Fermentation product for promoting growth and development of children and preparation method thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicine fermentation, and particularly relates to a fermented product for promoting growth and development of children and a preparation method thereof. The fermentation raw materials comprise the following components in parts by weight: 40-60 parts of cynanchum auriculatum, 15-25 parts of perilla seeds, 20-40 parts of rehmannia, 20-35 parts of pinenut kernels and 40-55 parts of burdock roots. The fermentation raw materials are subjected to extraction process treatment according to the properties of the raw materials, and after uniform fermentation, the obtained fermentation product can increase the body length, the body weight and the bone mineral density through rat experiment verification, and the effect of promoting growth and development is achieved.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine fermentation technology, specifically relating to a fermentation product that promotes children's growth and development and its preparation method. Background Technology

[0002] In Traditional Chinese Medicine (TCM), the spleen and stomach primarily function to digest and absorb nutrients, then transport these nutrients throughout the body. Since growth and development require nutrition, a healthy spleen and stomach ensure sufficient nutrition, leading to healthy growth and development. Growth and development encompasses various aspects, such as height, weight, bone development, and even intellectual development.

[0003] Children and adolescents grow and develop rapidly and have a high demand for nutrition. When there is an unreasonable diet, bad eating habits (cold drinks, sugary / high-fat foods, spicy foods), or improper feeding by parents, it can lead to a weak spleen and stomach, which will affect growth and development.

[0004] Food-medicine homologous ingredients are those that can be used as both food and medicine raw materials. They can exert therapeutic or health-preserving effects to a certain extent, and can be used to regulate the body, prevent diseases, or assist in treatment through daily diet. Their application in children's daily health care is more easily accepted. Furthermore, microbial fermentation technology can transform the large molecules of traditional Chinese medicine into small molecules, making them more easily absorbed by the human body. Summary of the Invention

[0005] Therefore, the present invention provides a fermented substance that promotes children's growth and development, which can promote children's growth and development in three dimensions: height, weight, and bone density.

[0006] On the other hand, the present invention provides a method for preparing a ferment that promotes children's growth and development. This method is simple and suitable for industrial production.

[0007] The technical content of this invention is as follows:

[0008] In one aspect, the present invention provides a fermented product that promotes children's growth and development. The fermentation ingredients, by weight, include 40-60 parts of auricularia auricula-judae, 15-25 parts of perilla seeds, 20-40 parts of rehmannia root, 20-35 parts of pine nuts, and 40-55 parts of burdock root.

[0009] Preferably, the fermentation raw materials, by weight, include 45-55 parts of auricularia auricula-judae, 20-25 parts of perilla seeds, 25-40 parts of rehmannia root, 20-30 parts of pine nuts, and 40-50 parts of burdock root.

[0010] Further preferred fermentation ingredients, by weight, include 50 parts of auricularia auricula-judae, 25 parts of perilla seeds, 30 parts of rehmannia root, 25 parts of pine nuts, and 45 parts of burdock root.

[0011] In another aspect, the present invention also provides a method for preparing a ferment that promotes children's growth and development, comprising the following steps:

[0012] (1) The ear-leaf cowhide was steam-exploded to obtain ear-leaf cowhide explosive;

[0013] (2) Mix Rehmannia glutinosa and burdock root with water, add enzyme preparation for enzymatic hydrolysis, and obtain mixed enzymatic hydrolysate;

[0014] (3) The ear leaf cowhide de-explosion material, the mixed enzymatic hydrolysate, perilla seeds and pine nuts are mixed and subjected to enzyme inactivation extraction treatment to obtain an extract;

[0015] (4) Add a fermenting agent to the extract and ferment to obtain the fermented product.

[0016] Preferably, the steam explosion pressure in step (1) is 0.8-2.0 MPa and the pressure holding time is 50-80 s.

[0017] Preferably, step (1) before steam explosion further includes a step of pre-soaking the ear leaf cowhide disinfectant; specifically, the ear leaf cowhide disinfectant is mixed with water at a material-to-liquid ratio of 1:(5-10) and soaked for 4-7 hours.

[0018] Preferably, in step (2), the ratio of the total weight of Rehmannia glutinosa and Arctium lappa root to water is 1:(10-20); the amount of enzyme preparation added is 8%-10%; specifically, the amount of enzyme preparation added refers to the ratio of the weight of enzyme preparation to the total weight of Rehmannia glutinosa and Arctium lappa root.

[0019] More preferably, the ratio of the total weight of Rehmannia glutinosa and burdock root to water is 1:15; and the amount of enzyme preparation added is 9%.

[0020] Preferably, the enzyme preparation comprises cellulase and neutral protease; the weight ratio of the cellulase to the neutral protease is (3.5-6):(1.3-3.6).

[0021] More preferably, the weight ratio of the cellulase to the neutral protease is 4.5:2.25.

[0022] More preferably, the cellulase activity is 10,000-50,000 U / g, and the neutral protease activity is 50,000-400,000 U / g.

[0023] Preferably, the enzymatic hydrolysis temperature in step (2) is 40-55℃ and the enzymatic hydrolysis time is 3-5h.

[0024] Preferably, in step (3), the enzyme inactivation temperature is 100-125℃ and the enzyme inactivation time is 20-30min.

[0025] Preferably, the amount of fermenting agent added in step (4) is 0.08%-0.12%; more preferably, the amount of fermenting agent added is 0.1%; specifically, the amount of fermenting agent added refers to the ratio of the weight of the fermenting agent to the total weight of the ear-leaf ox-skin shavings, perilla seeds, rehmannia, pine nuts, and burdock root.

[0026] Preferably, the fermenting agent includes Bifidobacterium adolescentis and Lactococcus lactis.

[0027] More preferably, the weight ratio of Bifidobacterium adolescentis to Lactococcus lactis is (0.05-0.12):(0.04-0.11); even more preferably, the weight ratio of Bifidobacterium adolescentis to Lactococcus lactis is 1:1.

[0028] Preferably, the viable count of the Bifidobacterium adolescentis is 100-800 billion CFU / g, and the viable count of the Lactococcus lactis is 100-500 billion CFU / g.

