Pomegranate peel extract mixed lactobacillus acidophilus metagen as well as preparation method and application thereof

Abstract

The invention is suitable for the technical field of biological medicine, and provides a pomegranate peel extract mixed lactobacillus acidophilus metagen and a preparation method and application thereof, the pomegranate peel extract mixed lactobacillus acidophilus metagen comprises a pomegranate peel extract and a lactobacillus acidophilus metagen, and the mass ratio of the pomegranate peel extract to the lactobacillus acidophilus metagen is 1: 4. The synergistic antibacterial effect of the combination of the pomegranate peel extract and the lactobacillus acidophilus metagen is defined, the inhibition effect of the pomegranate peel extract and the lactobacillus acidophilus metagen on various common pathogenic bacteria in culture is verified, and the problem that the effect of a single substitute product is poor can be solved; animal experiments prove that the combined preparation has double improvement effects on the growth performance and the immune function of bred animals, and the resistance of the animals to pathogens is improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a pomegranate peel extract mixed with Lactobacillus acidophilus postbiotic and a preparation method and application thereof. BACKGROUND

[0002] In the livestock industry, the widespread use of antibacterial drugs is considered to be one of the main driving factors of antimicrobial drug resistance, however, the overuse and misuse of antibiotics can lead to the emergence and spread of antibiotic-resistant bacteria (ARB).

[0003] Pomegranate peel is rich in natural bioactive antibacterial substances such as polyphenols, flavonoids, alkaloids and organic acids, and has broad-spectrum antibacterial activity against gram-positive and gram-negative bacteria.

[0004] Probiotics, as an effective antibiotic substitute, have been widely studied and applied, among which Lactobacillus acidophilus is a gram-positive bacterium that widely exists in the natural environment, especially in the digestive system of animals. Lactobacillus acidophilus can metabolize to produce digestive enzymes, vitamins, hydrogen peroxide and bacteriocins, etc., inhibit the growth of pathogenic microorganisms, regulate the balance of intestinal flora, thereby promoting the digestion of nutrients and enhancing the immune function. The experimental results of Liangyu xue et al. showed that Lactobacillus acidophilus LA85 can regulate the Notch signaling pathway, promote the repair of intestinal mucosa, and can enhance the immune response of the body through the TLR4 / NF-kB signaling pathway; Postbiotics, as secondary metabolites produced by probiotics during growth, have more advantages than probiotics in terms of safety, stability, long shelf life, easy storage and transportation, etc. The research of Homayouni-Rad et al. showed that postbiotics can have better absorption capacity through metabolism, have high signal transduction capacity to various organs and tissues, and can produce various probiotic effects on animals. At the same time, it has been reported that Lactobacillus planta postbiotics can improve the antioxidant capacity of animals, improve the intestinal morphology, promote the growth of intestinal probiotics, reduce intestinal pathogenic bacteria, and regulate the intestinal flora structure.

[0005] Among existing antibiotic substitutes, single Chinese herbal medicine has limited bacteriostatic effect and mild immune-enhancing effect, and single postbiotic has insufficient protective effect in complex pathogenic environment, both of which are difficult to meet the needs of the breeding industry for efficient prevention and control. Therefore, it is necessary to provide a set of process-specific and replicable combined preparation scheme to fill the technical gap of the combination of Chinese herbal medicine and postbiotic in the breeding industry, and to provide a green, safe, efficient and feasible new strategy for antibiotic-free breeding. SUMMARY

[0006] The purpose of this invention is to provide a pomegranate peel extract mixed with Lactobacillus acidophilus as a postbiotic, aiming to solve the problems mentioned in the background art.

[0007] The present invention is implemented as follows: pomegranate peel extract mixed with Lactobacillus acidophilus postbiotic, including pomegranate peel extract and Lactobacillus acidophilus postbiotic, wherein the mass ratio of pomegranate peel extract to Lactobacillus acidophilus postbiotic is 1:4.

[0008] The preparation method of the pomegranate peel extract is as follows: the crushed pomegranate peel is dissolved in 80% methanol solution in a water bath at 38-42℃ for 3.8-4.2h, then filtered, and the filter residue is extracted twice more with the same amount of 80% methanol solution, with the extraction time shortened to 1.8-2.2h and 0.8-1.2h respectively. All extracts are mixed, the filtrate is concentrated under reduced pressure, and dried at a constant temperature for 11-13h.

