CN116694525A - Lactobacillus plantarum and application thereof in preparing bacteria agent for inhibiting or relieving or treating oral mucositis - Google Patents

Abstract

The application provides lactobacillus plantarum and application thereof in preparing an oral mucositis inhibiting or relieving or treating bacterial agent. Lactobacillus plantarum chen-02 with a preservation number of CGMCC No.23397, and fermented liquid powder formed by fermenting the lactobacillus plantarum chen-02 and aloe extract to prepare Cheng Yi raw bacterial agent, which can inhibit or relieve or treat oral mucositis caused by tumor chemoradiotherapy. The probiotics preparation is prepared into total nutrient formula powder, and comprises protein, fat, nucleotide, amino acid, carbohydrate, dietary fiber, compound vitamin, compound mineral, lactobacillus plantarum aloe co-fermentation powder. The lactobacillus plantarum aloe co-fermentation liquid provided by the application has the advantages that the oral mucositis score is reduced, the apoptosis level of the tongue mucosa epithelium is reduced, the phosphorylation level of the protein related to the P13K/AKT/FoxO1 pathway is reduced, and the obvious difference exists. The total nutrient formula powder disclosed by the application meets the requirements of tumor patients on amino acids, essential fatty acids, vitamins, minerals and trace elements, reduces the incidence rate of tumors, prolongs the survival time of patients, and has good clinical application prospects.

Description

Lactobacillus plantarum and application thereof in preparing bacteria agent for inhibiting or relieving or treating oral mucositis

Technical Field

The application relates to the technical field of medical application formula foods, in particular to lactobacillus plantarum and application thereof in preparing an oral mucositis inhibiting or relieving or treating bacterial agent.

Background

Cancer is a major health problem worldwide.

According to the report of the literature, the proportion of malnutrition in cancer patients can be up to 30% -80%, and the specific number is also influenced by factors such as the type of cancer, the treatment mode and the nutritional status of the patients. Not only can the metabolism rate of the patient be increased due to the tumor, the energy and nutrient substance consumption is accelerated, but also the appetite and the digestion capacity of the patient can be influenced by treatment methods such as radiotherapy, chemotherapy, operation and the like. Malnutrition affects the quality of life of the patient, exacerbates the incidence of adverse effects and complications of treatment, and may even lead to treatment failure and death. Nutritional supplementation is therefore a very important ring for cancer patients.

Chemoradiotherapy is one of the most commonly used methods for treating cancers at present, but side effects of chemoradiotherapy are not ignored. It kills cancer cells and also causes damage to normal cellular tissues, resulting in a series of adverse reactions including oral mucositis. About 40% to 70% of cancer patients are studied to develop oral mucositis during radiotherapy and chemotherapy, and serious patients even develop symptoms such as oral pain, oral ulcer, dysphagia and the like, which not only seriously affect the life quality of the patients, but also can cause complications such as malnutrition, infection and the like. Therefore, how to prevent and treat oral mucositis has become an important task during radiotherapy and chemotherapy.

The gel extracted from aloe leaf parenchyma contains a wide range of natural components, has remarkable anti-inflammatory, antioxidant, antibacterial, antifungal and immunostimulating activities, and shows the capability of improving ulcer, wound healing and the like. Aloe has been studied for its application in the treatment of radiation-related dermatitis, esophagitis, radiation burns, radiation ulcers, canker sores, oral mucositis caused by radiation during radiotherapy of head and neck tumors, and the like. Several clinical trials have shown that aloe-containing mouthwashes can prevent oral mucositis caused by radiation during radiotherapy in patients with head and neck cancer.

The probiotics are beneficial bacteria which can regulate the balance of intestinal flora and maintain the health of intestinal tracts in human bodies, are one of the most widely applied micro-ecological regulators (including probiotics, prebiotics and synbiotics), and are researched and found to have the effects of preventing, relieving and treating various diseases. With the deeper research on the mechanism of the probiotics, the varieties of the developed and produced probiotics products are wider and wider. In human body, the probiotics can directly or indirectly inhibit the growth of pathogenic bacteria, produce antibiotics substances, promote the immune regulation of immune system and the like, and inhibit the growth, recurrence and metastasis of tumors. Clinical studies show that the probiotics can repair the oral mucositis caused by radiotherapy and chemotherapy. In addition, probiotics can promote the synthesis of some essential vitamins in human bodies, such as vitamin B, K and the like, and supplement needed nutrition for cancer patients.

Under the background, the development of the probiotic-containing aloe fermentation powder and the preparation method thereof have important clinical significance, and are suitable for rehabilitation of patients suffering from oral mucositis caused by tumor chemoradiotherapy.

