Preparation and Application of Postbiotics of Lactiplantibacillus plantarum ZJUIDS15 for Preventing and Treating Colon Cancer

Abstract

The present invention belongs to the field of microorganisms, and particularly relates to the preparation and application of postbiotics of Lactobacillus plantarum ZJUIDS15 for preventing and treating colon cancer. The preparation method of the postbiotics of Lactobacillus plantarum ZJUIDS15 is as follows: after the fermentation supernatant of Lactobacillus plantarum ZJUIDS15 (CGMCC NO. 26120) is inactivated, it is concentrated by freezing to obtain the postbiotics of Lactobacillus plantarum ZJUIDS15. The postbiotics of Lactobacillus plantarum ZJUIDS15 have antibacterial activity and antioxidant capacity, and can relieve the peritoneal metastasis ability and liver metastasis ability of colorectal cancer cells after gavage treatment.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and particularly relates to the preparation and application of postbiotics of Lactobacillus plantarum ZJUIDS15 for preventing and treating colon cancer. Background Art

[0002] Colorectal cancer (CRC) is one of the most common cancer types globally and the second leading cause of cancer-related deaths worldwide. In recent years, the incidence of CRC has been high, and its prevention and treatment have received increasing attention. Although there are many current treatment methods for CRC, such as surgery, chemotherapy, and biotherapy, the success rate of cancer treatment remains difficult to predict, the mortality rate is high, and the recurrence rate of the prognosis of CRC patients is still very high. Therefore, there is an urgent need to find alternative anti-cancer drugs to improve the treatment of future colon cancer. It has been found that the gut microbiota structure is closely related to the occurrence and development of CRC.

[0003] Probiotics, as beneficial gut microorganisms, have functions such as regulating gut immunity, enhancing the gut barrier, and maintaining the balance of the gut microbiota structure, and play an important role in the prevention and adjuvant treatment of CRC. With the in-depth study of probiotics, it has been found that not only live bacteria can exert probiotic functions, but also inactivated bacterial cells, components released by the lysis of dead bacteria, and bacterial metabolites also have health-promoting effects.

[0004] Chinese Patent CN116396890A discloses the application of Lactobacillus plantarum ZJUIDS15 in preventing and treating colon cancer. This patent provides a Lactobacillus plantarum ZJUIDS15 with the function of preventing and treating colon cancer, with a preservation number of CGMCC NO.26120. This strain has the effect of preventing and treating colon cancer, can tolerate the gastrointestinal environment, the culture solution has no antibiotic resistance, inhibits harmful pathogenic bacteria in the intestine, and has strong antioxidant ability.

[0005] Chinese Patent CN107629987A discloses the application of Lactobacillus plantarum CCFM164 or fermented foods, which is characterized by being applied to improve the gut microbiota, reduce the abnormally elevated short-chain fatty acid levels in the intestine, alleviate colorectal inflammation, and inhibit the occurrence of colorectal cancer.

[0006] However, gut microorganisms suitable for preventing and treating colon cancer still need to be further developed. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide the preparation and application of postbiotics of Lactobacillus plantarum ZJUIDS15 for preventing and treating colon cancer.

[0008] To solve the above technical problems, the present invention provides a method for preparing postbiotics of Lactobacillus plantarum ZJUIDS15 for preventing and treating colon cancer: after inactivating the fermentation supernatant of Lactobacillus plantarum ZJUIDS15 (CGMCC NO.26120), it is concentrated by freezing (first by rotary evaporation and then by freezing) to obtain postbiotics of Lactobacillus plantarum ZJUIDS15.

[0009] As an improvement to the method for preparing postbiotics of Lactobacillus plantarum ZJUIDS15 of the present invention, the method for preparing the fermentation supernatant of Lactobacillus plantarum ZJUIDS15 is as follows: Inoculate one inoculation loop of Lactobacillus plantarum ZJUIDS15 into 100 mL of MRS liquid medium, place it in an incubator at 37 ± 0.5 °C and culture for 18 ± 0.5 h; then transfer it to 250 - 300 mL of sterilized (by autoclaving at 121 °C for 15 min) MRS liquid medium at an inoculation amount of 1 - 2% (volume%); place it in an incubator and culture at 37 ± 0.5 °C for 18 ± 0.5 h; centrifuge the obtained fermentation broth at 4000 - 10000 r / min for 15 - 20 min, and collect the supernatant.

