Seven-flavor jujube and poria fermenting concentrated liquid with improved sleep function and preparation method and application thereof

Abstract

The present application relates to the technical field of traditional Chinese medicine drinks, in particular to a seven-flavor jujube and tuckahoe fermentation concentrate with sleep improvement function and its preparation method and application. The seven-flavor jujube and tuckahoe fermentation concentrate is composed of seven homologous medicinal and food materials such as zizyphus jujuba mill and tuckahoe. The preparation is completed through crushing and sieving, enzymolysis, lactic acid bacteria fermentation, solid-liquid separation and concentration. The lactic acid bacteria used are one or two of Lactobacillus paracasei YYS-69, Lactobacillus plantarum BXM2 and Lactobacillus plantarum YYS-99. Through the synergistic process of "crushing + enzymolysis + compound fermentation", the contents of spinozine, total polysaccharide and total saponin in the fermentation concentrate are increased by 3.3, 1.3 and 1.6 times respectively compared with the unfermented case. The seven-flavor jujube and tuckahoe fermentation concentrate can significantly regulate insomnia-related genes and reverse oxidative stress damage of nerve cells, and has the activities of improving insomnia and intelligence and brain health. The preparation process is simple, the equipment requirement is low, it is suitable for industrial production, and can be applied to functional products such as food and health products, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine beverage technology, and in particular to a seven-ingredient jujube and lingzhi fermented concentrate with sleep-improving function, its preparation method and application. Background Technology

[0002] In Traditional Chinese Medicine (TCM), insomnia is often referred to as "insomnia," "inability to lie down," or "inability to close the eyes." Its core location is the heart, and its core pathological mechanism is generally attributed to an imbalance of Yin and Yang, and a disharmony between water and fire. The etiology and pathogenesis of this disease can be broadly categorized into two types: deficiency and excess. Excess insomnia is often caused by excessive fire, phlegm-dampness, or phlegm-heat: anger and resentment damage the liver, leading to impaired liver function, disordered Qi flow, and prolonged Qi stagnation transforming into fire, which in turn causes excessive fire in the heart and liver; spleen deficiency leads to internal dampness, causing fluids to condense into phlegm. Phlegm, fire, and dampness disturb the mind, resulting in excessive Yang failing to nourish Yin, thus causing insomnia. Deficiency insomnia is primarily due to blood deficiency and Yin deficiency: deficiency of both the heart and spleen leads to insufficient production of Qi and blood, resulting in insomnia because the mind is not nourished; deficiency of kidney Yin causes internal fire to rise and disturb the mind, leading to a disharmony between water and fire, ultimately causing insomnia.

[0003] With the fast pace of modern life, people are often under high-intensity work pressure, which easily leads to negative emotions such as worry and anxiety, thus depleting the Qi and blood of the heart and spleen. Therefore, insomnia due to deficiency of both heart and spleen is the most common clinical manifestation. The *Classified Treatments for Diseases* clearly states, "Excessive thinking injures the spleen, leading to spleen blood deficiency and chronic insomnia," confirming the close relationship between overthinking and insomnia due to deficiency of both heart and spleen. The heart governs blood vessels and the mind; excessive thinking and overwork easily deplete the Yin and blood of the heart and impair the spleen's digestive function. Spleen injury results in insufficient production of Qi and blood, ultimately leading to blood deficiency that cannot nourish the heart, causing insomnia. Based on this, targeting the pathogenesis of insomnia due to deficiency of both heart and spleen, a modified version of the Suanzaoren Tang (Ziziphus jujuba seed decoction) from the *Essential Prescriptions of the Golden Chamber* was prepared to improve insomnia, namely the Seven-Ingredient Ziziphus Jujuba and Poria Formula.

[0004] The original Suanzaoren Decoction is composed of Semen Ziziphi Spinosae, Rhizoma Chuanxiong, Rhizoma Anemarrhenae, Radix Glycyrrhizae, and Poria. Among them, Rhizoma Anemarrhenae and Rhizoma Chuanxiong are not both medicinal and edible herbs. Considering the safety and applicability of medication, the medicinal and edible herbs Lily and Rose with similar effects are selected for replacement: Lily has the effects of nourishing yin and moistening the lungs, clearing the heart and calming the mind, and Rose can promote qi circulation and relieve depression, regulate qi and activate blood circulation to regulate menstruation. At the same time, Longan Aril and Gastrodia Rhizome are added to the formula to enhance the efficacy. The最终形成的七味枣苓方由酸枣仁、茯苓、百合、龙眼肉、玫瑰花、天麻、生甘草组成。该方配伍严谨,重用酸枣仁养血补肝、宁心安神,为君药;茯苓健脾宁心、百合养阴清心、龙眼肉补益心脾养血,三者共为臣药,协同发挥养心血、补心脾、清心安神之效;天麻性平味甘,入肝经,可平抑肝阳、息风止痉、通络止痛,玫瑰花性温味甘微苦,入肝脾经,能行气解郁、调畅情志,二者同为佐药;生甘草调和诸药、益气补中,辅助茯苓增强健脾之力,为使药。全方共奏养心补血安神、疏肝补气健脾之功,针对性改善心脾两虚型不寐。

[0005] Traditional Chinese medicine fermentation is an important means of combining traditional Chinese medicine processing with modern biotechnology. Its core is to use microorganisms in the natural environment or artificially inoculated to change the properties of traditional Chinese medicine, reduce drug toxicity, improve the efficacy activity and utilization rate of traditional Chinese medicine resources through the growth and metabolism process of microorganisms. Among many fermenting microorganisms, lactic acid bacteria are widely used in the field of traditional Chinese medicine fermentation due to their high biological safety and wide market recognition. Applying lactic acid bacteria fermentation technology to traditional Chinese medicine processing can not only promote the dissolution and transformation of effective components in traditional Chinese medicine, optimize the functional activity of traditional Chinese medicine, but also借助乳酸菌调节肠道菌群平衡的作用,辅助中药功效的高效发挥,同时显著提高中药有效成分的生物利用度,让中药药效得到更充分的体现。