The Chinese patent application No. 201510722385.7 discloses a full-nutrition special formula food for tumor patients, and the patent provides a full-nutrition formula food which is eaten by tumor patients and has the effects of nourishing liver, clearing heat and detoxicating, promoting blood circulation by removing blood stasis, harmonizing liver and spleen, regulating qi and dissolving turbidity, lowering adverse qi and preventing vomiting and enhancing immunity, and a preparation method thereof, wherein the raw materials such as medicinal materials are dozens of raw materials, but the actual effect is not verified, the comprehensive nutritional requirements of tumor patients cannot be ensured, the number of the medicinal materials is excessive, whether side effects and adverse reactions are caused or not is difficult to determine, and the clinical application value is difficult to determine

A Chinese patent with application number 201610245338.2 discloses a full-nutrition formula food for tumors, which is claimed to be capable of carrying out immunoregulation on tumor patients, inhibiting the growth, recurrence and metastasis of the tumors, reducing postoperative complications of the tumors, increasing the tolerance of radiotherapy and chemotherapy of the tumors, and improving the life quality and the life span of the tumor patients. However, there is no enough evidence to verify the actual effect, the comprehensive nutrition requirements of the tumor patients cannot be guaranteed, and the clinical application value of the tumor patients cannot be determined easily.

The formula food has no evidence for relieving or treating tumor complications, and has no effect for relieving or treating oral mucositis caused by tumor radiotherapy and chemotherapy.

Disclosure of Invention

Aiming at the defects of the prior art, the application provides the total nutrient formula powder containing the lactobacillus plantarum aloe common fermentation liquid powder and suitable for the rehabilitation of patients suffering from oral mucositis caused by tumor chemoradiotherapy and the preparation method thereof, which can meet the overall requirements of cancer patients on various nutrients, completely replace daily foods to meet the overall nutritional requirements of the cancer patients, are easy to absorb, can effectively prevent or improve the malnutrition problem of the cancer patients, prevent the symptoms such as emaciation, weakness and weakness, strengthen the immunity of the patients through immunoregulation and the like, prevent or treat the oral mucositis caused by chemoradiotherapy and reduce the pain of the cancer treatment of the patients.

In order to achieve the above purpose, the present application provides the following technical solutions:

the first aspect of the application provides lactobacillus plantarum (Lactobacillus plantarum) chen-02 with the preservation number of CGMCC No.23397, which is preserved in China center for type culture Collection of microorganisms at 9 and 13 of 2021.

The application provides a probiotic preparation which is prepared from lactobacillus plantarum chen-02 and can inhibit or relieve or treat oral mucositis, wherein the probiotic preparation is lactobacillus plantarum aloe co-fermentation liquid powder, particularly freeze-dried powder, formed by fermenting lactobacillus plantarum chen-02 and aloe extract.

Further, the oral mucositis is oral mucositis caused by tumor chemoradiotherapy.

Further, the tumor is a head and neck cancer.

The third aspect of the application provides an application of the probiotic preparation in preparing a product for inhibiting or relieving or treating oral mucositis caused by tumor chemoradiotherapy, wherein the product is a medicament and the like, and can be pharmaceutically acceptable dosage forms including spray, tablets, capsules, oral liquid or freeze-dried powder and the like.

The fourth aspect of the application provides a total nutrient formula powder containing the probiotic preparation, which comprises the following components in parts by weight:

18-23 parts of protein;

17-24 parts of fat;

0.1 to 1 part of nucleotide;

3-6 parts of amino acid;

15-20 parts of carbohydrate;

dietary fiber 2-5 parts;

1-2 parts of compound vitamin;

5.5 to 6.5 portions of composite mineral matters;

3-5 parts of lactobacillus plantarum aloe co-fermentation liquid powder.

Further, the protein source comprises one or more of soy protein isolate, milk protein, hydrolyzed whey protein.

Further, the amino acids include one or more of leucine, arginine, glutamine.

Further, the fat comprises one or more of sunflower seed oil, corn oil, soybean oil, peanut oil and fish oil.

Further, the fat comprises medium chain triglyceride and omega-3 unsaturated fatty acid, wherein the omega-3 unsaturated fatty acid comprises DHA and EPA, and the mass part ratio of DHA to EPA is 1:0.6-2.

Further, the nucleotide source includes one or more of disodium 5 '-inosinate, disodium 5' -guanylate, disodium 5 '-uridylate, and disodium 5' -cytidylate.

Further, the dietary fiber source comprises one or more of fructooligosaccharides, inulin, and resistant dextrins.