[0010] As a further improvement to the method for preparing postbiotics of Lactobacillus plantarum ZJUIDS15 of the present invention: Inactivate the collected supernatant in a water bath at 85 ± 2 °C for 2 ± 0.1 h, and then rotary evaporate it to 1 / 3.5 - 1 / 4.5 of the original volume; then perform freeze concentration on the obtained fermentation supernatant concentrate to obtain postbiotics of Lactobacillus plantarum ZJUIDS15.

[0011] As a further improvement to the method for preparing postbiotics of Lactobacillus plantarum ZJUIDS15 of the present invention, the freeze concentration is as follows:

[0012] After pre-freezing the fermentation supernatant concentrate in an ultra-low temperature freezer at -80 °C for not less than 2 h, quickly take it out and put it into a vacuum freeze dryer for vacuum treatment. The vacuum degree is maintained at 0.1 P, and the freezing temperature is -55 °C. After complete freeze-drying (i.e., after freezing to constant weight, freeze-drying for about 48 h), postbiotics of Lactobacillus plantarum ZJUIDS15 are obtained.

[0013] Take it out and store it in a 4 °C refrigerator.

[0014] The present invention also simultaneously provides postbiotics of Lactobacillus plantarum ZJUIDS15 prepared by using any of the above methods.

[0015] As an improvement to the postbiotics of Lactobacillus plantarum ZJUIDS15 of the present invention: The postbiotics of Lactobacillus plantarum ZJUIDS15 have antibacterial activity and antioxidant ability, and can relieve the peritoneal metastasis ability and liver metastasis ability of colorectal cancer cells after intragastric administration.

[0016] As a further improvement of the postbiotics of Lactobacillus plantarum ZJUIDS15 of the present invention: preparing a live bacterial preparation with the function of preventing and treating colon cancer; preparing a product (such as a medicine) with the function of preventing and treating colon cancer; preparing a bacteriocin with the function of preventing and treating colon cancer.

[0017] That is, the present invention provides: the application of the postbiotics of Lactobacillus plantarum ZJUIDS15 in the preparation of pet milk powder in food, the application of the postbiotics of Lactobacillus plantarum ZJUIDS15 in the preparation of functional milk tablets in food, the application of the postbiotics of Lactobacillus plantarum ZJUIDS15 in the preparation of functional gummies in food, the application of the postbiotics of Lactobacillus plantarum ZJUIDS15 in the preparation of a live bacterial preparation with the function of preventing and treating colon cancer, the application of the postbiotics of Lactobacillus plantarum ZJUIDS15 in the preparation of a solid beverage with the function of preventing and treating colon cancer, and the application of the postbiotics of Lactobacillus plantarum ZJUIDS15 in the preparation of a bacteriocin with the function of preventing and treating colon cancer.

[0018] The present invention provides a postbiotic of Lactobacillus plantarum with the function of preventing and treating colon cancer. This postbiotic has the effect of preventing and treating colon cancer, inhibits harmful pathogenic bacteria in the intestine, and has strong antioxidant capacity. Therefore, the postbiotics of Lactobacillus plantarum ZJUIDS15 of the present invention can be widely used in the development of colon cancer-related functional products.

[0019] Aiming at the current lack of research on postbiotics in the prevention and treatment of colorectal cancer, the present invention provides a postbiotic of Lactobacillus plantarum ZJUIDS15 for preventing and treating colon cancer and its application. Through an animal experimental model, the prevention and treatment effect of the postbiotics of Lactobacillus plantarum ZJUIDS15 on colon cancer was investigated.

[0020] The postbiotics of the present invention is prepared based on the fermentation supernatant of Lactobacillus plantarum ZJUIDS15. This postbiotic has high antibacterial and antioxidant capacities, and is more stable and safe than probiotics, providing a basis for the development of anti-colon cancer functional products.