[0029] Preferably, the fermentation temperature in step (4) is 30-40℃ and the fermentation time is 24-72h.

[0030] In a final aspect, the present invention provides the application of the above-described ferment or the ferment obtained by the preparation method in the preparation of products that promote children's growth and development.

[0031] The effects of each ingredient in the fermentation product provided by this invention are as follows:

[0032] *Auricularia auricula-judae* (a plant belonging to the genus *Auricularia* in the family Asclepiadaceae) is a traditional Chinese medicine, specifically the tuberous root of this plant. In traditional Chinese medicine, *Auricularia auricula-judae* is sweet, bitter, and slightly warm in nature, and enters the spleen, stomach, and lung meridians. It is used to treat indigestion, abdominal pain, bloating, and ascites. Modern research indicates that it contains compounds such as acetophenones, terpenes, steroidal glycosides, organic acids, sugars, lignans, and coumarins.

[0033] Rehmannia glutinosa (raw) is the dried tuberous root of Rehmannia glutinosa, a plant in the Scrophulariaceae family. It is cold in nature and sweet in taste, and enters the heart, liver, and kidney meridians. It has the effects of clearing heat and cooling blood, nourishing yin and promoting body fluid production. Its chemical components mainly include iridoid glycosides (such as catalpol and rehmannia glycoside D), sugars (such as stachyose and rehmannia polysaccharides), phenylethanol glycosides (such as verbascoside and isorhabditis glycoside), ionones, nucleosides, organic acids, and various trace elements. Modern research shows that Rehmannia glutinosa has various pharmacological effects, including anti-inflammatory, antioxidant, osteoporosis prevention, and hypoglycemic effects.

[0034] Burdock root is a herbaceous plant belonging to the genus Arctium in the family Asteraceae. Its dried, mature fruit is called burdock seed, and its fleshy taproot is called burdock root. Burdock root has extremely high nutritional value, rich in polyphenols, flavonoids, sugars, amino acids, and other active substances. It has pharmacological effects such as anti-oxidation, anti-aging, anti-inflammation, antibacterial properties, and regulation of glucose and lipid metabolism.

[0035] Perilla seeds are rich in unsaturated fatty acids, protein, minerals, vitamins, and other beneficial components. Alpha-linolenic acid (ALA) is the most important active ingredient in perilla seeds (accounting for over 50% of the seed's fat content). It is an essential fatty acid that the human body cannot synthesize and must obtain from food. Perilla seeds can supplement nutrients and participate in the growth and development process.

[0036] Pine nuts are the kernels of the Korean pine, a plant belonging to the Pinaceae family. They are rich in unsaturated fatty acids, high-quality protein, minerals (calcium, phosphorus, magnesium, etc.), vitamin E, and dietary fiber, providing essential nutrients for growth and development.

[0037] Compared with the prior art, the technical solution provided by this invention has the following advantages:

[0038] 1. The fermented product provided by this invention is prepared by fermentation using *Auricularia auricula-judae* and *Arctium lappa* root as the main raw materials, and *Perilla frutescens* seeds, pine nuts, and *Rehmannia glutinosa* as auxiliary materials. The five raw materials are combined to invigorate the spleen and replenish qi, harmonize the stomach and promote digestion. The synergistic effect of these herbs enhances the overall effect, regulating the absorption and conversion efficiency of nutrients in the rat's digestive system, thereby promoting the rat's height, longitudinal bone extension (increase), and lateral bone strengthening (bone density), thus achieving the effect of promoting growth and development.

[0039] 2. In the preparation method of the fermented product provided by the present invention, the ear leaf bovine skin is treated by steam explosion, and the rehmannia root and burdock root are treated by enzymatic hydrolysis. After pretreatment according to the characteristics of the raw materials, they are mixed, inactivated and sterilized by fermentation to obtain the product. The pretreatment according to the characteristics of the raw materials can better release the effective ingredients, so that the final fermented product has better effects on promoting body weight and bone growth.

[0040] 3. In the preparation method of the fermentation product provided by the present invention, the applicant has creatively discovered that the fermentation product obtained by fermenting a compound strain of Bifidobacterium adolescentis and Lactococcus lactis can better promote growth and development. Attached Figure Description

[0041] Figure 1 Typical images of zebrafish body length in the blank group, Example 1 group, and positive control group;

[0042] Figure 2 The zebrafish body lengths were those of the blank group, Example 1 group, and positive control group; compared with the normal control group, *p<0.05, ***p<0.001. Detailed Implementation

[0043] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0044] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0045] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of the present invention are obtained through conventional commercial channels. Products from different manufacturers do not have a significant impact on the effectiveness.

[0046] The following herbs were purchased: *Auricularia auricula-judae*, *Rehmannia glutinosa*, *Arctium lappa* root, and *Rehmannia glutinosa* (processed). These were purchased from Anhui Jiahe Traditional Chinese Medicine Technology Co., Ltd.; pine nuts were purchased from Anhui Jishun Traditional Chinese Medicine Pieces Co., Ltd.; perilla seeds were purchased from Wenshui Shunkemei Biotechnology Co., Ltd.; and flaxseeds were purchased from Bozhou Renyi Traditional Chinese Medicine Sales Co., Ltd. *Bifidobacterium adolescentis* LJ10101-SA, with a viable count of 100 billion CFU / g, was purchased from Jiangsu Xinshenao Biotechnology Co., Ltd.; *Lactococcus lactis* LJ10602-SA, with a viable count of 100 billion CFU / g, was purchased from Jiangsu Xinshenao Biotechnology Co., Ltd. Cellulase was purchased from Shandong Longket Enzyme Preparation Co., Ltd., with an enzyme activity of 20,000 U / g; and neutral protease was purchased from Nanning Pangbo Bioengineering Co., Ltd., with an enzyme activity of 200,000 U / g.

[0047] Example 1

[0048] This embodiment provides a fermented product that promotes children's growth and development. By weight, the fermentation ingredients include: 50 parts of ear-leaf oxhide extract, 25 parts of perilla seeds, 30 parts of rehmannia root, 25 parts of pine nuts, and 45 parts of burdock root.

