CN114128787B

CN114128787B - Nostoc commune functional feed and preparation method thereof

Info

Publication number: CN114128787B
Application number: CN202111462076.2A
Authority: CN
Inventors: 李祖明
Original assignee: Beijing Union University
Current assignee: Beijing Union University
Priority date: 2021-12-02
Filing date: 2021-12-02
Publication date: 2022-07-08
Anticipated expiration: 2041-12-02
Also published as: CN114128787A

Abstract

The invention provides a preparation method of a watermifoil functional feed, which comprises the following steps: 1) cutting raw materials; 2) inoculating and stirring the materials; 3) bagging and sealing; 4) fermenting at constant temperature; 5) drying to prepare powder; 6) and mixing the live bacteria. The functional feed for the pulverum foxtail algae prepared by the invention is fermented by lactobacillus plantarum, so that the protein content, the fat content and the ash content in the pulverum foxtail algae are improved, and the cellulose content in the pulverum foxtail algae is reduced. The feed improves the nutritive value and palatability of the watermifoil and the watermifoil feed, and is convenient to store and transport; but also improves the structure of animal intestinal flora and promotes the generation of beneficial metabolites, and can replace 10 to 20 percent of conventional feed; and the problem of secondary pollution caused by the treatment of the culture wastewater by the watermifoil myriophyllum is solved, and the mutual unification of environmental protection and resource development and utilization is realized.

Description

Nostoc commune functional feed and preparation method thereof

Technical Field

The invention belongs to the technical field of feed processing, and relates to a watermifoil-rich watermifoil functional feed and a preparation method thereof.

Background

In recent years, global grain crisis is increasingly intensified, international grain price is rapidly increased, and feed grain accounts for 1/3 of the total grain yield in China, and 40.0% of cultivated land is used for feed production. With the rapid development of the livestock breeding industry in China, the shortage of protein and energy feed resources and overhigh breeding cost, the problem of the shortage of the feed resources always influences the sustainable development of the livestock breeding industry and becomes a great challenge in the livestock breeding industry in China, so that the development and utilization of non-grain feed are problems to be solved urgently in the current development of the livestock breeding industry.

Meanwhile, with the vigorous development of the breeding industry in China, the problem of environmental pollution is increasingly aggravated. The water pollution problem caused by the excessive discharge of the aquaculture wastewater seriously affects the living environment of people and even becomes a key factor for restricting the development of the aquaculture industry. The second pollution of the national census bulletin shows that the aquaculture emits 1000.53 million tons of Chemical Oxygen Demand (COD) and ammonia Nitrogen (NH) every year₄ ⁺-N)11.09, 59.63 and 11.97 of Total Nitrogen (TN) and phosphorus (TP), accounting for 93.8, 51.3, 42.1 and 56.5% of the total agricultural non-point source pollution. According to estimation, the annual excrement production amount of livestock and poultry breeding industry in China is 2.34 hundred million t, the urine production amount of livestock and poultry breeding industry in China is 1.63 hundred million t, only a few livestock and poultry breeding industries are subjected to pollution-free treatment and then discharged, and the random discharge of breeding wastewater causes serious eutrophication of surface water. Aiming at the current situation of pollution of the breeding industry in China, aiming at effectively solving the pollutionThe problem is that the development of high-efficiency and low-consumption ecological treatment technology is the trend of non-point source pollution treatment at present.

The Myriophyllum rubrum (Myriophyllum aquaticum) has high growth speed and super-strong nitrogen and phosphorus absorption capacity, and can convert the pollutants such as nitrogen, phosphorus and the like in the sewage into nutrient substances required by the Myriophyllum rubrum (Myriophyllum aquaticum) through roots, stems and leaves, thereby purifying the water quality environment. Meanwhile, the roots of the watery myriophyllum have the function of secreting oxygen, and can promote the growth of aquatic organisms. Research shows that the watermifoil wetland can absorb pure nitrogen by 1-2 t/hm every year²100-300 kg/hm of phosphorus pentoxide²The watery foxtail algae is obtained in time, the decomposed litters of the watery foxtail algae can release assimilated nitrogen and phosphorus into the environment to cause secondary pollution, and in addition, the watery foxtail algae has high-efficiency propagation and high phenotypic plasticity and can threaten the diversity of aquatic organisms in a growing place.

The watermifoil green foxtail algae has high nutritive value, the protein content is 17-22%, the protein content is equivalent to or higher than that of alfalfa, and the crude protein content of the watermifoil green foxtail algae is respectively 13.2%, 8.90% and 7.40% higher than that of common feed raw materials of corn, rice bran and wheat bran; rich amino acid content and relatively balanced composition, and can meet the growth requirement of livestock. The higher content of the crude fiber is the prominent characteristic in the nutrition component of the watermifoil green armyworm. Research shows that the content of heavy metals such as cadmium, chromium, lead, arsenic and the like in the dry matter of the pulchrous myriophyllum is far lower than the concentration requirement specified in the feed hygiene standard. The feed is the basis of poultry breeding industry and accounts for 65-70% of the total breeding cost. Therefore, the myriophyllum pratense has extremely high feed development and utilization values. However, the problem of poor palatability of the watermifoil as a feed still exists.

The microbial fermented feed is biological feed, such as silage, which is rich in nutrition and good in palatability and is formed by decomposing part of macromolecular substances such as polysaccharide, protein and fat through the fermentation action of microorganisms from raw materials such as plant agricultural and sideline products and generating micromolecular substances such as organic acid and soluble small peptide. The microbial fermentation feed has high use value and low price in animal production, and has wide market prospect. However, the acidity is increased and the activity of the fermenting microorganism is decreased with the progress of the microbial fermentation, and when the fermentation process is finished, the activity of the fermenting microorganism is basically lost, and the feed enters a stable storage period. When animals eat microbial fermented feed, the feed part is mainly utilized, and the microorganisms are difficult to utilize due to activity reduction and even loss. And the microbial fermented feed needs to keep higher water content in order to meet the fermentation conditions, needs a specific storage space and is not beneficial to conventional storage and transportation.

Disclosure of Invention

In order to solve the problems, the invention aims to provide a preparation method of the watery foxtail algae powder functional feed, the watery foxtail algae powder functional feed produced by the method improves the palatability of the watery foxtail algae powder feed, is beneficial to maintaining the balance of animal intestinal flora and promoting the production of beneficial metabolites, simultaneously solves the problem that the watery foxtail algae powder is easy to cause secondary pollution after the wastewater is treated, and realizes the mutual unification of environmental protection and resource development and utilization.

The invention also aims to provide the myriophyllum rubrum functional feed prepared by the method.

In order to realize the aim, the invention aims to provide a preparation method of a watermifoil functional feed, which comprises the following steps:

1) cutting raw materials: cleaning the harvested fresh powder myriophyllum viridis, removing mildewed and deteriorated parts, airing until the water content is 55-65%, and then cutting into small sections of 1 +/-0.5 cm;

2) inoculating and stirring materials: will be 1 × 10⁹～9×10⁹Uniformly spraying a bacterial solution of the Lactobacillus plantarum BW2013 with the concentration of CFU/mL on the watery green armyworm according to the inoculation amount of 5-15 w/w%, then adding sucrose according to 5-10 w/w% of the watery green armyworm weight, and uniformly mixing to obtain a mixture;

the sucrose is solid and is directly mixed with the watermifoil.

3) Bagging and sealing: filling the mixture into a sterile plastic package bag, exhausting air by using a small vacuum sealing machine, and sealing;

4) constant-temperature fermentation: placing the sealed sterile plastic packaging bag into a constant-temperature incubator at 30-35 ℃ for standing and fermenting for 4-10 days;

5) drying to prepare powder: putting the fermented mixture into a freeze dryer for freeze drying, collecting and grinding the mixture into powder to obtain powder of the myriophyllum viridis fermented freeze-dried powder;

6) and (3) viable bacteria mixing: adding 1 × 10 per gram of powder-myriophyllum viridis fermented lyophilized powder⁸～9×10⁸Adding a bacterial solution of Lactobacillus plantarum BW2013 into the CFU viable bacteria in proportion, and uniformly mixing to obtain the powder L.Lvjingtze functional feed.

As mentioned above, the classification of lactobacillus plantarum BW2013 was named: lactobacillus plantarum BW 2013; the preservation unit is as follows: china general microbiological culture Collection center, addresses are: western road No. 1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is as follows: 9/5 days 2014, with a preservation number of: CGMCC No. 9642.

As mentioned above, the preparation method of the lactobacillus plantarum BW2013 bacterial liquid comprises the following steps: inoculating lactobacillus plantarum BW2013 seed liquid into an MRS liquid culture medium according to the inoculation amount of 5-10% w/w, and performing static culture at 37 ℃ for 18 hours; centrifuging at 8000r/min for 15 min; collecting thalli and discarding supernatant; the thalli is washed twice by sterile PBS; resuspend to 1X 10 concentration with sterile PBS⁹～9×10⁹CFU/mL of bacterial liquid.