[0006] Although the technology of lactic acid bacteria fermenting traditional Chinese medicine has been applied to a certain extent, and there are also relevant studies on traditional Chinese medicine compositions for insomnia, in the existing technology, the research reports on preparing traditional Chinese medicine combined fermented products with both insomnia-improving activity and intelligence-boosting and brain-strengthening activity are extremely scarce. At present, there is no mature technical scheme that can combine the fermentation advantages of lactic acid bacteria with the efficacy of traditional Chinese medicine for regulating insomnia of the syndrome of deficiency of both the heart and spleen to meet the clinical and market demands for safe, efficient and multi-functional traditional Chinese medicine fermented products. Summary of the Invention

[0007] To solve the technical problem in the existing technology that there is a lack of a mature technology for combining the fermentation advantages of lactic acid bacteria with the efficacy of traditional Chinese medicine for regulating insomnia of the syndrome of deficiency of both the heart and spleen to prepare a traditional Chinese medicine fermented product with both insomnia improvement and intelligence-boosting and brain-strengthening activities, the present invention provides a method for preparing a fermented and concentrated solution of Qiwei Zaoling, comprising the following steps:

[0008] S1. Grind the Seven-Flavor Jujube and Poria Formula into powder and sieve it to obtain the Seven-Flavor Jujube and Poria Formula powder. Add water and mix evenly. Then add cellulase and pectinase and enzymatically hydrolyze at 35~55℃ for 0.5~4.5h. After inactivating the enzymes, cool down.

[0009] S2. Add lactic acid bacteria powder to the enzymatic hydrolysate and ferment at 35-45℃ for 10-50 hours. After inactivation, the initial fermentation liquid is obtained.

[0010] S3. The initial fermentation liquid is subjected to solid-liquid separation to obtain the fermentation liquid of seven-flavor jujube and lingzhi.

[0011] S4. Concentrate the fermented liquid of the seven-flavor jujube and lingzhi at 60-70℃ for 6-10 times to obtain the fermented concentrated liquid of the seven-flavor jujube and lingzhi.

[0012] The Seven-Ingredient Jujube and Poria Formula consists of jujube seed, poria, lily bulb, longan pulp, rose, gastrodia, and raw licorice.

[0013] The lactic acid bacteria powder contains Lactobacillus paracasei ( Lactobacillus paracasei YYS-69, Lactobacillus plantarum ( Lactobacillus plantarum BXM2, Lactobacillus plantarum ( Lactiplantibacillus plantarum One or two of YYS-99; wherein, the preservation number of Lactobacillus paracasei YYS-69 is CGMCC NO.25837; the preservation number of Lactobacillus plantarum BXM2 is CGMCC NO.16436; and the preservation number of Lactobacillus plantarum YYS-99 is CGMCC NO.25838.

[0014] In some embodiments, in step S1, the sieve aperture is 0.05~0.85mm when the Seven-Flavor Jujube and Poria Formula is pulverized and sieved.

[0015] In some embodiments, in step S1, the mass ratio of the Seven-Flavor Jujube and Poria Cocos Powder to water is 1:(15~40).

[0016] In some embodiments, in step S1: the amount of cellulase added is 0.2% to 0.6% of the water mass; the amount of pectinase added is 0.1% to 0.3% of the water mass.

[0017] In some embodiments, in step S2, the amount of lactic acid bacteria powder added is 0.005% to 0.03% of the water mass.

[0018] Furthermore, when the lactic acid bacteria powder is composed of Lactobacillus paracasei YYS-69 and Lactobacillus plantarum BXM2, the mass ratio of Lactobacillus paracasei YYS-69 to Lactobacillus plantarum BXM2 is (1~5):(1~5).

[0019] In some embodiments, the enzyme inactivation is performed at 85-90°C for 10-20 minutes; the inactivation is performed at 85-100°C for 10-20 minutes.

[0020] The present invention also provides a seven-flavor jujube and lingzhi fermentation concentrate, which is prepared by the method described above for preparing the seven-flavor jujube and lingzhi fermentation concentrate; the seven-flavor jujube and lingzhi fermentation concentrate contains a spinosin content of not less than 8 mg / 100g, a total polysaccharide content of not less than 1200 mg / 100g, and a total saponin content of not less than 20 mg / 100g.

[0021] The present invention also provides a seven-flavor jujube and lingzhi fermented powder, which is prepared by mixing, drying, pulverizing and sieving the seven-flavor jujube and lingzhi fermented concentrate as described above with a carrier.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention employs a synergistic process of "pulverization + enzymatic hydrolysis + compound fermentation." By precisely controlling the pore size of the Qiwei Zao Ling Fang (Seven-Ingredient Jujube and Poria Formula), enzymatic hydrolysis parameters, and fermentation conditions, the content of spinosin, total polysaccharides, and total saponins in the fermentation concentrate is significantly increased. The content of spinosin in the fermentation concentrate is no less than 8 mg / 100g, the content of total polysaccharides is no less than 1200 mg / 100g, and the content of total saponins is no less than 20 mg / 100g, effectively overcoming the limitations of traditional water extraction processes in terms of insufficient release of active ingredients. The compound fermentation system of Lactobacillus paracasei YYS-69 and Lactobacillus plantarum BXM2 produces a synergistic effect. Combined with an optimal enzymatic hydrolysis time of 2 hours and a suitable fermentation duration of 20 hours, it further enhances the dissolution and accumulation of target components, with significantly better results than single-strain fermentation and some process combination schemes.

