La28 and lp45 composite probiotic fermentation preparation of dry type blood tonifying aigong method, product and application

Abstract

This invention belongs to the field of traditional Chinese medicine fermentation technology, specifically relating to a method for preparing a blood-tonifying and dehydrating donkey-hide gelatin based on the fermentation of a compound probiotic strain (La28 and LP45), the product itself, and its applications. This method uses donkey-hide gelatin as raw material, and through directional fermentation with compound probiotics, it efficiently degrades large-molecule collagen into easily absorbed small-molecule peptides and free amino acids. Under the same raw material conditions, the fermentation product contains significantly higher levels of small-molecule peptides and free amino acids than unfermented donkey-hide gelatin, and also introduces new specific active products such as D(+)-phenyllactic acid and cyclic dipeptides. The compound probiotics consist of *Lactobacillus acidophilus* La28 and *Lactobacillus plantarum* LP45. In vitro experiments show that this fermentation product can significantly alleviate inflammation-related dryness, heatiness, and stomach upset caused by excessive richness; it can also increase hemoglobin synthesis and enhance the blood-tonifying effect. The resulting fermented donkey-hide gelatin can be used alone or in combination with single medicinal materials or classic prescriptions, and has broad application prospects in the preparation of health products and medicines that tonify blood, reduce dryness, nourish yin and moisten the lungs, and improve intestinal absorption.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine fermentation technology, specifically relating to a method for preparing a dryness-reducing and blood-tonifying donkey-hide gelatin based on the fermentation of La28 and LP45 compound probiotics, the product, and its application. Background Technology

[0002] Donkey-hide gelatin was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), listed as a superior-grade herb. It is sweet in taste and neutral in nature, entering the lung, liver, and kidney meridians. It possesses the effects of nourishing blood and stopping bleeding, nourishing yin and moistening the lungs, enhancing immunity, combating fatigue, anti-aging, and promoting bone healing. Clinically, it is often used for blood deficiency syndromes, consumptive hemoptysis, hematochezia, yin deficiency with irritability and insomnia, and lung deficiency with dry cough. It is also widely used in women's health maintenance and rehabilitation. With the continuous development of modern traditional Chinese medicine, the fermentation of Chinese herbal medicines has gradually become a hot topic in the medical field.

[0003] Donkey-hide gelatin's main components are macromolecular collagen, various amino acids, and trace elements such as zinc, iron, and calcium. Traditional donkey-hide gelatin has two major drawbacks: (1) Large molecular collagen is difficult to be naturally hydrolyzed into small molecular peptides and free amino acids in the body. The intestinal absorption rate is low, and the undigested components are retained, causing discomfort such as greasiness, internal heat, and sexual discomfort. It may cause side effects such as greasiness and stomach upset. (2) Conventional processing and extraction techniques cannot reduce the digestive burden from the source, and the blood-tonifying effect cannot be maximized.

[0004] Traditional Chinese medicine fermentation combines traditional processing and fermentation with modern bioengineering techniques. It utilizes probiotic enzyme systems to modify and degrade the structure of medicinal components, improving bioavailability, enhancing properties and flavor, and increasing efficacy. Currently, there are no mature processes or products that utilize specialized probiotic complexes for targeted fermentation of donkey-hide gelatin to achieve the dual goals of reducing dryness and enhancing blood-nourishing effects. Summary of the Invention

[0005] (a) Technical problems to be solved In view of the above-mentioned shortcomings and deficiencies of the existing technology, this invention provides a method for preparing a dryness-reducing and blood-tonifying donkey-hide gelatin based on the fermentation of a La28 and LP45 compound probiotic. Through specific probiotic compound fermentation, the dryness and greasiness of donkey-hide gelatin can be significantly reduced, while its blood-tonifying efficacy and intestinal absorption efficiency can be improved. This provides a stable, controllable, and industrially scalable fermentation process, achieving efficient conversion of large-molecule collagen into small-molecule peptides / free amino acids. This makes the fermentation product suitable for products related to reducing dryness, tonifying blood, nourishing yin, improving intestinal microecology, and alleviating internal heat. This invention can degrade large molecules at the source, reduce dryness, and enhance blood-tonifying effects, achieving efficient and gentle utilization of donkey-hide gelatin.