As mentioned above, the temperature in step 4) was 30 ℃ and fermentation was carried out for 5 d.

The invention also provides the pulverum foxtail algae functional feed prepared by the preparation method.

The lactobacillus plantarum BW2013 used in the fermentation disclosed by the invention not only has probiotic characteristics and a protective effect on the integrity of intestinal mucosa of colitis mice, but also has a good effect on the fermentation of plant agricultural and sideline products and other raw materials. Therefore, the lactobacillus plantarum BW2013 is applied to preparing the watery green watery foxtail algae functional feed, not only is favorable for feed production, but also is favorable for healthy growth of edible feed animals, and can achieve the effect of killing two birds with one stone.

According to the preparation method of the watermifoil powder-green watermifoil functional feed, provided by the invention, watermifoil powder-green watermifoil with poor palatability is subjected to lactobacillus plantarum fermentation, the protein content, the fat content and the ash content are increased through fermentation, the cellulose content in the watermifoil powder-green watermifoil is reduced, the palatability is improved, the moisture content of the watermifoil powder-green watermifoil feed is reduced through a freeze-drying technology, the growth and the propagation of harmful bacteria are inhibited, and the storage and the transportation are convenient; meanwhile, freeze-drying can reduce the number of live bacteria and influence the probiotic effect of the lactobacillus plantarum, so that a proper amount of live bacteria of the lactobacillus plantarum BW2013 are supplemented in the freeze-dried powder, the improvement of the intestinal flora structure and the generation of beneficial metabolites are facilitated, and the healthy growth of the intestinal tracts of animals is promoted.

The invention has the beneficial effects that:

the invention provides a powder foxtail algae functional feed and a preparation method thereof, the functional feed is prepared by uniformly mixing powder foxtail algae fermentation freeze-dried powder and lactobacillus plantarum BW2013 viable bacteria liquid, the powder foxtail algae functional feed prepared by the method is convenient to store and transport, has better palatability and rich nutrient components, ensures a certain viable bacteria quantity of probiotic lactobacillus plantarum, is beneficial to the probiotic function, improves the intestinal flora structure of animals and promotes the generation of beneficial metabolites, can replace 10-20% of conventional feed, ensures the resource utilization of the powder foxtail algae after the culture wastewater is treated, reduces the sewage treatment cost and the feed cost, avoids secondary pollution, is beneficial to the environmental protection and sustainable and healthy development of the culture industry, and realizes the mutual unification of the environmental protection and resource development and utilization.

Drawings

FIG. 1 is a colon tissue section of each group of DSS modeling mice.

FIG. 2 is a graph showing the results of body weight changes of mice fed with different feeds; wherein CK is a control group; n is a Chalcospira group; NG is a group of Nostoc fargesii and Lactobacillus plantarum; f is fermented powder myriophyllum viridis group; FG is a fermentation powder myriophyllum viridis + lactobacillus plantarum group.

FIG. 3 is a graph showing the variation of biochemical indicators in mice fed with different feeds; wherein, (a) ALP: alkaline phosphatase; (b) AST: glutamic-oxalacetic transaminase; (c) and (3) CRE: creatinine; (d) UREA: urea; (e) CHO: cholesterol; (f) GLU: blood sugar. CK: a control group; n: a watercress armstrong group; NG: the watermifoil and lactobacillus plantarum group; f: fermenting powder of myriophyllum viridis group; FG: the fermented powder of the armyworm-green algae and the lactobacillus plantarum group.

Fig. 4 is a graph showing the results of changes in the intestinal length of mice fed with different feeds, wherein CK: a control group; n: a watercress armstrong group; NG: the group consisting of ulexia glauca and lactobacillus plantarum; f: fermenting powder of myriophyllum viridis group; FG: the fermented powder of the armyworm-green algae and the lactobacillus plantarum group.

Fig. 5 is a distribution plot of intestinal flora at the phylum level in mice fed with different feeds, wherein CK: a control group; n: a watercress armstrong group; NG: the group consisting of ulexia glauca and lactobacillus plantarum; f: fermenting powder of Myriophyllum rubrum group; FG: the fermentation powder of myriophyllum viridis + lactobacillus plantarum group. The abscissa is the group and the ordinate relative abundance is the relative abundance.

Fig. 6 is a cluster thermogram analysis of intestinal flora at genus level in mice fed with different feeds, where CK: a control group; n: a watercress armstrong group; NG: the group consisting of ulexia glauca and lactobacillus plantarum; f: fermenting powder of myriophyllum viridis group; FG: the fermented powder of the armyworm-green algae and the lactobacillus plantarum group.

Detailed Description

The following detailed and complete description of the embodiments of the present invention is provided to enable those skilled in the art to more easily understand the advantages and features of the present invention, and to clearly and clearly define the scope of the present invention.

The probiotic preparation is a microbial preparation prepared from beneficial flora members or promoting substances thereof, and can supplement, adjust or maintain the micro-ecological balance in the gastrointestinal tract of animals, promote the gastrointestinal tract peristalsis, accelerate the digestion and absorption of nutrient substances, and enhance the immune function of animal organisms, thereby achieving the purposes of preventing and treating animal diseases, promoting the health of animals and improving the production performance of animals. The probiotic preparation as a safe and reliable feeding additive has profound significance for the healthy development of animal husbandry, and is widely applied to livestock and poultry breeding production practice at present.

Materials:

MRS solid medium was purchased from Beijing Dingguo Changsheng Biotechnology, Inc., produced by Beijing Luqiao technology, Inc., under product number CM 188.

MRS liquid medium was purchased from Beijing Dingguo Changsheng Biotechnology, Inc., produced by Beijing Luqiao technology, Inc., under product number CM 187.

3. Alpha-amylase was purchased from Changsheng biotechnology, Limited liability company, Beijing ancient cooking, under product number A109181-100G.

4. Pepsin obtained from the gastric mucosa of pigs was purchased from Changshan biotechnology, Limited liability company, Beijing Ding Guo, under product number EP 233-25G.

Pancreatin was purchased from Changshan biotechnology, Limited liability company, Beijing Ding Guo, under product number DH 355-4.

Caco2 cells were human colon cancer cells purchased from Beijing coordination cell resource center.

7. Green streptomycin mixed liquor, namely double-antibody purchasing Beijing Ding Guoshang biotechnology Limited liability company with product number GA3502-100ML

8. Fetal bovine serum was purchased from Changsheng biotechnology Limited liability company, Beijing ancient China, product number S-FBS-050.

DMEM culture solution is purchased from Changsheng biotechnology Limited liability company of Beijing Ding, and has a product number of GD3103-500 ml.

10. Dextran Sulfate Sodium (DSS) was purchased from the beijing dingguosheng biotechnology limited liability company, product number 0216011080.

11. Phosphate buffered saline formulation (PBS): 8g NaCl, 0.2g KCl, 1.44g Na₂HPO₄,0.24g KH₂PO₄Diluting to 1L, pH7.4, sterilizing at 121 deg.C for 30min to obtain sterile PBS.

12. The ox bile is purchased from Changsheng biotechnology Limited liability company in Beijing ancient cooking, and is numbered S0900000.

13. The trypsin digestive juice is purchased from Changsheng biotechnology Limited liability company of Beijing Ding Guo, and has the product number of GP3108-100ML

14. Myeloperoxidase (MPO) kit, purchased from Changsheng biotechnology Limited liability company, Beijing Ding, under product number A044.

15. Mouse D-lactic acid ELISA kit purchased from Beijing Ding Guoshang biotechnology Limited liability company, product number ZK-4868

16. Diamine oxidase ELISA kit purchased from Beijing Dingguo Changsheng biotechnology Limited liability company, product number XFSW3303

FITC Rapid labeling kit, product number JK-R4846, purchased from Changsheng Biotechnology Limited liability company of Beijing ancient cooking

18. Mouse IL-1. beta. ELISA test kit, Shanghai Sigma reagent company, product number RAB 0275.

19. Mouse IL-6ELISA test kit, Shanghai Sigma reagent company, product number RAB 0309.

20. Mouse IL-17ELISA test kit, Shanghai Sigma reagent company, product number RAB 0263.

21. Mouse TNF-alpha ELISA test kit, Shanghai Sigma reagent company, product number RAB 0477.

22. Mouse IL-10ELISA test kit, Shanghai Sigma reagent company, product number RAB 0244.

BALB/c mice, 6 weeks old, SPF grade, 18-22 g, purchased from Wintonlihua laboratory animals technologies, Inc., Beijing

24. The lactobacillus-free mouse feed is purchased from Shanghai Si Laike laboratory animal, Inc., and is sterilized by radiation before feeding.

ICR male mice, 4 weeks old, purchased from experimental animal technology ltd, vindolizhihua, beijing.