[0024] In particular, the fermented concentrate of seven-ingredient jujube and lingzhi provided by this invention can significantly inhibit the expression of the inflammatory factor TNF-α gene in PC12 cells, promote the expression of the sedative and tranquilizing GABA gene and the circadian rhythm-regulated PER gene, and achieve efficient regulation of key genes related to insomnia; at the same time, it can reverse H2O2-induced oxidative stress damage to nerve cells, exert a neuroprotective effect by downregulating the expression of the pro-apoptotic gene bax and upregulating the expression of the anti-apoptotic gene bcl-2, and has a clear effect on improving insomnia and enhancing intelligence and brain function. Its activity is significantly better than that of traditional water extraction process and products of single enzymatic hydrolysis or fermentation treatment.

[0025] Furthermore, in terms of process and application value, the process design of this invention is simple and efficient, requiring only a combination of conventional operations such as pulverization, enzymatic hydrolysis, fermentation, solid-liquid separation, and concentration. It has low requirements for production equipment, and the process parameters are clearly defined and highly controllable, facilitating large-scale industrial production. The resulting fermentation broth, based on the compatibility of medicinal and edible raw materials and safe lactic acid bacteria fermentation, has wide applicability and can be widely used in various functional products such as food and health products. It provides a safe and efficient new product option for insomnia intervention and brain health maintenance, and has significant market application prospects. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a comparison chart of the spinosin content in different seven-ingredient jujube and lingzhi formulas of the present invention;

[0028] Figure 2 This is a comparison chart of the total polysaccharide content of different seven-ingredient jujube and lingzhi formulas of the present invention;

[0029] Figure 3 This is a comparison chart of the total saponin content of different seven-ingredient jujube and poria formulas of the present invention;

[0030] Figure 4 This is a comparison of the relative expression levels of TNF-α in different seven-ingredient jujube and poria formulas of the present invention;

[0031] Figure 5 This is a comparison diagram of the relative expression levels of the GABA gene in different seven-ingredient jujube and scutellaria formulas of this invention;

[0032] Figure 6 This is a comparison diagram of the relative expression levels of the PER gene in different seven-ingredient jujube and lingzhi formulas of this invention;

[0033] Figure 7 This is a comparison diagram of the relative expression levels of the Bax gene in different Seven-Flavor Jujube and Poria Formulas of this invention;

[0034] Figure 8 This is a comparison diagram of the relative expression levels of the bcl-2 gene in different seven-ingredient jujube and poria formulas of this invention. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments, and the technical features designed in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] This invention provides a seven-ingredient jujube and scutellaria fermented concentrate with sleep-improving function, which is prepared by the following steps:

[0037] S1. After pulverizing the Seven-Flavor Jujube and Poria Formula, pass it through a 0.05~0.85mm sieve to obtain the Seven-Flavor Jujube and Poria Formula powder. Add water at a mass ratio of 1:(15~40), mix evenly, then add 0.2%~0.6% water mass of cellulase and 0.1%~0.3% water mass of pectinase. Enzymatically hydrolyze at 35~55℃ for 0.5~4.5h, then inactivate the enzyme at 85~90℃ for 10~20min, and then cool down.

[0038] S2. Add 0.005%~0.03% water mass of lactic acid bacteria powder to the enzymatic hydrolysate, ferment at 35~45℃ for 10~50h, and then inactivate at 85~100℃ for 10~20min to obtain the initial fermentation broth;

[0039] S3. The initial fermentation liquid is subjected to solid-liquid separation to obtain the fermentation liquid of seven-flavor jujube and lingzhi.

[0040] S4. Concentrate the fermented liquid of the seven-flavor jujube and lingzhi at 60-70℃ for 6-10 times to obtain the fermented concentrated liquid of the seven-flavor jujube and lingzhi.

[0041] The Seven-Flavor Jujube and Poria Formula consists of jujube seed, poria, lily bulb, longan pulp, rose, gastrodia elata, and raw licorice root; the lactic acid bacteria powder contains one or two of Lactobacillus paracasei YYS-69, Lactobacillus plantarum BXM2, and Lactobacillus plantarum YYS-99, and when the lactic acid bacteria powder is composed of Lactobacillus paracasei YYS-69 and Lactobacillus plantarum BXM2, the mass ratio of Lactobacillus paracasei YYS-69 to Lactobacillus plantarum BXM2 is (1~5):(1~5).

[0042] It should be noted that:

[0043] Lactobacillus paracasei ( Lactobacillus paracasei YYS-69 was deposited on September 28, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 25837; Lactobacillus plantarum ( Lactobacillus plantarum BXM2, deposited on September 6, 2018, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 16436; *Lactobacillus plantarum* (BXM2), was deposited on September 6, 2018, at CGMCC NO. 16436. Lactiplantibacillus plantarum YYS-99 was deposited on September 28, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.25838.

[0044] The cellulase was Nanning Pangbo cellulase with an enzyme activity of 20,000 Units / g; the pectinase was Novozymes pectinase with an enzyme activity of 11,010 Units / g.

[0045] Example 1

[0046] This embodiment provides a seven-ingredient jujube and poria cocos fermentation concentrate, which is prepared by the following steps:

[0047] S1. After pulverizing the Seven-Flavor Jujube and Poria Formula, pass it through a 0.85mm sieve to obtain the Seven-Flavor Jujube and Poria Formula powder. Take 90g of the Seven-Flavor Jujube and Poria Formula powder and add it to 1.8kg of water. Mix well, then add 3.6g of cellulase and 1.8g of pectinase. Enzymatically hydrolyze at 50℃ for 2h, then inactivate the enzyme at 90℃ for 20min. Allow it to cool naturally to obtain the enzymatic hydrolysate.