[0006] (II) Technical Solution In a first aspect, the present invention provides a method for preparing a desiccant-reducing and blood-tonifying type based on fermentation of a La28 and LP45 compound probiotic, comprising the following steps: S1. Ingredients: Dissolve donkey-hide gelatin in water or an aqueous solvent by heating, add a fermentable carbon source, sterilize, and obtain fermentation substrate liquid; S2. Inoculation: Cool to ≤40℃, add compound probiotics, and mix well; the compound probiotics contain 1-3×10 11 CFU / g of Lactobacillus acidophilus La28 and 6-10×10 11 The dosage of CFU / g Lactobacillus plantarum LP45 is 20g-40g / ton of fermentation substrate liquid; S3. Fermentation: Ferment at 30℃-42℃ for 1-3 days to obtain fermented donkey-hide gelatin product.

[0007] According to a preferred embodiment of the present invention, in S1, the mass ratio of donkey-hide gelatin to water is 1:8-10.

[0008] According to a preferred embodiment of the present invention, in S1, the aqueous solvent is purified water, deionized water, or water for injection; the fermentable carbon source includes one or more of glucose, sucrose, maltose, fructooligosaccharides, and inulin; and the amount of fermentable carbon source added is 0.5%-5% of the mass of the fermentation substrate liquid.

[0009] Preferably, the amount of fermentable carbon source added is 1%-3% of the volume of the fermentation substrate liquid.

[0010] Preferably, in S1, the fermentable carbon source is glucose, and the amount added is 2% of the mass of the fermentation substrate liquid.

[0011] Preferably, in S1, the sterilization conditions are sterilization at 100℃-121℃ for 10-60 minutes, and more preferably sterilization at 105℃ for 30 minutes.

[0012] Preferably, in S3, the fermentation temperature is 35℃-39℃, and more preferably 37℃±1℃.

[0013] Preferably, in S3, the fermentation time is 36-48 hours.

[0014] Preferably, in S2, the ratio of live Lactobacillus acidophilus La28 to Lactobacillus plantarum LP45 in the compound probiotics is 1:1. Preferably, the addition amount is 30g / ton of fermentation substrate liquid.

[0015] Secondly, the present invention provides a donkey-hide gelatin product, which is prepared by any of the above-mentioned methods. The donkey-hide gelatin product contains small molecule peptides, free amino acids, D(+)-phenyllactic acid and cyclic dipeptide components. The small molecule peptides include proline-glycine dipeptide, and the free amino acids include L-proline. In the product obtained by fermentation of an equal amount of donkey-hide gelatin, the total content of small molecule peptides and free amino acids is higher than the corresponding content in the donkey-hide gelatin before fermentation.

[0016] Thirdly, the present invention provides the application of the above-mentioned donkey-hide gelatin product in the preparation of functional foods or medicines that reduce dryness, replenish blood, nourish yin and moisten the lungs, improve intestinal absorption, balance the intestinal flora structure, and reduce greasiness and stomach upset.

[0017] Preferably, the donkey-hide gelatin product can be used alone or in combination with other medicinal and edible raw materials / Chinese herbal fermentation liquids, such as astragalus, red dates, etc.; the donkey-hide gelatin product can be an oral liquid.

[0018] (III) Beneficial Effects This invention utilizes specific compound probiotics to directionally ferment donkey-hide gelatin, achieving a significant increase in the content of small molecule peptides and free amino acids under the same raw materials, while making the degradation of macromolecules easier to absorb; it reduces the dryness and greasiness of the gelatin from the source, inhibits inflammation, and improves the intestinal microecology; at the same time, it significantly promotes the proliferation of hematopoietic cells, inhibits apoptosis, and enhances hemoglobin synthesis, achieving a comprehensive technical effect of making it safer to reduce dryness, more efficient to replenish blood, and faster to absorb. Attached Figure Description

[0019] Figure 1 This presents the experimental results of the effects of the donkey-hide gelatin fermentation broth of the present invention on the NO secretion level and iNOS mRNA expression in Raw264.7 cells.

[0020] Figure 2 This presents the experimental results of the effect of the donkey-hide gelatin fermentation broth of the present invention on the expression of the inflammation-related gene COX-2 in Raw264.7 cells.

[0021] Figure 3 The results show the experimental effects of the donkey-hide gelatin fermentation broth of this invention on the survival rate of TF-1 cells.

[0022] Figure 4 The results show the effects of the donkey-hide gelatin fermentation broth of this invention on the pro-apoptotic gene BAX in TF-1 cells.