The lactobacillus plantarum BW2013 used in the invention is derived from fermented Chinese cabbage, is preserved and given a gift by white Shihui researchers in the ecological environment research center of Chinese academy of sciences, and is authorized to be used.

The classification and the name of the lactobacillus plantarum BW2013 are as follows: lactobacillus plantarum BW 2013; the preservation unit is as follows: china general microbiological culture Collection center, addresses are: beijing, Haoyang district, Xilu No. 1, Ministry of microbiology, China academy of sciences, 3; the preservation date is as follows: 9/5 days 2014, with a preservation number of: CGMCC No. 9642.

The invention provides a method for culturing lactobacillus plantarum BW2013 stage by stage, which comprises the following steps:

activating strains: selecting Lactobacillus plantarum BW2013 preserved on the inclined plane, streaking and separating on an MRS solid culture medium, and culturing at 37 ℃ for 48-72h to obtain an activated single colony.

Preparing a seed solution: and inoculating the activated single colony in 50mL of MRS liquid culture medium, and performing static culture at 37 ℃ for 20h to obtain seed liquid.

Preparing fermentation liquor: inoculating the seed solution into MRS liquid culture medium according to the inoculation amount of 10% (w/w), performing static culture at 37 ℃ for 18h, and calculating the bacterial concentration by a plate counting method.

Preparing bacterial liquid: centrifuging the fermentation liquor at 8000r/min for 15 min; collecting thalli and discarding supernatant; the thalli is washed twice by sterile PBS; resuspend with sterile PBS and calculate the concentration of the bacterial suspension by plate counting.

Example 1: probiotic properties of Lactobacillus plantarum BW2013

1. In vitro simulation gastrointestinal tract digestion experiment

1) Preparation of artificial saliva

6.2g NaCl，2.2g KCl，0.22g CaCl₂，1.2g NaHCO₃Dissolving in 1L deionized water, and autoclaving. Cooling to about 20 deg.C, adding 3.0g/L alpha-amylase, adjusting pH to 6.9, and filtering with 0.22 μm microporous membrane for sterilization.

2) Preparation of artificial gastric juice

3.0g NaCl，1.1g KCl，0.15g CaCl₂，0.60g NaHCO₃Preparing into 1L solution, and sterilizing with high pressure steam; cooling to about 20 deg.C, adding pepsin 3.0g/L obtained from pig gastric mucosa, adjusting pH to 2.0 with 10N HCl, and filtering with 0.22 micrometer microporous membrane for sterilization.

3) Preparation of artificial small intestine liquid

5.0g NaCl，0.60g KCl，0.30g CaCl₂，0.60g NaHCO₃Dissolving in 1L deionized water, and autoclaving. Cooling to about 20 deg.C, adding 3.0g/L bilis Bovina, 1.0g/L pancreatin and 3.0g/L pepsin, and filtering with 0.22 micrometer microporous membrane for sterilization.

4) Digestion process

The entire digestion process was carried out at 37 ℃ and the entire process was maintained at 50rpm/min to simulate gastrointestinal motility. 5mL of artificial saliva (pH6.9) was added to 2.34X 10¹⁰Suspending the bacterial liquid of the Lactobacillus plantarum BW2013 of CFU/mL for 5min, centrifuging for 15min at the rotating speed of 8000rpm/min to enable the bacterial bodies to sink, suspending with sterile water, centrifuging again under the same conditions, and cleaning with sterile water twice; the washed cells were resuspended in 10mL of gastric juice (adjusted to final pH3.0) and incubated for 2 h. Centrifuging at 8000rpm/min for 15min to precipitate thallus, suspending with sterile water, centrifuging under the same conditions, and cleaning with sterile water twice; the washed cells were further suspended in 10mL of small intestine solution (1M NaHCO3 was added to adjust the total pH to 7.6) and incubated for 2 h.

And finally, gradually diluting the bacterial suspension by 10 times of phosphate buffer solution, coating the bacterial suspension on an MRS solid culture medium plate, and culturing at 37 ℃ for 48-72h for counting. The experimental result shows that 2.34 is multiplied by 10⁹The viable bacteria concentration of the CFU/mL lactobacillus plantarum BW2013 after in-vitro simulated digestion is 6.78 multiplied by 10⁷CFU/mL, survival rate of 2.90%.

Caco2 cell adhesion assay

The Caco2 cells were cultured in DMEM medium containing diabody (streptomycin cocktail) and serum (fetal bovine serum). After the cells grew well (the culture dish was full), the cells were digested with trypsin digest for about 1min at room temperature. Counting with a hemocytometer, diluting with DMEM to a concentration of 5X 10⁵one/mL. 1mL of Caco2 cells suspension was added dropwise to a 6-well plate until a monolayer of cells grew. After washing the cells with sterile PBS, the Lactobacillus plantarum BW2013 biomass was diluted to 2.34X 10 with DMEM without double antibody and serum⁹CFU/mL, 1mL diluted bacterial suspension and 1mL DMEM with 20% fetal bovine serum in a petri dish with 5% CO at 37₂Incubate at-95% for 1 h. Washed 3 times with PBS and unadhered fines washed awayAnd (3) adding a proper amount of pancreatin into each hole for 5min to enable Caco2 cells to shed, then diluting the Caco2 cells by using sterile PBS, and counting the concentration of the thalli before and after adhesion by using a dilution plating method.

After counting, 2.34 is multiplied by 10⁹The viable bacteria concentration of the CFU/mL lactobacillus plantarum BW2013 after cell adhesion experiment is 2.40 multiplied by 10⁷The adhesion rate was 1.03%.

As can be seen from the in vitro gastrointestinal tract digestion simulation experiment and the Caco2 cell adhesion experiment, the lactobacillus plantarum BW2013 has the probiotic property, has certain acid resistance and bile salt resistance, can enter the intestinal tract through the gastrointestinal tract digestive system, and can colonize in the intestinal tract, so that the probiotic effect is exerted. It can be seen that lactobacillus plantarum BW2013 can be used as a probiotic.

Example 2 probiotic effect of Lactobacillus plantarum BW2013

Preparation of DSS: preparing a dextran sodium sulfate solution with the mass fraction of 2.5%, wherein the solvent is phosphate buffer solution.

2. Preparation of lactobacillus plantarum BW2013 live bacterial suspension: inoculating Lactobacillus plantarum BW2013 into 500ml of MRS liquid culture medium according to the inoculation amount of 10% (w/w), standing and culturing at 37 ℃ for 18h, and centrifuging at 8000r/min for 15min to collect thalli; the bacterial pellets were resuspended in phosphate buffer and prepared to 2.34X 10⁸CFU/ml low dose bacterial suspension L (DL group), 2.34X 10⁹CFU/ml medium dose bacterial suspension M (DM group), 2.34X 10¹⁰CFU/ml high-dose bacterial suspension H (DH group), NC (normal control group) (sterile PBS), D group (model group) successfully molded by DSS, and PC (mesalamine positive control group).

3. Molding: after 72 BALB/c mice were fed with the Lactobacillus-free mouse feed for one week, they were randomly assigned to 6 groups of 12 mice each. The dose of lactobacillus plantarum BW2013 per mouse was 400 μ L/d, and the detailed grouping is shown in table 1.

TABLE 1 animal Experimental groups

The NC group is a normal control group, and the D group is a model group which is successfully molded by DSS; the PC group is a positive control group, and DL, DM and DH are respectively a low, medium and high dose group of lactobacillus plantarum intragastric administration.

Weighing 1 time per day during molding, weighing once per week during intragastric administration, and continuously administrating for 28 d. During the experiment the experimental mice were fed freely, with a 12 hour light/dark cycle.

DSS model test and biochemical index measurement

DSS is a modeling reagent commonly used for animal experiments and medical researches on colitis, and mice successfully modeled can have the phenomena of weight loss, splenomegaly, diarrhea, loose stool or hematochezia and the like; secondly, a higher disease activity index indicates a higher degree of colitis damage. Therefore, in a lactobacillus plantarum intervened colitis mouse test model, when the mouse weight is obviously increased, the spleen quality is reduced and the disease activity index is reduced compared with a control group, the lactobacillus plantarum has obvious improvement effect on the damage of colitis mice. The body weight change of each group of mice after 28 days of feeding is shown in Table 2 below.

Table 2 mouse body weight changes

Note: significant p <0.05 compared to group D, very significant p <0.01 compared to group D

As can be seen from table 2, the difference between the group D and the group NC is significant (p <0.05), indicating that 2.5% of DSS successfully constructed the mouse model of acute colitis. Compared with the D (the weight is reduced by 2.93g), the weight reduction of the mice in the low, medium and high dose groups (the weight is reduced by 2.49g, 1.71g and 1.88g respectively) by the lactobacillus plantarum is obvious. Compared with the model group, the weight reduction degree of the mice in the medium-dose group is remarkably slowed down, and the weight reduction degree of the mice in the high-dose group is greatly different. The model is proved to have adverse effect on the growth of the mice, wherein the inhibition effect of the lactobacillus plantarum middle and high dose groups on the weight reduction of the colitis mice has obvious effect.

