Pentosicoccus and postbiotic compound thereof for tonifying qi and nourishing face and improving anemia

Abstract

The application discloses a pentosus pentosaceus and postbiotic compound thereof for tonifying qi, nourishing face and improving anemia, and belongs to the technical field of microbial fermentation. The application screens a pentosus pentosaceus, obtains the bacterial body and postbiotic by culture, and forms a compound of the postbiotic and extract of semen ziziphi spinozae. The compound exhibits a synergistic effect, specifically, compared with the iron deficiency model group, the movement and various blood indexes of rats are improved, and are close to the normal rats of the control group. The indexes of rats in the compound group are better than those of rats fed with probiotics, and rats fed with postbiotic or semen ziziphi spinozae alone, which indicates that the compound can promote the recovery of iron content and protein indexes in the blood of rats more quickly. The application has practical value for comprehensive utilization of the pentosus pentosaceus and postbiotic, and also has important value for tonifying qi, nourishing face and improving anemia.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a Pentosacchariphyte, which is used to replenish qi, improve complexion, and alleviate anemia, and its post-biotic complex. Background Technology

[0002] Anemia is a common blood disorder, with a global prevalence of up to 20%, affecting the health of over one billion people. The main characteristic of anemia is a decrease in the number of red blood cells in the peripheral blood, leading to insufficient oxygen supply to tissues and organs. The primary symptom is fatigue, and it can trigger various symptoms affecting multiple organs and systems, including headaches, tinnitus, fainting, muscle weakness, shortness of breath, palpitations, digestive discomfort, menstrual irregularities, and decreased sexual function.

[0003] Pediococcus pentosaceus ( Pediococcus pentosaceus Lactobacillus is a Gram-positive lactic acid bacterium widely found in fermented foods, animal and plant cultures, and the human gut. It is acid- and bile-resistant, and can produce active substances such as lactic acid and bacteriocins through metabolism, making it important in the food industry and feed additives.

[0004] Sour jujube ( Ziziphus jujuba var.spinosa (Bunge) Hu ex HFChow. is a woody plant belonging to the genus Ziziphus of the family Rhamnaceae. Sour jujube gets its name from its sweet and sour flesh. It originated in China, with distribution in most of northern China, as well as Jiangsu and Anhui provinces. It is also commonly cultivated in Asia, Europe, and the Americas. Sour jujube seeds contain fatty oils and proteins and can be eaten or used medicinally. According to the *Ben Cao Zai Xin* (New Compilation of Materia Medica), sour jujube seeds have the following effects: calming the liver and regulating qi, moistening the lungs and nourishing yin, warming the middle jiao and promoting diuresis, astringing qi and stopping sweating, strengthening the will and calming the mind, and improving hearing and vision.

[0005] Although there is some understanding of the effects of Pediococcus pentosaceus and Ziziphus jujuba var. spinosa, the specific effects of using these two substances together to replenish qi, nourish the complexion, and improve anemia have long been unknown. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to provide a Pentosacchariphyte and its post-biotic complex that can replenish qi, nourish the complexion, and improve anemia.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a *Pediococcus pentosaceus* strain that invigorates qi, nourishes the complexion, and improves anemia, wherein the *Pediococcus pentosaceus* is *Pediococcus pentosaceus* (… Pediococcus pentosaceus WZA-PP62 is deposited at the China Center for Type Culture Collection, accession number CCTCC NO:M 20241417.

[0009] In a second aspect, the present invention provides the application of the aforementioned *Pediococcus pentosaceus* in the preparation of products for replenishing qi and nourishing the complexion, and improving anemia, wherein the viable count of *Pediococcus pentosaceus* in the product is greater than 1 × 10⁻⁶. 7 cfu / mL.

[0010] In a third aspect, the present invention provides a Pediococcus pentosaceus post-biotic complex that invigorates qi, nourishes the complexion, and improves anemia. The Pediococcus pentosaceus post-biotic complex is composed of post-biotics of Pediococcus pentosaceus and jujube seed extract in a mass ratio of (0.5-5):(0.5-5).

[0011] The metagenes of Pediococcus pentosaceus were prepared by the following method:

[0012] The *Pediococcus pentosus* was activated and inoculated into a fermentation medium for fermentation at a temperature of 30-45℃ for 4-40 hours. The fermentation product was centrifuged and the supernatant was freeze-dried to obtain the metagenes of *Pediococcus pentosus*.

[0013] Furthermore, the fermentation medium is composed of the following raw materials in parts by weight: 5-15 parts beef extract powder, 5-12 parts peptone, 10-30 parts glucose, 1-10 parts yeast powder, 0.05-1 part magnesium sulfate, 1-5 parts diammonium citrate, 1-5 parts dipotassium hydrogen phosphate, 2-12 parts sodium acetate, 0.01-1 part manganese sulfate, 0.5-5 parts Tween 80, 800-1200 parts distilled water, pH: 5-7.