[0021] It should be emphasized that: Patent CN116396890A discloses that live Lactobacillus plantarum ZJUIDS15 has the effect of preventing and treating colon cancer in vivo, specifically manifested in the impact on the peritoneal metastasis of colon cancer cells, but does not show the alleviating effect on the liver metastasis ability of colorectal cancer cells. The most common target organ for hematogenous metastasis of colon cancer is the liver, and about more than 50% of patients will eventually develop liver metastasis. At present, radical surgical resection is the only method to cure colon cancer, but 20% - 50% of patients still develop liver metastasis after surgery, and the 5-year survival rate is less than 10%. However, the postbiotics of Lactobacillus plantarum ZJUIDS15 by gavage in the present invention can not only reduce the peritoneal metastasis of colorectal cancer cells but also alleviate the liver metastasis ability of colorectal cancer cells. In addition, there are some problems in the actual use of live bacteria, such as the change of their probiotic functions due to strain variation, and the decrease in the number of live bacteria during non-low-temperature storage and use. Postbiotics are collectively referred to as the metabolite components of probiotics after processing, including bacterial cells and metabolites. In contrast, postbiotics have a stable composition, safe dosage parameters, and a long food storage period (up to 5 years if used as raw materials or nutritional supplements for food and beverages). Postbiotics can provide food manufacturers with technical advantages such as easy storage, low transportation costs, and longer shelf life. Postbiotics not only have the probiotic functions of probiotics but also eliminate the management of live bacteria, and have various application forms, showing broad prospects in the development of new products with health functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The following further elaborates on the specific implementation manners of the present invention in conjunction with the drawings.

[0023] Figure 1 Postbiotics prepared from the culture supernatant of Lactobacillus plantarum ZJUIDS15;

[0024] Figure 2 Postbiotics of Lactobacillus plantarum ZJUIDS15 by gavage inhibits tumor peritoneal metastasis; Ctrl represents gavage with PBS buffer, and ZJUIDS15 supernatant represents gavage with ZJUIDS15 postbiotic solution;

[0025] Figure 2 In:

[0026] A shows a representative image of bioluminescence results;

[0027] B statistically analyzes the fluorescence signal results of each group. **p < 0.01; the statistical method of Mann-Whitney (B) is used. The 6 mice in each group are biological replicates within the animal experimental group.

[0028] Figure 3The postbiotics prepared from the culture supernatant of Lactobacillus plantarum ZJUIDS15 inhibited tumor liver metastasis by gavage; Ctrl represents gavage with PBS buffer, and ZJUIDS15 supernatant represents gavage with the postbiotic solution of ZJUIDS15;

[0029] Figure 3 In:

[0030] A shows the tumor metastasis in the liver tissues of mice in each group at the end of the experiment (scale bar = 1 cm);

[0031] B performs statistical analysis on the weights of the liver tissues of mice in each group;

[0032] C shows representative H&E staining images of the liver (scale bar = 200 μm); **p < 0.01; The statistical method of Mann-Whitney (B) is used. Detailed implementation manners

[0033] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0034] Example 1. Preparation of postbiotics of Lactobacillus plantarum ZJUIDS15

[0035] 1.1 Preparation of the fermentation supernatant of Lactobacillus plantarum ZJUIDS15

[0036] One loopful of Lactobacillus plantarum ZJUIDS15 was inoculated into 100 mL of MRS liquid medium and cultured in an incubator at 37 °C for 18 h. Then, it was transferred to 300 mL of sterilized (autoclaved at 121 °C for 15 min) MRS liquid medium at an inoculation amount of 1% (v / v). It was cultured in a bacterial incubator at 37 °C for 18 h. The obtained fermentation broth was centrifuged at 4000 r / min for 15 min to collect the supernatant. The supernatant after water bath inactivation (85 °C, 2 h) was rotary evaporated to 1 / 4 of the original volume, and the obtained concentrated fermentation supernatant was stored in a refrigerator at 4 °C for later use.

[0037] 1.2 Freeze concentration

[0038] The concentrated fermentation supernatant obtained in step 1.1 was vacuum freeze-concentrated. Specifically, the concentrated fermentation supernatant was pre-frozen in an ultra-low temperature refrigerator at -80 °C for no less than 2 h, and then quickly taken out and placed in a vacuum freeze dryer for vacuum pumping. The vacuum degree was maintained at about 0.1 Pa, the freezing temperature was -55 °C, and freeze-drying was carried out for 48 h until complete freeze-drying (i.e., frozen to constant weight), which was the postbiotics of Lactobacillus plantarum ZJUIDS15; after taking out, it was stored in a refrigerator at 4 °C.

[0039] Example 2: In vivo prevention and treatment effect of postbiotics of Lactobacillus plantarum ZJUIDS15 on colon cancer

[0040] 2.1 Preparation of samples

[0041] The postbiotics of Lactobacillus plantarum ZJUIDS15 prepared in Example 1 were configured into a ZJUIDS15 postbiotics solution with a concentration of 0.1 g / mL using PBS buffer at pH 7.2 - 7.4 for subsequent experiments.