Taking the experimental mice continuously administrated for 28 days in the steps, weighing after last intragastric administration, fasting for 12h, taking blood from eyeballs and killing all the mice by using a cervical dislocation method. Collecting blood by heparin sodium anticoagulation, centrifuging at 3000r/min for 10min, and preserving supernatant at 0 deg.C for later use. Dissecting a mouse, taking out the colon, the caecum and the caecum content, respectively weighing 0.1-0.2 g of colon tissue, adding 9 times of physiological saline, preparing 10% tissue homogenate in a homogenizer, centrifuging at 3000r/min for 10min, and respectively taking supernatant fluid and preserving at low temperature of 0 ℃ for later use.

The Myeloperoxidase (MPO) activity, the Fluorescein Isothiocyanate (FITC) fluorescence intensity, the D-lactic acid and the diamine oxidase (DAO) activity were measured by biochemical kits, respectively. The results are shown in tables 3 and 4.

MPO activity, Myeloperoxidase (MPO), is released into the extracellular fluid under inflammatory conditions, causing an increase in the level of MPO in tissues, and thus the level of inflammatory infiltration of neutrophils can be observed by measuring the level of MPO.

FITC fluorescence intensity, D-lactic acid level and DAO activity in mouse serum are indexes related to permeability of intestinal mucosa of mice, and the recovery degree of colitis injured mice and the effect of lactobacillus plantarum on the integrity of intestinal mucosa of mice can be obtained through the change of the indexes.

TABLE 3 Effect of Lactobacillus plantarum BW2013 on colitis mouse Disease Activity Index (DAI), spleen weight, Colon Length, Colon tissue MPO Activity, hemoglobin content

As can be seen from Table 3, the DAI value of group D was 8.21, spleen was enlarged, the length of colon was decreased by 40.34%, and the colon tissue was congested and enlarged compared with that of group NC; the hemoglobin concentration is reduced by 22.8 percent, which indicates that the acute colitis mouse model is successfully made.

The spleen weight and DAI value of the mice of the low, medium and high dose group and the positive group (intragastric mesalamine) of the intragastric lactobacillus plantarum BW2013 are respectively reduced by 37.4-55.34 percent and 69.8-89.3 percent, and are obviously reduced. Compared with a model group, the colon length and the hemoglobin concentration are respectively improved by 42.88-61.24 percent and 14.81-27.22 percent, and are obviously improved. Compared with a model control group, the MPO activity level is reduced by 36.17-59.95 percent and is obviously reduced. It can be seen that, in addition to the significant difference in MPO activity in the low dose group, the low, medium and high dose groups were very significantly different in colitis mouse Disease Activity Index (DAI), spleen weight, colon length and hemoglobin content, compared to the model group. The lactobacillus plantarum BW2013 has an obvious improvement effect on the symptoms of the acute colitis mouse.

TABLE 4 Effect of Lactobacillus plantarum BW2013 on FITC-dextran fluorescence intensity, D-lactic acid and DAO Activity in colitis mice

As can be seen from Table 4, compared with NC group, FITC fluorescence intensity, D-lactic acid level and DAO activity in serum of mice in group D were improved by 441.7%, 42.2% and 39.01%, indicating that colitis mice are successfully modeled.

The FITC fluorescence intensity, the D-lactic acid level and the DAO activity in serum of the mice of the low, medium and high dose group and the positive group (intragastric mesalamine) of the Lactobacillus plantarum BW2013 are respectively reduced by 30.69-54.77%, 17.35-29.45% and 13.87-26.13% compared with the model group, and are obviously reduced. As can be seen, FITC-dextran fluorescence intensity, D-lactic acid and DAO activity of the colitis mice in the low dose group were significantly reduced, and those in the medium and high dose groups were significantly reduced, compared with the model group. This shows that lactobacillus plantarum BW2013 has good effects in improving colitis mouse intestinal mucosa injury, reducing intestinal permeability and enhancing intestinal mucosa integrity. With the increase of the gavage concentration of lactobacillus plantarum BW2013, the FITC fluorescence intensity, the D-lactic acid level and the DAO activity in the serum of the mice show obvious differences, wherein the high-dose group of mice has better inflammation improvement effect and better growth signs compared with other groups; this result also coincides with the trend of the positive test results, and is closer.

The expression levels of IL-1. beta., IL-6, IL-17, TNF-. alpha.and IL-10 were measured by a biochemical kit, respectively, and the results are shown in Table 5.

TABLE 5 Effect of Lactobacillus plantarum BW2013 on Colon tissue inflammatory factor levels in colitis mice

As can be seen from Table 5, the colon tissue of mice in group D showed increased expression levels of IL-1 β, IL-6, IL-17 and TNF- α in 210.73%, 490.26%, 146.72% and 369.21% as compared to that in group NC, indicating that the molding of colitis mice was successful.

The levels of IL-1 beta, IL-6, IL-17 and TNF-alpha in colon tissues of mice in the low, medium and high dose groups and the positive group (intragastric mesalazine) of the lactobacillus plantarum BW2013 are respectively reduced by 19.14-36.21%, 13.59-44.02%, 26.04-33.62% and 27.92-57.36%, and the expression levels of IL-1 beta, IL-6, IL-17 and TNF-alpha are remarkably reduced (p is less than 0.001). And the expression level of the anti-inflammatory factor IL-10 in colon tissues in the lactobacillus plantarum BW2013 low, medium and high dose groups is improved by 20.31-32.09% compared with that in the model control group. Therefore, the inflammation degree of the colon tissues of the mice in the lactobacillus plantarum BW2013 treatment group and the positive control group has an extremely obvious improvement effect; the mucosa abscission degree and the mucosa integrity of the colon tissue are greatly improved. The colon tissue was sectioned for visualization (using hematoxylin-eosin staining) as shown in fig. 1. Compared with the NC group, the group D mice have serious colonic inflammatory cell infiltration, intestinal mucosa shedding and incomplete crypt structures; the colon morphology of the lactobacillus plantarum treatment groups (DL group, DM group and DH group) is recovered to a certain degree, inflammatory cells are partially infiltrated, and the damage of the colon mucosa is improved. The colon morphology of the PC group is also recovered to a certain degree, inflammatory cells are partially infiltrated, and the damage of the colon mucosa is also improved.

From tables 2 to 5 and fig. 1, it is demonstrated that lactobacillus plantarum 2013 has a very significant improvement effect on the degree of inflammation of colon tissues of mice, and can relieve colitis damage to colon tissues by protecting the integrity of intestinal mucosa of animals.

Example 3: preparation of powder myriophyllum clavatum functional feed

1. Preparation of the feed

1) Cutting of raw materials

The harvested fresh Myriophyllum viridis (Myriophyllum aquaticum) is cleaned, the samples are guaranteed to be free of mildew and deterioration, the cleaned samples are aired, the water content of the cleaned samples is reduced to 55-65%, and then the cleaned samples are cut into small sections of 1 +/-0.5 cm.

2) Inoculation mixing material

Will be 1 × 10⁹Uniformly spraying the bacterial liquid of the Lactobacillus plantarum BW2013 with the concentration of CFU/mL on the aired and cut pulverum vulgare according to the inoculation amount of 10% (w/w), then adding 6% (w/w) of sucrose solid, uniformly mixing, and preparing into a mixtureAnd (5) mixing materials.

3) Packaging and sealing

And filling the mixture into a sterile plastic package bag, exhausting air by using a small vacuum sealing machine, and sealing.

Filling the mixture into sterile plastic bags according to the bagging amount of 500g of the mixture per bag, exhausting air by using a small vacuum device, and sealing. The bagging amount is determined according to the size of the sterile plastic packaging bag, 500g of sterile plastic packaging bag can be contained in the sterile plastic packaging bag, air can be discharged after bagging and sealing is carried out, and an anaerobic fermentation environment is created for lactobacillus plantarum BW 2013.

4) Constant temperature fermentation

In order to examine the influence of different temperatures on the fermentation of the watermifoil and green foxtail algae, different air-exhaust sealed sterile plastic packaging bags filled with the mixture are grouped. Wherein, divide into 30 ℃ and 35 ℃ according to the temperature group, and each group is put into the constant temperature incubator respectively and is stood and fermented 1, 3, 5, 7, 10 days, is 10 processing groups: fermenting at 30 deg.C for 1d, fermenting at 30 deg.C for 3d, fermenting at 30 deg.C for 5d, fermenting at 30 deg.C for 7d, fermenting at 30 deg.C for 10d, fermenting at 35 deg.C for 1d, fermenting at 35 deg.C for 3d, fermenting at 35 deg.C for 5d, fermenting at 35 deg.C for 7d, and fermenting at 35 deg.C for 10 d.

