CN118104775A - Lactic acid bacteria fermented raspberry juice with effect of preventing colon cancer as well as preparation and application thereof - Google Patents
Lactic acid bacteria fermented raspberry juice with effect of preventing colon cancer as well as preparation and application thereof Download PDFInfo
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- CN118104775A CN118104775A CN202410534405.7A CN202410534405A CN118104775A CN 118104775 A CN118104775 A CN 118104775A CN 202410534405 A CN202410534405 A CN 202410534405A CN 118104775 A CN118104775 A CN 118104775A
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- lactobacillus
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- lactobacillus plantarum
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Abstract
The invention belongs to the fields of food biotechnology and microbial fermentation application, and particularly relates to lactobacillus fermentation raspberry juice with a colon cancer prevention effect, and preparation and application thereof. The raspberry juice is prepared by mixing lactobacillus plantarum NCU0011098 and lactobacillus acidophilus NCU426 with raspberry pulp as main raw materials and performing lactic acid fermentation, and the obtained fermented raspberry juice has obviously improved inhibition capability to Fusobacterium nucleatum, prevention and treatment effects to colon cancer, antioxidation capability and the like. The raspberry juice can be widely applied to the field of prevention and/or treatment of colon cancer, in particular to the application in preparing foods, health products and medicines for preventing and/or treating colon cancer.
Description
Technical Field
The invention belongs to the fields of food biotechnology and microbial fermentation application, and particularly relates to lactobacillus fermentation raspberry juice with a colon cancer prevention effect, and preparation and application thereof.
Background
Raspberry is a common name of fruits of plants in the genus of Rubus in the order of Rosales, rosaceae, has wide distribution in China and various varieties, is rich in nutrient substances, comprises saccharides, proteins, organic acids, amino acids, vitamins, mineral elements and the like, has active functional components of anthocyanin, ellagic acid, superoxide dismutase and the like, and has various remarkable dietary therapy values and pharmacological functions of resisting aging, preventing cancer and beautifying Yan Jiangshen and the like for human bodies. Raspberry is internationally called as third generation fruit and is an important resource for producing health care products and medicines. In addition, some researches indicate that raspberries have health care effects of improving vascular functions, reducing blood fat, resisting aging and cancer, reducing inflammation, improving immunity and the like.
At present, the development of raspberries is mainly focused on fermenting raspberry fruit wine by yeast, and the development of technology for fermenting raspberry juice by functional probiotics is relatively few. The invention develops the probiotic fermented raspberry beverage by taking raspberries as raw materials, and researches the efficacy of the probiotic fermented raspberry beverage in preventing colon cancer so as to fill the technical blank of the functional probiotic fermented raspberry juice beverage in preventing colon cancer, and has potential application prospect.
Disclosure of Invention
Aiming at the problems, the invention provides the lactobacillus fermented raspberry juice which is rich in nutrition, good in flavor, safe and sanitary, has an antioxidation effect, and has the prevention and treatment effects on colon cancer through in vivo and in vitro experiments. The invention has no essence, pigment, preservative and other components which are unfavorable to the health of human body, retains the original nutritional components of the juice, prolongs the shelf life of the juice, and is an excellent health care product.
The invention provides a preparation method of lactobacillus fermented raspberry juice, which is obtained by fermenting raspberry pulp serving as a raw material by lactobacillus plantarum and lactobacillus acidophilus, and comprises the following steps of:
(1) Selecting fresh, mature and full raspberries without mildew and rot, cleaning and homogenizing to obtain raspberry pulp for later use;
(2) The lactobacillus fermented raspberry juice comprises the following raw materials in parts by weight: 20-50 parts of raspberry pulp, 50-80 parts of purified water, 4-8 parts of glucose and 0.05-0.2 part of sodium iso-vitamin C, uniformly stirring, filtering, pasteurizing and rapidly cooling to room temperature;
further, the raspberry pulp is selected from one or more of raspberry pulp, blackberry pulp, huang Shumei pulp and purple raspberry pulp;
(3) Adding 0.001-1 part of compound lactobacillus direct vat set starter, and fermenting at 28-38 ℃ for 24-72 h;
Further, the compound lactobacillus direct vat set starter comprises lactobacillus plantarum and lactobacillus acidophilus;
Furthermore, in the compound lactobacillus direct vat set starter, the addition ratio of the viable count of the lactobacillus plantarum to the viable count of the lactobacillus acidophilus is 1:1;
Further, the bacterial activity number in the compound lactobacillus direct vat set starter is (1-9) multiplied by 10 10 CFU/g;
Preferably, the bacterial activity in the compound lactobacillus direct vat set starter is 1X 10 10 CFU/g;
Preferably, the lactobacillus plantarum is specifically lactobacillus plantarum (Lactiplantibacillus plantarum) NCU0011098, and the lactobacillus plantarum has the capability of inhibiting Fusobacterium nucleatum and preventing and treating colon cancer in self-fermented pickle. The strain NCU0011098 was deposited in China general microbiological culture Collection center, address: the collection number of the microbial institute of China academy of sciences is CGMCC No.28774 in the Korean area North Star, west Lu No.1, no. 3 of Beijing city.
Preferably, the lactobacillus acidophilus is lactobacillus acidophilus (Lactobacillus acidophilus) NCU426 with a preservation number of CGMCC No.15572;
preferably, the fermentation conditions are fermentation at 35 ℃ 48 h;
(4) Sterilizing at 85-121deg.C and 5-30 min, and packaging or refrigerating at 4deg.C to obtain lactobacillus fermented raspberry juice.
The second technical scheme provided by the invention is the lactobacillus fermented raspberry juice prepared by the method in one of the technical schemes.
The third technical scheme provided by the invention is the application of the lactobacillus fermented raspberry juice, particularly the application in preventing and/or treating colon cancer, and more particularly the application in preparing foods, health care products and medicines for preventing and/or treating colon cancer.
Advantageous effects
(1) The lactobacillus fermented raspberry juice provided by the invention does not need to be added with essence, pigment and other additives which are unfavorable to human health, and does not need to be added with potassium sorbate and other preservatives which are unfavorable to human health, so that the quality guarantee period of the juice is prolonged, and the putrefaction is prevented. In addition, the invention does not need to add any enzyme preparation, has simple operation, saves cost, maintains the original nutritional ingredients of the fruit juice, has thick taste and is sweet and sour.
(2) Compared with the unfermented raspberry, the content of flavone, polyphenol and organic acid in the fermented raspberry juice is obviously improved;
(3) The lactobacillus fermented raspberry juice prepared by the invention has excellent antioxidation capability, and has better performance in DPPH free radical, hydroxyl free radical, ABTS free radical scavenging capability and total reducing capability.
(4) The lactobacillus fermentation raspberry juice extract prepared by the invention has better inhibition activity on cancer cells HT-29, and the IC 50 value is 9.41+/-0.22 mug/mL;
(5) The lactobacillus fermented raspberry juice prepared by the invention has remarkable inhibition effect on common pathogenic bacteria in intestinal tracts and clostridium nucleatum related to colon cancer pathogenic bacteria;
(6) The lactobacillus fermented raspberry juice prepared by the invention has the effect of preventing colon cancer: after the colon cancer model mice ingest the fermented raspberry juice, the development of colon cancer is inhibited, which is mainly characterized in that the number and the size of colon tumors of colon cancer model mice are obviously reduced, intestinal inflammation is relieved, and the content of fecal short-chain fatty acid is increased;
(7) The invention has simple manufacturing process, easy control, high standardization degree, stable product quality and easy realization of large-scale industrial production.