[0042] 2.2 Effect of intragastric administration of ZJUIDS15 postbiotics solution (0.1 g / mL) on peritoneal metastasis of colorectal cancer cells

[0043] Murine CT26-luc (luciferase-labeled) colorectal cancer cells were injected into the abdominal cavity of each BALB / c mouse at a cell amount of 1×10 6 cells / 300 μl (n = 6). From the day of modeling, each group of mice was intragastrically administered with 300 μl of ZJUIDS15 postbiotics solution (0.1 g / mL) or PBS buffer (pH 7.2 - 7.4) every day, and the degree of peritoneal metastasis in mice was detected using a live imaging instrument. After 7 days of intragastric administration, as Figure 2 shown, in the mice intragastrically administered with ZJUIDS15 postbiotics, the fluorescence signal in the abdominal cavity decreased significantly, indicating that the ZJUIDS15 postbiotics solution can alleviate the peritoneal metastasis ability of colorectal cancer cells.

[0044] 2.3 Effect of intragastric administration of ZJUIDS15 postbiotics solution (0.1 g / mL) on liver metastasis of colorectal cancer cells

[0045] Murine MC38-luc (luciferase-labeled) colorectal cancer cells were injected into the spleen of anesthetized C57 mice at a cell amount of 2×10 6 cells / 100 μl to construct a liver metastasis model. After the spleen injection was completed, the spleen was removed, and the wound was sutured and disinfected. On the 7th day after modeling, small animal live imaging was performed to evaluate the modeling situation. Mice with similar tumor growth were randomly divided into 2 groups (n = 6), and then each group of mice was intragastrically administered with 300 μl of ZJUIDS15 postbiotics solution (0.1 g / mL) or PBS buffer (pH 7.2 - 7.4) every day. After 16 days of intragastric administration, the mice were sacrificed by cervical dislocation, and then the liver tissues of the mice were taken out, photographed and weighed. As Figure 3As shown in A, in the control group (Ctrl) treated with PBS by gavage, liver metastasis in mice was severe (liver metastases of colorectal cancer cells were marked with red dotted lines). In the group gavaged with postbiotics prepared from the culture supernatant of Lactobacillus plantarum ZJUIDS15, the liver metastasis ability of colorectal cancer cells was significantly inhibited. The liver weight of mice in the group gavaged with postbiotics prepared from the culture supernatant of Lactobacillus plantarum ZJUIDS15 was significantly lighter ( Figure 3 B). Figure 3 C shows representative H&E staining pictures of the liver tissues of mice in each group, where tumor metastases appear dark purple. These results suggest that gavage treatment with the ZJUIDS15 postbiotic solution (postbiotics prepared from the culture supernatant of Lactobacillus plantarum ZJUIDS15) can alleviate the liver metastasis ability of colorectal cancer cells.

[0046] It should be noted that: Patent CN116396890A only reported that: the live bacteria of Lactobacillus plantarum ZJUIDS15 have the in vivo prevention and treatment effect on colon cancer, specifically manifested in the effect on the peritoneal metastasis of colon cancer cells, and did not show the alleviating effect on the liver metastasis ability of colorectal cancer cells. That is, when the live bacteria of ZJUIDS15 are used instead of the ZJUIDS15 postbiotic solution and operated according to step 2.3 above, the obtained results have no significant difference from the control group (Ctrl).

[0047] Example 3. Verification of the antioxidant capacity of the postbiotic solution of Lactobacillus plantarum ZJUIDS15

[0048] 1. Preparation of postbiotic solution

[0049] Refer to Example 1 to prepare the postbiotics of Lactobacillus plantarum ZJUIDS15. Use PBS solvent (pH 7.2 - 7.4) for reconstitution at a mass ratio of 1:20 to prepare the postbiotic solution, which is used as the test sample for verification of antioxidant capacity.