5) Drying to obtain powder

And putting the fermented samples of each treatment group which are divided according to the conditions into a freeze dryer for freeze drying, and collecting and grinding the samples to obtain the freeze-dried fermented powder of the pulverum vulgare.

6) Live bacteria blend

Before use, the content of the fermented freeze-dried powder of the myriophyllum viridis in a ratio of 1.6 multiplied by 10 per gram of the fermented freeze-dried powder⁸Adding bacterial liquid of lactobacillus plantarum BW2013 into the CFU lactobacillus plantarum BW2013 viable bacteria in proportion, and uniformly mixing to obtain the watery foxtail algae powder functional feed.

Example 4: determination of fermentation index of feed

After the step of constant-temperature fermentation 4) of example 1 was completed, each treatment group was sampled by quartering method based on the amount of sample actually required for each index measurement, and the sensory evaluation was performed to measure fermentation indexes such as pH, organic acid (acetic acid, propionic acid, and lactic acid), dry matter, crude protein content, and the like.

1) Sensory evaluation

According to the evaluation criteria (trial) of silage quality promulgated by the Ministry of agriculture (J.) Chinese feeds 1996,000(021): 5-7), the fermented product of the Chalcore Vulva (equivalent to silage) is subjected to sensory evaluation from the aspects of smell, color, mildew and texture.

From the sense, the stem and leaf structures of the fermented powder of the myriophyllum viridis of each treatment group are basically intact, no mildew occurs, the color is yellow green, no hand sticking phenomenon occurs, and the fermented powder has good texture and obvious acid fragrance, which indicates that the fermentation is successful.

2) Determination of pH value

The pH value was measured with a pH meter according to the method of section 5.1.3.2 of GB5009.237-2016, national food safety standards for determination of pH value of food, and the quality of fermentation samples was evaluated.

3) Determination of organic acid content

Respectively preparing leaching liquor from the samples of each treatment group, and determining the content of organic acid, wherein the specific processes and parameters are as follows:

preparing a leaching solution: adding 180mL of sterile water into 20g of fermentation sample, mixing uniformly, leaching in a refrigerator at 4 ℃ for 24h, filtering for 1 time by using 4 layers of nylon cloth, and filtering for 1 time by using filter paper to obtain filtrate, namely the leaching solution. And then measuring the content of organic acids (acetic acid, propionic acid and lactic acid) in the leaching solution by adopting a high performance liquid chromatography method to evaluate the quality of the fermentation sample.

And (3) measuring the content of organic acid: a chromatographic column: X-Aqua (250 mm. times.4.6 nm, 5 μm); a detector: a UV detector; detection wavelength: 220 nm; column temperature: 30 ℃; flow rate: 0.8 mL/min; mobile phase A: 0.1% trifluoroacetic acid; mobile phase B: methanol (elution condition: 5% -17.5%, 0-10 min); sample introduction amount: 10 μ L.

4) Determination of the Dry matter content

And (3) respectively taking 100g of samples from each treatment group, putting the samples into a 65-DEG C oven, drying the samples until the weight of the samples is constant, and measuring the dry matter content of the samples.

5) Crude protein content determination

And (3) determining the crude protein of the dried sample by using a full-automatic protein determinator according to a Kjeldahl method.

pH, dry matter, crude protein, each during fermentationOrganic acid content and ammonia Nitrogen (NH)₃-N/N) are shown in Table 6.

TABLE 6 fermentation quality of Chaetomium globosum

In the table, a, b, c, d and e are marked by significant difference letters, and if one letter with the same mark is marked, the difference is not significant, and if the letter with different marks is marked, the difference is significant.

As can be seen from Table 6, in the fermentation process of the Nostoc commune, the pH value shows the trend of firstly decreasing and then increasing and then tending to be stable along with the increase of the fermentation time, and the pH value starts to rise again after 5d and reaches the minimum value of 4.11 after the fermentation at 30 ℃, and the pH value does not change obviously any more after 5d and tends to be stable after 7 d. The lactobacillus plantarum BW2013 can utilize sugar and sucrose in the watery foxtail algae and reduce the pH value by secreting organic acid, when the pH value is reduced to be below 4.2, most bacteria stop breeding, and the lactobacillus plantarum BW2013 possibly dominates the survival. The change of each organic acid is different, the content of lactic acid increases along with the fermentation time, the lactic acid content reaches the maximum value at the 7 th fermentation time and the 10 th fermentation time at the 30 ℃ and the 35 ℃ and has no significant difference with the lactic acid content at the 5 th fermentation time at the 30 ℃; acetic acid content continues to increase with fermentation time; the propionic acid content reaches the maximum value at 30 ℃ fermentation 10d, but has no significant difference with the propionic acid content at 30 ℃ fermentation 5d, and reaches the maximum value at 35 ℃ fermentation 7 d. The increase in the content of each organic acid lowers the pH to inhibit the growth of some harmful microorganisms, and especially the production of lactic acid improves the flavor of the fermented sample and increases the nutritional value. The dry matter content reached a maximum at 30 ℃ fermentation 5d, whereas the crude protein content reached a maximum at 30 ℃ fermentation 5 days and 7 d. The contents of dry matter and crude protein are in positive correlation with the nutritional value of the fermented feed, and the higher content of dry matter and crude protein indicates the nutritional loss of the fermented feedThe loss is less. NH of 35 ℃ fermentation samples₃The value of-N/N increases with the fermentation time, and becomes stable after 5 days, NH of the sample fermented at 30 DEG C₃the-N/N value is increased along with the fermentation time, the change difference of 3 rd to 7 th days is not obvious, and the value is continuously increased after the change, generally, the ammonia Nitrogen (NH) in the fermented feed is considered₃The value of-N)/total nitrogen (N) should be less than 7 if NH₃An N/N value higher than 7 indicates that the amount of protein decomposed by the microorganisms during fermentation is relatively large, resulting in an excessively high protein loss in the fermented sample. In combination with the above data and sensory evaluation, the preferred fermentation temperature is 30 ℃ and the preferred fermentation time is 5 days.

Example 5: determination of nutrient content of feed

The content of 6 basic nutrient components is determined by selecting fermented powder myriophyllum rubrum and unfermented powder myriophyllum rubrum under the better fermentation condition (the fermentation temperature is 30 ℃ and the fermentation time is 5 d). The ash content is measured according to GB 5009.4-2016 (first method); the content of the crude fiber is measured according to GB/T5009.10-2003; the protein content is determined by adopting a trace Kjeldahl method according to GB 5009.5-2016 (first method) of determination of protein in food; the content of crude fat is measured by Soxhlet extraction according to GB/T5009.6-2016 (second method); the phosphorus content is measured according to GB 5009.87-2016 (first method); the calcium content was measured according to GB 5009.268-2016 (second method), the results of which are shown in Table 7.

TABLE 7 nutritional ingredients of Nostoc commune and its fermentation products

Nutrient composition	All-grass of Nostoc commune	Fermented powder of watermifoil	Trend of change	P value
					Crude protein	18.1±1.20g/100g	20.5±0.80g/100g	↑13.3％	0.045*
Crude fat	3.00±0.14g/100g	4.60±0.22g/100g	↑53.3％	0.000**
					Ash content	12.4±0.90g/100g	15.9±0.72g/100g	↑28.2％	0.006**
Coarse fiber	13.2±0.80g/100g	11.2±0.70g/100g	↓15.2％	0.031*
					Phosphorus (P)	653±6.00mg/100g	657±4.00mg/100g	↑<0.01％	0.391
Calcium carbonate	2.07±0.03g/100g	2.09±0.04g/100g	↑<0.01％	0.510

Wherein denotes a p-value <0.05, significant differences; indicates p value <0.01, with very significant differences. Carrying out statistical test analysis by using an independent sample T test method to obtain a P value; the variation trend refers to the variation trend of the nutrient components of the fermented powder myriophyllum viridis compared with the unfermented powder myriophyllum viridis; the method comprises fermenting Chaetomium globosum under a better fermentation condition and fermenting Chaetomium globosum, repeating the determination method for 3 times, and taking the average value of the values in the second row and the third row of the table as +/-standard deviation.

As can be seen from Table 2, the fermented powder of Foliumisalanx spicata has a crude protein content of 20.5 + -0.80 g/100g, a lipid content of 4.60 + -0.22 g/100g, and an ash content of 15.9 + -0.72 g/100g, which are respectively significantly higher than those of Foliumisalanx spicata. However, the content of crude fiber of the myriophyllum pratense after fermentation is 11.2 +/-0.70 g/100g, which is reduced by 15.2 percent, and the difference is obvious. Since the crude fiber is used as a nutrient source for microbial growth. Crude fiber content is indirectly related to animal digestibility and feed intake. After fermenting the watermifoil, the contents of phosphorus and calcium are slightly increased but are not obvious. Therefore, the fermentation can improve the nutritional quality of the watermifoil green watermifoil.