[0023] Figure 5 The results show the effects of the donkey-hide gelatin fermentation broth of this invention on the hemoglobin content synthesized in TF-1 cells. Detailed Implementation

[0024] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0025] This invention uses specific compound probiotics to ferment donkey-hide gelatin products, which, compared to donkey-hide gelatin before fermentation, have the following effects: 1. Enhances dryness-reducing and blood-nourishing functions Fermentation significantly reduces the "greasy and drying" properties of donkey-hide gelatin, alleviating discomfort caused by excessive heat. The fermentation process reduces the digestive burden from the source, eliminating the dryness-related problems associated with traditional donkey-hide gelatin, such as excessive heat, bloating, dry mouth, and constipation, making it a gentler tonic. Furthermore, the fermentation process enhances its blood-nourishing effects and improves hematopoietic capacity. It promotes the proliferation and differentiation of erythroid progenitor cells, inhibits hematopoietic cell apoptosis, and significantly increases hemoglobin synthesis, resulting in a blood-nourishing effect superior to unfermented donkey-hide gelatin.

[0026] In addition, fermentation enhances the anti-inflammatory and "dryness-reducing" effects, significantly reduces the release of the inflammatory factor NO, inhibits the high expression of iNOS and COX-2 genes, alleviates the inflammatory response in the body, and achieves the effect of nourishing yin and moisturizing dryness.

[0027] 2. Change the ingredient composition to enhance absorption. The efficient degradation of macromolecules and the significant enhancement of small-molecule nutrients result in a higher content of small-molecule peptides and free amino acids in fermented donkey-hide gelatin of the same quality. Macromolecular collagen is directionally hydrolyzed into easily absorbed small-molecule peptides and free amino acids.

[0028] Improved bioavailability leads to faster and more complete intestinal absorption. The smaller molecular size allows for direct absorption without secondary digestion in the body, significantly increasing nutrient utilization and reducing raw material waste.

[0029] It produces novel active substances and enhances functional fermentation to generate specific metabolites such as D(+)-phenyllactic acid and cyclic dipeptides, which have antibacterial, anti-inflammatory and intestinal regulating effects.

[0030] 3. Effects on improving gut microbiota Regulates the balance of gut microbiota and inhibits harmful bacteria. Probiotics and their metabolites improve the gut microecology, enhance digestive enzyme activity, and reduce abnormal intestinal fermentation.

[0031] Reduces the burden on the stomach and intestines, avoiding the greasy and irritating effects of traditional donkey-hide gelatin. Solves the problems of indigestion, stomach upset, and loss of appetite associated with traditional donkey-hide gelatin, making it more suitable for the elderly, children, and people with weak digestive systems.

[0032] It enhances the body's overall tolerance. Long-term use has no drying side effects; it is a gentle tonic suitable for a wider range of people.

[0033] The donkey-hide gelatin fermentation process of this invention is stable and controllable, suitable for industrial production, with mild conditions, clear parameters, good repeatability, and no toxic or harmful substances, complying with food / health product / pharmaceutical production standards. Furthermore, it preserves all the nutrients of donkey-hide gelatin, without damaging the active ingredients; low-temperature fermentation does not result in the loss of amino acids, trace elements, etc., thus ensuring complete nutrient retention. The fermented donkey-hide gelatin product can be formulated alone into oral liquids, powders, or tablets, or it can be combined with fermented Chinese medicinal herbs such as jujubes, goji berries, mulberries, and astragalus, or other classic formulas, to achieve synergistic enhancement of efficacy.

[0034] The following description is based on preferred embodiments, comparative examples, and effect verification of the present invention.

[0035] Example 1 This embodiment describes a method for preparing donkey-hide gelatin fermentation liquid, and the preparation process is as follows: 10 kg of donkey-hide gelatin was melted in 80 L of 50°C hot water, and 1.8 kg of glucose was added to obtain a mixture. The mixture was sterilized at 105°C for 30 minutes, then cooled to 40°C to obtain a sterilized solution. 2.7 g of a compound probiotic powder was added to the sterilized solution, and fermentation was carried out at 37°C for 40 hours to obtain a fermentation broth. The compound probiotic mixture consisted of Lactobacillus acidophilus La28 and Lactobacillus plantarum LP45. Finally, the mixture was filtered, bottled, and sterilized to obtain the donkey-hide gelatin fermentation broth.