[0050] 2. Determination of DPPH free radical scavenging ability

[0051] Prepare VC solutions with different concentration gradients (0 - 30 μg / ml) using deionized water. Add 100 μL of the test sample (or VC standard solution) and 100 μL of 0.2 mM DPPH ethanol solution (prepared with absolute ethanol, stored in the dark at 4°C and used freshly) to the enzyme-linked immunosorbent assay (ELISA) plate. After shaking well, keep it in the dark at room temperature for 30 min, and measure the absorbance of the solution at 517 nm; use 100 μL of absolute ethanol instead of 100 μL of DPPH ethanol solution as the blank group; use 100 μL of PBS buffer (pH 7.2 - 7.4) (or MRS liquid medium) instead of 100 μL of the test sample as the control group, and zero with a mixture of 100 μL of PBS buffer (or MRS liquid medium) and absolute ethanol. Each sample is repeated in 3 parallels, and the average value is calculated.

[0052] DPPH free radical scavenging ability (%) = [1 - (As - Ab) / Ac] × 100%

[0053] Where: As - absorbance of the sample group; Ab - absorbance of the blank group; Ac - absorbance of the control group.

[0054] Table 1 Antioxidant properties of postbiotics of Lactobacillus plantarum ZJUIDS15

[0055] Index Postbiotics solution of Lactobacillus plantarum ZJUIDS15 DPPH free radical scavenging rate / % 92.4±1.02

[0056] It should be emphasized that: Patent CN116396890A uses live bacteria. A higher concentration of live bacteria does not necessarily mean better antioxidant effects, and a bacterial solution with too high a concentration may even have an adverse impact on the human body; while the present invention uses postbiotics, and the concentration can be adjusted according to actual needs.

[0057] Example 4, Confirmation of the pathogen inhibitory ability of Lactobacillus plantarum ZJUIDS15

[0058] 1. Preparation of postbiotic solution

[0059] Refer to Example 1 to prepare postbiotics of Lactobacillus plantarum ZJUIDS15. Use PBS solvent (pH 7.2 - 7.4) to reconstitute at mass ratios of 1:5, 1:10, 1:20, 1:30, 1:40, and 1:50 to prepare postbiotic solutions with different concentrations as test samples for antibacterial ability verification.

[0060] 2. Determination of antibacterial activity

[0061] The international common agar diffusion method was used to determine the antibacterial activity of lactic acid bacteria. The four indicator strains (Escherichia coli, Salmonella, Staphylococcus aureus, and Listeria monocytogenes) stored in frozen state were activated 2 - 3 times on LB solid medium. Single colonies after activation were separately picked and inoculated into LB medium, and cultured at 37°C for 18 h. Bacterial cells were collected by centrifugation and resuspended in physiological saline to make the concentration reach 10 8 CFU / mL. The indicator bacteria suspension was added to the sterilized LB solid medium cooled to about 55°C at a ratio of 1% (v / v), mixed evenly and then poured into petri dishes (15 mL / dish). After condensation, the previously placed sterile Oxford cups were removed. The postbiotic solution of Lactobacillus plantarum ZJUIDS15 was added to the cup wells (200 μL / well), and PBS (pH 7.2 - 7.4) was used as the blank control. Cultured at 37°C, the diameter of the antibacterial zone was measured after 24 h. Strains with obvious antibacterial zones around the small holes were selected, and the diameter of the antibacterial zone was measured, with each measurement repeated three times.

[0062] As shown in Table 2, the postbiotics solution of Lactobacillus plantarum ZJUIDS15 has a certain inhibitory effect on Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, and Listeria monocytogenes. Moreover, the antibacterial ability varies at different reconstitution ratios and decreases as the postbiotic concentration decreases.

[0063] Table 2 Results of the inhibitory ability of postbiotics solutions at different concentrations against pathogenic bacteria

[0064]

[0065] Staphylococcus aureus is the most common pathogenic bacterium in human purulent infections. Some Escherichia coli can cause severe diarrhea and septicemia, and some Salmonella species can also cause human food poisoning. Listeria monocytogenes is defined by the World Health Organization as one of the four major foodborne pathogenic bacteria, and the fatality rate of the diseases it causes is as high as 20 - 30%. Bacteriocins, organic acids, hydrogen peroxide and other antibacterial products in lactic acid bacteria postbiotics can inhibit the growth of these pathogenic bacteria alone or in combination. The postbiotics of Lactobacillus plantarum ZJUIDS15 provided by the present invention have a certain antagonistic effect on these four pathogenic bacteria and have the potential to develop antibacterial products.