(8) The invention discovers that lactobacillus plantarum NCU0011098 with the effects of inhibiting Fusobacterium nucleatum and preventing and treating colon cancer is obtained, and after lactobacillus plantarum NCU0011098 and lactobacillus acidophilus NCU426 are used for carrying out mixed fermentation on raspberries, the inhibition capability of the obtained fermented raspberry juice on the Fusobacterium nucleatum, the prevention and treatment effect on colon cancer, the antioxidation capability and the like are all obviously improved.
Drawings
Figure 1 shows the body weight change of mice.
Fig. 2 shows the colon length change in mice.
FIG. 3 shows the distribution of the number of intestinal tumors in mice.
FIG. 4 shows the intestinal tumor size distribution in mice.
Figure 5 shows the total short chain fatty acid content of the mouse faeces.
FIG. 6 shows the short chain fatty acid content of mouse faeces.
FIG. 7 shows the cytokine content of colon tissue of mice.
FIG. 8 shows the inhibitory activity of Lactobacillus plantarum NCU0011098 on the migration of colon cancer cells HCT-116
Wherein A1, A2 and A3 are migration areas of HCT-116 after the control group is treated for 0h, 24 h and 48h respectively; b1, B2 and B3 are migration areas of HCT-116 after NCU0011098 group treatment for 0h, 24 h and 48h respectively; c1, C2 and C3 are migration areas of HCT-116 after CICC21801 group treatment for 0h, 24 h and 48h, respectively.
FIG. 9 shows the effect of Lactobacillus plantarum NCU0011098 on survival in mice.
FIG. 10 shows the effect of Lactobacillus plantarum NCU0011098 on colon length in mice.
FIG. 11 shows the effect of Lactobacillus plantarum NCU0011098 on HE staining (x 5) of colon tissue sections of mice
Wherein, A-F are respectively a normal group, a model group, a positive control group, a low-dose group, a medium-dose group and a high-dose group.
FIG. 12 shows the effect of Lactobacillus plantarum NCU0011098 on the number of intestinal tumors in mice.
FIG. 13 shows the effect of Lactobacillus plantarum NCU0011098 on the total short chain fatty acid content of mouse faeces.
FIG. 14 shows the effect of Lactobacillus plantarum NCU0011098 on different short chain fatty acid levels in mouse faeces.
FIG. 15 shows the effect of Lactobacillus plantarum NCU0011098 on the cytokine content of colon tissue in mice.
FIG. 16 is a diagram showing the morphology of the cells of Lactobacillus plantarum NCU0011098 (gram stain).
Detailed Description
In order to make the objects, technical solutions and advantages of the present patent more apparent, the present patent will be described in further detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the present invention.
Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any method or material similar or equivalent to those described may be used in the present invention.
The lactobacillus plantarum related in the invention and the embodiment is lactobacillus plantarum (Lactiplantibacillus plantarum) NCU0011098, which is preserved in the China general microbiological culture Collection center, address: the collection number of the microbial institute of China academy of sciences is CGMCC No.28774 in the Korean area North Star, west Lu No. 1, no. 3 of Beijing; the lactobacillus plantarum NCU0011098 has the following properties:
(1) Acid resistance: the survival rate after treatment of 3 h in a pH 2.5 environment is 91.93%;
(2) Bile salt resistance: the survival rate is as high as 96.97% after 4h is treated in an environment with the concentration of bile salt of 0.3%;
(3) Adhesion ability to colon cancer cells HT-29: the adhesion rate of colon cancer cells HT-29 is 62.09%;
(4) Inhibitory Activity against proliferation of colon cancer cell HT-29: after 24 h of the fermentation supernatant and colon cancer cells HT-29 are incubated together, the inhibition rate of HT-29 proliferation is 46.77%;
(5) Inhibitory Activity against colon cancer cell HCT-116 migration: the fermentation supernatant can slow down the migration capacity of colon cancer cells HCT-116, and the migration inhibition rate reaches 90.23% after 48 h;
(6) Inhibition ability against Fusobacterium nucleatum: the inhibition rate of the fermentation supernatant to the fusobacterium nucleatum 24h reaches 95.26 percent, and the inhibition rate of the broken thalli and intracellular metabolites thereof to the fusobacterium nucleatum 24h reaches 69.63 percent;
(7) Prevention and treatment effects on colon cancer model mice:
① Slow down the shortening of colon length in colon cancer model mice:
② Reducing the number of tumors at the colon part of the mouse;
③ Relieving the damage degree of colon epithelium;
④ Reducing colonic inflammatory factors;
⑤ Increasing the content of fecal short-chain fatty acid;
⑥ Improving survival rate of colon cancer mice.
The lactobacillus acidophilus referred in the present invention and examples is lactobacillus acidophilus (Lactobacillus acidophilus) NCU426, which was deposited in the general microbiological center of the chinese microbiological bacterial culture collection center, address: the collection number of the microbial institute of China academy of sciences is CGMCC No.15572 in the Korean area North Star, west Lu No.1, no. 3 of Beijing city. Lactobacillus acidophilus NCU426 has been published in chinese invention patent application 201810978365.X (CN 109136129A).
Unless otherwise indicated, some of the experimental or assay methods employed in the present invention are as follows (the remaining not explicitly recited methods are all routine assays or detection methods in the art):
(1) The organic acid detection method comprises the following steps:
The organic acid content was detected using an Agilent 1260 high performance liquid chromatograph. Taking 1mL fermented sample, centrifuging (4 ℃, 10000 ℃, rpm, 10, min), taking supernatant, passing through a 0.22 μm water-based filter membrane, and then loading to a machine for testing. Chromatographic column: BIO-RAD Aminex.HPX-87H ion exchange column C18 (5 μm,300 mm ×7.8 mm); mobile phase: 6 mmol/L of dilute sulfuric acid; a detector: ultraviolet detector, wavelength: 210 nm; column temperature: 35. the temperature is lower than the temperature; sample injection amount: 20. mu L; flow rate: 0.5 mL/min; external standard quantification was performed using organic acid standards (lactic acid, succinic acid, malic acid, citric acid, tartaric acid).
(2) Determination of total phenol and total flavone content
The method for measuring total phenols comprises the following steps: folin-Ciocalteu colorimetric assay was used. 0.5 mL Folin-ciocalteu reagent (50%) was mixed with 1.0 mL diluted sample. Na 2CO3 (5%, 1.0 mL) was then added and 1 min was mixed. After incubation (25 ℃,60 min), the absorbance of the mixture was measured at 760 nm.
The method for measuring total flavonoids comprises the following steps: the colorimetric determination of NaNO 2-Al(NO3)3 was carried out by taking 1: 1mL diluted sample into a 10: 10 mL volumetric flask, adding sodium nitrite solution (0.3 mL, 10%) and shaking, incubating for 6: 6 min, adding aluminum nitrate solution (0.3 mL, 10%), incubating for 6: 6 min, adding NaOH solution (2 mL, 8%), and fixing the volume to 10: 10 mL with 60% ethanol, shaking, standing for 15: 15min, and measuring the absorbance at 510: 510 nm.
(3) The method for detecting the antioxidant capacity comprises the following steps:
The total antioxidant capacity and total reducing capacity of the fermented raspberry juice were determined using DPPH, ABTS, FRAP and a hydroxyl radical detection kit.
(4) The method for detecting the IC 50 of the colon cancer cell HT-29 comprises the following steps:
Weighing 5.0 g of homogenized fermented raspberry juice, adding 80% ethanol, ultrasonically extracting for 30min g, centrifuging to obtain supernatant, and concentrating by rotary evaporation for later use. And re-dissolving the mixed solution by using RPMI complete culture medium and preparing the mixed solution into the solutions to be tested with different concentrations. The inhibition rate of the sample to colon cancer cell proliferation is detected by adopting a CCK-8 method, and the IC 50 value of the fermented raspberry juice to HT-29 is calculated according to the inhibition rate.