[0036] Example 2 The difference between this embodiment and Example 1 is that glucose is replaced with maltose at 2.5% of the fermentation substrate mass. The compound probiotics consist of Lactobacillus acidophilus La28 and Lactobacillus plantarum LP45. Preferably, the addition amount is 30g / ton of fermentation substrate, and fermentation is carried out for 36 hours. The fermentation volume is the same as in Example 1, yielding the fermentation broth.

[0037] Comparative Example 1 The difference between this comparative example and Example 1 is that the mixed probiotic powder added in Example 1 was replaced with an equal amount of single probiotic powders for fermenting donkey-hide gelatin. These included *Bifidobacterium longum* subsp. *longum* L693, *Lactobacillus paracasei* YMC1069, *Lactobacillus acidophilus* La28, *Streptococcus salivarius* subsp. *thermophilus* S709, *Bifidobacterium breve* CCFM1151, *Lactobacillus paracasei* L578, *Lactobacillus plantarum* LP45, *Bifidobacterium animalis* subsp. *thermophilus* JMCC0025, *Bifidobacterium bifidum* B11, *Lactobacillus helveticus* L551, *Streptococcus salivarius* subsp. *thermophilus* S131, *Lactobacillus rhamnosus* LR519, *Lactobacillus casei* CICC 6117, and *Lactobacillus fermentum* CGMCC 11058. The results are shown in Table 1.

[0038] Table 1:

[0039] Table 1 shows that probiotic fermentation significantly alters the chemical composition of donkey-hide gelatin, noticeably increasing the content of amino acids and their derivatives, as well as fatty acids and their derivatives. Single strains exhibit significant functional limitations. Lactobacillus acidophilus La28 demonstrates outstanding conversion ability for fatty acid derivatives, but its effect on amino acid enhancement is only moderate. Lactobacillus plantarum LP45 and Lactobacillus rhamnosus LR519 possess excellent enzymatic hydrolysis capabilities for donkey-hide gelatin collagen, significantly increasing the content of amino acids and their derivatives, but their effect on improving fatty acid components is poor. Other single strains show relatively low increases in both types of active ingredients, resulting in limited overall modification effects.

[0040] When *Lactobacillus acidophilus* La28 and *Lactobacillus rhamnosus* LR519 are combined, the two strains exhibit significant competition in fermentation metabolism and deficiencies in environmental adaptability. *Lactobacillus rhamnosus* LR519 possesses only excellent collagenase hydrolysis capabilities, but its fatty acid derivative conversion ability is extremely poor. Furthermore, it has poor adaptability to the acid-producing fermentation environment of *Lactobacillus acidophilus* La28, and its activity is easily inhibited. The two strains cannot achieve efficient synergy; instead, they mutually deplete fermentation substrates and their respective environments, leading to a significant decrease in fatty acid modification effects and failing to achieve simultaneous synergistic effects on both amino acids and fatty acids.

[0041] In Example 1 of this invention, a co-fermentation of *Lactobacillus acidophilus* La28 and *Lactobacillus plantarum* LP45 was used to achieve complementary bacterial functions. This resulted in a 38.6% increase in amino acid derivatives and a 32.4% increase in fatty acid derivatives, simultaneously enhancing protein enzymatic hydrolysis and optimizing fatty acid composition. The overall modification effect was significantly superior to all single probiotic fermentation groups. Furthermore, the co-fermentation of *Lactobacillus acidophilus* La28 and *Lactobacillus plantarum* LP45 in donkey-hide gelatin showed a significant increase in glycine-arginine and L-proline in the fermentation product.

[0042] Example 3 This embodiment verifies the drying function of the fermentation broth in Example 1 above. The experimental material used includes RAW264.7 macrophages. The experimental content and results are as follows: (1) Culture of RAW264.7 cells: Mouse macrophages RAW 264.7 were grown in complete culture medium (containing 10% fetal bovine serum, 100 U / mL streptomycin and 100 U / mL penicillin). The cell culture flasks were placed in a 37 ℃, 5% CO2 incubator. When the cell number reached 70-80%, the cells were passaged. The cells were gently scraped with a cell scraper, centrifuged at 1100 rpm for 4 min, the supernatant was discarded, and the cells were resuspended in complete culture medium and cultured at 37 ℃, 5% CO2. When the cells were passaged to the 3rd-4th generation, they were counted with a counting chamber and the cell concentration was adjusted to 1×10⁻⁶. 5 Units / mL are available for use.