[0049] Its preparation method is as follows:

[0050] (1) Add 50 parts of ear leaf cowhide to 250 parts of water and soak for 5 hours. Then put it into the material bin of the steam explosion machine, introduce saturated steam, select 1.0 MPa pressure, select 60s pressure holding time, and complete the depressurization in a very short time to achieve steam explosion and obtain ear leaf cowhide explosion material.

[0051] (2) Mix 30 parts of Rehmannia glutinosa, 45 parts of Arctium lappa root and 1125 parts of water, add 4.5 parts of cellulase and 2.25 parts of neutral protease, and enzymatically hydrolyze at 50℃ for 4 hours to obtain a mixed enzymatic hydrolysate;

[0052] (3) Mix the ruptured bovine bark, mixed enzyme hydrolysate, 25 parts pine nuts, and 25 parts perilla seeds, adjust the pH to 5.0, and extract at 120℃ for 25 minutes to obtain the extract.

[0053] (4) After the temperature of the extract decreases, add 0.09 parts of Bifidobacterium adolescentis and 0.09 parts of Lactococcus lactis and ferment at 35°C for 60 hours to obtain the fermented product.

[0054] The viable counts of Bifidobacterium adolescentis and Lactococcus lactis were 100 billion CFU / g; the cellulase activity was 20,000 U / g; and the neutral protease activity was 200,000 U / g.

[0055] Example 2

[0056] This embodiment provides a fermented product that promotes children's growth and development. By weight, the fermentation ingredients include: 40 parts of ear-leaf oxhide extract, 18 parts of perilla seeds, 20 parts of rehmannia root, 28 parts of pine nuts, and 40 parts of burdock root.

[0057] Its preparation method is as follows:

[0058] (1) After soaking 40 parts of ear leaf cowhide in 400 parts of water for 7 hours, put it into the material bin of the steam explosion machine, introduce saturated steam, select 1.0 MPa pressure, select 60s pressure holding time, complete the depressurization in a very short time, realize steam explosion to obtain ear leaf cowhide explosion material;

[0059] (2) Mix 20 parts of Rehmannia glutinosa, 40 parts of Arctium lappa root and 600 parts of water, add 3.5 parts of cellulase and 1.3 parts of neutral protease, and enzymatically hydrolyze at 40℃ for 5 hours to obtain a mixed enzymatic hydrolysate;

[0060] (3) Mix the ear leaf cowhide de-explosion material, mixed enzyme hydrolysate, 18 parts of perilla seeds and 28 parts of pine nuts, adjust the pH to 5.0, and extract at 125℃ for 20 minutes to obtain the extract;

[0061] (4) After the temperature of the extract decreases, add 0.05 parts of Bifidobacterium adolescentis and 0.07 parts of Lactococcus lactis and ferment at 30°C for 72 hours to obtain the fermented product.

[0062] The viable counts of Bifidobacterium adolescentis and Lactococcus lactis were 100 billion CFU / g; the cellulase activity was 20,000 U / g; and the neutral protease activity was 200,000 U / g.

[0063] Example 3

[0064] This embodiment provides a fermented product that promotes children's growth and development. By weight, the fermentation ingredients include: 45 parts of ear-leaf oxhide extract, 23 parts of perilla seeds, 25 parts of rehmannia root, 35 parts of pine nuts, and 55 parts of burdock root.

[0065] Its preparation method is as follows:

[0066] (1) After soaking 45 parts of ear leaf cowhide in 360 parts of water for 5 hours, put it into the material bin of the steam explosion machine, introduce saturated steam, select 1.0 MPa pressure, select 60 s pressure holding time, complete the depressurization in a very short time, realize steam explosion to obtain ear leaf cowhide explosion material;

[0067] (2) Mix 25 parts of Rehmannia glutinosa, 55 parts of Arctium lappa root and 1600 parts of water, add 6 parts of cellulase and 2 parts of neutral protease, and enzymatically hydrolyze at 55℃ for 3 hours to obtain a mixed enzymatic hydrolysate.

[0068] (3) Mix the ear leaf cowhide de-explosion material, mixed enzyme hydrolysate, 23 parts of perilla seeds and 35 parts of pine nuts, adjust the pH to 5.0, and extract at 100℃ for 30 minutes to obtain the extract;

[0069] (4) After the temperature of the extract decreases, add 0.12 parts of Bifidobacterium adolescentis and 0.04 parts of Lactococcus lactis and ferment at 40°C for 24 hours to obtain the fermented product.

[0070] The viable counts of Bifidobacterium adolescentis and Lactococcus lactis were 100 billion CFU / g; the cellulase activity was 20,000 U / g; and the neutral protease activity was 200,000 U / g.

[0071] Example 4

[0072] This embodiment provides a fermented product that promotes children's growth and development. By weight, the fermentation ingredients include: 55 parts of ear-leaf oxhide extract, 20 parts of perilla seeds, 40 parts of rehmannia root, 30 parts of pine nuts, and 50 parts of burdock root.

[0073] Its preparation method is as follows:

[0074] (1) After soaking 55 parts of ear leaf cowhide in 330 parts of water for 6 hours, put it into the material chamber of the steam explosion machine, introduce saturated steam, select 1.0 MPa pressure, select 60s pressure holding time, complete the depressurization in a very short time, realize steam explosion, and obtain ear leaf cowhide explosion material.

[0075] (2) Mix 40 parts of Rehmannia glutinosa, 50 parts of Arctium lappa root and 1080 parts of water, add 5 parts of cellulase and 2.65 parts of neutral protease, and enzymatically hydrolyze at 48℃ for 3.5h to obtain mixed enzymatic hydrolysate;

[0076] (3) Mix the ear leaf cowhide de-explosion material, mixed enzyme hydrolysate, 20 parts of perilla seeds and 30 parts of pine nuts, adjust the pH to 5.0, and extract at 110℃ for 28 minutes to obtain the extract;

[0077] (4) After the temperature of the extract decreases, add 0.12 parts of Bifidobacterium adolescentis and 0.09 parts of Lactococcus lactis and ferment at 33°C for 48 hours to obtain the fermented product.