CCK-8 method: preparing a cell suspension (1-5.0X10 5 cells/mL) with a certain concentration by using an RPMI culture medium containing 10% FBS, adding a 96-well culture plate, culturing by 100 mu L of each well, and adding 10% solutions to be tested with different concentrations after culturing by 24: 24 h; each group was provided with 6 parallel wells, and the culture was continued for 24 h in a cell incubator at 37℃with 5% CO 2%, after which 10. Mu.L of CCK-8 solution was placed into each well, incubation was continued for about 1.1 h, absorbance (OD value) at 450. 450nm was measured using a multifunctional microplate reader, and the inhibition ratio of cell proliferation was calculated. The wells without the test solution were used as control (A c) and the wells without the cell suspension were used as blank (A b)
Wherein a c is absorbance of a control group, a t is absorbance of a different treatment group after 24 h, and a b is absorbance of a blank group.
(5) The antibacterial activity detection method comprises the following steps:
The bacteriostatic activity of the fermented raspberry juice on escherichia coli, staphylococcus aureus and fusobacterium nucleatum is measured by adopting a perforation method.
Centrifuging the fermented raspberry juice sample (4deg.C, 10000 g,10 min), and filtering the supernatant with 0.22 μm filter membrane. The prepared various indicator bacteria suspensions are inoculated into sterilized LB (Luria-Bertani, escherichia coli and staphylococcus aureus) and TSA (trypticase soy agar, fusobacterium nucleatum) solid culture media (10 7-108 CFU/mL) respectively, the culture media are poured into a sterile culture dish after shaking evenly, after the culture media are cooled and solidified, punching operation is carried out on each flat plate by using a puncher (diameter 6 mm), and three holes are formed in each flat plate. 200 mu L of the prepared fermented raspberry juice sample solution is added into each hole, incubated at 37 ℃ for 12-16 h, and the diameter of a bacteriostasis ring is measured by using a vernier caliper. The indicator bacteria are respectively escherichia coli CMCC44350, staphylococcus aureus CMCC26003 and clostridium nucleatum ATCC25586.
(6) Preventive effect on colon cancer
60C 57BL/6 mice (6-8 weeks old, male) were purchased and randomly divided into 6 groups of 10. The samples were divided into a model group, a normal group, a positive control group, example 1, comparative example 1, and comparative example 2.
After all mice were acclimatized for one week, the mice were intraperitoneally injected with azomethane (Azoxymethane, AOM) (10 mg/kg, sigma) in addition to normal group (saline replacement), and after 7 days of normal drinking water, 2.5% dextran sodium sulfate (Dextran Sulfate Sodium Salt, DSS) (MW 36000-50000,MP Bio medicals) was added to drinking water for 7 days, after which normal drinking water was restored for 14 days. One week of drinking water containing 2.5% dss and two weeks of normal drinking water were taken as one cycle, and the experiment was ended after 3 consecutive cycles.
During the above experiment, the examples and comparative examples were followed daily on day 10 to initiate preventive intervention of colon cancer by each of the gastric fermented raspberry juice (10 mL/kg body weight) and until the end of the experiment, the positive control group was filled with 5-aminosalicylic acid (75 mg/kg body weight) daily, and the model group and normal group mice were filled with 200 μl of PBS daily.
The growth environment of the mice is 23-25 ℃, the humidity is 55-60%, and 12 h are alternately illuminated. Mice were observed daily for status, weighed and recorded weekly. Collecting the feces of each group of mice after the experiment is finished, and detecting the content of short chain fatty acid in the feces; the number and the size of tumors in colon tissues are counted, and related inflammatory factors are measured.
The method for detecting the content of the fecal short-chain fatty acid comprises the following steps:
The feces were homogenized with ultrapure water at a ratio of 1:5 (mg/. Mu.L), acidified with sulfuric acid, then added with 2 volumes of diethyl ether, sonicated at low temperature to give 10 min, vortexed to give 5 min, centrifuged (12000 r/min,4 ℃,5 ℃ C., min) and allowed to stand for 10 min, the upper organic phase was filtered through a 0.22 μm filter membrane, assayed for short chain fatty acid content on-machine using GC-QTOF/MS, and 2-ethylbutyric acid was used as an internal standard, mixed with the supernatant to a concentration of 1 mmol/L.
GC-QTOF/MS (Agilent, USA) using DB-Wax column (Agilent 122-7062, 60m X1250 μm X0.25 μm, USA) with helium as carrier gas at a flow rate of 1 mL/min; the sample injection volume is 1 mu L, and the split ratio is 10:1; the temperature was raised as follows: the initial temperature was 60 ℃, equilibrated at 2 min ℃, warmed to 140 ℃ at 7.5 ℃/min, held at 2 min, then warmed to 200 ℃ at 60 ℃/min, held at 3 min. The temperature of the sample inlet is set to be 250 ℃, the temperature of the ion source is set to be 230 ℃, the temperature of the quaternary rod is set to be 150 ℃, and the energy of the ion source is set to be 70 eV; the analysis adopts a full scanning mode, and the ion scanning range is 20-350 amu. The concentration (mu mol/g) of each short chain fatty acid was calculated by the external standard method.
Preparation of short-chain fatty acid standard solution and preparation of standard curve: respectively weighing acetic acid, propionic acid, butyric acid, isobutyric acid and isovaleric acid, dissolving in deionized water to obtain standard solutions with different concentrations, and performing GC analysis after the standard solutions are treated in the same way according to a sample method.
The invention will be further illustrated by the following examples.
Example 1: lactic acid bacteria fermented raspberry juice
Fresh, mature, full and mildew-free raspberries are selected, cleaned and pulped for later use. 35 parts of raspberry pulp, 70 parts of purified water, 6 parts of glucose and 0.2 part of sodium iso-vitamin C are added according to the following weight parts. Uniformly stirring, filtering, pasteurizing at 95 ℃ to 20min, rapidly cooling to room temperature, adding 0.01 part of compound lactobacillus direct-vat starter (1×10 10 CFU/g, lactobacillus plantarum: lactobacillus acidophilus=1:1 (viable count ratio)), uniformly mixing, and fermenting at 35 ℃ to 48 h; sterilizing at 95deg.C, sterilizing at min, and packaging or cold preserving at 4deg.C to obtain fermented raspberry juice.
The fermentation strain used in this example was:
Lactobacillus plantarum (Lactiplantibacillus plantarum) NCU0011098 with a preservation number of CGMCC No.28774;
Lactobacillus acidophilus (Lactobacillus acidophilus) NCU426 with a preservation number of CGMCC No.15572.
Comparative example 1:
this comparative example was used as a comparative example of example 1, and in the seed fermentation step, 0.01 part of lactobacillus plantarum NCU0011098 lactobacillus starter was added to perform direct fermentation (1×10 10 CFU/g), and the other conditions were the same as in example 1.
Comparative example 2:
This comparative example was used as a comparative example of example 1, and in the seed fermentation step, 0.01 part of lactobacillus acidophilus NCU426 lactic acid bacteria starter was added to perform direct fermentation (1X 10 10 CFU/g), and the other conditions were the same as in example 1.