[0009] The preparation method of the Lactobacillus acidophilus postbiotic is as follows: Lactobacillus acidophilus is inoculated into MRS liquid medium for activation, anaerobic incubation at 37°C overnight, the bacterial solution is centrifuged for 8-12 min, the bacterial cells are washed several times with PBS, the supernatant is discarded, PBS is added, and the mixture is sonicated for 20 min.

[0010] Another objective of this invention is to provide a method for preparing a postbiotic by mixing pomegranate peel extract with Lactobacillus acidophilus, comprising the following steps:

[0011] (1) Preparation of pomegranate peel extract and Lactobacillus acidophilus postbiotic;

[0012] (2) Add pomegranate peel extract and Lactobacillus acidophilus post-biotic at a mass ratio of 1:4 and then sonicate.

[0013] Another objective of this invention is to provide the application of pomegranate peel extract mixed with Lactobacillus acidophilus postbiotic in the preparation of growth-promoting immunoprotective agents.

[0014] Another objective of this invention is to provide a feed additive comprising the above-mentioned pomegranate peel extract mixed with Lactobacillus acidophilus postbiotic.

[0015] This invention, for the first time, clarifies the synergistic antibacterial mechanism of pomegranate peel extract combined with Lactobacillus acidophilus postbiotic, verifying its inhibitory effect on a variety of common livestock pathogens and solving the problem of poor efficacy of single alternative products. Animal experiments confirm that the combined formulation has a dual effect of improving the growth performance and immune function of livestock, enhancing their resistance to pathogens. It provides a set of well-defined and reproducible preparation and application schemes for the combined formulation, filling the technological gap in the application of traditional Chinese medicine and postbiotics in the livestock industry, and providing a green, safe, efficient and feasible new strategy for antibiotic-free farming. Attached Figure Description

[0016] Figure 1 The antibacterial diameter measurement results provided in the embodiments of the present invention are A. veroniTH0426, A. veroni 33, A. hydrophilaTPS, A. caviae AC-CY, S. aureus, E. coli, S. Castellani, and S. enterica, respectively.

[0017] Figure 2 The results of serum SOD content determination provided in the embodiments of the present invention;

[0018] Figure 3 The results of serum LZM content determination provided in the embodiments of the present invention;

[0019] Figure 4 The results of serum IgM content determination provided in the embodiments of the present invention;

[0020] Figure 5 The serum IL-6 level provided in the embodiments of the present invention;

[0021] Figure 6 The serum IL-1β level provided in the embodiments of the present invention;

[0022] Figure 7 The serum IL-10 level provided in the embodiments of the present invention;

[0023] Figure 8 The serum TNF-α level provided in the embodiments of the present invention;

[0024] Figure 9 The serum cytokine TGF-α level provided in the embodiments of the present invention. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0026] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0027] Example 1: Identification of the main chemical components of pomegranate peel:

[0028] The chemical components in the pomegranate peel herbal solution were identified by high-resolution liquid chromatography-mass spectrometry. The preparation method of the pomegranate peel herbal solution was as follows: 20g of pomegranate peel herbal medicine (purchased from Fubaicao Pharmacy) was dissolved in 200mL of ultrapure water at a ratio of 1:10 and soaked for 24h. After sterilization, the solution was filtered and then filtered under reduced pressure. The supernatant was concentrated under reduced pressure using a rotary evaporator (80℃). Finally, the volume was adjusted to 10mL to obtain the pomegranate peel herbal solution, which was stored at 4℃ for later use.

[0029] Take 200 µL of pomegranate peel herbal solution, add 800 µL of methanol, vortex mix for 10 min, centrifuge at 13000 rpm for 10 min, collect the supernatant, and analyze it using high-resolution mass spectrometry and chromatography, respectively. The high-resolution LC-MS data were initially processed using CD3.3 (Compound Discoverer 3.3) (Thermo Fisher) and then compared with the mzCloud database. The results are shown in Table 1.

[0030] Table 1

[0031]

[0032] The main chemical components in pomegranate peel are phenolic compounds, primarily gallic acid, cinnamic acid, salicylic acid, syringic acid, chlorogenic acid, catechin, caffeic acid, and ellagitannins.

[0033] Example 2: A pomegranate peel extract mixed with Lactobacillus acidophilus postbiotic, the preparation method of which includes the following steps:

[0034] (1) Preparation of pomegranate peel extract: Powdered pomegranate peel (purchased from Fubaicao Pharmacy) was dissolved in 20 times the amount of 80% methanol solution in a 40℃ water bath for 4 hours, and then filtered through a 0.45μm filter membrane. The filter residue was extracted twice more with the same amount of 80% methanol solution, with the extraction time shortened to 2 hours and 1 hour respectively. All extracts were mixed, and the filtrate was concentrated under reduced pressure using a rotary evaporator (40℃). The product was then dried at a constant temperature for 12 hours. The process parameters were verified by three sets of parallel experiments. Compared with conventional extraction methods (such as 60% methanol and room temperature extraction), the yield of target active ingredients (phenols and flavonoids) was increased by more than 25%, and the activity retention rate was more than 95%.