[0078] The viable counts of Bifidobacterium adolescentis and Lactococcus lactis were 100 billion CFU / g; the cellulase activity was 20,000 U / g; and the neutral protease activity was 200,000 U / g.

[0079] Example 5

[0080] This embodiment provides a fermented product that promotes children's growth and development. By weight, the fermentation ingredients include: 60 parts of ear-leaf oxhide extract, 15 parts of perilla seeds, 35 parts of rehmannia root, 20 parts of pine nuts, and 45 parts of burdock root.

[0081] Its preparation method is as follows:

[0082] (1) After soaking 60 parts of ear leaf cowhide in 540 parts of water for 4 hours, put it into the material chamber of the steam explosion machine, introduce saturated steam, select 1.0 MPa pressure, select 60s pressure holding time, complete the depressurization in a very short time, realize steam explosion, and obtain ear leaf cowhide explosion material;

[0083] (2) Mix 35 parts of Rehmannia glutinosa, 45 parts of Arctium lappa root and 1440 parts of water, add 4 parts of cellulase and 3.6 parts of neutral protease, and enzymatically hydrolyze at 53℃ for 4.5h to obtain a mixed enzymatic hydrolysate;

[0084] (3) Mix the ear leaf cowhide de-explosion material, mixed enzyme hydrolysate, 15 parts of perilla seeds and 20 parts of pine nuts, adjust the pH to 5.0, and extract at 121℃ for 23 minutes to obtain the extract;

[0085] (4) After the temperature of the extract decreases, add 0.10 parts of Bifidobacterium adolescentis and 0.11 parts of Lactococcus lactis and ferment at 38°C for 65 hours to obtain the fermented product.

[0086] The viable counts of Bifidobacterium adolescentis and Lactococcus lactis were 100 billion CFU / g; the cellulase activity was 20,000 U / g; and the neutral protease activity was 200,000 U / g.

[0087] Comparative Example 1

[0088] This comparative example provides a fermented product that differs from Example 1 only in that the fermentation raw materials do not include pine nuts. Specifically, it consists of 50 parts of *Auricularia auricula-judae*, 25 parts of perilla seeds, 30 parts of *Rehmannia glutinosa*, and 45 parts of burdock root. However, the total weight of *Auricularia auricula-judae*, perilla seeds, *Rehmannia glutinosa*, and burdock root is the same as that of Example 1.

[0089] Its preparation method is as follows:

[0090] (1) Add 50 parts of ear leaf cowhide to 250 parts of water and soak for 5 hours. Then put it into the material bin of the steam explosion machine, introduce saturated steam, select 1.0 MPa pressure, select 60s pressure holding time, and complete the depressurization in a very short time to achieve steam explosion and obtain ear leaf cowhide explosion material.

[0091] (2) Mix 30 parts of Rehmannia glutinosa, 45 parts of Arctium lappa root and 1125 parts of water, add 4.5 parts of cellulase and 2.25 parts of neutral protease, and enzymatically hydrolyze at 50℃ for 4 hours to obtain a mixed enzymatic hydrolysate;

[0092] (3) Mix the ear leaf cowhide de-explosion material, mixed enzyme hydrolysate, and 25 parts of perilla seeds, adjust the pH to 5.0, and extract at 120℃ for 25 minutes to obtain the extract;

[0093] (4) After the temperature of the extract decreases, add 0.09 parts of Bifidobacterium adolescentis and 0.09 parts of Lactococcus lactis and ferment at 35°C for 60 hours to obtain the fermented product.

[0094] The viable counts of Bifidobacterium adolescentis and Lactococcus lactis were 100 billion CFU / g; the cellulase activity was 20,000 U / g; and the neutral protease activity was 200,000 U / g.

[0095] Comparative Example 2

[0096] This comparative example provides a fermented product, which differs from Example 1 only in that the fermentation raw materials are replaced with flax seeds instead of perilla seeds. Specifically, the fermentation raw materials are 50 parts of auricularia auricula-judae, 25 parts of flax seeds, 30 parts of rehmannia glutinosa, 25 parts of pine nuts, and 45 parts of burdock root; the rest is the same as in Example 1.

[0097] Comparative Example 3

[0098] This comparative example provides a fermented product, which differs from Example 1 only in that the raw Rehmannia glutinosa is replaced with processed Rehmannia glutinosa in the fermentation raw materials. Specifically, it consists of 50 parts of Auricularia auricula-judae, 25 parts of Perilla frutescens seeds, 30 parts of processed Rehmannia glutinosa, 25 parts of pine nuts, and 45 parts of burdock root.

[0099] Its preparation method is as follows:

[0100] (1) Add 50 parts of ear leaf cowhide to 250 parts of water and soak for 5 hours. Then put it into the material bin of the steam explosion machine, introduce saturated steam, select 1.0 MPa pressure, select 60s pressure holding time, and complete the depressurization in a very short time to achieve steam explosion and obtain ear leaf cowhide explosion material.

[0101] (2) Mix 30 parts of Rehmannia glutinosa, 45 parts of Arctium lappa root and 1125 parts of water, add 4.5 parts of cellulase and 2.25 parts of neutral protease, and enzymatically hydrolyze at 50℃ for 4 hours to obtain a mixed enzymatic hydrolysate;

[0102] (3) Mix the ruptured bovine bark, mixed enzyme hydrolysate, 25 parts pine nuts, and 25 parts perilla seeds, adjust the pH to 5.0, and extract at 120℃ for 25 minutes to obtain the extract.

[0103] (4) After the temperature of the extract decreases, add 0.09 parts of Bifidobacterium adolescentis and 0.09 parts of Lactococcus lactis and ferment at 35°C for 60 hours to obtain the fermented product.

[0104] The viable counts of Bifidobacterium adolescentis and Lactococcus lactis were 100 billion CFU / g; the cellulase activity was 20,000 U / g; and the neutral protease activity was 200,000 U / g.