Experimental example 1:
In order to verify the beneficial effects of the invention, the relevant indexes of the final fermentation products of the embodiment 1 and the comparative embodiment 2 are detected, and the method mainly relates to the IC50 of the fermentation product on colon cancer cells HT-29, the inhibition rate on pathogenic bacteria and the prevention and treatment effect on colon cancer mice (the specific experimental method is referred to the 'part of experiments or determination methods adopted by the invention are as follows'). After the lactobacillus plantarum NCU0011098 and the lactobacillus acidophilus NCU426 were subjected to mixed fermentation (example 1), compared with the lactobacillus plantarum and lactobacillus acidophilus which are fermented independently, the in vitro inhibition of colon cancer cells and the prevention of colon cancer of a model mouse are both obviously improved. The specific results are as follows:
(1) Inhibition rate of IC50 and pathogenic bacteria of colon cancer cell HT-29:
TABLE 1 influence of different fermentation strains on antitumor and bacteriostatic Activity of fermented Raspberry juice
Note that: the "unfermented example 1" refers to: all the raw materials are uniformly mixed with the starter, directly sterilized without fermentation, and then aseptically canned or refrigerated at 4 ℃.
As shown in table 1, compared with comparative example 1 and comparative example 2, the compound lactic acid bacteria fermented raspberry juice prepared in example 1 has the lowest IC50 value for colon cancer cells HT-29 and the highest inhibition rate for pathogenic bacteria, and can effectively inhibit cancer cell proliferation and pathogenic bacteria proliferation, indicating that the compound lactic acid bacteria fermented raspberry juice prepared in example 1 has excellent anticancer activity and antibacterial activity.
Comparison results of example 1 and comparative examples 1 and 2 show that: compared with single-strain fermentation, the IC50 of colon cancer cells is obviously reduced and the inhibition rate of pathogenic bacteria is obviously improved after the lactobacillus plantarum NCU0011098 and lactobacillus acidophilus NCU426 are mixed for fermentation, so that the lactobacillus plantarum NCU0011098 and lactobacillus acidophilus NCU426 generate a synergistic effect, and the anticancer activity of the fermented raspberry juice and the inhibition effect on food-borne pathogenic bacteria are obviously improved.
The compound lactobacillus fermentation raspberry juice prepared in the embodiment 1 has strong inhibition capability to food-borne pathogenic bacteria staphylococcus aureus and escherichia coli, and can be the effect of organic acid, antibacterial peptide and other antibacterial substances generated in the fermentation process. In addition, research shows that the clostridium nucleatum is closely related to colon cancer, can cause intestinal inflammation, promotes the occurrence and development of digestive tract diseases such as colon cancer and the like, and is a potential target for treating colon cancer. As shown in table 1, lactobacillus plantarum NCU0011098 and lactobacillus acidophilus NCU426 each have a certain capacity of inhibiting clostridium nucleatum, but the capacity of the composite lactobacillus fermented raspberry juice prepared in example 1 after mixed fermentation for inhibiting clostridium nucleatum is obviously improved compared with that of single bacteria fermentation, and unexpected technical effects are obtained.
In conclusion, the compound lactobacillus fermented raspberry juice prepared in the embodiment 1 has better capabilities of inhibiting food-borne pathogenic bacteria, fusobacterium nucleatum and preventing colon cancer.
(2) Body weight and colon length changes in mice during the experiment
As can be seen from FIG. 1, AOM/DSS resulted in a decrease in body weight in mice compared to the blank. During the experiment, the weight reduction percentage of the mice in the treatment group of the example 1 is obviously lower than that of the mice in the treatment group of the comparative example 1, the mice in the treatment group of the comparative example 2 and the mice in the positive control group, which shows that the lactobacillus fermented raspberry juice prepared in the example 1 can effectively slow down the weight reduction of the mice caused by AOM/DSS. And the effect of the example 1 is better than that of the comparative example 1 and the comparative example 2, which shows that the lactobacillus plantarum NCU0011098 and the lactobacillus acidophilus NCU426 can effectively improve the capability of the fermented raspberry juice to inhibit the weight loss of mice caused by AOM/DSS after mixed fermentation.
Colon length change as shown in fig. 2, the average length of colon of model group mice after AOM/DSS treatment was significantly lower than that of normal mice (9.14 cm), 5.22: 5.22 cm. The lactic acid bacteria prepared in the stomach filling example 1 significantly improves the colon shortening caused by AOM/DSS after fermenting raspberry juice, and the average length is 7.87 cm, which is obviously higher than that of the comparative examples 1 and 2 (6.36-6.76 cm). In conclusion, the lactobacillus fermented raspberry juice prepared in example 1 can relieve weight loss and colon shortening caused by AOM/DSS, can relieve occurrence and development of colon cancer, and has certain prevention and alleviation effects on colon cancer. And the effect of the example 1 is better than that of the comparative example 1 and the comparative example 2, which shows that the lactobacillus plantarum NCU0011098 and lactobacillus acidophilus NCU426 can effectively improve the capability of the fermented raspberry juice to inhibit the colon shortening of mice caused by AOM/DSS after mixed fermentation.
(3) Number and size of mouse tumors
The number and size of AOM/DSS induced tumors were measured, as shown in FIGS. 3 and 4, with almost no tumor tissue observed in the mice in the blank group; the average tumor number on the colon of the model group mice was 10.46, and its size was a bias in the range of >2 mm; after the lactobacillus fermented raspberry juice prepared in example 1 is treated, the average tumor number is significantly reduced by 5.31 compared with the model group, and the tumor size is reduced, mainly focusing on the range < 1mm and 1-2 mm. The results show that the lactobacillus fermented raspberry juice prepared in example 1 can obviously inhibit the generation of colon tumor of mice caused by AOM/DSS. And the effect is better than that of raspberry juice prepared by single-bacteria fermentation in comparative example 1 and comparative example 2.
(4) Short chain fatty acid content in mouse feces
The short chain fatty acid is saturated fatty acid with less than 6 carbon atoms, can maintain the growth and reproduction of normal colon epithelial cells, and researches show that the short chain fatty acid has the effects of relieving and treating intestinal diseases such as colon inflammation, and can be used as a nutrient substance to be absorbed and utilized by intestinal epithelial cells of a body, such as maintaining the integrity of intestinal barrier, repairing damaged intestinal mucosa, intestinal peristalsis, regulating intestinal immunity and the like. Wherein, the butyric acid not only can maintain the metabolism of normal colon epithelial cells, but also can inhibit the proliferation of colon cancer cells, induce the apoptosis and differentiation of colon cancer cells, and increase the barrier function of intestinal wall by regulating the expression quantity of the tight junction protein, maintain the stable environment in the intestinal tract and relieve the damage of the intestinal barrier. In a word, the short chain fatty acid can effectively prevent, repair and relieve colon inflammation and colon cancer.
As shown in fig. 5 and 6, after mice in the model group are induced by AOM/DSS, the total short-chain fatty acid content in the feces is significantly reduced, and after the lactobacillus fermented raspberry juice of example 1 is ingested, the total short-chain fatty acid content in the feces of the mice is significantly increased, which is significantly higher than that of the comparative example. From the aspect of different short-chain fatty acid contents, the reduction of the short-chain fatty acid in the mouse feces is obviously relieved after the lactobacillus fermented raspberry juice of the embodiment 1 is taken, which shows that the lactobacillus fermented raspberry juice of the embodiment 1 has a beneficial effect on increasing the short-chain fatty acid, thereby relieving intestinal inflammation and barrier damage and inhibiting the occurrence and development of colon cancer. And after the raspberry juice of example 1 is taken up, the content of short chain fatty acid is higher than that of raspberry juice produced by single-bacteria fermentation of comparative examples 1 and 2.
(5) Cytokine content in colon tissue of mice
As shown in FIG. 7, in the AOM/DSS induced model group, the production of proinflammatory cytokines (IL-6, IL-8, IL-1β) was significantly increased at the colon part of mice, while the fermentation of raspberry juice by gastric lavage lactic acid bacteria reduced the levels of the proinflammatory cytokines in mice and increased the levels of anti-inflammatory factor IL-10. These results indicate that the compound lactobacillus fermented raspberry juice can reduce the level of inflammatory factors in mice and reduce the inflammatory level of colon parts, thereby playing an anti-inflammatory role to relieve colon cancer.