Further, the vitamin complex in each 70g of the total nutrient formula comprises the following components: vitamin A, 450-660 mug; vitamin D, 4-5 mug; vitamin E, 60-80 mug; vitamin K, 30-40 mug; vitamin B10.6-0.8 mg; vitamin B2, 0.6-0.8 mg; vitamin B6, 0.7-1.0 mg; vitamin B12, 1.4-1.8 mug; 150-170 mg of vitamin C; biotin, 10-25 mug; nicotinamide 5.6-7.2 mg; pantothenic acid, 2.68-4.95 mg; folic acid 104-215 mug.

The fifth aspect of the present application provides a method of preparing a total nutritional formula comprising the steps of:

(1) Weighing the components according to the proportion, adding 1000 parts by weight of pure water at 20-25 ℃ into a preparation tank, and adding the compound vitamins and the compound minerals into the pure water through a mixer under high-speed stirring to fully dissolve or disperse the compound vitamins and the compound minerals in the pure water; adding protein, fat, carbohydrate, dietary fiber, amino acid and nucleotide to uniformly mix; finally, adding food additives to dissolve or disperse the food additives in the pure water, and stirring and reacting to obtain mixed nutrient solution;

(2) Spray drying the mixed nutrient solution to obtain nutrient powder;

(3) Mixing 3-5 parts by weight of lactobacillus plantarum aloe co-fermentation liquid powder with nutrition powder to obtain the total nutrition formula powder suitable for rehabilitation of tumor patients.

Further, nitrogen is introduced to protect the reaction in the step (1).

The carbohydrate, the composite mineral and the food additive are all materials acceptable or required in the field, for example, the carbohydrate can be sugar, cereal (such as rice, wheat, corn, barley, oat, sorghum, etc.), fruit (such as sugarcane, melon, watermelon, banana, grape, etc.), dried fruit, dried bean, rhizome vegetable (such as carrot, sweet potato, etc.); the compound mineral can be food-grade adjuvants containing elements such as calcium, magnesium, zinc, selenium and ferrum, and the food additive can be edible essence, sweetener, acidity regulator, emulsifier, etc.

The formula powder is convenient to take and comprehensive in nutrition. Wherein, the lactobacillus plantarum aloe co-fermentation powder plays an important role in preventing, relieving and treating oral mucositis generated by radiotherapy and chemotherapy of patients, and is one of the important raw materials of the formula powder. Further, lactobacillus plantarum chen-02 plays a key role in the lactobacillus plantarum co-fermentation powder. Prior to the application, lactobacillus plantarum aloe co-fermentation powder and lactobacillus plantarum subspecies chen-02 have not been used in the fields of preventing, relieving and treating oral mucositis generated by radiotherapy and chemotherapy, regulating the immune level of cancer patients and the like. The technical staff of the application researches and discovers that lactobacillus plantarum chen-02, aloe extract and chen-02 lactobacillus plantarum-aloe co-fermentation liquid powder have therapeutic effects on oral mucositis, and the lactobacillus plantarum aloe co-fermentation liquid powder has more obvious therapeutic effects than any other modes, so that the lactobacillus plantarum aloe co-fermentation liquid powder has better beneficial effects on the oral mucositis.

The application has the beneficial effects that: the oral mucositis score of rats in the experimental group using lactobacillus plantarum aloe co-fermentation broth decreased, the level of apoptosis of the tongue mucosa epithelium decreased, and the PI3K/AKT/FoxO1 pathway-related protein phosphorylation level decreased, with a significant difference, compared to the control group model. And can regulate intestinal flora of rats. Patients taking the lactobacillus plantarum aloe co-fermentation broth powder infusion have decreased oral mucositis scores, and serum albumin levels are increased, and BMI of the patients is increased, compared with the patients in the control group. The solid beverage has comprehensive nutrition, can effectively improve the symptoms of emaciation, malnutrition, limb weakness and the like of a tumor patient, meets the requirements of the tumor patient on amino acid, essential fatty acid, vitamin, mineral substances and trace elements, reduces the incidence of tumor, prolongs the survival time of the tumor patient, and has good clinical application prospect.

Drawings

FIG. 1 shows TUNEL staining results of example 4.

FIG. 2 shows the HE staining results of example 4.

FIG. 3 is a Western Blot result of example 4.

FIG. 4 is the high throughput sequencing results of example 5; wherein, chao index and Simpson index in A show oral flora alpha diversity, B is oral flora beta diversity analysis result, C is Wien diagram result of five groups of oral flora, and D is the composition of oral flora portal horizontal species of each group.

FIG. 5 is an overall experimental procedure of example 6.