[0048] S2. Inoculate 0.36g of mixed Lactobacillus paracasei YYS-69 and Lactobacillus plantarum BXM2 bacterial powder into the enzymatic hydrolysate, ferment at 37℃ for 20h, then inactivate at 100℃ for 20min, and cool naturally to obtain the initial fermentation liquid.

[0049] S3. The initial fermentation liquid is subjected to solid-liquid separation to obtain the fermentation liquid of seven-flavor jujube and lingzhi.

[0050] S4. Concentrate the fermented liquid of the seven-flavor jujube and lingzhi at 60-70℃ for 6-10 times to obtain the fermented concentrated liquid of the seven-flavor jujube and lingzhi.

[0051] Preferably, the mass ratio of Lactobacillus paracasei YYS-69 to Lactobacillus plantarum BXM2 in the mixed bacterial powder is 1:1.

[0052] Example 2

[0053] This embodiment provides a seven-ingredient jujube and lingzhi fermented concentrate, the preparation steps of which differ from those of Example 1 only in that the mixed bacterial powder in step S2 is replaced with an equal mass of Lactobacillus paracasei YYS-69 bacterial powder. All other components, proportions, operating steps, and process parameters remain consistent with those of Example 1.

[0054] Example 3

[0055] This embodiment provides a seven-ingredient jujube and lingzhi fermented concentrate, the preparation steps of which differ from those of Example 1 only in that the mixed bacterial powder in step S2 is replaced with an equal mass of Lactobacillus plantarum BXM2. All other components, proportions, operating steps, and process parameters remain consistent with those of Example 1.

[0056] Example 4

[0057] This embodiment provides a seven-ingredient jujube and lingzhi fermented concentrate, the preparation steps of which differ from those of Example 1 only in that the mixed bacterial powder in step S2 is replaced with an equal mass of Lactobacillus plantarum YYS-99. All other components, proportions, operating steps, and process parameters remain consistent with those of Example 1.

[0058] Example 5

[0059] This embodiment provides a seven-ingredient jujube and scutellaria fermented concentrate, the only difference between its preparation steps and those of Example 1 is that the fermentation time in step S2 is 10 hours. All other components, proportions, operating steps, and process parameters remain the same as in Example 1.

[0060] Example 6

[0061] This embodiment provides a seven-ingredient jujube and scutellaria fermented concentrate, the only difference between its preparation steps and those of Example 1 is that the fermentation time in step S2 is 30 hours. All other components, proportions, operating steps, and process parameters remain the same as in Example 1.

[0062] The present invention also provides the following comparative examples 1 to 5:

[0063] Comparative Example 1

[0064] This comparative example provides a seven-ingredient jujube and scutellaria fermented concentrate, the preparation steps of which differ from those of Example 1 only in that: in step S1, cellulase and pectinase are not added for enzymatic hydrolysis, and instead, extraction is carried out at 100°C for 1 hour; and step S2 is omitted, without microbial fermentation. The remaining components, proportions, operating steps, and process parameters are consistent with those of Example 1.

[0065] Comparative Example 2

[0066] This comparative example provides a seven-ingredient jujube and scutellaria fermentation concentrate, the preparation steps of which differ from those of Example 1 only in that: in step S1, cellulase and pectinase are not added for enzymatic hydrolysis, and instead, extraction is carried out at 100°C for 1 hour. All other components, ratios, operating steps, and process parameters are consistent with those of Example 1.

[0067] Comparative Example 3

[0068] This comparative example provides a seven-ingredient jujube and scutellaria fermentation concentrate, the preparation steps of which differ from those of Example 1 only in that step S2 is omitted, and microbial fermentation is not performed. All other components, proportions, operating steps, and process parameters remain consistent with those of Example 1.

[0069] Comparative Example 4

[0070] This embodiment provides a seven-ingredient jujube and scutellaria fermentation concentrate. The only difference between this concentrate and Example 1 is that the fermentation step is omitted, and the enzymatic hydrolysis temperature in step S1 is 52°C, and the hydrolysis time is 0.5 h. All other components, proportions, operating steps, and process parameters remain the same as in Example 1.

[0071] Comparative Example 5

[0072] This embodiment provides a seven-ingredient jujube and scutellaria fermented concentrate. The only difference between this concentrate and Example 1 is that the fermentation step is omitted, and the enzymatic hydrolysis temperature in step S1 is 52°C, and the hydrolysis time is 3.5 hours. All other components, proportions, operating steps, and process parameters remain the same as in Example 1.

[0073] Based on the above embodiments and comparative examples, the present invention also conducted the following performance tests:

[0074] Test 1: Effects of different preparation processes on the content of spinosin, total polysaccharides, and total saponins in the fermented concentrate of Seven-Flavor Jujube and Poria cocos

[0075] 1.1 Effects of different preparation processes on the content of spinosin in the fermented concentrate of Seven-Flavor Jujube and Poria cocos

[0076] The content of spinosin in the fermented concentrates of seven-ingredient jujube and lingzhi prepared by different processes was detected, and the results are shown in [the table below]. Figure 1 Different letters indicate statistically significant differences between groups (p < 0.05). The determination of spinosin in the fermented concentrate of Qiwei Zao Ling was conducted according to the standard detection method specified in the 2020 edition of the Pharmacopoeia.

[0077] Comparing Example 1 with Examples 2-4, it can be seen that the content of spinosin in the fermentation concentrate of Example 1, which uses two strains of bacteria for compound fermentation, is significantly higher than that of the single-strain fermentation group. This indicates that there is a synergistic effect between BXM2 and YYS-69 strains, which can more efficiently promote the release and accumulation of spinosin in Qiwei Zao Ling Fang. The compound fermentation process has obvious advantages over the single-strain fermentation process.