Example 6: feeding effect of powder green myriophyllum functional feed

1. Feed preparation

According to the nutrient standard of the experimental mouse maintenance feed of GB14924.3-2010 'nutrient components of the experimental animal compound feed', the measured nutrient components of the Pink-Vulva and the fermented Pink-Vulva are combined, and the proper addition proportion is calculated to achieve the same nutrient standard and heat as the control group maintenance feed. In this example, two SPF-level mouse feeds prepared by adding 10% of Pink Vulva virescens or 10% of Spodoxa virens fermented to a conventional SPF-level mouse feed manufactured by Beijing Huafukang Biotechnology GmbH were used. Fermenting the pulverum vulgare pers at 30 deg.C for 5 days.

2. Experimental protocol

4 week old ICR male mice 60, randomly divided into 5 groups, each group of 12. The animal house was acclimatized for 1 week, and then the formal experiment was started. The details of the 5 components are as follows:

control group (CK): feeding a maintenance feed for a common mouse;

myriophyllum glauca group (N): feeding fodder containing 10% of unfermented powder of Selaginella vulgaris;

watermifoil myriophyllum + lactobacillus plantarum group (NG): combining the probiotic properties of Lactobacillus plantarum BW2013 of example 2, feeding with 10% addition of unfermented powder Myriophyllum rubrum + 1.6X 10⁸Feed of CFU/g lactobacillus plantarum BW2013 bacterial liquid

Fermented powder myriophyllum viridis group (F): feeding fodder containing 10% fermented powder of Selaginella vulgaris;

fermented powder myriophyllum clavatum + lactobacillus plantarum group (FG): feeding watermifoil and 1.6 × 10⁸CFU/g feed of Lactobacillus plantarum BW 2013.

2. Feeding management

Feeding conditions are as follows: the SPF animal house is free to eat and drink water with the temperature of 20-22 ℃, the humidity of 40-60% and 12h alternating light and shade. Continuously feeding for 5 weeks.

3. Body weight measurement

The body weight of the mice was measured and recorded every week, and the results of the body weight change of the mice are shown in fig. 2.

As can be seen from fig. 2, the body weight of each group of mice steadily increased during the 5-week feeding, and there was no significant difference in the body weight of the mice between the groups before the 4-week (including the 4-week); at week 5, the body weight of the mice in group F was significantly increased (4.86% increase) compared to the body weight of the mice in group N, and the differences between the remaining groups were insignificant.

This indicates that feeding the feed containing watermifoil has no adverse effect on the body weight of mice compared to the ordinary feed, indicating that watermifoil can replace part of the ordinary feed. The weight of the mice in the F group is obviously increased compared with the weight of the mice in the N group, which shows that the weight of the mice fed with the feed containing the fermented powder of the myriophyllum viridis is more beneficial to the weight increase of the mice compared with the feed containing the powder of the myriophyllum viridis. The said powder or fermented powder can be used to replace partial feed.

4. Biochemical index determination

Before killing the mouse, the eyeball is picked to draw blood, the blood is collected in a 1.5mL centrifuge tube, the centrifuge is carried out for 15min at the temperature of 4 ℃ at the speed of 3000r/min, and the supernatant is taken and put in a new 1.5mL centrifuge tube, namely the serum. The results of measuring the contents of aspartate Aminotransferase (AST), alkaline phosphatase (ALP), UREA (UREA), Creatinine (CRE), Cholesterol (CHO), and blood Glucose (GLU) by a full-automatic biochemical analyzer are shown in FIG. 3.

As can be seen from fig. 3, the results of the liver function-related index detection in the blood biochemical indices of the mice in each group are shown in fig. 3(a) and fig. 3(b), and there is no significant difference between the AST and ALP indices in each group. The results of the renal function-related index tests are shown in fig. 3(c) and 3(d), and there was no significant difference between UREA and CRE among the groups.

The two indices GLU, CHO represent the blood glucose and cholesterol levels of the mice, respectively. As can be seen from fig. 3(e) and 3(F), there was no significant difference between the groups of blood cholesterol levels, and the blood glucose levels of the NG group and F group mice were significantly lower than that of the CK group.

From the above results, it can be seen that feeding the feed containing the myriophyllum pulchrum has no adverse effect on the blood biochemical indexes of the mice compared with the common feed, and that feeding the feed added with unfermented myriophyllum pulchrum and lactobacillus plantarum BW2013 or feeding the fermented powder myriophyllum pulchrum may have the effect of reducing blood sugar compared with feeding the common feed. The said powder or fermented powder can be used to replace partial feed.

5. Determination of intestinal Length

The length of the intestinal tract and the length of the small intestine of the mouse can reflect the digestion and absorption capacity of the intestinal tract to nutrients. After the mouse was dissected, the intestinal tract of the mouse was removed, and the length of the entire intestinal tract and small intestine were measured, and the results are shown in fig. 4.

As can be seen from fig. 4, there was no significant difference between the total intestinal length and the small intestinal length of each group of mice, and the small intestinal lengths of the NG group and the FG group were slightly increased by 0.64% and 1.23%, respectively, compared to the CK group.

Compared with the common feed, the feed containing the watermifoil has no adverse effect on the digestion and absorption capacity of the nutrient substances of the mice when being fed, and the watermifoil can replace part of the feed; the feed added with unfermented powder of the myriophyllum viridissimum and the lactobacillus plantarum BW2013 or the feed added with the fermented powder of the myriophyllum viridissimum and the lactobacillus plantarum BW2013 is beneficial to improving the digestion and absorption capacity of nutrient substances of mice. The said powder or fermented powder can replace partial feed.

6. Sequencing and analysis of intestinal flora 16S rDNA

Taking feces before slaughtering a mouse aseptically, and sending the feces sample to an Illumina NovaSeq sequencing platform of Tianjin Nuo grass genesis company for extracting and quality identifying of feces microorganism genome DNA, 16S rDNA sequencing and subsequent bioinformatics analysis, wherein the basic process comprises the following steps:

1) computer-loading process

In the process from sample to data acquisition, DNA extraction and detection, PCR amplification, purification of PCR products, library preparation and library examination are carried out.

2) Sequencing data quality control

Splicing the original Data (Raw Data), filtering the interference Data (Dirty Data) and finally obtaining effective Data (Clean Data).

3) OTU clustering and species annotation

In order to research the species composition of each sample, OTUs (operational taxomic units) are clustered by 97% of consistency (Identity) of all samples, then species annotation is carried out, namely, in order to obtain the species classification information corresponding to each OTU, an RDP classifier Bayes algorithm is adopted to carry out Taxonomic comparison analysis on OTU representative sequences with certain similarity level, and the community composition of each sample is counted respectively at each classification level, namely, domain, gkndom, phylum, class, order, family, genus and species.

As shown in FIG. 5, the distribution of the intestinal flora in the mice was observed at the phylum level, and as can be seen from FIG. 5, the distribution of the intestinal flora in the mice of each group was observed at the phylum level. The Bacteroidetes and Firmicutes in the intestinal tract of each group of mice account for more than 95% of the total intestinal flora, and in addition, a small amount of actinomycetes, Verrucomicrobia, Proteobacteria, deferobacteria and the like. The bacteroidetes proportions of the N group, the NG group, the F group and the FG group are increased, and the firmicutes proportion is reduced compared with the CK group. Meanwhile, compared with the CK group, the proportion of proteobacteria and actinomycetoma of each group is reduced. The proportion of melainabiaceae in the intestinal flora of mice in the FG group increased to 0.22% compared to the level of melainabiaceae in the CK group (0.02%).

The distribution of the mouse intestinal flora at the genus level is shown in FIG. 6. From the species annotation and abundance information of all samples at the genus level, cluster analysis was performed from both the sample and species levels and plotted as a heatmap as shown in fig. 6. As can be seen from FIG. 6, the intestinal flora of the CK group of mice mainly aggregated belong to Muribacterium, Bifidobacterium, Lactobacillus, Lachnospira, Enterobacter; the genera in which the intestinal flora of the N groups of mice mainly gathers are Angelakissella, Blautia, Mucispiralium, Turcibacter, Desulfovibrio and Acetactifacto; the genera mainly aggregated by the intestinal flora of the mice in the NG group are Bilophila, Akkermansia, Ruminiclostridium and Alloprovella; the F group mice have the main gathered genera of intestinal flora such as Bacteroides, Marvinbryantia and butylricicoccus; the genera in which the intestinal flora of FG group mice mainly aggregated are unidentified _ Ruminococcaceae, Candidatus _ Saccharomyces, Butyricimonas, and Parabacteroides. It can be seen that the main groups of aggregating genera are not identical.