Detailed Description

In order that the application may be understood more fully, a more particular description of the application will be rendered by reference to specific embodiments thereof which are illustrated in the appended claims. This application may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete

EXAMPLE 1 acquisition of Lactobacillus plantarum chen-02, a key ingredient in the formulation

1. Sample processing method

Fresh fecal samples from normal gastrointestinal populations were collected in EP tubes, 1g of the samples were taken, and 3-5ml of sterile PBS was added. After fully mixing, centrifuging (200 rpm,5 min), respectively diluting 100-107 times, respectively plating/scribing on MRS solid culture medium (lactobacillus plantarum) on a sterile operation table, culturing for 24-48 hours, taking 200-400 colony-growing plates, picking 40-50 single colonies, activating and culturing on MRS liquid culture medium for 24-48 hours, and respectively carrying out glycerol sterilization, DNA extraction and identification work and the like.

2. Extraction of genomic DNA

(1) The isolated single bacteria were inoculated into the respective liquid culture media.

(2) 2mL of the bacterial suspensions were placed in 2mL centrifuge tubes, centrifuged (800rmp, 2 min), and the supernatants were discarded.

(3) To the pellet, 600. Mu.L of lysate (lysis buffer: 500mM NaCl,50mM Tris-HCl, pH8.0, 50mM EDTA,4% SDS), 200. Mu.L of Tris-saturated phenol and 0.3-0.4g of glass beads were added, and the mixture was shaken 30s for 3 times until the cells were thoroughly suspended, and centrifuged (8000 rmp,1 min).

(4) The supernatant was removed to a new 1.5mL centrifuge tube, 250. Mu.L of 10M ammonium acetate was added, and the mixture was left on ice for 10min and centrifuged (8000 rmp,1 min).

(5) The supernatant from the organic layer was applied to a DNA adsorption column, and the supernatant was centrifuged (8000 rmp,1 min).

(6) The pellet was washed 1 time with 600 μl of 75% ethanol and repeated one time.

(7) After 8000rmp air-throwing for 2min, transferring the DNA adsorption column to a new EP tube, and airing for 30min.

(8) To the dried centrifuge tube, 40 μl of TE (ph=8.0) was added.

(9) The extracted DNA was sent to the company for sequencing and the results were analyzed.

Lysis buffer: 500mM NaCl,50mM tris-HCl, pH8.0, 50mM EDTA,4% SDS.

TE buffer: 1M tris-HCl Buffer (Ph 8.0) 5ml,0.5M EDTA (Ph 8.0) 1ml, adding 400ml MilliQ water to the beaker, mixing uniformly, fixing the volume of the solution to 500ml, sterilizing at high temperature under high pressure, and preserving at room temperature.

3. Determination of Lactobacillus plantarum chen-02 Strain

According to the sequencing result, the separated strain is a new clostridium butyricum subspecies chen-02, and the preservation number is CGMCC No.23397.

Example 2 Lactobacillus plantarum chen-02 and Aloe extract fermentation to form Lactobacillus plantarum Aloe co-fermentation broth powder A method for obtaining Lactobacillus plantarum Aloe co-fermentation broth powder:

the bottom of the aloe leaf is cut and thoroughly washed, then its edges are removed, and a viscous and transparent gel is obtained by peeling the leaf. The gel was mashed with a pulper and then centrifuged at 5000×g for 20 minutes to obtain aloe supernatant as aloe extract. Lactobacillus plantarum chen-02 was cultured at 37℃for 24 hours using de Man-Rogosa-Sharpe (MRS) medium, and then Lactobacillus chen-02 was added to aloe extract with +5% glucose to a final concentration of 5X 106 cfu/ml, followed by further culturing for 48 hours. The aloe broth was obtained by centrifuging the fermented supernatant at 5000×g for 20 minutes.

Example 3

The application relates to a powder tumor total nutrient formula powder, which comprises the following nutrient components in parts by weight in each 70g of powder: 18-23 g of protein; 17-24 g of fat; 0.1-1 g of nucleotide, 3-6 g of amino acid and 15-20 g of carbohydrate; 2-5 g of dietary fiber; 1-2 g of compound vitamin; 5.5-6.5 g of composite mineral; 3-5 g of lactobacillus plantarum aloe co-fermentation powder.

The preparation method of the total nutrient formula powder comprises the following steps:

(1) Weighing the components according to the proportion, adding 1000 parts by weight of pure water at 20-25 ℃ into a preparation tank, and adding the compound vitamins and the compound minerals into the pure water through a mixer under high-speed stirring to fully dissolve or disperse the compound vitamins and the compound minerals in the pure water; adding protein, fat, carbohydrate, dietary fiber, amino acid and nucleotide to uniformly mix; finally, adding food additives to dissolve or disperse the food additives in pure water, introducing nitrogen for protection in the whole reaction process, and stirring to react to obtain mixed nutrient solution;

(2) Spray drying the mixed nutrient solution to obtain nutrient powder;

(3) Mixing 3-5 parts by weight of lactobacillus plantarum aloe co-fermentation liquid powder with nutrition powder to obtain the total nutrition formula powder suitable for rehabilitation of tumor patients.