[0078] Comparative studies of Examples 1, 5, and 6 show that fermentation time is one of the key parameters affecting the content of spinosin. If the fermentation time is too short, the microbial metabolism will be insufficient, while if it is too long, it may lead to the decomposition or loss of spinosin, both of which will cause its content to decrease. In this invention, 20 hours is the appropriate fermentation time. Within this time, the microbial fermentation and metabolic activities are sufficient, which can effectively promote the accumulation of spinosin.

[0079] As can be seen from the comparison between Example 1 and Comparative Example 1, the combined process of "pulverization + enzymatic hydrolysis + compound fermentation" adopted in this invention can significantly increase the content of spinosin in the fermented concentrate of Seven Flavors Jujube and Poria cocos compared with the traditional water extraction process. The traditional water extraction process is difficult to fully release the spinosin in the raw materials, while the combined process of this invention can effectively break through the structural barrier of the raw materials and greatly improve the extraction efficiency and yield of the target components.

[0080] Comparing Example 1 and Comparative Example 2, it can be seen that the enzymatic hydrolysis process is an important guarantee for increasing the content of spinosin. On the basis of raw material crushing, enzymatic hydrolysis can further break the structural limitations of raw materials, release spinosin in the fermentation system, and thus effectively increase its content.

[0081] Comparing Example 1 and Comparative Example 3, it can be seen that the fermentation process is the core and key step in increasing the content of spinosin. Adding fermentation treatment on the basis of "pulverization + enzymatic hydrolysis" can further significantly increase the content of spinosin, which fully verifies the necessity and important role of the fermentation process in the process of this invention.

[0082] Furthermore, comparing Example 1 with Comparative Examples 4 and 5, it was found that the enzymatic hydrolysis time has a significant impact on the extraction effect of Spinosin, and it is not necessarily better to have a longer or shorter time. In this invention, 2 hours is the optimal enzymatic hydrolysis time, under which the enzymatic hydrolysis reaction is most complete, which can maximize the efficient extraction of Spinosin.

[0083] 1.2 Effects of different preparation processes on the total polysaccharide content in the fermented concentrate of Seven-Flavor Jujube and Poria cocos

[0084] The total polysaccharide content in the fermented concentrates of seven-ingredient jujube and lingzhi prepared by different processes was determined, and the results are shown in [the table below]. Figure 2 Different letters indicate statistically significant differences between groups (p < 0.05). The determination of total polysaccharides in the Qiwei Zao Ling fermented concentrate was carried out according to the detection methods in "Detection Methods for Efficacy Components in Health Foods".

[0085] Comparing Examples 1 with Examples 2-4, it can be seen that, compared with the single-strain fermentation treatment in Examples 2, 3, and 4, the total polysaccharide content in the fermentation concentrate of Example 1, which uses a dual-strain fermentation process of BXM2 and YYS-69, is significantly higher than that of the single-strain fermentation group. This result indicates that there is a significant synergistic effect between the BXM2 and YYS-69 strains. This compound fermentation mode can more efficiently increase the accumulation of total polysaccharides in the fermentation concentrate of Qiwei Zao Ling, fully demonstrating that the compound fermentation process is superior to the single-strain fermentation process.

[0086] Comparing Examples 1 with Examples 5 and 6, it is shown that 20 hours is the appropriate fermentation time to promote the accumulation of total polysaccharides. The strain can fully complete the metabolic process and efficiently promote the synthesis and accumulation of total polysaccharides. If the fermentation time is too short (such as 10 hours in Example 5), the strain metabolism is incomplete and the total polysaccharide synthesis is insufficient. If the fermentation time is too long (such as 30 hours in Example 6), it may cause problems such as degradation of metabolites and decreased strain activity. Both of these will lead to a decrease in the total polysaccharide content. (Note: Example 5 is pulverization + enzymatic hydrolysis (enzymatic hydrolysis time 2 hours, temperature 52℃) + fermentation (BXM2+YYS-69, temperature 37℃, time 10 hours), and Example 6 is pulverization + enzymatic hydrolysis (enzymatic hydrolysis time 2 hours) + fermentation (BXM2+YYS-69, temperature 37℃, time 30 hours). The only difference between the two and Example 1 is the fermentation time.)

[0087] Comparative Example 1 and Comparative Example 1 show that the process route of pulverization, enzymatic hydrolysis and compound fermentation adopted in this invention has a breakthrough advantage in increasing the total polysaccharide content of the fermented concentrate of Seven Flavors Jujube and Poria cocos compared with the traditional simple water extraction process, and the effect of increasing the total polysaccharide content is extremely significant.

[0088] Comparing Example 1 and Comparative Example 2, it can be seen that the enzymatic hydrolysis process is an important key step in increasing the total polysaccharide content of the fermentation broth. On the basis of raw material crushing, enzymatic hydrolysis can effectively destroy the cell structure of the raw materials and further release the bound polysaccharide components in the fermentation system, thereby significantly increasing the detection content of total polysaccharides.

[0089] Comparing Example 1 and Comparative Example 3, it can be seen that the fermentation process is the core and key step in increasing the total polysaccharide content. Under the premise that other process conditions are the same (both use pulverization, Example 1 adds enzymatic hydrolysis + compound fermentation, while Comparative Example 3 only uses enzymatic hydrolysis), the compound fermentation treatment can further significantly increase the total polysaccharide content on the basis of the basic process, highlighting the core role of compound fermentation in the entire process system (Note: Comparative Example 3 is pulverization + fermentation (BXM2+YYS-69, temperature 37℃, time 20h), which differs from Example 1 only from whether enzymatic hydrolysis is performed, highlighting the core value of the fermentation process).