[0014] Furthermore, the jujube seed extract is prepared by the following method:

[0015] Take jujube seeds, wash them, dry them in an oven at 70℃, and pulverize them to pass through a 400-mesh sieve to obtain jujube seed powder. Extract the jujube seed powder in 70% v / v ethanol at a mass-to-volume ratio of 1g:5ml, at an extraction temperature of 30℃, for 10 hours each time, for a total of 2 extractions. Combine the extracts obtained each time, concentrate them under reduced pressure at 50℃, and then dry them in an oven at 75℃ to obtain jujube seed extract.

[0016] In a fourth aspect, the present invention provides the application of the aforementioned Pediococcus pentosaceus postbiotic complex in the preparation of products for replenishing qi and nourishing the complexion, and improving anemia.

[0017] The beneficial effects of this invention are:

[0018] (1) This invention obtains Pediococcus pentosaceus through screening. This bacterium has the characteristics of acid resistance and bile salt resistance, and can replenish qi and nourish the skin, and improve anemia. The survival rate of the strain is as high as 91% after 5 hours in simulated gastric juice, and the survival rate is 89% after 5 hours in 1% bile salt.

[0019] (2) In this invention, Pediococcus pentosacchari was freeze-dried to obtain the postgenetic of Pediococcus pentosacchari. The postgenetic was combined with Ziziphus jujuba var. spinosa extract to form a complex, namely Pediococcus pentosacchari postgenetic complex.

[0020] (3) In this invention, the Pediococcus pentosaceus postbiotic complex was used in experiments. The results showed that, compared with the iron-deficient model group, the motor indicators and various blood indicators of rats in each experimental group were improved, approaching those of normal rats in the control group. Among them, the complex group performed best: in terms of motor indicators, the rate of spontaneous activity in the complex group decreased by 0.23%; in terms of blood indicators, SI was 73.9±8.7 mg / L, SF was 240.2±27.4 ng / mL, Hb was 140.9±9.7 g / L, and RBC was 6.69±0.51 (10) 12 The MCV was 63.6 ± 1.9 fL. It is evident that after treatment with the complex group, rats showed improved hemoglobin levels, sufficient blood and qi, better external observation, and improved health status, including exercise and blood quality. The complex group treatment exhibited a synergistic effect, superior to feeding probiotics or feeding postbiotics or jujube seed alone. The Pediococcus pentosaceus postbiotic complex could more quickly promote the recovery of iron content and protein levels in rat blood, demonstrating better ability to replenish qi and improve complexion, and alleviate anemia. Detailed Implementation

[0021] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0022] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0023] The experimental materials used in the embodiments of this invention, unless otherwise specified, are all conventional experimental materials in the art and can be purchased through commercial channels. The jujube seed used in this invention is listed in the 2025 edition of the Chinese Pharmacopoeia, is grade one, and is used after sieving to remove the shell and broken kernels. The normal feed used in this invention is a maintenance diet for rats and mice purchased from Spiford (Beijing) Biotechnology Co., Ltd.

[0024] The preservation information of *Pediococcus pentosaceus* used in this invention is as follows:

[0025] Chinese name: Pediococcus pentosaceus WZA-PP62;

[0026] Latin name: Pediococcus pentosaceus WZA-PP62;

[0027] Accession number: CCTCC NO:M 20241417;

[0028] Deposit date: July 1, 2024;

[0029] Preservation institution: China Center for Type Culture Collection (CCTCC);

[0030] Location of collection: Wuhan University, Wuhan, China.

[0031] Example 1: Screening of Pediococcus pentosaceus

[0032] 1. Isolation and initial screening of bacterial strains:

[0033] Collect 10 g of naturally fermented cheese samples. Take 1 g of each sample and place them separately into 50 mL centrifuge tubes containing 9 mL of sterile physiological saline. Shake for 15 min to mix thoroughly, let stand for 25 s, and then take 2 mL of the suspension from each sample and incubate at 80 ℃ for 10 min. Take 1 mL of each suspension and perform a 10-fold serial dilution. Then, select a suitable gradient and spread the suspension onto MRS agar medium (self-prepared) containing 0.3% CaCO3. Incubate at 37 ℃ for 48 h. Single colonies with calcium dissolution zones are streaked for purification. After the single colony morphology is stable, number and store them.

[0034] 2. Strain screening: acid resistance and bile salt tolerance

[0035] Probiotics must be able to survive in the gut to exert specific biological activities. Therefore, they need to enter the gut through the stomach, which contains acidic gastric juice. At the same time, the gut contains a high concentration of bile acids, so bile acid tolerance is also an important selection criterion for probiotic microorganisms.