[0066] Example 5: Preparation of pet postbiotic milk powder using the postbiotics of Lactobacillus plantarum ZJUIDS15

[0067] 1. Preparation of the postbiotics of Lactobacillus plantarum ZJUIDS15

[0068] Refer to Example 1 to prepare the postbiotics of Lactobacillus plantarum ZJUIDS15.

[0069] 2. Preparation of pet formula powder

[0070] Initial raw material selection: milk powder, fish meal, bone meal, grains, vegetable oil, additives: vitamins, trace elements, functional factors, others;

[0071] Automatic batching: Put the obtained material raw materials into the material bin according to the formula;

[0072] Crushing: Crush the weighed materials through a crusher;

[0073] Mixing: Add vegetable oil and trace elements to the crushed materials and mix them evenly in a mixer;

[0074] Extrusion: Make the mixed materials into granular materials through an extruder;

[0075] Drying: Dry the mixed materials through a dryer, and control the temperature at 65 - 70 degrees;

[0076] Classification and screening: Pass the materials through a grading sieve, and control the particle size at 2.5 - 5 mm.

[0077] 3. Preparation of Postbiotics Formula Powder for Pets

[0078] Mix the postbiotics prepared in Step 1 and the pet feed prepared in Step 2 evenly at a ratio of 10:100. After the product is filled, store it in the warehouse for sale.

[0079] Example 6: Preparation of Postbiotic Nutritional Milk Tablets Using Postbiotics of Lactobacillus plantarum ZJUIDS15

[0080] 1. Preparation of Postbiotics of Lactobacillus plantarum ZJUIDS15

[0081] Refer to Example 1 to prepare the postbiotics of Lactobacillus plantarum ZJUIDS15.

[0082] 2. Processing Technology Flow of Postbiotic Milk Tablets

[0083] Ingredient preparation → Mixing → Granulation → Drying → Screening → Tabletting.

[0084] 3. Key Operation Points

[0085] (1) Ingredient preparation: 100 g of full-fat sweet milk powder, 20 g of postbiotic powder, 15 g of microcrystalline cellulose (MCC), 10 g of xylitol, and the dosage of magnesium stearate is 0.5% of the total mass of dry granules.

[0086] (2) Mixing: Weigh the raw and auxiliary materials according to the dosage of the milk tablet formula, put them into a blender, and mix them by the equal increment addition method. The mixing and stirring time of the ingredients is about 10 minutes.

[0087] (3) Granulation: When all components in the milk tablets are fully mixed evenly, add an appropriate amount of 70% alcohol as a wetting agent, and stir and mix while adding to prepare soft materials. When the soft materials reach the standard of "forming a ball when held in the hand and dispersing when gently pressed", stop adding the wetting agent, and use a granulator equipped with a 20-mesh nylon sieve to prepare wet granules.

[0088] (4) Drying: The wet granules are dried in a vacuum at 50 °C, and turned over every 0.5 h during the drying process to ensure the same drying degree of the granules.

[0089] (5) Screening: When the water content of the wet granules is less than 3%, stop drying, and use a granulator to force the dry granules through a 16-mesh sieve for screening.

[0090] (6) Tabletting: Weigh the mass of the dry granules, add an appropriate amount of magnesium stearate as a lubricant, mix evenly, adjust the pressure, and use a rotary tabletting machine equipped with a suitable tabletting die to tablet, and then the postbiotic milk tablets are obtained.

[0091] Example 7: Preparation of Postbiotic Nutritional Gummy Candies Using Postbiotics of Lactobacillus plantarum ZJUIDS15

[0092] 1. Preparation of postbiotics solution of Lactobacillus plantarum ZJUIDS15

[0093] Refer to Example 1 to prepare postbiotics of Lactobacillus plantarum ZJUIDS15, and configure the postbiotics solution according to a mass ratio of 40%.

[0094] 2. Processing technological process of postbiotics gummy candies

[0095]

[0096] 3. Key operation points

[0097] (1) Boiling of sugar solution: Add isomaltitol and maltitol (addition amount 80%) into the sugar boiling pan, stir and boil until the sugar solution is difficult to break when lifted, reaching the end point of sugar boiling (temperature about 110°C).

[0098] (2) Preparation of colloid: Soak pectin (addition amount 9.0%) with 1.5 times its weight of water, place it in a constant temperature water bath at 85°C and stir to dissolve and melt it for standby.

[0099] (3) Mixed boiling: Cool the sugar solution to 70°C, mix it evenly with the colloid solution, and finally add the postbiotics solution (addition amount 10%), citric acid (addition amount 0.15%), etc. and mix, then keep warm and stand still.