As can be seen from fig. 5 and 6, bacteroidides and firmutes are two major members of the intestinal tract, and studies have shown that higher abundance bacteroides and lower abundance firmutes are associated with the beneficial effects of apple pectin (a type of cellulose) on the gut microbiota of diet-induced obese rats. Proteobacteria is a group of bacteria that cause chronic colitis and is reported to be in low relative abundance in healthy hosts. The Faecalibacterium contains anti-inflammatory and other important functional strains, and higher abundance of Faecalibacterium is related to the enhancement of epithelial defense function in piglets supplemented with lactobacillus plantarum ZLP 001. In one study on fecal transplantation, Bacteroides and Parabacteroides, among the species that contribute primarily to donor transplantation, may be key species for maintaining the donor microflora. Bacteroides can break down food and produce bioactive substances and energy, which contain beneficial species such as Bacteroides vulgatus and Bacteroides coprocolla. Vulgatus exhibits a role in mediating inflammation inhibition and ultimately preventing colitis induction in a mouse model of IBD. Coprocola can produce extracellular enzymes that help the host break down some complex plant polysaccharides, such as cellulose and hemicellulose. Parabacteroidides containing beneficial species such as Parabacteroidides goldsteinii, ingestion of live Parabacteroides goldsteinii by high fat diet fed mice reduces obesity symptoms and increases gut integrity, inflammation and insulin resistance levels. Furthermore, Lactobacillus delbrueckii is used in some studies as a probiotic that can efficiently hydrolyze casein and modulate the intestinal immune system. Asticcacaulis excentricus can be used as a biopesticide, which can produce protein toxins and is deficient in protease which degrades the toxins, thereby killing mosquitoes and possibly causing damage to animal organisms.

The significance of intestinal flora differential species was analyzed and the fold difference between the group mice intestinal differential phyla and genus levels was compared, as shown in table 8.

TABLE 8 fold difference (FC) between groups of mice intestinal Difference phylum, genus and species

Wherein, CK: a control group; n: a watercress armstrong group; NG: the watermifoil and lactobacillus plantarum group; f: fermenting powder of Myriophyllum rubrum group; FG: the fermented powder of the armyworm-green algae and the lactobacillus plantarum group. The former vs the latter means that the former is compared with the latter, and the fold difference FC is equal to the relative abundance of the former bacteria/the relative abundance of the latter bacteria, and +∞ represents that the relative abundance of the latter bacteria is 0. ns means no significant difference; denotes p <0.05, difference is significant; indicates p <0.01, with very significant differences.

Table 8 is the fold difference between the dissimilarity gates, genera and species between each control group. Compared with the CK group, the relative abundance of Bacteroides, Faecalibacterium, Lactobacillus delbruueckii, Bacteroides vulgatus and Bacteroides coprocolla in the intestinal tract of the mice in the F group is obviously increased, and the relative abundance of Firmicutes is obviously reduced; the intestinal tracts of N groups of mice have significantly reduced Asticcacaulis excentricus; meanwhile, Parabacteroides goldsteinii increased significantly in the N-, NG-, F-and FG-groups. In addition, the relative abundance of Faecalibacterium, Bacteroides vulgatus and Bacteroides coprocolla in the intestines of the mice in group F was significantly increased compared to that in group N, while the relative abundance of asticacaculis excentricus in the intestines of the mice in group F was higher than that in group N, but was not significantly changed compared to group CK. The reason for the occurrence of the good changes is that the watermifoil or the fermentation product thereof replaces part of common feed, which shows that the watermifoil replaces part of feed to reduce obesity symptoms, enhance the integrity of intestinal tracts and reduce inflammation and insulin resistance level; the fermented powder myriophyllum procumbens replacing part of the feed has the advantages of replacing part of the feed with the myriophyllum procumbens, and can promote the production of bioactive substances and energy in a host body, enhance the epithelial defense function, regulate the intestinal immune system and reduce the occurrence of colitis.

Compared with the group N, the Faecalibacterium in the intestinal tracts of the mice in the group NG is obviously increased, which shows that the function of defending the intestinal epithelium of the mice can be enhanced by adding lactobacillus plantarum BW2013 into the feed containing the watermifoil armyworm. Compared with the group F, the intestinal tract of the FG group mice is obviously reduced in Proteobacteria, which shows that the content of Proteobacteria in the intestinal tract of the mice can be reduced by adding the lactobacillus plantarum BW2013 into the feed containing the fermented powder of the myriophyllum rubrum, and the occurrence probability of chronic colitis can be reduced.

Compared with CK group, Bacteroides, Faecalibacterium and Parabacteroides goldsteinii in intestinal tract of NG group mice are increased remarkably, which shows that feeding of Nostoc commune and Lactobacillus plantarum BW2013 is beneficial to enhancing the intestinal tract epithelial defense function, and has the effects of reducing obesity symptoms, enhancing the integrity of the intestinal tract, and reducing inflammation and insulin resistance level; in the intestinal tracts of FG group mice, Bacteroides, Parabacteroides goldsteinii, Lactobacillus delbruueckii, Bacteroides vulgatus and Bacteroides coprococola are obviously increased, and Firmicutes and Proteobacteria are obviously reduced, which indicates that the simultaneous feeding of Zymus exigua and Lactobacillus plantarum BW2013 can promote the generation of bioactive substances and energy in a host body, regulate an intestinal immune system and reduce the occurrence of colitis besides the functions brought by feeding of Zymus exigua.

These results indicate that the watery green foxtail algae or its fermentation product can be used to replace part of the feed and have a favorable effect on the intestinal flora of mice, and that the fermented watery green foxtail algae replaces part of the feed better than watery green foxtail algae. In addition, the lactobacillus plantarum BW2013 is added on the basis that the watery green foxtail algae or the fermentation product thereof replaces part of the feed, so that the intestinal flora structure is better improved, especially the lactobacillus plantarum BW2013 is added on the basis that the watery green foxtail algae replaces part of the feed, so that the intestinal flora structure is better improved, and the intestinal health is better facilitated. In conclusion, feeding the mice with the foxtail algae pulchrum, the fermented foxtail algae pulchrum, the foxtail algae pulchrum containing lactobacillus plantarum BW2013 and the fermented foxtail algae pulchrum containing lactobacillus plantarum BW2013 has beneficial improvement effects on the intestinal flora structure and is beneficial to intestinal health, wherein the feeding of the fermented foxtail algae pulchrum replaces part of feed, and the addition of the lactobacillus plantarum BW2013 is the optimal choice.

7. Metabolic analysis

1) Metabolite extraction

100mg of liquid nitrogen is respectively taken to grind the cecal content samples of each group of mice, the cecal content samples are placed in an EP tube, 500 mu L of 80 percent methanol water solution containing 0.1 percent formic acid is added, vortex oscillation is carried out, ice bath standing is carried out for 5min, centrifugation is carried out at 15000rpm and 4 ℃ for 10min, a certain amount of supernatant is taken and added with mass spectrum water to be diluted until the methanol content is 53 percent, the mixture is placed in a centrifuge tube with a 0.22 mu m filter membrane for 15000g and centrifugation at 4 ℃ for 10min, filtrate is collected, and sample injection LC-MS is carried out for analysis.

2) Conditions of liquid chromatography

A chromatographic column: superil Gold column (C18), column temperature: 40 ℃; flow rate: 0.2mL/min, positive mode: a mobile phase A: 0.1% formic acid, mobile phase B: methanol, negative mode: mobile phase A: 5mM ammonium acetate, pH 9.0, mobile phase B: methanol; the chromatographic gradient elution procedure is shown in table 9.

TABLE 9 liquid chromatography gradient elution conditions

3) Conditions of Mass Spectrometry

Selecting m/z 70-1050 for scanning range; the ESI sources are set as follows: spray V oltage:3.2 kV; a shear gas flow rate of 35 arb; 10arb as Aux Gas flow rate; capillary temp. 320 deg.C, Polarity: positive; negative; MS/MS two-stage scanning is data-dependent scans.

4) Data analysis

Firstly, performing spectrogram processing and database searching on original data by using Compound discover 3.1 software to obtain qualitative and quantitative results of metabolites, and then performing quality control on the data to ensure the accuracy and reliability of data results. The metabolites were then subjected to Principal Component Analysis (PCA), partial least squares discriminant analysis (PLS-DA), etc., to analyze the differences between the two groups of metabolites. Finally, the biological significance of the metabolite representation is explored by analysis of the metabolic pathways involved in the metabolite.

The intestinal flora metabolites of the mice are changed differently under different feeding modes. Analysis and comparison of the groups of significantly altered metabolites revealed that some bioactive substances such as aspartic acid, threonine, vitamin a, myricetin, gallic acid, luteolin, lysophosphatidylglycerol 18:1(LPG18:1), 9-oxo-10, 12-octadecadienoic acid (9-oxo-ODA), etc., were found as shown in table 10.

TABLE 10 differential metabolite analysis

Wherein, CK: a control group; n: a watercress armeniaca group; NG: the group consisting of ulexia glauca and lactobacillus plantarum; f: fermenting powder of Myriophyllum rubrum group; FG: the fermented powder of the armyworm-green algae and the lactobacillus plantarum group. The former vs the latter means that the former is compared with the latter, and the fold difference FC is equal to the relative metabolite content of the former/the relative metabolite content of the latter. ns means no significant difference; denotes p < 0.05; denotes p < 0.01.