[0105] Comparative Example 4

[0106] This comparative example provides a fermented product that differs from Example 1 only in that the fermentation raw materials do not include burdock root. Specifically, it consists of 50 parts of *Auricularia auricula-judae*, 25 parts of perilla seeds, 30 parts of Rehmannia glutinosa, and 25 parts of pine nuts. However, the total weight of *Auricularia auricula-judae*, perilla seeds, Rehmannia glutinosa, and pine nuts is the same as that of *Auricularia auricula-judae*, perilla seeds, Rehmannia glutinosa, pine nuts, and burdock root in Example 1.

[0107] Its preparation method is as follows:

[0108] (1) Add 50 parts of ear leaf cowhide to 250 parts of water and soak for 5 hours. Then put it into the material bin of the steam explosion machine, introduce saturated steam, select 1.0 MPa pressure, select 60s pressure holding time, and complete the depressurization in a very short time to achieve steam explosion and obtain ear leaf cowhide explosion material.

[0109] (2) Mix 30 parts of Rehmannia glutinosa with 1125 parts of water, add 4.5 parts of cellulase and 2.25 parts of neutral protease, and enzymatically hydrolyze at 50℃ for 4 hours to obtain the enzymatic hydrolysate;

[0110] (3) Mix the ear leaf cowhide de-explosion material, enzyme hydrolysate, 25 parts pine nuts and 25 parts perilla seeds, adjust the pH to 5.0, and extract at 120℃ for 25 minutes to obtain the extract;

[0111] (4) After the temperature of the extract decreases, add 0.09 parts of Bifidobacterium adolescentis and 0.09 parts of Lactococcus lactis and ferment at 35°C for 60 hours to obtain the fermented product.

[0112] The viable counts of Bifidobacterium adolescentis and Lactococcus lactis were 100 billion CFU / g; the cellulase activity was 20,000 U / g; and the neutral protease activity was 200,000 U / g.

[0113] Comparative Example 5

[0114] This comparative example provides a fermented product, which differs from Example 1 only in that the preparation method of *Auricularia auricula-judae* is changed to enzymatic hydrolysis. Specifically, by weight, the fermentation raw materials include: 50 parts of *Auricularia auricula-judae*, 25 parts of perilla seeds, 30 parts of Rehmannia glutinosa, 25 parts of pine nuts, and 45 parts of burdock root.

[0115] Its preparation method is as follows:

[0116] (1) Mix 50 parts of auricularia auricula-judae, 30 parts of Rehmannia glutinosa, 45 parts of Arctium lappa root with 1375 parts of water, add 7.5 parts of cellulase and 3.75 parts of neutral protease, and enzymatically hydrolyze at 50℃ for 4 hours to obtain a mixed enzymatic hydrolysate.

[0117] (2) Mix the mixed enzyme hydrolysate, 25 parts of pine nuts and 25 parts of perilla seeds, adjust the pH to 5.0, and extract at 120℃ for 25 minutes to obtain the extract;

[0118] (3) After the temperature of the extract decreases, add 0.09 parts of Bifidobacterium adolescentis and 0.09 parts of Lactococcus lactis and ferment at 35°C for 60 hours to obtain the fermented product.

[0119] The viable counts of Bifidobacterium adolescentis and Lactococcus lactis were 100 billion CFU / g; the cellulase activity was 20,000 U / g; and the neutral protease activity was 200,000 U / g.

[0120] Comparative Example 6

[0121] This comparative example provides a fermented product that differs from Example 1 in that the fermentation raw materials do not include *Auricularia auricula-judae*, but are specifically 25 parts of perilla seeds, 30 parts of rehmannia root, 25 parts of pine nuts, and 45 parts of burdock root; however, the total weight of perilla seeds, rehmannia root, pine nuts, and burdock root is the same as the total weight of *Auricularia auricula-judae*, perilla seeds, rehmannia root, pine nuts, and burdock root in Example 1.

[0122] Its preparation method is as follows:

[0123] (1) Mix 30 parts of Rehmannia glutinosa, 45 parts of Arctium lappa roots with 1125 parts of water, add 4.5 parts of cellulase and 2.25 parts of neutral protease, and carry out enzymatic hydrolysis at 50 °C for 4 h to obtain a mixed enzymatic hydrolysate;

[0124] (2) Mix the mixed enzymatic hydrolysate, 25 parts of pine nuts, and 25 parts of perilla seeds, adjust the pH to 5.0, and carry out enzyme inactivation extraction at 120 °C for 25 min to obtain an extract;

[0125] (3) Wait for the temperature of the extract to decrease, add 0.09 parts of Bifidobacterium adolescentis and 0.09 parts of Lactococcus lactis cremoris, and ferment at 35 °C for 60 h to obtain a fermented product.

[0126] Among them, the viable count of Bifidobacterium adolescentis is 100 billion CFU / g, and the viable count of Lactococcus lactis cremoris is 100 billion CFU / g; the enzyme activity of cellulase is 20,000 U / g, and the enzyme activity of neutral protease is 200,000 U / g.

[0127] Comparative Example 7

[0128] This comparative example provides a fermented product, which is different from Example 1 in that the starter only includes 0.18 parts of Lactococcus lactis cremoris; the rest is the same as Example 1.

[0129] Experimental Example 1 Experiment on the efficacy of promoting growth and development of zebrafish

[0130] 1 Experimental animals

[0131] Zebrafish are all raised in fish-raising water at 28 °C (water quality: add 200 mg of instant sea salt to every 1 L of reverse osmosis water, the conductivity is 450 - 550 μS / cm; pH is 6.5 - 8.5; hardness is 50 - 100 mg / L CaCO3), provided by the fish-raising center of Hangzhou Huante Biotechnology Co., Ltd., and the license number for the use of experimental animals is: SYXK (Zhe) 2022 - 0004. The feeding management meets the requirements of international AAALAC certification (certification number: 001458), and the IACUC ethical review number: IACUC - 2025 - 12243 - 01.

[0132] 2 Experimental samples

[0133] The supernatant obtained by centrifuging the fermented product of Example 1, and the solvent is standard dilution water.

[0134] Positive control: Swisse "Calcium&VitaminD", milky white tablets, batch number 33152A, Swisse, and the solvent is standard dilution water.

[0135] 3 Instruments, consumables and reagents

[0136] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); Precision electronic balance (CP214, OHAUS, America).