[0100] (4) Pouring and forming: Before pouring, apply an appropriate amount of release oil on the mold for easy demolding during pouring.

[0101] (5) Drying and dehydration: After the sample is demolded, place it in a drying room and dry for 36h. Note that pectin is easily affected by temperature and melts, so the temperature needs to be strictly controlled.

[0102] Example 8. Preparation of postbiotics powder with the efficacy of preventing and treating colon cancer using postbiotics of Lactobacillus plantarum ZJUIDS15

[0103] 1. Preparation of postbiotics of Lactobacillus plantarum ZJUIDS15

[0104] Refer to Example 1 to prepare postbiotics of Lactobacillus plantarum ZJUIDS15.

[0105] 2. Preparation of Lactobacillus plantarum ZJUIDS15 bacterial sludge

[0106] Pick a single colony of plant lactobacillus ZJUIDS15 and inoculate it in 50mL MRS liquid medium, and place it in a 37°C incubator to cultivate for 18h. Activate it again in 250mL MRS liquid medium at 5% inoculum, and place it in a 37°C incubator to cultivate for 24h. Finally, the activated plant lactobacillus ZJUIDS15 is cultured in a 10L fermenter at a high density anaerobic culture with a 5% inoculum, and cultivated for 18h at 37°C and pH 6.8. Afterwards, centrifuge for 15min at 8000r / min and 4°C, discard the supernatant, collect the bacterial precipitation, and rinse the bacterial body twice with sterile phosphate buffer (pH 7.0). You can get the plant lactobacillus ZJUIDS15 bacterial mud.

[0107] 3. Preparation of Protective Agent

[0108] The freeze-dried protective agent contains 15% skim milk powder, 5% trehalose, 3% sodium glutamate, 1% glycerol, and 0.5% cysteine hydrochloride. Water is used as the solvent. Sterilize at 110°C for use.

[0109] 4. Preparation of probiotic powder containing Lactobacillus plantarum ZJUIDS15 postbiotics with the efficacy of preventing and treating colon cancer

[0110] The prepared Lactobacillus plantarum ZJUIDS15 postbiotic powder and the Lactobacillus plantarum ZJUIDS15 bacterial precipitate were fully mixed in a ratio of 1:1:4. Pre-freeze at -40°C for 5 hours to freeze evenly on the inner wall of the container, and then vacuum freeze-dry. After drying for 18 to 20 hours, a probiotic powder containing Lactobacillus plantarum ZJUIDS15 postbiotics with the effect of preventing and treating colon cancer can be obtained. After rehydration with physiological saline, it was washed twice, and the number of live bacteria in the Lactobacillus plantarum ZJUIDS15 powder was measured to be 1.0×10 11 ~1×10 12 CFU / g.

[0111] Example 9: Postbiotic solid beverage using Lactobacillus plantarum ZJUIDS15 with the function of preventing and treating colon cancer

[0112] 1. Preparation of Lactobacillus plantarum ZJUIDS15 postbiotics: Prepare Lactobacillus plantarum ZJUIDS15 postbiotics with reference to Example 1.

[0113] 2. The formula of solid beverage is as follows: edible corn starch, white sugar (10%), oligofructose (6%), oligosaccharide

[0114] Maltose (9%), citric acid, flavoring, postbiotic powder (2-5%)

[0115] 3. Mixed packaging: After adding according to the formula and mixing evenly, it is packaged in 10 / 20 g per small packet, and 10 - 20 small packets are packaged into a large packet.

[0116] Example 10 uses postbiotics bacteriocin of Lactobacillus plantarum ZJUIDS15 with the function of preventing and treating colon cancer

[0117] 1. Preparation of a postbiotic solution of Lactobacillus plantarum ZJUIDS15 with the function of preventing and treating colon cancer

[0118] Refer to Example 1 to prepare postbiotics of Lactobacillus plantarum ZJUIDS15, and redissolve it in PBS solvent (pH 7.2 - 7.4) at a mass ratio of 1:20. Adjust the pH value to 6.0 with 2M NaOH solution, and the obtained postbiotic solution (pH 6.0) is stored in a 4°C refrigerator for standby.