[0036] Survival rates were recorded after treating the strains in simulated gastric fluid at pH 3 for 0 h, 2 h, 3 h, 4 h, and 5 h; survival rates were also recorded after treatment at bile salt concentrations of 0.1%, 0.3%, 0.6%, 0.8%, and 1%. Strains with higher survival rates were selected for further identification. The survival rate of the strains in simulated gastric fluid after 5 hours was as high as 91%, and the survival rate in 1% bile salt solution after 5 hours was 89%.

[0037] 3. Strain identification:

[0038] The strain was sent to the Henan Provincial Engineering Technology Research Center for Industrial Microbial Strains for 16S rDNA gene sequence identification. The nucleotide sequence is shown in SEQ ID NO:1. The identification results showed that the strain belongs to Pediococcus pentosaceus. Pediococcus pentosaceus Subsequently, strain WZA-PP62 was biopreserved.

[0039] Example 2: Preparation of metagenes from Pediococcus pentosaceus

[0040] The preparation method is as follows:

[0041] (1) The *Pediococcus pentosaceus* preserved in Example 1 was activated in a microaerobic environment (3%-5% O2, 10% CO2) to obtain an activated *Pediococcus pentosaceus* bacterial solution. The activation temperature was 37°C and the activation time was 24 h.

[0042] (2) The activated Pediococcus pentosaceus broth obtained in step (1) was inoculated into the fermentation medium at an inoculation rate of 5 wt% to obtain the Pediococcus pentosaceus fermentation broth. The fermentation temperature was 37℃, and the fermentation time was 26h. The composition of the fermentation medium used was as follows: beef extract powder: 8g, peptone: 10g, glucose: 20g, yeast powder: 4g, magnesium sulfate: 0.2g, diammonium citrate: 2g, dipotassium hydrogen phosphate: 2g, sodium acetate: 5g, manganese sulfate: 0.04g, Tween 80: 1g, distilled water to make up to 1L, pH: 5.7.

[0043] (3) Take the fermentation broth of Pediococcus pentosacchari obtained in step (2), centrifuge at 25°C, freeze-dry the supernatant to obtain the metagene of Pediococcus pentosacchari.

[0044] The Pediococcus pentosacchari cell pellet was prepared according to a viable cell count of 5 × 10⁻⁶. 8 After quantifying the cfu / ml concentration, store it in a refrigerator at 4°C for later use.

[0045] Example 3: Preparation of Pediococcus pentosaceus postbiotic complex

[0046] Preparation of Ziziphus jujuba seed extract

[0047] Take jujube seeds, wash them, dry them in an oven at 70℃, and pulverize them to a 400-mesh sieve to obtain jujube seed powder. Take 10g of jujube seed powder and add 50ml of 70% v / v ethanol for extraction at 30℃ for 10h. Filter to obtain the extract. Add another 50ml of 70% v / v ethanol to the filtered precipitate for a second extraction at 30℃ for 10h. Filter to obtain the extract. Combine the two extracts, concentrate them under reduced pressure at 50℃, and then dry them in an oven at 75℃ to obtain jujube seed extract.

[0048] Preparation of the complex

[0049] The Pediococcus pentosaceus postgenetic complex was obtained by mixing the Pediococcus pentosaceus postgenetic complex prepared in Example 2 and the Ziziphus jujuba var. spinosa extract prepared in Example 3 at a mass ratio of 1:1.

[0050] Experimental Example 1: Animal Model Experiment

[0051] Eight-week-old SD rats, half male and half female, were randomly divided into six groups of six each. One group served as the control group, fed with a normal diet. The other five groups were fed a low-iron diet, supplemented with small-scale tail vein bloodletting to establish an iron deficiency anemia model. During the modeling period, all rats drank deionized water and were fed for three weeks, with day and night light and shadow patterns varying according to natural circadian rhythms. The low-iron diet was formulated according to the AOAC recommended feed formula, specifically: 54 wt% corn starch, 40 wt% skim milk powder, 1 wt% salt, and 5 wt% edible oil. The feed was made into 5 mm diameter sticks, dried, and then used.