[0035] (2) Preparation of Lactobacillus acidophilus post-genetic agents: Lactobacillus acidophilus CCTCC AB 2010208 (purchased from China National Culture Collection Center) was inoculated into MRS liquid medium for activation and anaerobic incubation overnight at 37°C. The bacterial culture was centrifuged for 10 min (4°C, 4000 r / min), the bacterial cells were washed 3 times with PBS, the supernatant was discarded, PBS was added, and the cells were sonicated for 20 min. The cells were pretreated with 250 W low power for the first 5 min (to loosen the cell wall), lysed with 350 W medium power for the middle 10 min (to release metabolites), and finished with 250 W low power for the last 5 min (to avoid excessive fragmentation).

[0036] (3) Pomegranate peel extract and Lactobacillus acidophilus post-biotic were ultrasonically broken up at a mass ratio of 1:4 (pre-treatment with 250W low power for the first 5 minutes, medium power of 350W for the middle 10 minutes, and low power of 250W for the last 5 minutes). During the ultrasonic process, "bacterial disruption + polyphenol compounding" was achieved simultaneously, reducing subsequent mixing steps.

[0037] Performance Analysis:

[0038] 1. Determination of inhibition diameter: The inhibition zone diameter of the single pomegranate peel extract group and the pomegranate peel extract mixed with Lactobacillus acidophilus followed by a prebiotic group against 8 pathogens (all pathogens used were publicly available strains that could be purchased from the China General Microbiological Culture Collection Center (CGMCC)) was determined using the agar diffusion method. A concentration of 100 μL was used. 6 CFU / mL Aeromonas veroni (TH0426) bacterial suspension was dropped into agar plates, spread evenly, and perforated. Then, 200 μL of the solution was added to each well, and the plates were incubated at 37°C for 18-24 h. The size of the inhibition zone was measured and recorded (the treatment method for the other 7 pathogens was the same).

[0039] The results are as follows Figure 1 As shown in Table 2:

[0040] Table 2

[0041]

[0042] It can be seen that both the single pomegranate peel extract group and the pomegranate peel extract combined with Lactobacillus acidophilus and postbiotic group showed certain antibacterial activity against 8 pathogens. Among them, the antibacterial effect of the combined group was significantly better than that of the single pomegranate peel extract group.

[0043] 2. MIC Determination: Using the two-fold dilution method, the original concentrations of the single pomegranate peel extract group and the pomegranate peel extract combined with Lactobacillus acidophilus and biotic group were diluted to 200, 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 mg / mL, respectively. LB liquid culture medium control and negative control were set up. Then, 50 μL of A. veroni (TH0426) bacterial suspension was added to test tubes and incubated in a 28℃ constant temperature shaker for 24 h. After incubation, the tubes were observed. The minimum inhibitory concentration (MIC) was determined to be clear with no obvious bacterial growth. The results are shown in Table 3.

[0044] Table 3

[0045]

[0046] 3. MBC Determination: After determining the MIC, take 100 μL of the diluted solution with a concentration ≥ MIC, spread it on LB medium, and then incubate at 28℃ for 24 h. After incubation, observe whether colonies are formed. If no colonies grow on the medium, it can be determined as the minimum bactericidal concentration (MBC). The results are shown in Table 4.

[0047] Table 4

[0048]

[0049] 4. Animal experimental grouping and feeding plan: Before the experiment, the rat cages inside and out were completely disinfected, and bedding was placed. The bedding was changed regularly every week. One-month-old male Kunming rats of uniform size, health and activity were selected and acclimated to a basic diet until they ate normally and their survival rate was stable. The selected mice were randomly divided into 4 groups: control group, pomegranate peel extract group, Lactobacillus acidophilus post-biotic group, and pomegranate peel extract mixed with Lactobacillus acidophilus post-biotic group, with 20 mice in each group. Each group was set up with three replicates. The mice were gavaged twice a day at a fixed time. The gavage volume was 3% of the mouse's body weight. The gavage reagents were aqueous solutions with a concentration of 50 mg / mL. The mice were immunized continuously for four weeks.