The protein source comprises one or more of soy protein isolate, milk protein, hydrolyzed whey protein. The amino acid comprises one or more of leucine, arginine and glutamine. The fat comprises one or more of sunflower seed oil, corn oil, soybean oil, peanut oil and fish oil; the fat comprises medium chain triglyceride and omega-3 unsaturated fatty acid, the omega-3 unsaturated fatty acid comprises DHA and EPA, the mass part ratio of DHA to EPA is 1:0.6-2, and the nucleotide source comprises one or more of disodium 5 '-inosinate, disodium 5' -guanylate, disodium 5 '-uridylate and disodium 5' -cytidylate. The dietary fiber source comprises one or more of fructooligosaccharides, inulin, and resistant dextrins.

The compound vitamin in each 70g of the total nutrient formula powder comprises the following components: vitamin A, 450-660 mug; vitamin D, 4-5 mug; vitamin E, 60-80 mug; vitamin K, 30-40 mug; vitamin B10.6-0.8 mg; vitamin B2, 0.6-0.8 mg; vitamin B6, 0.7-1.0 mg; vitamin B12, 1.4-1.8 mug; 150-170 mg of vitamin C; biotin, 10-25 mug; nicotinamide 5.6-7.2 mg; pantothenic acid, 2.68-4.95 mg; folic acid 104-215 mug.

The carbohydrate can be one or more of sugar, cereal (such as rice, wheat, corn, barley, oat, sorghum, etc.), fruit (such as sugarcane, melon, watermelon, banana, grape, etc.), dried fruit, dried bean, rhizome vegetable (such as carrot, sweet potato, etc.); the compound mineral can be food-grade adjuvants containing elements such as calcium, magnesium, zinc, selenium and ferrum, and the food additive can be edible essence, sweetener, acidity regulator, emulsifier, etc.

EXAMPLE 4 improving Effect of Lactobacillus plantarum aloe Co-fermentation liquid as the main ingredient in the formula powder on rat nutritional status and oral mucositis

1. Grouping and feeding animals

Animals were randomly divided into five groups, with 8 weeks of age, body weight (220±10) g, 30 SPF grade SD male rats, n=6 per group:

group A: a control group, which received no chemotherapy, was treated with 1.0ml of physiological saline for 10 days;

group B: model group, receiving radiotherapy and chemotherapy, and treating with 1.0ml physiological saline for 10 days;

group C: aloe group, receiving radiotherapy and chemotherapy, treated with 1.0ml aloe for 10 days;

group D: the lactobacillus plantarum culture solution group is subjected to radiotherapy and chemotherapy, and is treated by 1.0ml of lactobacillus plantarum culture solution for 10 days.

Group E: aloe lactobacillus plantarum broth (AFB) group, receiving radiotherapy and chemotherapy, treating with 1.0ml aloe lactobacillus plantarum broth for 10 days;

2. test method

During irradiation, groups B, C, D and E were each treated with the same volumes of saline, aloe vera broth, aloe vera and lactobacillus plantarum broth, and the rats were rubbed on their oral cavity once a day for 10 days (significant improvement to oral mucositis).

(1) Careful observation was made during the experiment, and changes in body weight, feed intake, were recorded daily for all rats.

(2) Oral mucositis scores of rats were recorded daily during the experiment. The lingual back surface oral mucositis score (1983) was evaluated for each rat using the scoring system previously developed by Parkins et al, and survival was calculated (table 1).

Table 1 scoring criteria for oral mucositis

(3) Blood collection: on day 10 of the experiment, blood samples were collected from the heart following light ether anesthesia.

Monitoring indexes: blood inflammatory factors TNF-alpha, IL-1 beta, IL-6;

(4) After sacrifice, take its tongue specimen: at the end of the experiment, rats were anesthetized, sacrificed by cervical removal, the tongue was exposed for photographing, and tongue mucosa specimens were collected. The tongues of each group of rats were collected with a sterile centrifuge tube.

The detection method and the index are as follows: part of the tongue tissue was used for HE staining and was blindly examined by the same pathologist using predetermined histological healing scoring criteria. The criteria are as follows (table 2):

TABLE 2 histological evaluation scoring criteria for ulcer healing

Scoring of	Histological manifestations
		1	There was epithelial necrosis but no signs of inflammation
2	Inflammatory reactions have begun without new capillary proliferation
		3	The inflammatory response is very pronounced with little capillary increase on the basis of ulcers but no epithelialization of the surface
4	The inflammatory response is reduced, new capillary proliferation has reached the surface, and the surface begins to epithelialize
		5	Epithelialization completion

Part of the lingual mucosa specimens were stained by TUNEL for apoptosis of lingual mucosa epithelial cells.