Experimental example 2:
The present invention also measured the organic acid content and antioxidant activity of the fermented raspberry juice of example 1, comparative example 2, as follows
(1) Influence on the organic acid content
TABLE 2 influence of different fermentation broths on the organic acid content (g/L) of fermented raspberry juice
As shown in Table 2, the organic acid content of the raspberry juice increased after fermentation, mainly lactic acid and citric acid. The lactic acid content of example 1 was 17.15 times that of unfermented and the citric acid content was 1.76 times that of unfermented. The overall organic acid content was increased relative to the comparative example 1, with higher levels of lactic acid and citric acid being increased, which resulted in a softer, more fragrant sour taste of the product. Research shows that the organic acid can promote digestion, stimulate gastrointestinal peristalsis, relieve constipation, regulate intestinal flora and the like, and has a certain prevention effect on chronic diseases of colon. Therefore, the composite lactobacillus fermented raspberry juice prepared by lactobacillus plantarum NCU0011098 and lactobacillus acidophilus NCU426 has potential colon cancer prevention capability.
(2) Influence of antioxidant Activity
TABLE 3 Effect of different fermentation broths on antioxidant Activity of fermented Raspberry juice
Oxidative stress is a negative effect of free radical generation in vivo, and is closely connected with inflammation, and various diseases such as tumor can be caused under long-term inflammation state. Therefore, reducing inflammation in vivo is an important step in preventing tumors. Research shows that the food-borne antioxidant can remove free radicals in vivo, reduce the occurrence of oxidation reaction, protect gastrointestinal mucosa cells from oxidative damage, reduce the level of inflammation markers in vivo, reduce inflammation, and improve the antioxidant capacity of intestines and stomach, thereby reducing the risk of cancer.
The results of antioxidant activity of lactobacillus fermented raspberry juice are shown in Table 3, and lactobacillus plantarum NCU0011098 and lactobacillus acidophilus NCU426 prepared in example 1 were higher in DPPH radical scavenging rate, hydroxyl radical scavenging rate, ABTS, FRAP content than in unfermented example 1 and comparative example.
In addition, the total phenol and total flavone contents of the compound lactobacillus fermented raspberry juice prepared in example 1 are high, and the total phenol and total flavone contents are respectively as high as 3.59 mg/mL and 2.30 mg/mL.
In conclusion, the composite lactobacillus fermented raspberry juice prepared from the lactobacillus plantarum NCU0011098 and the lactobacillus acidophilus NCU426 has excellent antioxidant activity and potential colon cancer prevention capability.
Example 2A lactic acid bacterium fermented raspberry juice
Fresh, mature, full and mildew-free raspberries are selected, cleaned and pulped for later use. 20 parts of raspberry pulp, 80 parts of purified water, 4 parts of glucose and 0.05 part of sodium iso-vitamin C are added according to the following weight parts. Uniformly stirring, filtering, pasteurizing at 95 ℃ to 20min, rapidly cooling to room temperature, adding 0.001 part of compound lactobacillus direct vat set starter (1×10 10 CFU/g, lactobacillus plantarum: lactobacillus acidophilus=1:1), uniformly mixing, and fermenting at 28 ℃ to 72 h; sterilizing the fermented raspberry beverage at 85deg.C, sterilizing at min, and packaging or cold preserving at 4deg.C to obtain fermented raspberry juice.
The fermentation strain used in this example was:
Lactobacillus plantarum (Lactiplantibacillus plantarum) NCU0011098 with a preservation number of CGMCC No.28774;
Lactobacillus acidophilus (Lactobacillus acidophilus) NCU426 with a preservation number of CGMCC No.15572.
Example 3A lactic acid bacteria fermented raspberry juice
Fresh, mature, full and mildew-free raspberries are selected, cleaned and pulped for later use. 50 parts of raspberry pulp, 50 parts of purified water, 8 parts of glucose and 0.1 part of sodium iso-vitamin C are added according to the following weight parts. Uniformly stirring, filtering, pasteurizing at 95 ℃ to 20 min, rapidly cooling to room temperature, adding 1 part of compound lactobacillus direct-vat starter (1×10 10 CFU/g, lactobacillus plantarum: lactobacillus acidophilus=1:1), uniformly mixing, and fermenting at 38 ℃ to 24 h; sterilizing the fermented raspberry beverage at 121deg.C, sterilizing at 5 min, and packaging or cold preserving at 4deg.C to obtain fermented raspberry juice.
The fermentation strain used in this example was:
Lactobacillus plantarum (Lactiplantibacillus plantarum) NCU0011098 with a preservation number of CGMCC No.28774;
Lactobacillus acidophilus (Lactobacillus acidophilus) NCU426 with a preservation number of CGMCC No.15572.
Example 4: related Properties of Lactobacillus plantarum NCU0011098
1. Isolation, screening and identification of Lactobacillus plantarum NCU0011098
The lactobacillus plantarum NCU0011098 of the invention is obtained: cleaning conventional fresh cabbage purchased from market, putting into pickle jar rinsed with boiled water in advance, adding cold boiled water containing 4% (w/v) edible salt, immersing, and naturally fermenting at normal temperature for 7 days. In the process, taking out a pickle liquid sample, carrying out gradient dilution (10 -1、10-2、10-3、10-4、10-5), coating on an MRS solid culture medium added with 0.04% (w/v) bromocresol purple, inversely culturing at 37 ℃ for 48h, selecting strains with yellow color development rings around colonies and different colony morphology sizes, carrying out secondary streaking, repeatedly streaking for 2-3 times, and storing the purified strains at-80 ℃ for later use by using 25% (v/v) glycerol. Then, the cancer cell proliferation, migration inhibition capability, acid and bile salt resistance capability, adhesion capability and pathogenic bacteria inhibition capability are measured to obtain a strain with better performance, and finally DNA extraction and PCR are carried out to carry out 16S rRNA sequencing, and the strain is identified as lactobacillus plantarum and named as lactobacillus plantarum (Lactiplantibacillus plantarum) NCU0011098. The bacterial form of NCU0011098 is shown in FIG. 16.
The lactobacillus plantarum NCU0011098 has been deposited at the China general microbiological culture Collection center, address: the collection number of the microbial institute of China academy of sciences is CGMCC No.28774 in the Korean area North Star, west Lu No. 1, no. 3 of Beijing city.
The 16S rRNA sequence of the Lactobacillus plantarum NCU0011098 is as follows (SEQ ID NO. 1):
GGCGTGGGCGGGGTGCTATACATGCAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCGTAAGTGACAGGT.
2. Acid and bile salt resistance of lactobacillus plantarum NCU0011098
The cell-activated NCU0011098 (4 ℃, 7000 Xg, 5 min) was collected, centrifuged, washed twice with sterile PBS, resuspended in MRS medium pH 2.5, incubated at 37℃for 3h, and the viable count was determined at 0, 1, 2, 3h, three replicates for each sample. Meanwhile, the acid resistance of the lactobacillus plantarum with CICC21801 is measured under the same conditions.
NCU0011098 cells were collected and washed as described above, then resuspended in MRS at pH 8.0 containing 0.3% bovine bile salts, incubated at 37℃for 4 h, and the viable count of the cultures was determined at 0,2,4 h, three replicates per sample. Meanwhile, the bile salt resistance of the lactobacillus plantarum when being CICC21801 is measured under the same conditions. The lactobacillus survival rate is calculated as follows:
Where N 1 is the number of viable bacteria after incubation and N 0 is the initial number of bacteria.