[0090] Furthermore, comparing Example 1 with Comparative Examples 4 and 5, it can be seen that the optimal enzymatic hydrolysis parameter for increasing the total polysaccharide content in this invention is when the enzymatic hydrolysis time is controlled at 2 hours. Under this time condition, the enzymatic hydrolysis reaction is sufficient and there is no problem of excessive degradation, and the enzymatic hydrolysis effect reaches the best, which can provide maximum support for the efficient extraction of total polysaccharides (Note: Comparative Example 4 is pulverization + enzymatic hydrolysis (enzymatic hydrolysis time 0.5 hours, temperature 52°C), and Comparative Example 5 is pulverization + enzymatic hydrolysis (enzymatic hydrolysis time 3.5 hours, temperature 52°C). The only difference between the two and Example 1 is the enzymatic hydrolysis time, which can accurately compare the effect of enzymatic hydrolysis time).

[0091] 1.3 Effects of different preparation processes on the total saponin content in the fermented concentrate of Seven-Flavor Jujube and Poria cocos

[0092] The total saponin content in the fermented concentrates of seven-flavor jujube and lingzhi prepared by different processes was determined, and the results are shown in [the table below]. Figure 3 Different letters indicate statistically significant differences between groups (p < 0.05). The determination of total saponins in the Qiwei Zao Ling fermented concentrate was conducted according to the testing methods in the "Technical Guidelines for the Testing and Evaluation of Physicochemical and Hygienic Indicators of Health Foods (2020 Edition)".

[0093] Comparing Example 1 with Examples 2-4, it can be seen that Example 1, which uses a compound lactic acid bacteria fermentation process, has a more significant effect on increasing the total saponin content in the fermented concentrate of Seven Flavors Jujube and Poria. This indicates that compound lactic acid bacteria fermentation is more advantageous than single lactic acid bacteria fermentation in promoting the generation or dissolution of total saponins.

[0094] Comparative studies of Examples 1 and 5 and 6 show that fermentation time is one of the key factors affecting the total saponin content. The 20-hour fermentation time determined in this invention is the preferred condition. If the fermentation time is too short, the fermentation will be insufficient and the total saponin content will not reach its peak. If the fermentation time is too long, it may cause the degradation of total saponins, which will also reduce the total saponin content and is not conducive to the retention of the target components.

[0095] Comparing Example 1 and Comparative Example 1, it can be seen that the traditional water extraction process has a low extraction efficiency for total saponins. The pulverization, enzymatic hydrolysis, and compound fermentation combination process of the present invention can significantly increase the total saponin content in the fermented concentrate of Seven-Flavor Jujube and Poria cocos, and has obvious technical advantages and better extraction effect compared with the traditional water extraction process.

[0096] Comparing Example 1 and Comparative Example 2, it can be seen that, based on the fermentation of compound lactic acid bacteria, the addition of enzymatic hydrolysis can further increase the total saponin content in the fermentation concentrate of Qiwei Zao Ling, which fully demonstrates that there is a synergistic effect between the enzymatic hydrolysis process and the fermentation process, and the combination of the two can more effectively promote the accumulation of total saponins.

[0097] Comparing Example 1 and Comparative Example 3, it can be concluded that fermentation is one of the core key processes for increasing the total saponin content. Further fermentation with compound lactic acid bacteria on the basis of enzymatic hydrolysis can effectively promote the generation or dissolution of total saponins, thereby significantly increasing the final content of total saponins in the fermentation broth.

[0098] Furthermore, a comparison of Example 1 with Comparative Examples 4 and 5 revealed that the enzymatic hydrolysis time has a significant impact on the total saponin content in the fermented concentrate of Seven Flavors Jujube and Poria. The 2-hour enzymatic hydrolysis time used in this invention is the optimal condition. If the enzymatic hydrolysis time is too short, the effect of enzymatic hydrolysis in promoting the dissolution of total saponins cannot be fully utilized, while if the enzymatic hydrolysis time is too long, the total saponin content will decrease, and neither can achieve the ideal extraction effect.

[0099] In summary, this invention employs a pulverization process, combined with specific enzymatic hydrolysis conditions, a specific combination of compound lactic acid bacteria, and specific fermentation conditions. Through a dedicated process that integrates pulverization, enzymatic hydrolysis, and compound fermentation, a concentrated fermented liquid of seven-flavor jujube and lingzhi is produced. This process has significant advantages over traditional processes and single or partial process steps, significantly increasing the content of spinosin, total polysaccharides, and total saponins in the concentrated fermented liquid, and achieving a high level of total saponin content. This process not only provides reliable technological support for the preparation of concentrated fermented liquid of seven-flavor jujube and lingzhi with high spinosin content, but also lays a solid technological foundation for improving the overall efficacy of the concentrated fermented liquid of seven-flavor jujube and lingzhi.

[0100] Test 2: The insomnia-improving activity of fermented concentrated extracts of seven-ingredient jujube and lingzhi prepared by different processes.

[0101] The effects of different fermentation concentrates of Ziziphus jujuba and Poria cocos prepared by different processes on the expression levels of TNF-α, GABA, and PER genes in PC12 cells were investigated. The results are shown in [Figure number missing]. Figures 4-6 The culture method for PC12 cells and the PCR detection method for the above-mentioned gene expression levels were performed in accordance with the routine cell biology detection specifications and the standard procedure for PCR detection of gene expression levels.

[0102] Comparing the results of the blank group with those of Examples 1-6 and Comparative Examples 1-5, it can be seen that after the fermented concentrate of seven-flavor jujube and lingzhi prepared by different processes was applied to PC12 cells, the expression levels of the above three types of key genes related to insomnia were significantly different from those of the blank group (p<0.05), proving that the fermented concentrate of seven-flavor jujube and lingzhi prepared by different processes of the present invention can effectively regulate the expression of key genes related to insomnia in PC12 cells.