And (3) detecting the expression conditions of IL-1 beta, IL-6 and TNF-alpha mRNA in the lingual mucosa tissues by qRT-PCR on part of lingual mucosa specimens.

Detecting the change of PI3K/AKT/FoxO1 signal paths (PI 3K, AKT, p-AKT, foxO1 and p-FoxO 1) of part of lingual membrane samples through Western blot; NF- κB, p-NF- κB, TLR4 expression.

3. Experimental results

(1) Symptoms of oral mucositis

(1) Scoring, see table 3 below:

TABLE 3 oral mucositis symptom scoring criteria

(2) TUNEL staining, see figure 1;

(3) HE staining, see fig. 2;

(2) PI3K/AKT/FoxO1 signaling pathway related protein expression is shown in FIG. 3.

From the oral mucositis score and TUNEL staining pathological results, the lactobacillus plantarum aloe co-fermentation liquid has obvious treatment effect on the oral mucositis of rats and reduces the apoptosis of tongue mucous membrane epithelium. The lactobacillus plantarum aloe co-fermentation liquid is used for treating rats for ten days, so that the oral mucositis score of the rats can be reduced, and the pathological changes can be relieved. From the Western blot result, the phosphorylation level of the protein related to the PI3K/AKT/FoxO1 signal path of the rat is reduced after treatment, which suggests that the lactobacillus plantarum aloe co-fermentation liquid has an inhibition effect on the occurrence and the development of malignant tumors.

EXAMPLE 4 Effect of the principal component of the formula-Lactobacillus plantarum Aloe Co-fermentation broth on rat colonic permeability, oral and intestinal flora

1. Grouping and feeding animals

Animals were randomly divided into five groups, with 8 weeks of age, body weight (220±10) g, 30 SPF grade SD male rats, n=6 per group:

group A: a control group, which received no chemotherapy, was treated with 1.0ml of physiological saline for 10 days;

group B: model group, receiving radiotherapy and chemotherapy, and treating with 1.0ml physiological saline for 10 days;

group C: aloe group, receiving radiotherapy and chemotherapy, treated with 1.0ml aloe for 10 days;

group D: the lactobacillus plantarum culture solution group is subjected to radiotherapy and chemotherapy, and is treated by 1.0ml of lactobacillus plantarum culture solution for 10 days.

Group E: aloe lactobacillus plantarum broth (AFB) group, receiving radiotherapy and chemotherapy, treating with 1.0ml aloe lactobacillus plantarum broth for 10 days;

2. test method

6 rats in each group were randomly selected and pre-treatment and 7 days, 14 days, 21 days later intraoral and fresh fecal samples were collected for DNA extraction of microorganisms and high throughput sequencing analysis of bacterial diversity.

3. Test results

Oral high-throughput sequencing is shown in fig. 4, and the high-throughput sequencing result shows that lactobacillus plantarum aloe co-fermentation liquid has a regulating effect on oral flora of rats.

The results show that: group a and four groups B, C, D, E rats were significantly different in alpha diversity (a).

PCoA analysis results (B) show that the E group is obviously separated from the B group, the C group and the D group, and the obvious difference exists.

Venn panel (C) shows five sets of consensus ASV sequences 166, unique sequences of set E, 446, set A1110, set B807, set C512, and set D714.

(3) Results of changes in portal horizontal flora.

The results show that: proteus (Proteus), achromobacter (Firmides), actinobactylodes (Actinobactylodes), bacteroides (Bacteroides) are the most common gates of group A (relative abundance of 34.83, 32.92, 27.91,1.29, respectively), group B (relative abundance of 51.02, 25.7, 19.82,2.35, respectively), group C (relative abundance of 45.38, 35.71, 18.27,0.13, respectively) group D (relative abundance of 37.59, 41.19, 19.81,0.20, respectively), group E (relative abundance of 37.37, 47.04, 14.81,0.04, respectively).

Further analysis found that actinomycota in patients generally decreased and proteobacteria generally increased compared to healthy people; compared with the lactobacillus plantarum (group C) before treatment (group B), the aloe extract (group D) further descends, the firmicutes door further ascends in rats after treatment, and the two groups have obvious differences; in the rats treated with lactobacillus plantarum aloe co-fermentation broth (group E), the decline of the firmicutes and the rise of the proteus are improved, and no significant difference from before treatment is observed.

In addition, the aloe extract (group D) also showed a decrease in phylum of phylum defenses in rats after treatment compared to pre-treatment (group B) with statistical differences. But the differences between the 5 groups of bacteroides are not significant.