As can be seen from Table 10, L-aspartic acid and L-threonine are converted as metabolites from the decomposition of dietary proteins and endogenous proteins by intestinal microorganisms. L-aspartic acid is a major fuel in the gut for ATP production by intestinal cells, protecting the intestinal barrier from lipopolysaccharide damage. The increase of the amount of threonine can promote mucin synthesis, thereby strengthening the close interaction between the small intestine microbiota and metabolism, and helping to promote the intestinal function perfection and immune development. Vitamin a deficiency is a worldwide public health problem that can lead to night blindness and permanent blindness, requiring extensive supplementation from the diet. Vitamin A can regulate intestinal flora, relieve inflammatory reaction, and enhance intestinal epithelial barrier of necrotizing enterocolitis. Myricetin, gallic acid and luteolin belong to flavonoid compounds, which are widely present in fruits and vegetables. Research shows that myricetin has certain curative effect on colon cancer, inflammation and other diseases. Gallic acid has cardiovascular protection, immunoregulation and intestinal tract protection effects. Luteolin exhibits anti-cancer activity in cancer cell lines and in vivo models. Lysophosphatidylglycerol (LPG) is a lysophospholipid that is important in physiological processes. Oleate-treated macrophages are reported to reduce the levels of LPG, and treatment with WY-14643 and/or pioglitazone (two drugs of metabolic disease) can reduce or even eliminate this negative effect. 9-oxo-ODA can promote fatty acid oxidation in vivo as PPAR alpha agonist, thereby inhibiting accumulation of triglyceride.

Compared with CK group, the content of aspartic acid, threonine, vitamin A, myricetin, gallic acid and luteolin is obviously increased in the intestinal tracts of mice in N group, NG group, F group and FG group. Compared with the group N, the contents of vitamin A, myricetin, gallic acid and luteolin in the intestinal tracts of the mice in the group F are obviously increased; the LPG18:1 content in NG group was significantly increased compared to N group; the LPG18:1 content in the FG group was significantly increased compared to the F group. Furthermore, the 9-oxo-ODA content in FG group was significantly increased compared to F group.

The results show that the content of aspartic acid, threonine, vitamin A, myricetin, gallic acid and luteolin in the intestinal tract can be increased by feeding the powder or fermented powder of the myriophyllum viridis to the mice, and the substances have the functions of resisting inflammation, enhancing the intestinal barrier, regulating the immunity and the like, so that the intestinal tract health can be promoted. The contents of vitamin A, myricetin, gallic acid and luteolin in the intestinal tract of the mice fed with the fermented powder myriophyllum viridis substitute feed are obviously higher than those of the mice fed with the powder myriophyllum viridis substitute feed, which shows that compared with the mice fed with the powder myriophyllum viridis substitute feed, the fermented powder myriophyllum viridis substitute feed has more obvious beneficial effect on the intestinal tract. The addition of lactobacillus plantarum BW2013 in the watery foxtail algae or the fermentation product thereof instead of the feed can restore the content of LPG18:1, thereby having beneficial effect on intestinal metabolism. In addition, the addition of lactobacillus plantarum BW2013 on the basis of feeding fermented powder myriophyllum viridis instead of feed can increase the content of 9-oxo-ODA in intestinal tracts and play a role in reducing fat accumulation. In conclusion, the 4 kinds of the pulverous myriophyllum functional feed can generate metabolites beneficial to the mouse body, and is beneficial to the intestinal health of the mouse; the effect of feeding the watermifoil alternative feed added with the lactobacillus plantarum BW2013 is the best.

The results of the analysis of the intestinal differential metabolic pathway are shown in Table 11.

TABLE 11 differential metabolism enrichment pathways

Wherein, CK: a control group; n: a watercress armstrong group; NG: the group consisting of ulexia glauca and lactobacillus plantarum; f: fermenting powder of myriophyllum viridis group; FG: the fermented powder of the armyworm-green algae and the lactobacillus plantarum group. The former vs the latter means a metabolic pathway significantly enriched in the former compared to the latter. ns indicates no significant difference.

As can be seen from table 11, the metabolic pathway of histidine metabolism alone was significantly enriched in N and NG group mice relative to the CK group; three metabolic pathways, namely porphyrin and chlorophyll metabolism, aminobenzoate degradation and monobactein biosynthesis, in the F group of mice are significantly enriched; histidine metabolism, porphyrin and chlorophyll metabolism, taurine and hypotaurine metabolism, and glycine, serine and threonine metabolism in FG group mice were significantly enriched. In general, porphyrin and chlorophyll metabolism in FG group and F group mice are prominent, and histidine metabolism in NG group and N group mice is prominent in two feeding modes. The metabolic pathway for unsaturated fatty acid biosynthesis in mice of group F was significantly enriched compared to group N. The supplementation of lactobacillus plantarum BW2013 on the basis of edible fermentation powder of the L.pulcherrima can obviously enrich the metabolic pathway of arginine and proline metabolism in mice; the supplementation of lactobacillus plantarum BW2013 on the basis of edible pulegonus elongatus can obviously enrich four metabolic pathways of a sulfur relay system, degradation of aromatic compounds, tyrosine metabolism and nitrotoluene degradation in a mouse body.

The embodiment shows that the lactobacillus plantarum BW2013 used for fermentation is not only animal probiotics and can improve the intestinal characteristics of animals, but also can be used for fermentation of plant agricultural and sideline products and other raw materials, and has the effect of killing two birds with one stone. The powder-containing Vulpes viridis feed improves nutritive value and palatability of Vulpes viridis feed, and is convenient for storage and transportation. After the functional feed prepared by using the lactobacillus plantarum BW2013 fermented powder of the myriophyllum elatinoides and mixing with the lactobacillus plantarum BW2013 live bacteria is fed to a mouse, the weight of the mouse is not obviously changed, the serum biochemical index is basically not obviously influenced, the total length of an intestinal tract and the length of a small intestine are not obviously different, and the feed can replace 10 percent of the feed; the functional feed has beneficial improving effect on intestinal flora structure and metabolite of mice, and is beneficial to intestinal health and healthy growth of mice. Therefore, the watermifoil and myriophyllum pratense functional feed provided by the invention is rich in nutrient components, ensures the activity of probiotic lactobacillus plantarum BW2013, and has good effects of regulating intestinal flora and improving metabolites for animals. And the problem of secondary pollution caused by the treatment of the culture wastewater by the watermifoil myriophyllum is solved, and the mutual unification of environmental protection and resource development and utilization is realized.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A preparation method of a watermifoil functional feed is characterized by comprising the following steps:

2) inoculating and stirring materials: will be 1 × 10⁹～9×10⁹Uniformly spraying a bacterial solution of the Lactobacillus plantarum BW2013 with the concentration of CFU/mL on the watery green armyworm according to the inoculation amount of 5-15% w/w, then adding sucrose according to 5-10% w/w of the watery green armyworm weight, and uniformly mixing to obtain a mixture;

4) constant-temperature fermentation: placing the sealed sterile plastic packaging bag into a constant-temperature incubator at the temperature of 30-35 ℃ for standing and fermenting for 4-10 days;

6) and (3) live bacteria mixing: adding 1 × 10 per gram of powder-myriophyllum viridis fermented lyophilized powder⁸～9×10⁸Adding bacterial liquid of Lactobacillus plantarum BW2013 into the CFU viable bacteria in proportion, and uniformly mixing to obtain a powder myriophyllum functional feed;

wherein the classification of the lactobacillus plantarum BW2013 is named as: lactobacillus plantarum BW 2013; the preservation unit is as follows: china general microbiological culture Collection center, addresses are: beijing, Haoyang district, Xilu No. 1, Ministry of microbiology, China academy of sciences, 3; the preservation date is as follows: 9/5/2014, with a collection number of: CGMCC No. 9642.

2. The preparation method of the watery foxtail algae functional feed in claim 1, wherein the preparation method of the lactobacillus plantarum BW2013 bacterial liquid comprises the following steps: inoculating lactobacillus plantarum BW2013 seed liquid into an MRS liquid culture medium according to the inoculation amount of 5-10% w/w, and performing static culture for 18 hours at 37 ℃; centrifuging at 8000r/min for 15 min; collecting thalli and discarding supernatant; the thalli is washed twice by sterile PBS; resuspend to 1X 10 concentration with sterile PBS⁹～9×10⁹CFU/mL of bacterial liquid.

3. The method for preparing the watermifoil functional feed according to claim 1, wherein the temperature in the step 4) is 30 ℃ and the fermentation is carried out for 5 days.

4. A watermifoil-containing functional feed prepared by the preparation method according to any one of claims 1 to 3.