[0119] 2. Ammonium sulfate precipitation

[0120] Slowly stir and add the pre-weighed solid powder of (NH4)2SO4 to the postbiotic solution (pH 6.0) until the final saturation of ammonium sulfate is 60%. Stir the mixture with a magnetic stirrer during the addition process. Then the sample is placed at 4°C overnight for sufficient precipitation. Centrifuge at 4°C for 20 min at a speed of 10000 r / min to separate the precipitate. Dissolve the precipitate with an adequate amount of 50 mmol / L pH 6.0 phosphate buffer solution (PBS); obtain the crude bacteriocin solution after ammonium sulfate treatment; finally, store it in a 4°C refrigerator for use.

[0121] 3. G-25 Sephadex gel chromatography

[0122] Take the crude bacteriocin solution obtained after ammonium sulfate treatment, filter it through a 0.22 μm filter membrane, and then slowly add it to G-25 Sephadex gel chromatography. Elute with ultrapure water at a flow rate of 1.0 mL / min. At the same time, detect and collect protein components at 280 nm, and use BaCl2 to detect whether there is salt residue in the collected protein components. The protein components that do not produce precipitation with BaCl2 are the purified bacteriocin solution, which is collected. Using 4 pathogenic bacteria (Escherichia coli, Salmonella, Staphylococcus aureus, and Listeria monocytogenes) as indicator bacteria, the agar diffusion Oxford cup method is used to determine its antibacterial activity.

[0123] 4. Freeze concentration

[0124] The bacteriocin solution collected after the dextran gel chromatography is concentrated by vacuum freezing, specifically: the bacteriocin solution is pre-frozen in a -80°C ultra-low temperature refrigerator for not less than 2 hours, and then quickly taken out and placed in a vacuum freeze dryer for vacuum treatment, and the vacuum degree is maintained at about 0.1P. After concentrating to 1 / 5 of the original volume, the bacteriocin is taken out under reduced pressure and placed in a 4°C refrigerator, and is slowly dissolved in its frozen state; the bacteriocin prepared by the postbiotics of Lactobacillus plantarum ZJUIDS15 can be obtained.

[0125] Compared with the bacteriocin process of CN116396890A, the bacteriocin preparation process of the present invention simplifies the preparation process, omits the process of bacterial culture and the like, and can be directly prepared from the postbiotic raw materials and then re-dissolved.

[0126] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. Application of postbiotics of Lactobacillus plantarum ( Lactobacillus plantarum ) ZJUIDS15 in the preparation of a product with the function of preventing and treating colon cancer, characterized in that: The preparation method of postbiotics of Lactobacillus plantarum ZJUIDS15 is as follows: after inactivating the fermentation supernatant of Lactobacillus plantarum ZJUIDS15 in a water bath at 85±2°C for 2±0.1 h, it is concentrated by freezing to obtain postbiotics of Lactobacillus plantarum ZJUIDS15; The preparation method of the fermentation supernatant of Lactobacillus plantarum ZJUIDS15 is: inoculate 1 loopful of Lactobacillus plantarum ZJUIDS15 into 100 mL of MRS liquid medium, place it in an incubator at 37±0.5°C and culture for 18±0.5 h; then transfer it to the sterilized 250 - 300 mL of MRS liquid medium at an inoculation amount of 1 - 2%; place it in the incubator and culture at 37±0.5°C for 18±0.5 h; the obtained fermentation broth is centrifuged at 4000 - 10000 r / min for 15 - 20 min, and the collected supernatant is the fermentation supernatant of Lactobacillus plantarum ZJUIDS15; The postbiotics of Lactobacillus plantarum ZJUIDS15 has the ability to alleviate the peritoneal metastasis and liver metastasis of colorectal cancer cells.

2. The application according to claim 1, wherein: After inactivating the collected supernatant in a water bath, it is then rotary evaporated to 1 / 3.5 - 1 / 4.5 of the original volume; then the concentrated fermentation supernatant is concentrated by freezing to obtain postbiotics of Lactobacillus plantarum ZJUIDS15.

3. The application according to claim 2, wherein The freezing concentration is as follows: The concentrated fermentation supernatant is pre-frozen in a -80°C ultra-low temperature refrigerator for not less than 2 h, then quickly taken out and placed in a vacuum freeze dryer for vacuum pumping. The vacuum degree is maintained at 0.1P, and the freezing temperature is -55°C. After complete freeze-drying, the postbiotics of Lactobacillus plantarum ZJUIDS15 is obtained.

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