Example 6 prospective, double blind, placebo random control experiment

1. Purpose of experiment

The food prepared in example 1 was evaluated for improvement of oral mucositis and nutritional status of patients undergoing radiotherapy and chemotherapy by a clinical double-blind placebo randomized controlled experiment.

2. Materials and methods

1. Study object

(1) Study population inclusion criteria

(1) Age 55-75 years old;

(2) receiving or being receiving radiotherapy (or/and) chemotherapy treatment in the last month;

(3) oral mucositis is diagnosed.

(4) No special disease history, no special medicine history and no special allergy history;

(2) Exclusion criteria

(1) Patients who cannot cooperate due to mental disorder and other diseases;

(2) patients who cannot be orally taken or who are allergic to lactobacillus plantarum.

(3) Drugs that are forbidden using clinical protocols, such as antibiotics, other drugs affecting the intestinal flora, etc., may be needed during clinical studies;

(4) patients taking anti-inflammatory drugs;

(3) Midway exit standard

(1) During the period of taking the product, medicines forbidden by using a clinical experiment scheme, such as antibiotics, other medicines affecting intestinal flora and the like, are needed to be taken during clinical study;

(2) the illness state needs to take other oral cavity membranitis treatment medicines or other lactobacillus plantarum products.

(4) Case of falling off

All patients who were screened for eligibility and entered the trial failed to complete the full observation of the clinical trial for a number of reasons were considered as abscission cases. Common reasons for shedding are:

(1) serious adverse events occur;

(2) the subject can not take the product according to the prescribed dosage, times (stopping taking for more than 2 times continuously) and treatment courses, and the curative effect is unknown because of no treatment;

(3) the patient automatically withdraws.

2. Finally, 85 patients with radiotherapy and chemotherapy treatment and diagnosed oral mucositis are selected, 20 patients in normal population are divided into two groups by adopting an envelope method.

Experiment 1 group (T1 group): it is taken orally with warm water (70 g, 3/day).

Experiment 2 (T2): other formulations (70 g, 3/day) were taken with warm water.

Control group (group C): placebo (70 x 3/day), taken with warm water.

Healthy group (group H) was also used as a normal population data control.

Wherein: the placebo is common milk powder, and the other formula powder is yapequanan

The product is a total nutrient formula powder, and the probiotics is Lactobacillus plantarum subspecies chen-02 > 10 ⁶ CFU, the bacterium is in the list of edible strains issued by the country, belongs to food-grade probiotics, the safety of the bacterium is fully ensured, and no obvious adverse reaction is found.

In this example, all eligible patients had signed informed consent by their own and family members and were approved by the ethics committee.

3. Study procedure and related examination

Before starting: baseline assessment of age, sex, body Mass Index (BMI) and oral mucositis scores of all patients according to a relevant scale, and collection of 10mL blood of all individuals for biochemical and routine examination; treatment period: patients were dosed with powder in groups and received follow-up.

Treatment period: the group took different powders and received follow-up.

Follow-up content: three groups of T1, T2 and C in the experiment are followed, and group H of normal group is only subjected to 1 evaluation, 1 blood collection and 1 fecal collection. Three groups T1, T2 and C in the experiment are subjected to total clinical impression (CGI) and oral mucositis scoring again in twenty days of continuous powder administration, and 10mL of blood of an individual is collected for biochemical and routine examination; adverse reactions reported by individuals were recorded according to the TESS scale throughout the treatment period.

4. Endpoint index

All follow-up patients except those who were unable to complete the study were at the end of follow-up at the end of the twenty-day following powder administration of the study.

The main measurement index is as follows:

1. oral mucosa inflammatory severity: evaluation criteria for oral mucositis using CTCAE5.0

2. Nutritional status: BMI, serum albumin

3. Side reaction: adverse event scale (Treatment Emergent Symptom Scale, TESS) was used.

5. Combination therapy

Avoiding mixing with other probiotics mixture.

6. Statistical analysis

1. Sample content estimation

The maximum sample size collected as much as possible.

2. Statistics and analysis of study data

SPSS 19.0 statistical software is adopted to analyze data such as baseline data, symptom improvement, fecal sequencing result and the like, t test is used for measuring p value of metering data, chi-square test is used for calculating x of counting data ² The value, P < 0.05, is statistically significant for the difference.

8. Experimental results

The whole experimental flow of this example is shown in fig. 5.

(1) And finally, the data statistics are included in the T1 group 29 people, the T2 group 28 people, the control group number is 28 people, and the healthy group 20 people.

Basic information for oral mucositis score and serum albumin, including patient basic information, is shown in table 4 below:

TABLE 4 basic information statistics of oral mucositis score and serum Albumin

There were no significant differences in baseline data between the experimental and control groups.