[0043] (2) NO secretion detection Cells in the logarithmic growth phase were taken at 4 × 10⁻⁶. 5 The cells were seeded at a density of 1 mL / well in 12-well plates, and incubated for 24 hours. Three groups were set up: Control group: Add 1 mL of complete culture medium; Model group: Add 1 mL of LPS to a final concentration of 1 μg / mL; Experimental group: The fermentation broth of donkey-hide gelatin included three dosage groups: low, medium and high, and the final concentration of LPS was 1 μg / mL.

[0044] Before stimulation with LPS (lipopolysaccharide), pre-drug administration was performed. The experimental groups received the appropriate drug concentration, while the blank and model groups received an equal volume of culture medium without antibiotics. After incubation for 30 minutes, the supernatant was discarded, and the corresponding drug was administered. The final serum concentration in each group was 10%. After 24 hours of incubation, the supernatant was collected, centrifuged to remove residual cells, and the NO content was determined according to the instructions of the NO content detection kit.

[0045] (3) Determination of iNOS and COX-2 gene expression levels Cells were plated and drugged according to the NO secretion assay method. After LPS stimulation for 4 hours, the supernatant was discarded, and 200 μL of cell lysis buffer was added to each well. The cells were then pipetted several times to ensure thorough contact between the lysis buffer and the cells. The lysed samples were centrifuged at 10,000 g for 3-5 minutes, and the supernatant was used for RNA extraction, reverse transcription, qPCR, and other procedures.

[0046] (4) Experimental results like Figure 1 Figure 1 shows the experimental results of the effects of donkey-hide gelatin fermentation broth on NO secretion levels (Figure a) and iNOS mRNA expression (Figure b) in Raw264.7 cells. As shown in the figures, under normal physiological conditions, NO in the body is synthesized by eNOS, and its content is low. Its main function is to dilate blood vessels and regulate immune balance, without causing discomfort. However, when inflammation occurs in the body, inflammatory factors induce a large expression of iNOS. iNOS continuously catalyzes the production of excessive NO, at which point NO transforms from a "physiological regulator" into a "pro-inflammatory damaging factor." Compared to the blank control group, the NO release level increased after intervention with unfermented donkey-hide gelatin and donkey-hide gelatin fermentation broth in macrophages. However, the NO release level after intervention with donkey-hide gelatin fermentation broth was 45% lower than that after intervention with unfermented donkey-hide gelatin, and the iNOS gene expression level after intervention with donkey-hide gelatin fermentation broth was relatively reduced by 58%.

[0047] like Figure 2 The figure shows the experimental results of the effect of donkey-hide gelatin fermentation broth on the expression of the inflammation-related gene COX-2 in Raw264.7 cells. As can be seen from the figure, compared with the donkey-hide gelatin group, treatment with the donkey-hide gelatin fermentation broth significantly inhibited the high expression level of COX-2 mRNA in LPS-induced macrophages (large molecules that are difficult to absorb when consuming donkey-hide gelatin accumulate in the intestines, causing inflammation), thereby reducing the production of PGE2 and exerting a cooling effect. The relative COX-2 gene expression level decreased by 81% after treatment with the donkey-hide gelatin fermentation broth.

[0048] Example 4 In this embodiment, high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) was used to qualitatively detect the small molecule metabolites of the compound probiotic fermented donkey-hide gelatin of this invention. The specific method is as follows: (1) Method setting Liquid phase conditions: Waters ACQUITY HSS T3 column as the chromatographic column, 0.1% formic acid aqueous solution and acetonitrile as the mobile phase, flow rate 0.2 mL / min, column temperature 40°C Mass spectrometry conditions: Positive and negative ion modes are acquired separately in two runs, the spray voltage is 3800V in positive ion mode and 3200V in negative ion mode, the sheath gas flow rate is 45 Arb, and the auxiliary gas flow rate is 10 Arb.

[0049] (2) Sample treatment The sample is treated with a mixed solution of methanol and acetonitrile, followed by centrifugation and filtration to obtain the test solution.

[0050] (3) Data processing The supporting mass spectrometry workstation of the instrument is used for data acquisition, peak integration, qualitative and quantitative analysis, Excel and SPSS software are used for statistical analysis of data, all experiments are performed in triplicate, and the results are expressed as "mean ± standard deviation".