[0050] Mouse blood sample collection: Three mice were randomly selected from each of the control group, pomegranate peel group, Lactobacillus acidophilus post-biotic group, and pomegranate peel extract mixed with Lactobacillus acidophilus post-biotic group. Blood was collected from the orbital vein after anesthesia (once every 7 days, for a total of 4 times). The collected whole blood was placed flat in a 37°C incubator for 40-50 minutes, and then incubated at 4°C for 12 hours. The separated serum was centrifuged for 10 minutes (4°C, 4000 r / min), and the supernatant was collected and stored at -80°C.

[0051] The following tests were performed respectively:

[0052] Growth performance assessment: At 0 days and 28 days, three mice were randomly selected from each experimental group for weighing and measurement to calculate various growth indicators, using the following formula:

[0053] Weight gain rate (WGR%) = 100 × (final average weight - initial average weight) / initial average weight;

[0054] Specific growth rate (SGR%) = 100 × (final average weight - initial average weight) / number of feeding days;

[0055] Immunological markers and cytokine assays: Serum levels of immunoglobulin M (IgM), lysozyme (LZM), superoxide dismutase (SOD), and cytokines such as IL-6, IL-1β, IL-10, TNF-α, and TGF-α were measured using ELISA.

[0056] Protection against viral challenge: Mice were fed for 28 days and fasted for 48 hours before being challenged to detect their survival rate after immunization. Ten mice were randomly selected from each experimental group, and 0.2 mL (1×10⁻⁶) of the solution was administered. 6 A. veroni (TH0426) bacterial culture (CFU / mL) was injected into the peritoneal cavity, and the patients were observed continuously for 7 days. The immune protection rate against challenge was calculated using the following formula:

[0057] RPS = (1 - Mortality rate in the immunized group / Mortality rate in the control group) × 100%;

[0058] Statistical analysis of data: The data results were analyzed for significance using SPSS 22, and the required graphs were plotted using Graph Prism 9 (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

[0059] The results are as follows:

[0060] Table 5 shows the growth performance results of the control group, pomegranate peel group, Lactobacillus acidophilus post-biotic group, and pomegranate peel extract mixed with Lactobacillus acidophilus post-biotic group after a 28-day immunization cycle.

[0061] Table 5

[0062]

[0063] As shown in the table, the weight gain rate and specific growth rate of the pomegranate peel group, the Lactobacillus acidophilus post-biotic group, and the pomegranate peel extract mixed with Lactobacillus acidophilus post-biotic group were significantly different from those of the blank control group. Among them, the weight gain rate and specific growth rate of the pomegranate peel extract mixed with Lactobacillus acidophilus post-biotic group were higher than those of the other immunized groups. This indicates that the pomegranate peel extract mixed with Lactobacillus acidophilus post-biotic group has a better effect on the growth performance of mice.

[0064] SOD content in serum: Detection of SOD activity in serum, such as... Figure 2 As shown, the results indicated that after 28 days of gavage, the SOD levels in the pomegranate peel group, post-biotic group, and combined group were significantly higher than those in the control group (P < 0.001). Among them, the SOD content in the combined group increased significantly from the start of immunization to 28 days of immunization, increasing by 22% compared to the single pomegranate peel group and by 18% compared to the single post-biotic group, demonstrating a clear advantage in antioxidant capacity.

[0065] LZM content in serum: LZM activity in serum, such as Figure 3 As shown, the results indicated that within 28 days of immunization, the levels of LZM in the pomegranate peel group, post-biotic group, and combined group were significantly higher than those in the control group (P < 0.001). Among them, the levels in the pomegranate peel group and the Lactobacillus acidophilus post-biotic group were similar (P < 0.05), while the pomegranate peel extract mixed with Lactobacillus acidophilus post-biotic group showed a significant upward trend at 14 days of immunization, indicating higher LZM activity.

[0066] Serum IgM levels: Changes in serum IgM activity are as follows Figure 4 As shown in the data, there were significant differences in all three immunization groups compared to the control group (P < 0.001). Among them, the serum IgM concentration was highest in the pomegranate peel extract mixed with Lactobacillus acidophilus post-biotic group, which was 20% higher than the single pomegranate peel group and 16% higher than the single post-biotic group. This indicates that pomegranate peel + Lactobacillus acidophilus post-biotic can better promote the secretion of serum IgM and has a certain degree of immune protection for mice.