[0052] After blood tests confirmed successful establishment of the iron deficiency anemia model, one group was selected as the iron deficiency model group; the remaining groups were used as experimental groups and fed in the following manner: Postbiotic group, supplemented with postbiotics of *Pediococcus pentosaceus* prepared in Example 2; Ziziphus jujuba seed group, supplemented with *Ziziphus jujuba seed* extract prepared in Example 3; Probiotic group, supplemented with *Pediococcus pentosaceus* prepared and preserved in Example 2 (1 ml was considered 1 g); Complex group, supplemented with *Pediococcus pentosaceus* postbiotic complex prepared in Example 3. Each supplement group was administered via gavage at a dose of 3 mg / kg according to animal body weight. The iron deficiency model group and the control group received an equal volume of physiological saline. In addition to being fed the same normal diet, rats were administered gavage once daily until the end of the 14-day experiment. Motor and blood parameters of the experimental animals were tested using the following methods:

[0053] Motor activity indicators: Spontaneous activity of mice in each group was measured in a quiet environment under natural light. After placing the mice in the spontaneous activity testing device, they were allowed to acclimatize for 10 minutes before the number of spontaneous movements was measured over 5 minutes. The rate of decrease in spontaneous activity was calculated as follows:

[0054] Reduction rate of spontaneous activity (%) = (Number of spontaneous activities before experiment - Number of spontaneous activities after experiment) / Number of spontaneous activities before experiment × 100%

[0055] Blood parameters: Blood samples were collected from each group after the 14-day trial. Serum iron (SI) was measured using a biochemical reagent kit; serum ferritin (SF) was measured using ELISA. Hemoglobin (Hb), red blood cell count (RBC), and mean red blood cell volume (MCV) were measured using a fully automated blood cell analyzer. The results are shown in Table 1 below:

[0056] Table 1 Results of exercise and blood parameters

[0057]

[0058] External observations showed that control group mice had shiny fur, normal appetite, and agile movements; model group mice had dull fur that was prone to shedding, reduced appetite, and were lethargic, curled up, arched their backs, and drowsy. Further comparison of test indicators, as shown in Table 1, revealed that compared with the iron-deficiency model group, the motor indicators and various blood indicators of rats in each experimental group were improved, approaching those of normal rats in the control group. Among them, the complex group showed the best performance. In the complex group, the rate of reduction in spontaneous activity was 0.23%, SI was 73.9±8.7 mg / L, SF was 240.2±27.4 ng / mL, Hb was 140.9±9.7 g / L, and RBC was 6.69±0.51 (10). 12 The MCV was 63.6 ± 1.9 fL. It is evident that after treatment with the complex group, rats showed improved hemoglobin levels, sufficient blood and qi, better external observation, and improved health status, including exercise and blood quality. The complex group treatment exhibited a synergistic effect, superior to feeding probiotics or feeding postbiotics or jujube seed alone. The Pediococcus pentosaceus postbiotic complex could more quickly promote the recovery of iron content and protein levels in rat blood, demonstrating better ability to replenish qi and improve complexion, and alleviate anemia.

[0059] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A post-biotic complex of *Pediococcus pentosaceus* that invigorates qi, nourishes the complexion, and improves anemia, characterized in that... The Pediococcus pentosaceus postgenetic complex is composed of postgenetic agents of Pediococcus pentosaceus and Ziziphus jujuba seed extract in a mass ratio of (0.5-5):(0.5-5). The Pediococcus pentosaceus is Pediococcus pentosaceus ( Pediococcus pentosaceus WZA-PP62, deposited at the China Center for Type Culture Collection, accession number CCTCC NO:M 20241417; The metagenes of Pediococcus pentosaceus were prepared by the following method: The *Pediococcus pentosus* was activated and then inoculated into a fermentation medium for fermentation at a temperature of 30-45℃ for 4-40 hours. The fermentation product was centrifuged and the supernatant was freeze-dried to obtain the metagenes of *Pediococcus pentosus*. The jujube seed extract is prepared by the following method: Take jujube seeds, wash them, dry them in an oven at 70℃, and pulverize them to pass through a 400-mesh sieve to obtain jujube seed powder. Extract the jujube seed powder in 70% v / v ethanol at a mass-to-volume ratio of 1g:5ml, at an extraction temperature of 30℃, for 10 hours each time, for a total of 2 extractions. Combine the extracts obtained each time, concentrate them under reduced pressure at 50℃, and then dry them in an oven at 75℃ to obtain jujube seed extract.

2. The Pediococcus pentosaceus postbiotic complex according to claim 1, characterized in that, The fermentation medium is composed of the following raw materials in parts by weight: 5-15 parts beef extract powder, 5-12 parts peptone, 10-30 parts glucose, 1-10 parts yeast powder, 0.05-1 part magnesium sulfate, 1-5 parts diammonium citrate, 1-5 parts dipotassium hydrogen phosphate, 2-12 parts sodium acetate, 0.01-1 part manganese sulfate, 0.5-5 parts Tween 80, 800-1200 parts distilled water, pH: 5-7.

3. The application of the Pentosaccharide Pediococcus postbiotic complex according to claim 1 or 2 in the preparation of products for replenishing qi and nourishing the complexion and improving anemia.

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