[0137] Methylcellulose (batch number C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China).

[0138] 4. Determination of Maximum Detectable Concentration (MTC)

[0139] Wild-type AB strain zebrafish, 3 days post-fertilization (3 dpf), were randomly selected and placed in beakers, with 30 zebrafish per beaker. They were treated with water-soluble solutions as described in Example 1 (concentrations of 3.91, 7.81, 15.6, 31.2, and 62.5 μL / mL), with a normal control group also included. Each beaker had a volume of 20 mL. The solution was changed daily at a water temperature of 28°C. After three days of treatment, the effect of the samples on the mortality rate of the zebrafish was determined to establish the mean criterion (MTC).

[0140] 5. Experiment on the efficacy of promoting growth and development

[0141] Wild-type AB strain zebrafish (3dpf) were randomly selected and placed in beakers, with 30 zebrafish in each beaker. The sample from Example 1 was administered in water at concentrations of 3.91, 7.81, and 15.6 μL / mL. A normal control group and a positive control group (250 μg / mL Swisse Calcium & Vitamin D) were also included, with the solution changed daily. After treatment at 28℃ for 3 days, 10 zebrafish from each group were randomly selected and photographed under a dissecting microscope. Data were analyzed and collected using NIS-ElementsD 3.20 advanced image processing software. The body length of the zebrafish was analyzed to evaluate the effect of the sample on promoting growth and development.

[0142] 6 Experimental Results

[0143] 6.1 Determination of Maximum Detectable Concentration (MTC)

[0144] The MTC results are shown in Table 1. As can be seen from Table 1, when the sample concentration in Example 1 was 31.2 μL / mL, the zebrafish were in a more severe condition compared with the normal control group, but no deaths occurred; when the concentration was 62.5 μL / mL, all the zebrafish died; therefore, the MTC of Example 1 was 15.6 μL / mL.

[0145] Table 1. Effects of different concentrations of Example 1 on zebrafish survival.

[0146]

[0147] 6.2 Results of promoting growth and development

[0148] See the effect of promoting growth and development Figure 1 and Figure 2 ,Depend on Figure 1 and Figure 2 It can be seen that when the concentration of Example 1 is 3.91 μL / mL, there is no significant difference in body length compared with the normal control group; when the concentration is 7.81 μL / mL, the body length is 3.90 mm, which is significantly higher than the body length of 3.82 mm in the normal control group. This indicates that the fermentation product provided by the present invention has the effect of promoting growth and development within a certain concentration range.

[0149] Experiment Example 2: Rat Experiment

[0150] 1. Laboratory animals

[0151] SPF-grade SD rats, 8 weeks old, 30 females and 15 males, weighing 180-300g. Rats had free access to water, and were kept in an environment with a temperature of 22-26℃, humidity of 40-70%, 12h light / 12h dark, and were acclimatized for 7 days.

[0152] 2 samples

[0153] The fermentation supernatant obtained from the solid-liquid separation of the fermentation products in Examples 1-5 and Comparative Examples 1-7 was used as the test sample.

[0154] 3 Experimental Groups

[0155] Rats were housed together overnight. The following morning, sperm was found under a microscope in a smear of the vaginal irrigation fluid from the female rats, indicating that conception occurred on day 1. After conception, the female rats were randomly divided into a low-protein diet group (8% crude protein compared to the standard diet group, Beijing Keao Feed Co., Ltd.) and a standard diet group (20% crude protein, Beijing Keao Feed Co., Ltd.). Pregnant rats in both the low-protein diet group and the standard diet group were fed the standard diet after giving birth.

[0156] At 14 days of age, female pups were removed from both the low-protein diet group and the standard diet group, and the pups were weaned at 21 days of age and fed with standard diet. The dwarfism model was defined as follows: newborn pups from the low-protein diet group whose post-weaning length was 2 standard deviations shorter than the average length of newborn pups from the standard diet group. Ten pups from the standard diet group were randomly selected using a random number table for weighing and labeled as the normal group. The dwarfism model pups were then randomly divided into four groups using a random number table: model group, Examples 1-5, Comparative Examples 1-7, and Vitamin D group (Swisse "Calcium & Vitamin D", milky white tablets, 300 tablespoons, Swisse), with 10 pups in each group.

[0157] 4. Administration method

[0158] The normal group and the model group were given sterile saline, 1 mL / 100 g. Examples 1-5, Comparative Examples 1-7, and the Vitamin D group were given the corresponding samples. The dosage for Examples 1-5 and Comparative Examples 1-7 was calculated to be 9 mL / kg using the body surface area conversion factor method. The medication was administered once daily at 8:00 AM for three consecutive weeks.

[0159] The vitamin D group (Swisse "Calcium & Vitamin D", mini calcium, 300 tables, 1.5g / tablet, of which calcium citrate content is 165mg / tablet) was administered at a dose of 4 tablets per day for humans, and the rat dosage was calculated to be 540mg / kg based on the body surface area conversion method. The drug was administered once daily at 8:00 AM for three consecutive weeks.

[0160] 5. Indicator Detection and Methods

[0161] Body length: The rat was immobilized and its body was stretched out. The distance from the tip of the nose to the anus was measured as the body length.

[0162] Body weight: The rat was placed on an electronic balance and its body weight was measured when it was at rest.

[0163] Bone mineral density: After anesthetizing rats with 0.8% sodium pentobarbital via intraperitoneal injection, the rats were placed on the table of a dual-energy X-ray absorptiometry (DXA) bone densitometer. The rats were scanned by the instrument, and their bone mineral density was recorded. The average value was taken from three scans.

[0164] 6 Experimental Results

[0165] The effects of each group of samples on the body weight and length of rats are shown in Tables 2 and 3.

[0166] Table 2 shows that the initial body weight of each group before drug administration was significantly lower than that of the normal group, indicating that the growth rate of the dwarf rats obtained by the present invention was lagging and their development was delayed. At the end of drug administration, compared with the model group, the final body weight of Examples 1-5 was significantly higher, and there was no significant difference in the final body weight between Example 1 and the normal group, indicating that the fermentation product provided by the present invention can significantly increase the body weight of dwarf rats.