[0051] The test results show that fermentation-specific metabolites such as D(+)-phenyllactic acid and cyclic dipeptides are newly added in the product, and such substances are not detected in unfermented Colla Corii Asini. The above newly added products have definite antibacterial and anti-inflammatory activities, can inhibit the proliferation of harmful intestinal bacteria and optimize the structure of intestinal flora; at the same time, they improve the intestinal microecology, enhance the activities of digestive enzymes such as protease and amylase, promote the efficient metabolism of nutrients, and fundamentally avoid the "dryness" manifestation similar to "excessive internal heat" caused by indigestion.

[0052] Specifically, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) is used for qualitative component analysis of the Colla Corii Asini solution before fermentation and the Colla Corii Asini solution after fermentation. Chromatographic conditions: Waters ACQUITY HSS T3 column as the chromatographic column, 0.1% formic acid aqueous solution and acetonitrile as the mobile phase. Mass spectrometry conditions: Electrospray ionization (ESI), scanning in positive / negative ion mode. Through comparison of differential peaks between groups, the characteristic peaks of D(+)-phenyllactic acid and cyclic dipeptides are not detected in the Colla Corii Asini solution before fermentation, and new characteristic peaks of D(+)-phenyllactic acid and cyclic dipeptides appear in the Colla Corii Asini solution after fermentation, which are confirmed as fermentation-specific newly added products by combining the secondary mass spectrometry fragment information and comparing with reference standards.

[0053] Antibacterial activity test: Using the agar well diffusion method, it is found through detection that the fermented Colla Corii Asini solution shows good bacteriostatic effect on common harmful intestinal bacteria such as Escherichia coli, Staphylococcus aureus and Salmonella, which helps to improve the intestinal microenvironment, increase the abundance of beneficial bacteria and reduce the abundance of harmful bacteria, while the original water extract of Colla Corii Asini has no obvious bacteriostatic effect.

[0054] Digestive enzyme activity assay: Six 6-8 week old SPF-grade ICR mice or C57BL / 6 mice were used and acclimatized for 3 days before the experiment began. They were divided into two groups of 3 mice each, and were administered either the fermented donkey-hide gelatin broth prepared in Example 1 or the aqueous extract of donkey-hide gelatin by gavage, with each mouse receiving 5 mg of either broth or extract daily for 5 consecutive days. On day 6, feces were collected to detect the activities of protease, amylase, and lipase. The results showed that, compared with the control group, the fermented donkey-hide gelatin broth improved the enzyme activities of protease, amylase, and lipase in the mouse intestines, which is beneficial for promoting intestinal metabolic function.

[0055] Example 5 This embodiment verifies the cell viability-enhancing and hematopoietic-replenishing effects of the fermentation broth from Example 1. The experimental material used included: human erythroid leukemia cell line (TF-1). The experimental content and results are as follows: TF-1 cell culture: TF-1 cells were grown in complete culture medium (containing 10% fetal bovine serum, 100 U / mL streptomycin, 100 U / mL penicillin, and GM-CSF). Cell flasks were placed in an incubator at 37°C and 5% CO2. When the cell count reached 95%, the cells were passaged, centrifuged at 250×g for 4 min, the supernatant was discarded, and the cells were resuspended in complete culture medium and cultured at 37°C and 5% CO2. After 3-4 passages, cell counts were performed using a counting chamber, and the cell concentration was adjusted to 1×10⁻⁶ cells / mL. 5 Units / mL are available for use.

[0056] (1) Measurement of cell viability The study included a blank group containing only culture medium and no cells, a control group (normally cultured TF-1 cells with a drug concentration of 0 μg / mL), and a drug-treated group (normal TF-1 cells with different concentrations of donkey-hide gelatin fermentation broth added), with 6 replicates per group.

[0057] Take a cell suspension in the logarithmic growth phase, count the cells using an automated cell counter, and then dilute to a cell density of 2 × 10⁻⁶. 4 100 μL of cells / well was seeded into each well of a 96-well plate and cultured in an incubator. Both cells and drugs were diluted with complete culture medium, free of GM-CSF. The next day, the cell supernatant was discarded, and 100 μL of the corresponding drug-containing culture medium was added to each well (for the control group, only culture medium was added). The plates were incubated for 24 hours. Under dark conditions, 10 μL of CCK-8 was added to each well. The 96-well plate was wrapped with aluminum foil, shaken on a shaker for 10 minutes, and then placed in an incubator. After 4 hours, the OD was measured using a microplate reader. 450 .