TABLE 4 acid resistance of Lactobacillus plantarum
TABLE 5 Lactobacillus plantarum tolerance to bile salts
The results show that the survival rate of lactobacillus plantarum NCU0011098 after being treated by 3 h in the environment of pH 2.5 is still more than 90 percent, and the survival rate after being treated by 4h in the environment of 0.3 percent of bile salt concentration is more than 95 percent. Therefore, the lactobacillus plantarum NCU0011098 has stronger acid resistance and bile salt resistance.
3. Adhesion capability of Lactobacillus plantarum NCU0011098 to colon cancer cell HT-29
The cultured HT-29 cells were digested with pancreatin-EDTA digests, followed by resuspension of the cells with DMEM complete broth and adjustment of cell concentration to 1.0X10 5 cells/mL, mounted in 6 well tissue culture plates, 2 mL cells per well, incubated in CO 2 incubator (5% CO 2, 95% air) at 37℃to differentiated monolayers, and the fluid was changed every two days. The DMEM medium in each well of the plate was discarded, the plate was washed 2 times with sterile PBS, 1 mL of Lactobacillus plantarum NCU0011098 bacterial suspension (10 8 CFU/mL, HT-29 cells: bacterial count. Gtoreq.1:100) resuspended in DMEM incomplete medium was added, and incubated at 37℃for 2h (2 h belonging to the adaptation phase, lactic acid bacteria did not grow, and generally 4-6 h did not enter the logarithmic phase). After the incubation was completed, the mixture of wells in the tissue culture plate was discarded, and washed 5 times with sterile PBS buffer to remove non-adherent Lactobacillus plantarum. 0.5 mL pancreatin-EDTA digest and 0.5 mL PBS buffer were added separately and incubated 5 min at 37 ℃ to digest the cells, then the cell digests were diluted in a gradient and plated on MRS plates to calculate the number of bacteria that adhered. Meanwhile, the adhesion capability of colon cancer cells HT-29 when the lactobacillus plantarum is CICC21801 is measured.
Adhesion = (number of lactic acid bacteria after 2 h/number of initial inoculations) ×100%.
TABLE 6 adhesion Capacity of lactic acid bacteria to colon cancer cells HT-29
The result shows that the adhesion rate of the lactobacillus plantarum NCU0011098 to colon cancer cells HT-29 is 62.09%, which is obviously higher than that of lactobacillus plantarum CICC21801, and has stronger adhesion capability.
4. Inhibition ability of lactobacillus plantarum NCU0011098 against common pathogenic bacteria
The antibacterial activity of the fermentation supernatant of lactobacillus plantarum NCU0011098 against Escherichia coli, staphylococcus aureus, salmonella typhimurium and Fusobacterium nucleatum was determined by a punching method and a co-culture method.
Inhibition ability assay for E.coli, staphylococcus aureus and Salmonella typhimurium: lactobacillus plantarum NCU0011098 (and lactobacillus plantarum CICC 21801) were serially passaged 2 times (inoculated into fresh MRS medium at an inoculum size of 2%), and incubated at 37℃for 24: 24 h), centrifuged (4 ℃,7000 Xg, 10 min) and the supernatant was filtered through a 0.22 μm filter membrane for use. The prepared various indicator bacteria suspensions are inoculated into sterilized LB solid culture medium (10 7-108 CFU/mL) respectively, the culture medium is poured into a semi-solid sterile culture dish after shaking, after the culture medium is cooled and solidified, punching operation is carried out on LB plates by using a puncher (diameter is 6 mm), and three holes are formed in each plate. 200. Mu.L of the prepared lactobacillus plantarum NCU0011098 fermentation supernatant was added to each well, incubated at 37℃for 12-16: 16h, and the diameter of the inhibition zone was measured using a vernier caliper. The indicator bacteria are respectively escherichia coli CMCC44350, staphylococcus aureus CMCC26003 and salmonella typhimurium ATCC13311. Meanwhile, the inhibition capability of the lactobacillus plantarum to pathogenic bacteria is measured by adopting the same method when the lactobacillus plantarum is CICC 21801.
Inhibition ability assay for Fusobacterium nucleatum: lactobacillus plantarum NCU0011098 is subjected to continuous passage for 2 times and then centrifuged (4 ℃,7000 Xg, 10 min), and the supernatant is filtered by a 0.22 mu m filter membrane for later use; NCU0011098 thalli are washed twice by PBS and then are suspended in equal proportion (1-2 multiplied by 10 8 CFU/mL), ultrasonic is carried out for 5 s under the condition of ultrasonic power of 280-350 w, 8 s is stopped, and total ultrasonic is circulated for 30min for later use. Activating the freeze-dried powder of the clostridium nucleatum twice with a trypticase soytone liquid culture medium, culturing in an anaerobic workstation at 37 ℃, and passaging twice to recover the activity of the strain. Inoculating activated clostridium nucleatum (1-2 multiplied by 10 8 CFU/mL) into a fresh liquid culture medium according to the proportion of 5% (v/v), respectively adding 10% (v/v) of lactobacillus fermentation supernatant and lactobacillus broken cells, respectively measuring the value of 24-h-bacteria liquid OD 600 for culture, calculating the inhibition rate of the activated clostridium nucleatum according to the following formula, and taking the clostridium nucleatum without adding the lactobacillus fermentation supernatant or the broken cells as a control group. Meanwhile, the inhibition capability of the lactobacillus plantarum to the fusobacterium nucleatum when the lactobacillus plantarum is CICC21801 is determined by adopting the same method. Each group was repeated three times and the indicator was fusobacterium nucleatum ATCC25586. The inhibition results were calculated according to the following formula:
Wherein A c is the post-control absorbance of 24 h; a c0 is the absorbance of control group 0h, and a t is the absorbance after treatment group 24 h; a t0 is treatment group 0h absorbance;
TABLE 7 inhibition of common pathogenic bacteria by lactic acid bacteria
The pathogenic bacteria result shows that the fermentation supernatant of the lactobacillus plantarum NCU0011098 has strong inhibition capability on three common food-borne pathogenic bacteria such as escherichia coli, staphylococcus aureus and salmonella typhimurium.
In addition, research shows that Fusobacterium nucleatum is closely related to colon cancer, can cause intestinal inflammation, increase proliferation, migration and invasion activity of colon cancer cells, promote occurrence and development of digestive tract diseases such as colon cancer and the like, and is a potential target for treating colon cancer. The inhibition capability test result of the invention on the clostridium nucleatum shows that the inhibition rate of the fermentation supernatant of the lactobacillus plantarum NCU0011098 on the clostridium nucleatum 24h reaches 95.26 percent, the inhibition rate of the broken thalli and intracellular metabolites thereof on the clostridium nucleatum 24h reaches 69.63 percent, and the invention can obviously inhibit the growth of the clostridium nucleatum, thus indicating that the lactobacillus plantarum NCU0011098 thalli and intracellular extracellular metabolites thereof have stronger inhibition capability on the clostridium nucleatum.
In conclusion, the lactobacillus plantarum NCU0011098 has certain prevention and treatment capability on food-borne pathogenic bacteria and colon cancer promoted by clostridium nucleatum.
5. Proliferation inhibition ability of lactobacillus plantarum NCU0011098 on colon cancer cells
The inhibition rate of proliferation of colon cancer cells HT-29 by Lactobacillus plantarum NCU0011098 was determined by CCK-8 method.
Preparing a fermentation supernatant: the lactobacillus suspension was centrifuged (4 ℃,7000×g,10 min) for 2 consecutive passages, the pH of the supernatant was adjusted to 7.0.+ -. 0.1 with NaOH, and filtered through a 0.22 μm filter membrane for use.