[0103] Specifically, comparing the results of Example 1 with Examples 2-4, it can be seen that the fermentation effect of the BXM2 and YYS-69 compound strain is significantly better than that of single strain fermentation. The compound strain can produce a significant synergistic effect, which can more significantly inhibit the expression of the inflammatory factor TNF-α, while effectively promoting the expression of the sedative and tranquilizing GABA gene and the circadian rhythm regulation-related PER gene, thus highlighting the core efficacy of improving insomnia.

[0104] The results of comparing Example 1 with Examples 5 and 6 show that the fermentation efficiency of the compound strain is optimal when the fermentation time is 20 hours, which can fully transform the effective components in the raw materials and make the fermentation broth have the best regulatory effect on the target genes. Too short a fermentation time will result in the strain not being fully utilized, while too long a fermentation time will easily cause the decomposition of the effective components. Both will reduce the biological activity of the fermentation broth.

[0105] Comparing Example 1 with Comparative Example 1, it can be seen that the combined process used in this invention can significantly enhance the regulatory ability of the Qiwei Zao Ling fermented concentrate on key genes related to insomnia compared with the traditional water extraction process, and the process has significant advantages.

[0106] Comparing Example 1 and Comparative Example 2, it can be seen that the introduction of the enzymatic hydrolysis step can significantly improve the regulatory effect of the fermented concentrate of Seven-Flavor Jujube and Poria on the target gene, which is an important component of the preferred process of this invention.

[0107] As can be seen from the comparison between Example 1 and Comparative Example 3, the fermentation step is the key step in enhancing the bioactivity of the fermented concentrate of Qiwei Zao Ling. The fermentation of compound strains can further enhance the regulatory effect on target genes and form a synergistic effect with the crushing and enzymatic hydrolysis steps, maximizing the potential of the raw materials to improve insomnia.

[0108] Furthermore, comparing Example 1 with Comparative Examples 4 and 5, it can be seen that longer enzymatic hydrolysis time is not necessarily better. 2 hours is the optimal enzymatic hydrolysis time in this experiment. Under this parameter, the enzymatic hydrolysis effect can be maximized, and the biological activity of the fermentation broth can be effectively improved.

[0109] In summary, the fermented concentrate of seven-ingredient jujube and lingzhi prepared by the present invention through a combined process of "pulverization + enzymatic hydrolysis + compound fermentation" can significantly inhibit the expression of the inflammatory factor TNF-α gene in PC12 cells, while significantly promoting the expression of the sedative-sedative GABA gene and the circadian rhythm-regulating PER gene. Its regulatory effect on key genes related to insomnia is significantly better than other process schemes and traditional water extraction processes, and it has excellent biological activity in improving insomnia, providing a highly efficient product and reliable process support for the intervention of insomnia.

[0110] Test 3: Brain-boosting and cognitive-enhancing activities of fermented concentrated extracts of seven-flavor jujube and lingzhi prepared by different processes.

[0111] The effects of different fermentation concentrates of Ziziphus jujuba and Poria cocos prepared by different processes on the expression levels of apoptosis-related genes (bax and bcl-2) in a H2O2-induced oxidative stress injury model of PC12 cells were investigated. The results are shown in […]. Figure 7 , 8 The PCR detection methods for the expression levels of the bax and bcl-2 genes were performed according to the standard procedure for real-time quantitative PCR. The expression levels of the blank group were normalized, and different letters represented statistically significant differences between groups (p < 0.05).

[0112] Comparison between the control group and the model group shows that H2O2 successfully induced oxidative stress damage in PC12 cells, the model was successfully constructed, and oxidative stress damage significantly upregulated the expression of the pro-apoptotic gene bax and downregulated the expression of the anti-apoptotic gene bcl-2.

[0113] Comparing the model group with Examples 1-6 and Comparative Examples 1-5, it can be seen that, compared with the model group, after treatment with Examples 1-6 and Comparative Examples 1-5, the expression level of the bax gene in PC12 cells was significantly reduced, and the expression level of the bcl-2 gene was significantly increased. This indicates that the products obtained by the relevant process of the present invention can effectively reverse the abnormal expression of genes related to H2O2-induced oxidative stress damage in PC12 cells, and have the effects of inhibiting cell apoptosis and protecting cells damaged by oxidative stress.

[0114] Specifically, comparing Example 1 with Examples 2-4, it can be seen that Example 1 uses a BXM2+YYS-69 compound microbial strain for fermentation, while Examples 2, 3, and 4 use a single microbial strain for fermentation. The expression levels of the bax gene in Examples 2-4 are significantly higher than those in Example 1, while the expression levels of the bcl-2 gene are significantly lower than those in Example 1. This indicates that the fermentation effect of the BXM2+YYS-69 compound microbial strain is significantly better than that of the single microbial strain, and the compound microbial strain can produce a synergistic effect, more effectively regulating the expression of the bax and bcl-2 genes.

[0115] Comparing Example 1 with Examples 5 and 6, it can be seen that the fermentation time needs to be controlled within a suitable range. The 20-hour fermentation time selected in this invention allows the compound strain to fully metabolize and produce active substances. Insufficient or excessive fermentation will reduce the product effect.

[0116] Comparing Example 1 with Comparative Example 1, it can be seen that the combined process of "pulverization + enzymatic hydrolysis + compound fermentation" used in this invention can significantly improve the regulatory effect of Qiwei Zao Ling Fang on gene expression in cells damaged by oxidative stress compared with the traditional water extraction process. The activity of the product from the traditional process is far lower than that of the product from the process of this invention.

[0117] Comparing Example 1 and Comparative Example 2, it can be seen that the enzymatic hydrolysis step is one of the key processes for improving the gene expression regulation effect of the fermented concentrate of Seven-Flavor Jujube and Poria, and can have a synergistic effect with the fermentation process to enhance activity.