[0058] (2) Determination of the expression level of the pro-apoptotic gene BAX Take cells in the logarithmic growth phase at 1×10 5Cells were seeded at a density of 1 mL / well in 12-well plates and incubated for 24 hours. Three groups were established: a control group (1 mL of complete culture medium), a model group (1 mL of EPO at a final concentration of 2 U / mL), and experimental groups (three dose groups of donkey-hide gelatin fermentation broth, with a final EPO concentration of 2 U / mL). Before EPO stimulation, cells were cultured for 24 hours in a medium containing 10% fetal bovine serum, 100 U / mL streptomycin, and 100 U / mL penicillin. The following day, donkey-hide gelatin fermentation broth and EPO were added, and cells were cultured for another 24 hours. Cells were then collected for RNA extraction, reverse transcription, and qPCR.

[0059] (3) Determination of hemoglobin content Cells were plated and drugged according to the method for detecting the expression level of the pro-apoptotic gene BAX. After 72 hours of EPO stimulation, cells were collected, and 200 μL of cell lysis buffer was added. The cells were then pipetted several times to ensure sufficient contact between the lysis buffer and the cells. The lysed sample was centrifuged at 10,000 g for 3-5 minutes, and the supernatant was collected to determine the hemoglobin content according to the instructions of the hemoglobin assay kit.

[0060] (4) Experimental results like Figure 3 The results show the effect of donkey-hide gelatin fermentation broth on the survival rate of TF-1 cells. CCK-8 assay results indicate that the donkey-hide gelatin fermentation broth significantly promotes the proliferation of TF-1 cells, increasing the survival rate of erythroid precursor cells by 15%.

[0061] like Figure 4 The image shows the effect of donkey-hide gelatin fermentation broth on the pro-apoptotic gene BAX in TF-1 cells. Under normal physiological conditions, the core expression characteristic of the pro-apoptotic gene BAX is constitutive expression at a basal low level. After intervention with donkey-hide gelatin aqueous extract, the relative expression level of the pro-apoptotic gene increased by 52%, accelerating cell death; while after intervention with fermented donkey-hide gelatin, the relative expression level of the pro-apoptotic gene decreased by 21%, which was 16% lower than that of competing products (commercially available similar fermented donkey-hide gelatin products).

[0062] The main reason why the aqueous extract led to an increase in the relative expression of the pro-apoptotic gene BAX is that cells are highly sensitive to external stimuli. After co-incubation with the aqueous extract of donkey-hide gelatin, the nutrients in the extract (mostly large-molecule intact collagen and high-molecular-weight glycoproteins) are poorly absorbed by cells, easily causing cellular stress, upregulating BAX, and resulting in cell death due to nutrient deficiency. However, when the donkey-hide gelatin fermentation broth provided in Example 1 is used for co-incubation with cells, the nutrients in the fermentation broth can be utilized by the cells to maintain cell growth. The fermented donkey-hide gelatin product of Example 1 can reduce BAX expression by 21%, inhibit cell apoptosis, protect hematopoietic cells, and significantly enhance the blood-tonifying effect of donkey-hide gelatin.

[0063] like Figure 5The image shows the effect of donkey-hide gelatin fermentation broth on the hemoglobin synthesis content of TF-1 cells. Erythroid progenitor cells can differentiate into erythrocytes and synthesize hemoglobin under the regulation of the hematopoietic factor EPO. After intervention with donkey-hide gelatin aqueous extract, the hemoglobin content decreased by 0.69%; after intervention with donkey-hide gelatin fermentation product, the hemoglobin content increased by 8.35%, which is 7.66% higher than that of competing products (commercially available similar fermented donkey-hide gelatin products). Therefore, the donkey-hide gelatin fermentation product prepared by the fermentation process of this invention is more conducive to hemoglobin synthesis and has a better blood-tonifying effect.

[0064] In summary, when the probiotic-fermented donkey-hide gelatin liquid described in this invention is used alone, the enzyme system produced by the probiotics during fermentation can efficiently degrade the large, indigestible collagen molecules in donkey-hide gelatin into small peptides and free amino acids that are easily absorbed by the intestines, significantly improving the bioavailability of the nutrients in donkey-hide gelatin. Simultaneously, it can generate novel active substances, further enhancing the traditional blood-nourishing effects of donkey-hide gelatin. This pre-digestion process alleviates the discomfort caused by the traditional donkey-hide gelatin's rich, greasy texture and indigestion, making its nourishing effects gentler and more tolerable. Therefore, the probiotic-fermented donkey-hide gelatin liquid can be made into functional foods or medicines such as oral liquids, or freeze-dried into solid preparations.