[0167] The difference between Comparative Example 1 and Example 1 is that pine nuts are not included. As shown in Table 2, the final body weight of the rats in Comparative Example 1 was significantly lower than that of the rats in Example 1. Moreover, Comparative Example 1 did not show a significant increase compared with the model group. This indicates that pine nuts have a significant impact on the body weight of dwarf rats. This is mainly because pine nuts contain a large amount of unsaturated fatty acids, have a high fat content and a very high calorie density, which can quickly convert excess calories into fat, leading to weight gain.

[0168] The difference between Comparative Example 2 and Example 1 is that perilla seeds were replaced with flax seeds, which also contain unsaturated fatty acids. As shown in Table 2, the final body weight of rats in Comparative Example 2 was significantly lower than that in Example 1, indicating that the perilla seeds, pine nuts, auricularia auricula-judae, rehmannia glutinosa, and burdock root selected in this invention can increase the body weight of dwarf rats more quickly.

[0169] The difference between Comparative Example 3 and Example 1 is that raw Rehmannia glutinosa is replaced with processed Rehmannia glutinosa. The difference between Comparative Example 4 and Example 1 is that Arctium lappa root is not included. The results show that the final body weight of rats in Comparative Examples 3-4 is significantly lower than that in Example 1, indicating that the present invention selects Rehmannia glutinosa and Arctium lappa root in combination with Auricularia auricula-judae, Perilla frutescens seeds and pine nuts to promote the increase of body weight in dwarf rats.

[0170] The difference between Comparative Example 5 and Example 1 lies in the replacement of the steam explosion process with an enzymatic hydrolysis process for the *Auricularia auricula-judae* (ear-leaf oxhide) decontamination. The steam explosion technology, under high temperature and high pressure conditions, allows high-temperature saturated steam to rapidly penetrate into plant cells. Through instantaneous pressure release, thermal energy is converted into mechanical energy, and acid-like hydrolysis occurs, causing cell rupture and structural damage in the plant tissue, facilitating the dissolution of chemical substances and enhancing biological activity. The enzymatic method relies on enzyme-catalyzed degradation of cell wall polysaccharides, releasing the effective components embedded in the cell matrix. The results show that the final body weight of the rats in Comparative Example 5 was significantly lower than that in Example 1, indicating that the steam explosion process can more effectively promote the dissolution of effective components such as steroidal glycosides and organic acids in the *Auricularia auricula-judae* decontamination, thus contributing to an increase in the final body weight of the rats.

[0171] The difference between Comparative Example 6 and Example 1 is that Comparative Example 6 does not include Auricularia auricula-judae. As shown in Table 2, the final body weight of the rats in Comparative Example 6 was significantly lower than that in Example 1, indicating that Auricularia auricula-judae, in combination with Perilla frutescens seeds, pine nuts, burdock root and Rehmannia glutinosa, can promote the increase of body weight in dwarf rats.

[0172] The difference between Comparative Example 7 and Example 1 is that the fermentation was carried out by a single strain. As shown in Table 2, the final body weight of rats in Comparative Example 7 decreased compared with that in Example 1, indicating that the co-fermentation of Lactococcus lactis and Bifidobacterium adolescentis was more effective in promoting the increase of body weight in dwarf rats.

[0173] Compared with the model group, the vitamin D group did not show a significant increase in final body weight; compared with Example 1, the final body weight decreased significantly, indicating that the vitamin D group was less effective in promoting the weight gain of dwarf rats than the fermentation product obtained in this application.

[0174] Table 2. Effects of each sample group on rat body weight

[0175]

[0176] Note: Compared with the normal group, #P<0.05, ##P<0.01; compared with the model group, +P<0.05, ++P<0.01; compared with Example 1, △P<0.05, △△P<0.01.

[0177] As shown in Table 3, at the end of the administration, the final body length of Examples 1-5 was significantly increased compared with the model group, and there was no significant difference between Examples 1-5 and the normal group, indicating that the fermentation product provided by the present invention can increase the height of dwarf rats.

[0178] Compared with Example 1: The difference between Comparative Examples 1-4 and Example 1 is that pine nuts were removed in Comparative Example 1, perilla seeds were replaced with flax seeds in Comparative Example 2, raw rehmannia root was replaced with processed rehmannia root in Comparative Example 3, burdock root was removed in Comparative Example 4, and Comparative Example 6 was the same as Example 1 except that it did not include auricularia auricula-judae. As shown in Table 3, the final body length of Comparative Examples 1-4 was significantly lower than that of Example 1, the final body length of Comparative Example 6 was lower than that of Example 1, and there was no significant difference in the final body length between Comparative Example 4 and the model group. This indicates that the five fermentation raw materials selected in this invention—Auricularia auricula-judae, Perilla frutescens seeds, Rehmannia glutinosa, pine nuts, and Arctium lappa root—have comprehensive nutritional value and a certain synergistic effect in promoting the increase of rat body length. The difference between Comparative Example 5 and Example 1 is that the steam explosion process of Auricularia auricula-judae was changed to an enzymatic hydrolysis process. The results show that the final body length of the dwarf rats in Comparative Example 5 was significantly lower than that in Example 1. This indicates that the steam explosion process can better promote the release of the effective components in Auricularia auricula-judae, and the released effective components have a greater impact on height, ultimately resulting in a better effect on increasing the final body length of the dwarf rats.

[0179] The difference between Comparative Example 7 and Example 1 is that the fermentation strain was a single strain. As shown in Table 3, the final body length of Comparative Example 7 was significantly lower than that of Example 1, indicating that the co-fermentation of Bifidobacterium adolescentis and Lactococcus lactis had a better effect on promoting the increase of rat body length. The final body length of the dwarf rats in the vitamin D group was not significantly different from that in Example 1, indicating that the fermentation product provided by the present invention can achieve the effect of promoting the increase of body length in dwarf rats in the vitamin D group.

[0180] Table 3. Effects of each sample group on rat body length

[0181]

[0182] Note: Compared with the normal group, #P<0.05, ##P<0.01; compared with the model group, +P<0.05, ++P<0.01; compared with Example 1, △P<0.05, △△P<0.01.