Preparation of crushed thalli: and centrifuging the lactobacillus suspension subjected to continuous passage 2 times (4 ℃,7000×g,10 min), collecting thalli, washing NCU0011098 thalli twice with PBS, carrying out equal-weight suspension (1-2×10 8 CFU/mL), carrying out ultrasonic treatment under the condition of ultrasonic power 280-350 w for 5 s, stopping 8 s, and carrying out cyclic total ultrasonic treatment for 30min for later use.
Preparing HT-29 cell suspension (1-5.0X10 5 cells/mL) with a certain concentration by using RPMI culture medium containing 10% fetal bovine serum, paving into 96-well culture plates, culturing at 100 mu L per well, adding 10% (v/v) of prepared NCU0011098 fermentation supernatant or crushed thalli after culturing at 24 h, and taking the group without adding the supernatant and the crushed thalli as a control group; each group was incubated with 6 parallel wells in a cell incubator at 37℃with 5% CO 2 for 24: 24 h. Then 10 mu L of CCK-8 solution is added into each hole, incubation is continued for about 1 h, absorbance (OD value) at 450: 450 nm is detected by using a multifunctional enzyme-labeled analyzer, and the inhibition rate of cell proliferation is calculated. Meanwhile, the proliferation inhibition capacity of the lactobacillus plantarum for colon cancer cells is measured by adopting the same method when the lactobacillus plantarum is CICC 21801.
Wherein A c is absorbance of a control group, A t is absorbance of a different treatment group after 24 h, and A b is absorbance of a blank group (RPMI medium containing 10% fetal bovine serum).
TABLE 8 proliferation inhibition ability of lactic acid bacteria on colon cancer cells HT-29
The CCK-8 result shows that after the fermentation supernatant of the lactobacillus plantarum NCU0011098 is incubated with colon cancer cells HT-29 for 24: 24 h, the proliferation of HT-29 is obviously inhibited, the inhibition rate is 46.77 percent, and the capacity of inhibiting the proliferation of colon cancer cells HT-29 in vitro is shown by the fermentation supernatant of the lactobacillus plantarum NCU 0011098.
6. Migration inhibition ability of Lactobacillus plantarum NCU0011098 on colon cancer cells HCT-116
The cultured HCT-116 was centrifuged by pancreatin digestion, the cell suspension was adjusted to 2-3X 10 5 cells/mL with RPMI medium containing 10% fetal bovine serum, the cells were inoculated into 24-well plates, one cell scratch insert was added to each well, each insert had two cell culture chambers separated by 500. Mu.m, and 70. Mu.L of cell suspension was added to each chamber. Cells were incubated at 37℃and 5% CO 2 for 24 hours. After 24 hours the cell density was examined under a microscope to form a fused cell layer, the inserts were removed, a 500 μm scratch was formed, the cell layer was washed with serum-free medium or PBS, and cell debris and non-adherent cells were removed. The culture was continued by adding 0.5 mL serum-free cell culture medium to each well of a 24-well plate and adding 50. Mu.L of the prepared NCU0011098 fermentation supernatant (see section 5 of this example) to the control group. After 0h, 24h and 48h, cell migration was observed under a microscope and a picture was taken (as shown in fig. 8), and the scratch area was analyzed by using Image J software, so as to evaluate the inhibition ability of lactic acid bacteria on cancer cell migration. Meanwhile, the migration inhibition capacity of the lactobacillus plantarum to HCT-116 when the lactobacillus plantarum is CICC21801 is determined by the same method.
TABLE 9 inhibition of the ability of colon cancer cell HCT-116 to migrate
The results show that lactobacillus plantarum NCU0011098 fermentation supernatant can slow down the migration capacity of colon cancer cells HCT-116, and the migration inhibition rate reaches 90.23% after 48: 48 h, so that the lactobacillus plantarum NCU has excellent colon cancer migration inhibition capacity.
7. Prevention and treatment effect of lactobacillus plantarum NCU0011098 on colon cancer mice
78C 57BL/6 mice (6-8 weeks old, male) were purchased and randomly divided into 6 groups of 13. The method is divided into a model group, a normal group, a positive control group, a lactobacillus plantarum low-dose group, a lactobacillus plantarum medium-dose group and a lactobacillus plantarum high-dose group.
After one week of adaptive feeding, other mice were intraperitoneally injected with AOM (10 mg/kg, sigma) in addition to the normal group (saline replacement), with normal drinking water for 7 days. 2.5% DSS (MW 36000-50000,MP Biomedicals) was then added to the drinking water for 7 consecutive days, followed by a 14 day recovery of normal drinking water. One week of drinking water containing 2.5% DSS and two weeks of normal drinking water were one cycle, and the experiment ended after 3 consecutive cycles, during which each group had free feeding.
During the test period described above: the Lactobacillus plantarum group was re-suspended with 200. Mu.L of each of Lactobacillus plantarum NCU0011098 bacterial suspensions in PBS at different doses (high dose: 1X 10 9 CFU/mL; medium dose: 1X 10 8 CFU/mL; low dose: 1X 10 7 CFU/mL) per day starting on day 10, until the end of the experiment; the positive control group was perfused with 5-aminosalicylic acid (75 mg/kg body weight) daily; mice in the model and normal groups were perfused with 200 μl of PBS daily; the growth environment of the mice is 23-25 ℃, the humidity is 55-60%, and 12h are alternately illuminated. Mice were observed daily for status, weighed and recorded weekly. Counting the death number of each group of mice after the experiment is finished, respectively collecting the faeces of each group of mice, and detecting the content of short chain fatty acid in the faeces; counting the length of colon and the number of tumors, analyzing pathological conditions of colon parts, and measuring inflammatory factors related to colon parts. The results were as follows:
(1) Effect of Lactobacillus plantarum NCU0011098 on survival of colon cancer mice
The results are shown in fig. 9, and colon cancer causes death of mice over time until the end of molding, with survival rate of only 68.42% in the model group. The survival rate of the mice in the interference group of the lactobacillus plantarum NCU0011098 is 73.33 percent, 80 percent and 92.86 percent respectively, so that the death rate of the mice is reduced. Therefore, lactobacillus plantarum NCU0011098 can reduce the death of mice caused by colon cancer to some extent.
(2) Effect of Lactobacillus plantarum NCU0011098 on colon length in colon cancer mice
Colon length changes as shown in fig. 10, the average length of the colon of model mice after AOM/DSS treatment was reduced to 5.32 cm, which is significantly lower than the normal mouse colon length (8.99 cm). The supplementation of lactobacillus plantarum NCU0011098 significantly improved the shortening of the colon in mice, whereas medium and high doses of lactobacillus had no significant difference in the changes in colon length in mice. Therefore, the lactobacillus plantarum NCU0011098 can improve colon shortening caused by AOM/DSS, so that the colon length of a mouse tends to be normal, and the lactobacillus plantarum NCU0011098 has certain prevention and alleviation effects on colon cancer.
(3) Effect of Lactobacillus plantarum NCU0011098 on colon pathology in colon cancer mice
The pathological results of the colon tissue of the mice are shown in fig. 11, the colon part of the normal group mice shows complete mucosal epithelial cells, a large number of cup-shaped cells can be seen in the mucosal layer, the large intestine glands are rich, the arrangement is orderly, and the colon crypt structure is complete. The submucosal space is uniform in size and free of inflammatory cell infiltration. The mucosal layer of the mice in the model group had altered crypt structure, crypt atrophy, upward migration, irregular crypt surface, goblet cell depletion, crypt abscess, inflammatory abnormal infiltration, and other pathological conditions (fig. 11). After the interference of the lactobacillus plantarum NCU0011098, pathological conditions such as the reduction of the goblet cells of the mucous membrane of the colon, the change of the crypt structure and the like are obviously relieved, so that the colon tissue structure is close to normal. Therefore, the lactobacillus plantarum NCU0011098 has the effects of relieving inflammatory infiltration of colon parts, preventing colon epithelium from further lesions and preventing colon cancer to a certain extent.