[0065] In practical use, donkey-hide gelatin fermentation liquid can be combined with single-herb fermentation liquids, and the combination scheme can be optimized according to the principles of harmonious properties and complementary effects. Donkey-hide gelatin is sweet and neutral in nature, and enters the lung, liver and kidney meridians. It is good at nourishing yin and blood, moistening dryness and stopping bleeding. When combining, it is best to select fermentation liquids of herbs with compatible properties and synergistic effects to achieve multiple effects such as replenishing blood, nourishing yin and moistening dryness, and improve the overall conditioning effect.

[0066] Furthermore, the fermented donkey-hide gelatin liquid can be combined with classic prescriptions, achieving a deep integration of traditional wisdom in formula compatibility with modern bio-fermentation technology. The fermented donkey-hide gelatin liquid is rich in probiotics and their metabolites, which can synergistically work with the medicinal components of classic prescriptions. While retaining the core efficacy of ancient formulas, it further enhances the absorption efficiency of components, reduces greasiness and dryness, and broadens the applicable scenarios, representing an important direction for the modernization and upgrading of classic prescriptions.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. These modifications or substitutions, or combinations of technical features in the above embodiments that do not conflict with each other, can be made in accordance with the manner described in the embodiments. These modifications, substitutions or combinations do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a desiccant-reducing and blood-tonifying type based on fermentation of La28 and LP45 compound probiotics, characterized in that, include: S1. Ingredients: Dissolve donkey-hide gelatin in water or an aqueous solvent by heating, add a fermentable carbon source, sterilize, and obtain fermentation substrate liquid; S2. Inoculation: Cool to ≤40℃, add compound probiotics, and mix well; the compound probiotics contain 1-3×10 11 CFU / g of Lactobacillus acidophilus La28 and 6-10×10 11 The dosage of CFU / g Lactobacillus plantarum LP45 is 20g-40g / ton of fermentation substrate liquid; S3. Fermentation: Ferment at 30℃-42℃ for 1-3 days to obtain fermented donkey-hide gelatin product.

2. The method according to claim 1, characterized in that, In S1, the mass ratio of donkey-hide gelatin to water is 1:8-10.

3. The method according to claim 1, characterized in that, In S1, the aqueous solvent is purified water, deionized water, or water for injection; the fermentable carbon source includes one or more of glucose, sucrose, maltose, fructooligosaccharides, and inulin; the amount of fermentable carbon source added is 0.5%-5% of the mass of the fermentation substrate liquid.

4. The method according to claim 3, characterized in that, The amount of fermentable carbon source added is 1%-3% of the volume of the fermentation substrate liquid.

5. The method according to claim 3, characterized in that, In S1, the fermentable carbon source is glucose, and the amount added is 2% of the mass of the fermentation substrate liquid.

6. The method according to claim 1, characterized in that, In S1, the sterilization conditions are 100℃-121℃ for 10-60 minutes.

7. The method according to claim 1, characterized in that, In S3, the fermentation temperature is 35℃-39℃ and the fermentation time is 36-48h.

8. The method according to claim 1, characterized in that, In S2, the ratio of live Lactobacillus acidophilus La28 to Lactobacillus plantarum LP45 in the compound probiotics is 1:

1.

9. A donkey-hide gelatin product prepared by the method according to any one of claims 1-8, characterized in that, The donkey-hide gelatin product contains small molecule peptides, free amino acids, D(+)-phenyllactic acid and cyclic dipeptides. The small molecule peptides include proline-glycine dipeptides, and the free amino acids include L-proline. In the product obtained by fermenting an equal amount of donkey-hide gelatin, the total content of small molecule peptides and free amino acids is higher than the corresponding content in the donkey-hide gelatin before fermentation.

10. The application of the donkey-hide gelatin product according to claim 9 in the preparation of functional foods or medicines that reduce dryness, replenish blood, nourish yin and moisten the lungs, improve intestinal absorption, balance the intestinal flora structure, and reduce greasiness and stomach upset; wherein the donkey-hide gelatin product is an oral liquid of donkey-hide gelatin.

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