[0118] Comparing Example 1 and Comparative Example 3, it can be seen that the fermentation step is one of the core steps of the process of the present invention, and enzymatic hydrolysis alone (without fermentation) cannot achieve the optimal gene regulation effect.

[0119] Furthermore, comparing Example 1 with Comparative Examples 4 and 5, it can be seen that the longer or shorter the enzymatic hydrolysis time is not necessarily better. The 2-hour enzymatic hydrolysis time selected in this invention is the optimal parameter, which can ensure sufficient enzymatic hydrolysis and avoid damage to the effective components, thereby maximizing the effect of Qiwei Zao Ling Fang in regulating gene expression.

[0120] This demonstrates that the combined "pulverization + enzymatic hydrolysis + fermentation" process used in this embodiment of the invention, resulting in a concentrated fermented broth of seven-ingredient jujube and lingzhi, can significantly reverse the abnormal expression of bax and bcl-2 genes related to H2O2-induced oxidative stress damage in PC12 cells. By downregulating pro-apoptotic genes and upregulating anti-apoptotic genes, it exerts a cytoprotective effect, thus providing an effective material basis for improving insomnia. The process parameter combination in Example 1 is the optimal scheme, enabling the fermentation broth to achieve the best gene regulatory activity, demonstrating significant advantages compared to other process parameter combinations and traditional processes.

[0121] In summary, the fermented concentrate of the seven-flavor jujube and lingzhi of the present invention is prepared by combined enzymatic hydrolysis and fermentation with specific microorganisms. Compared with the unfermented case, its content of spinosin, total polysaccharides and total saponins is increased by 3.3 times, 1.3 times and 1.6 times respectively. It also has the activity of improving insomnia and enhancing brain function. The preparation process can be completed by simple operations such as fermentation, enzymatic hydrolysis and filtration. The equipment requirements are low and the operation is easy. It is suitable for large-scale industrial production. Moreover, the fermented liquid can be applied to various functional products such as food and health products that have the effects of improving insomnia and enhancing brain function.

[0122] It should also be noted that the terms such as microfiltration, extraction, and enzymatic hydrolysis used in this invention are names of processing steps commonly used by those skilled in the art, and their names can accurately describe the processing steps, so they will not be repeated in this invention.

[0123] 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; and these modifications or substitutions 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 preparation method of seven-flavor jujube-ling fermentation concentrate, characterized by, It comprises the following steps: S1, crushing and sieving the Qimei Zaoling Decoction to obtain Qimei Zaoling Decoction powder, mixing with water, then adding cellulase and pectinase for enzymolysis at 35-55℃ for 0.5-4.5h, and then inactivating the enzyme and cooling down; S2, inoculating lactic acid bacteria powder into the enzymolysis solution, and then fermenting at 35-45℃ for 10-50h, and then inactivating to obtain a fermentation liquor; S3, performing solid-liquid separation on the fermentation liquor to obtain a Qimei Zaoling fermentation liquor; S4, concentrating the Qimei Zaoling fermentation liquor at 60-70℃ by 6-10 times to obtain the Qimei Zaoling fermentation concentrate; The content of spinozine in the Qimei Zaoling fermentation concentrate is not less than 8mg / 100g, the content of total polysaccharide is not less than 1200mg / 100g, and the content of total saponins is not less than 20mg / 100g. The Qimei Zaoling Decoction is composed of Jujubae Fructus, Poria cocos, Lilium brownii, Longan Arillus, Rosa rugosa, Gastrodia elata, and Glycyrrhiza uralensis; the mass ratio of the Qimei Zaoling Decoction powder to water is 1:(15-40); the addition amount of cellulase is 0.2%-0.6% of the mass of water; the addition amount of pectinase is 0.1%-0.3% of the mass of water; and the addition amount of lactic acid bacteria powder is 0.005%-0.03% of the mass of water. The lactic acid bacteria powder contains Lactobacillus paracasei ( Lactobacillus paracasei YYS-69, Lactobacillus plantarum ( Lactobacillus plantarum BXM2, Lactobacillus plantarum ( Lactiplantibacillus plantarum One or two of YYS-99; wherein, the preservation number of Lactobacillus paracasei YYS-69 is CGMCC NO.25837; the preservation number of Lactobacillus plantarum BXM2 is CGMCC NO.16436; and the preservation number of Lactobacillus plantarum YYS-99 is CGMCC NO.25838.

2. The preparation method of the seven-flavor jujube-ling fermented concentrate liquid according to claim 1, characterized in that, In step S1, the sieve aperture is 0.05-0.85mm when the Qimei Zaoling Decoction powder is crushed and sieved.

3. The preparation method of the seven-flavor jujube-ling fermented concentrate liquid according to claim 1, characterized in that, In step S2, when the lactic acid bacteria powder is composed of Lactobacillus paracasei YYS-69 and Lactobacillus plantarum BXM2, the mass ratio of Lactobacillus paracasei YYS-69 to Lactobacillus plantarum BXM2 is (1-5):(1-5).

4. The preparation method of the seven-flavor jujube-ling fermented concentrate liquid according to claim 1, characterized in that: The enzyme inactivation is performed at 85-90℃ for 10-20min, and the inactivation is performed at 85-100℃ for 10-20min.

5. A seven-flavor jujube poria fermented concentrate, characterized by: The preparation method of the Qimei Zaoling fermentation concentrate is prepared by the method of any one of claims 1-4; the content of spinozine in the Qimei Zaoling fermentation concentrate is not less than 8mg / 100g, the content of total polysaccharide is not less than 1200mg / 100g, and the content of total saponins is not less than 20mg / 100g.

6. A seven-flavor jujube-ling fermented powder, characterized by: The Qimei Zaoling fermentation concentrate is prepared by mixing, drying, crushing, and sieving the Qimei Zaoling fermentation concentrate of claim 5.

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