In addition, since the index related to metabolism of healthy people (group H) is not available, and the age is different from that of group C and groups T1 and T2, the baseline data of group H is not written in the table. During the trial, the follow-up lost C group n=2, T1 group n=1, T2 group n=2.

The detection result of the influence of the formula powder on the grading and nutrition conditions of the oral mucositis of the patient is shown in figure 2. Specific data are shown in table 5 below:

table 5 effects of formula powder on patient oral mucositis score and nutritional status detection results

The results show that: the oral mucositis score of the patients in the experimental group taking the formula powder is further reduced (3.3 vs 1.6, p < 0.05) compared with the patients in the control group, and the nutrition condition of the patients in the experimental group is better than that of the patients in the control group.

The increase in serum albumin levels, with a small increase in BMI (36.9 vs.40.0, p < 0.05), improved nutritional status and a significant difference between the two groups for each experimental group of patients taking the formulas compared to the control group of patients.

The total nutritional formula of this example is compared with other formulas of the prior art, see table 6 below:

table 6 comparison of the effects of prior art applications

Compared with the prior art, the application provided by the application can better improve the nutrition state of a patient, prevent malnutrition, prevent, relieve and treat oral mucositis and regulate the immune level of the patient.

Claims (10)

1. Lactobacillus plantarum (Lactobacillus plantarum) chen-02, characterized in that: the preservation number is CGMCC No.23397, and is preserved in China center for type culture Collection of microorganisms in 9 and 13 of 2021.

2. A probiotic preparation capable of inhibiting or alleviating or treating oral mucositis, characterized in that: the probiotic preparation is lactobacillus plantarum aloe co-fermentation liquid powder formed by fermenting lactobacillus plantarum chen-02 and aloe extract.

3. A probiotic formulation capable of inhibiting or alleviating or treating oral mucositis according to claim 2, characterized in that: the oral mucositis is oral mucositis caused by tumor chemoradiotherapy.

4. A probiotic formulation capable of inhibiting or alleviating or treating oral mucositis according to claim 3, characterized in that: the tumor is a head and neck cancer.

5. Use of a probiotic preparation according to claim 2 for the preparation of a product for inhibiting/alleviating or treating oral mucositis caused by tumor chemoradiotherapy.

6. A total nutritional formula comprising the probiotic formulation according to any one of claims 2 to 4, characterized in that it comprises the following components in parts by weight:

18-23 parts of protein;

17-24 parts of fat;

0.1 to 1 part of nucleotide;

3-6 parts of amino acid;

15-20 parts of carbohydrate;

dietary fiber 2-5 parts;

1-2 parts of compound vitamin;

5.5 to 6.5 portions of composite mineral matters;

3-5 parts of lactobacillus plantarum aloe co-fermentation liquid powder.

7. The total nutritional formula of claim 6, wherein: the protein source comprises one or more of soy protein isolate, milk protein, lactoferrin, hydrolyzed whey protein;

the fat comprises one or more of sunflower seed oil, corn oil, soybean oil, peanut oil and fish oil;

the nucleotide source comprises one or more of disodium 5 '-inosinate, disodium 5' -guanylate, disodium 5 '-uridylate and disodium 5' -cytidylate;

the amino acid comprises one or more of leucine, arginine and glutamine;

the dietary fiber source comprises one or more of fructo-oligosaccharide, inulin, resistant dextrin and pectin;

the compound vitamins comprise vitamin A, vitamin D, vitamin E, vitamin K, vitamin B2, vitamin B6, vitamin B12, vitamin C, biotin, nicotinamide, pantothenic acid and folic acid.

8. The total nutritional formula of claim 7, wherein: the fat comprises medium chain triglyceride and omega-3 unsaturated fatty acid, wherein the omega-3 unsaturated fatty acid comprises DHA and EPA, and the mass part ratio of DHA to EPA is 1:0.6-2.

9. The method of preparing the total nutrient formula powder as claimed in claim 6, wherein the method comprises the steps of:

(1) Weighing the components according to the proportion, adding 1000 parts by weight of pure water at 20-25 ℃ into a preparation tank, and adding the compound vitamin and the compound mineral substances into the pure water through a mixer under high-speed stirring to fully dissolve or disperse the compound vitamin and the compound mineral substances in the pure water; adding protein, fat, carbohydrate, dietary fiber, amino acid and nucleotide to uniformly mix; finally, adding food additives to dissolve or disperse the food additives in the pure water, and stirring and reacting to obtain mixed nutrient solution;

(2) Spray drying the mixed nutrient solution to obtain nutrient powder;

(3) Mixing 3-5 parts by weight of lactobacillus plantarum aloe co-fermentation liquid powder with nutrition powder to obtain the total nutrition formula powder suitable for rehabilitation of tumor patients.

10. The method of claim 9, wherein nitrogen is purged during the step (1).