[0067] Serum cytokine levels: The effects of different drugs administered by gavage on serum cytokine levels in mice, such as Figures 5-9 As shown in the figure, the analysis reveals that compared with the blank control group, the cytokine levels in the immune group were significantly increased (P < 0.001), and were promoted to varying degrees. The pomegranate peel and Lactobacillus acidophilus post-biotic group had a significant promoting effect on IL-10 and IL-1β (P < 0.01), while the pomegranate peel extract mixed with Lactobacillus acidophilus post-biotic group did not have a significant promoting effect, but it showed a significant increase in the promoting effect on IL-6, TNF-α, and TGF-α (P < 0.001).

[0068] The virus attack protection rate is shown in Table 6:

[0069] Table 6

[0070]

[0071] As shown in the table, the relative protection rates of the pomegranate peel group, the Lactobacillus acidophilus post-biotic group, and the pomegranate peel extract mixed with Lactobacillus acidophilus post-biotic group were 62.5%, 50%, and 75%, respectively, while the survival rate of the PBS group was 20%. The results show that all three immunization groups have different degrees of protective effects on mice, among which the pomegranate peel extract mixed with Lactobacillus acidophilus post-biotic group has the best immunoprotective effect.

[0072] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pomegranate peel extract mixed with Lactobacillus acidophilus postbiotic, characterized in that, The pomegranate peel extract and the Lactobacillus acidophilus postbiotic are in a mass ratio of 1:4; The pomegranate peel extract is prepared by the following method: the crushed pomegranate peel is dissolved in 80% methanol solution in a 38-42℃ water bath for 3.8-4.2 hours, and then filtered; the filter residue is extracted with the same amount of 80% methanol solution for 2 times, and the time is shortened to 1.8-2.2 hours and 0.8-1.2 hours; all the extract solutions are mixed, the filtrate is concentrated under reduced pressure, and dried at constant temperature for 11-13 hours. The Lactobacillus acidophilus postbiotic is prepared by the following method: the Lactobacillus acidophilus is inoculated into MRS liquid medium for activation, and incubated at 37℃ in an anaerobic environment overnight; the bacterial solution is centrifuged for 8-12 minutes, the bacterial body is washed with PBS for several times, the supernatant is discarded, and PBS is added for ultrasonic crushing for 20 minutes.

2. The Punica granatum extract-mixed Lactobacillus acidophilus postbiotic according to claim 1, characterized in that, In the preparation method of the pomegranate peel extract, the mass ratio of the crushed pomegranate peel and the 80% methanol solution is 1:

20.

3. The Punica granatum extract mixed Lactobacillus acidophilus postbiotic of claim 1, characterized by, In the preparation method of the lactobacillus acidophilus probiotics, the lactobacillus acidophilus is lactobacillus acidophilus (CCTCC AB 2010208). Lactobacillus acidophilus ) CCTCC AB 2010208.

4. The Punica granatum extract mixed Lactobacillus acidophilus postbiotic of claim 1, wherein, In the preparation method of the Lactobacillus acidophilus postbiotic, the bacterial solution is centrifuged at 4000r / min at 4℃.

5. The Punica granatum extract mixed Lactobacillus acidophilus postbiotic of claim 1, wherein, In the preparation method of the Lactobacillus acidophilus postbiotic, the ultrasonic crushing for 20 minutes is specifically as follows: the low power of 250W is used for pretreatment for the first 5 minutes, the middle power of 350W is used for crushing for the middle 10 minutes, and the low power of 250W is used for finishing for the last 5 minutes.

6. A method of preparing the extract of Punica granatum mixed with postbiotic of Lactobacillus acidophilus as claimed in any one of claims 1-5, characterized in that, The method comprises the following steps: (1) preparing the pomegranate peel extract and the Lactobacillus acidophilus postbiotic; (2) adding the pomegranate peel extract and the Lactobacillus acidophilus postbiotic in a mass ratio of 1:4, and performing ultrasonic crushing.

7. The method of claim 6, wherein the preparation of the Punica granatum extract-mixed Lactobacillus acidophilus postbiotic is characterized by, In step (2), the ultrasonic crushing is specifically as follows: the low power of 250W is used for pretreatment for the first 5 minutes, the middle power of 350W is used for crushing for the middle 10 minutes, and the low power of 250W is used for finishing for the last 5 minutes.

8. Use of the pomegranate peel extract mixed with the Lactobacillus acidophilus postbiotic according to any one of claims 1-5 in the preparation of a growth-promoting immune protective preparation.

9. A feed additive, characterized in that, The method comprises the following steps: (1) preparing the pomegranate peel extract and the Lactobacillus acidophilus postbiotic; (2) adding the pomegranate peel extract and the Lactobacillus acidophilus postbiotic in a mass ratio of 1:4, and performing ultrasonic crushing.

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