[0183] Bone mineral density (BMD) refers to the amount of bone mineral contained per unit area or unit volume of bone tissue, and is an important indicator of bone strength. Studies have shown that the increase in bone mineral density in children and adolescents is significantly correlated with their height and weight growth and development.

[0184] The effects of each group of samples on bone mineral density in rats are shown in Table 4. As can be seen from Table 4, at the end of the administration, compared with the model group, the bone mineral density of Examples 1-5 was significantly increased, indicating that the fermentation product provided by the present invention can improve the bone mineral density of dwarf rats.

[0185] Compared with Example 1, the differences between Comparative Examples 1-4 and Example 1 are as follows: pine nuts were removed in Comparative Example 1; perilla seeds were replaced with flax seeds in Comparative Example 2; raw rehmannia root was replaced with processed rehmannia root in Comparative Example 3; and burdock root was removed in Comparative Example 4. Comparative Example 6 differs from Example 1 in that it does not include *Auricularia auricula-judae*. As shown in Table 4, the bone density of Comparative Examples 1-4 was significantly lower than that of Example 1, and the bone density of Comparative Example 6 was significantly lower than that of Example 1. This indicates that the five fermentation ingredients selected in this invention—*Auricularia auricula-judae*, perilla seeds, rehmannia root, pine nuts, and burdock root—work synergistically to promote bone density in rats. The effect of increasing bone density was better in Comparative Example 5 than in Example 1. The difference between Comparative Example 5 and Example 1 is that the steam explosion process of the herbal medicine was changed to an enzymatic hydrolysis process. The results show that the bone density of Comparative Example 7 was significantly lower than that of Example 1, indicating that the steam explosion process can better promote the release of the effective components in the herbal medicine and thus have a better effect on increasing bone density in rats. The difference between Comparative Example 7 and Example 1 is that the fermentation strain was a single strain. As shown in Table 4, the bone density of Comparative Example 7 was significantly lower than that of Example 1, indicating that the co-fermentation of traditional Chinese medicine by Bifidobacterium adolescentis and Lactococcus lactis has a better effect on increasing bone density in rats.

[0186] Table 4. Effects of each sample group on bone mineral density in rats

[0187]

[0188] Note: Compared with the normal group, #P<0.05, ##P<0.01; compared with the model group, +P<0.05, ++P<0.01; compared with Example 1, △P<0.05, △△P<0.01.

[0189] Growth and development indicators involve three aspects: height, weight, and bone density. During the growth process, due to incomplete nutrition, there may be a rapid increase in weight or height, insufficient bone mineral deposition, and relatively low bone density, which makes the bones fragile and prone to fractures.

[0190] The five raw materials selected in this invention—Auricularia auricula-judae, Arctium lappa root, Rehmannia glutinosa, Perilla frutescens seed, and pine nut kernel—work synergistically with the raw material processing technology and the selection of fermentation strains to simultaneously promote increased bone density in rats, enabling them to withstand the load of height and weight gain and better achieve the effect of promoting rat growth and development. This prevents excessively rapid growth and development, resulting in only weight gain with relatively low bone density.

[0191] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A fermented product that promotes children's growth and development, characterized in that, By weight, it includes 40-60 parts of auricularia auricula-judae, 15-25 parts of perilla seeds, 20-40 parts of rehmannia root, 20-35 parts of pine nuts, and 40-55 parts of burdock root.

2. The fermented product for promoting children's growth and development according to claim 1, characterized in that, By weight, it includes 45-55 parts of auricularia auricula-judae, 20-25 parts of perilla seeds, 25-40 parts of rehmannia root, 20-30 parts of pine nuts, and 40-50 parts of burdock root.

3. The method for preparing the fermented product for promoting children's growth and development according to any one of claims 1-2, characterized in that, include: (1) Exploding the ear-leaf cowhide explosive to obtain ear-leaf cowhide explosive; (2) Mix Rehmannia glutinosa and burdock root with water, add enzyme preparation for enzymatic hydrolysis, and obtain mixed enzymatic hydrolysate; (3) The ear leaf cowhide de-explosion material, the mixed enzymatic hydrolysate, perilla seeds and pine nuts are mixed and subjected to enzyme inactivation extraction treatment to obtain an extract; (4) Add a fermenting agent to the extract and ferment to obtain the fermented product.

4. The method for preparing the fermented product for promoting children's growth and development according to claim 3, characterized in that, In step (2), the amount of enzyme preparation added is 8%-10%; the enzyme preparation includes cellulase and neutral protease, and the weight ratio of cellulase to neutral protease is (3.5-6):(1.3-3.6).

5. The method for preparing the fermented product for promoting children's growth and development according to claim 3, characterized in that, In step (4), the amount of fermenting agent added is 0.08%-0.12%; the fermenting agent includes Bifidobacterium adolescentis and Lactococcus lactis, and the weight ratio of Bifidobacterium adolescentis to Lactococcus lactis is (0.05-0.12):(0.04-0.11).

6. The method for preparing the fermented product for promoting children's growth and development according to claim 3, characterized in that, In step (1), the steam explosion pressure is 0.8-2.0 MPa and the pressure holding time is 50-80 s.

7. The method for preparing the fermented product for promoting children's growth and development according to claim 3, characterized in that, In step (2), the ratio of the total weight of Rehmannia glutinosa and Arctium lappa root to water is 1:(10-20).

8. The method for preparing the fermented product for promoting children's growth and development according to claim 3, characterized in that, In step (2), the enzymatic hydrolysis temperature is 40-55℃ and the enzymatic hydrolysis time is 3-5h.

9. The method for preparing the fermented product for promoting children's growth and development according to claim 3, characterized in that, In step (3), the enzyme inactivation temperature is 100-125℃ and the enzyme inactivation time is 20-30 min; In step (4), the fermentation temperature is 30-40℃ and the fermentation time is 24-72h.

10. The use of the fermentation product according to claims 1-2 or the fermentation product obtained by any of the preparation methods according to claims 3-9 in the preparation of products that promote children's growth and development.

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