(4) Effect of Lactobacillus plantarum NCU0011098 on colon cancer tumor development in colon cancer mice
The number of tumors in AOM/DSS-induced colon cancer mice model was determined, as shown in fig. 12, with an average number of tumors on the colon of model mice of 5.30±0.85, a significant decrease in the number of colon tumors in mice by intervention of lactobacillus plantarum NCU0011098, a decrease in the number of colon tumors in low-dose lactobacillus mice to 3.85±0.80, a number of colon tumors in medium-dose lactobacillus mice of 2.54±0.88, and a number of colon tumors in high-dose lactobacillus mice of 2±1. Compared with the model group, the average tumor number of colon of the mice in the lactic acid bacteria intervention group is obviously reduced. The result shows that the lactobacillus plantarum NCU0011098 can obviously inhibit the generation of colon tumor of mice caused by AOM/DSS, and has the effect of improving colon cancer symptoms of the mice.
(5) Effect of Lactobacillus plantarum NCU0011098 on colon cancer mice faeces short chain fatty acids
Short chain fatty acid (Short CHAIN FATTY ACID, SCFA) detection method: mouse feces were collected, homogenized with ultrapure water, acidified with sulfuric acid, extracted with 2 volumes of diethyl ether by vortexing, centrifuged (12000 r/min,4 ℃,5 min) and allowed to stand for 10. 10 min, the upper organic phase was filtered through a 0.22 μm filter membrane, the content of short chain fatty acids (acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid) was analyzed on-press using GC-QTOF/MS, and 2-ethylbutyric acid was used as an internal standard, which was mixed with the supernatant to a concentration of 1 mmol/L. GC-QTOF/MS (Agilent, USA) using DB-Wax column (Agilent 122-7062, 60 m X1250 μm X0.25 μm, USA) with helium as carrier gas at a flow rate of 1 mL/min; the sample injection volume is 1 mu L, and the split ratio is 10:1; the temperature was raised as follows: the initial temperature is 60 ℃, the temperature is kept at 2 min ℃, the temperature is increased to 140 ℃ at 7.5 ℃/min, the temperature is kept at 2 min, the temperature is increased to 200 ℃ at 60 ℃/min, and the temperature is kept at 3 min. The temperature of the sample inlet is set to be 250 ℃, the temperature of the ion source is set to be 230 ℃, and the energy of the ion source is set to be 70 eV; the analysis adopts a full scanning mode, and the concentration (mg/g) of each short chain fatty acid is calculated by an external standard method.
Short chain fatty acid is an important product of intestinal bacteria decomposing indigestible carbohydrate, can maintain the growth and reproduction of normal colon epithelial cells, and can effectively prevent, repair and relieve intestinal diseases such as colon inflammation, colon cancer and the like. Wherein the propionic acid and butyric acid can provide energy for colon inner wall cells, can inhibit proliferation of colon cancer cells, and have antiinflammatory and anticancer effects. The results in fig. 13 and 14 show that the total short-chain fatty acid content in the feces of the AOM/DSS induced colon cancer mouse model is significantly reduced, and the dry prognosis of lactobacillus plantarum NCU0011098 can significantly increase the total short-chain fatty acid content and the concentration of each short-chain fatty acid in the mouse feces, indicating that the intestinal flora composition and the short-chain fatty acid metabolic pathway are improved by supplementing lactobacillus plantarum NCU 0011098.
(6) Effect of Lactobacillus plantarum NCU0011098 on colon inflammatory factor in colon cancer mice
Sample treatment: the colon of the mouse was rinsed with pre-chilled PBS (0.01 m, ph=7.4), after removing residual blood from the tissue, PBS (m/v) was added at a ratio of 1:9, and the resulting slurry was thoroughly ground on ice, sonicated in an ice bath for 10min, and centrifuged (8000×g,10 min,4 ℃) to obtain the supernatant for use. Interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-1β (IL-1β) and tumor necrosis factor- α (TNF- α) were detected using ELISA kits.
Results As shown in FIG. 15, in the AOM/DSS induced colon cancer mouse model group, the production of proinflammatory cytokines (IL-6, IL-1 beta, TNF-alpha) at the colon part of the mice was significantly increased, and the anti-inflammatory factor (IL-10) was significantly decreased. The intervention of the lactobacillus plantarum NCU0011098 can obviously reduce the level of the colon pro-inflammatory factors of mice and increase the level of the anti-inflammatory factors. These results indicate that lactobacillus plantarum NCU0011098 can alleviate the level of inflammation at the colon site in a colon cancer model mouse, thereby exerting an anti-inflammatory effect to alleviate the occurrence and development of colon cancer.
The above examples merely represent a few embodiments of the present invention, which are described in more detail and are not to be construed as limiting the scope of the patent. It should be noted that, for a person skilled in the art, the above embodiments may also make several variations, combinations and improvements, without departing from the scope of the present patent. Therefore, the protection scope of the patent is subject to the claims.
Claims (7)
1. The preparation method of the lactobacillus fermented raspberry juice is characterized in that the raspberry juice is obtained by fermenting lactobacillus plantarum and lactobacillus acidophilus serving as raw materials, and the preparation method specifically comprises the following steps:
(1) Selecting fresh, mature and full raspberries without mildew and rot, cleaning and homogenizing to obtain raspberry pulp for later use;
(2) The lactobacillus fermented raspberry juice comprises the following raw materials in parts by weight: 20-50 parts of raspberry pulp, 50-80 parts of purified water, 4-8 parts of glucose and 0.05-0.2 part of sodium iso-vitamin C, uniformly stirring, filtering, pasteurizing, and rapidly cooling to room temperature;
(3) Adding 0.001-1 part of compound lactobacillus direct vat set starter, and fermenting at 28-38 ℃ for 24-72 h;
The compound lactobacillus direct-vat set starter comprises lactobacillus plantarum and lactobacillus acidophilus;
the lactobacillus plantarum is lactobacillus plantarum (Lactiplantibacillus plantarum) NCU0011098, and the preservation number is CGMCC No.28774;
the lactobacillus acidophilus is lactobacillus acidophilus (Lactobacillus acidophilus) NCU426, and the preservation number is CGMCC No.15572.
2. A method of preparing a lactic acid bacteria fermented raspberry juice as claimed in claim 1, wherein the raspberry pulp is selected from one or more of raspberry pulp, black raspberry pulp, huang Shumei pulp and purple raspberry pulp.
3. The method for preparing the lactobacillus fermented raspberry juice of claim 1, wherein the ratio of the viable count of lactobacillus plantarum to lactobacillus acidophilus in the compound lactobacillus direct vat set is 1:1.
4. The method for preparing the lactobacillus fermented raspberry juice of claim 1, wherein the number of bacterial activities in the compound lactobacillus direct vat set starter is (1-9) x 10 10 CFU/g.
5. The method for preparing the lactobacillus-fermented raspberry juice of claim 1, wherein the lactobacillus-fermented raspberry juice is obtained by sterilizing at 85-121 ℃ and 5-30 min ℃ and then aseptically canning or refrigerating at 4 ℃.
6. A lactobacillus fermented raspberry juice prepared by the method of any one of claims 1 to 5.
7. The use of the lactobacillus fermented raspberry juice of claim 6, in the preparation of food, health care products, medicaments for preventing and/or treating colon cancer.
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