal microorganisms.

[0192] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A highly active natural dietary supplement for improving children's intestinal flora, characterized in that: The highly active natural dietary supplement is an enzymatic hydrolysis product of natural substances; Among them, dietary fiber content is >400mg / g, total flavonoids content is >1mg / g, total polyphenols content is >1.5mg / g, and unsaturated fatty acid content is >30mg / g.

2. The highly active natural dietary supplement for improving children's intestinal flora according to claim 1, characterized in that: The enzymatic hydrolysis product of the natural substance has an enrichment rate of 7.9-10.9% higher for beneficial bacteria in children's intestines than inulin, and an inhibition rate of 14.5-14.8% higher for harmful bacteria in children's intestines than inulin; Among them, beneficial bacteria include Bifidobacterium and Weissella, and harmful bacteria include Klebsiella and some genera in the Enterobacteriaceae family.

3. A method for preparing a highly active natural dietary supplement for improving children's intestinal flora, characterized in that: include, Powdered natural substances are mixed with purified water to obtain a paste-like liquid, wherein the powdered natural substances include one or more of green banana powder and galactomannan powder; The paste liquid is mixed with a calcium chloride solution, heated in a water bath and stirred, and then α-amylase is added to perform enzymolysis to obtain an enzymolysis solution A; The enzymatic solution A is mixed with a sodium chloride solution, the pH is adjusted to be acidic, pepsin is added, and the mixture is heated in a water bath and stirred, and enzymatically treated to obtain an enzymatic solution B; The enzymatic solution B is mixed with a dipotassium hydrogen phosphate solution, and the pH is adjusted to be neutral, and then pancreatin is added, and the mixture is heated in a water bath and stirred, and enzymatically treated to obtain an enzymatic solution C; The enzymatic hydrolysate C is placed at low temperature to inhibit enzyme activity, and then freeze-dried and crushed to obtain a highly active natural dietary supplement.

4. The method for preparing a highly active natural dietary supplement for improving intestinal flora in children according to claim 3, characterized in that: The mass volume ratio of the powdered natural substance to pure water is 1:3-5.

5. The method for preparing a highly active natural dietary supplement for improving children's intestinal flora according to claim 3, characterized in that: The concentration of the calcium chloride solution is 1-3 mM, and the volume ratio of the calcium chloride solution to the paste liquid is 1:

1.

6. The method for preparing a highly active natural dietary supplement for improving children's intestinal flora according to claim 5, characterized in that: After the paste liquid is mixed with the calcium chloride solution, the temperature of water bath heating and stirring is 35-40° C., the rotation speed is 200-300 rpm, and the time is 10-30 min. α-amylase is added so that the enzyme activity of the enzymatic hydrolyzate A is 75-100 U / ml. The enzymatic hydrolysis conditions are temperature 30-45° C., pH 5-8, rotation speed 200-300 rpm, and time 5-15 min.

7. The method for preparing a highly active natural dietary supplement for improving children's intestinal flora according to claim 3, characterized in that: The concentration of the sodium chloride solution is 30-40 mM, and the volume ratio of the sodium chloride solution to the enzymatic hydrolysis solution A is 1-3:

1.

8. The method for preparing a highly active natural dietary supplement for improving children's intestinal flora according to claim 7, characterized in that: After the enzymatic solution A is mixed with the sodium chloride solution, pepsin is added to make the enzyme activity of the enzymatic solution B 50-70 U / ml, and the enzymatic hydrolysis conditions are temperature 30-45° C., pH 1-4, rotation speed 200-300 rpm, and time 1-3 hours.

9. The method for preparing a highly active natural dietary supplement for improving intestinal flora in children according to claim 3, characterized in that: The concentration of the dipotassium hydrogen phosphate solution is 45-55 mM, and the volume ratio of the enzymatic hydrolysis solution B is 1-3:

1.

10. The method for preparing a highly active natural dietary supplement for improving intestinal flora in children according to claim 9, characterized in that: After the enzymatic solution B is mixed with the dipotassium hydrogen phosphate solution, pancreatin is added to make the enzyme activity of the enzymatic solution C 90-110 U / ml, and the enzymatic hydrolysis conditions are temperature 30-45° C., pH 5-8, rotation speed 200-300 rpm, and time 1-3 hours.

Images