CN112205473A

CN112205473A - Yoghourt biotechnological beverage with functions of resisting oxidation and inhibiting growth of digestive tract cancer cells and preparation method thereof

Info

Publication number: CN112205473A
Application number: CN201910631432.5A
Authority: CN
Inventors: 钱佑; 张登豪; 张灏; 丘锦朋; 黄建诚; 凃鸿宇; 杜英齐; 吕美津; 许育铭
Original assignee: Yuyang Biomedical Co ltd
Current assignee: Yuyang Biomedical Co ltd
Priority date: 2019-07-12
Filing date: 2019-07-12
Publication date: 2021-01-12

Abstract

The invention discloses a yoghourt biotechnological beverage with functions of resisting oxidation and inhibiting growth of digestive tract cancer cells and a preparation method thereof, the yoghourt biotechnological beverage comprises 8 to 12 different strains, and the strains are fermented for 8 to 12 hours at 37 to 43 ℃ by using an animal milk product. The yoghourt biotechnological beverage has good sensory evaluation result, and can be added with sauce ingredients, solid ingredients or solid fruit ingredients to improve the overall edible sensory effect. The yogurt raw drink is also extracted by a solvent with increasing polarity, and the main amount components are identified in different extracts. The yogurt biotechnological beverage is proved to have the effects of scavenging free radicals, resisting oxidation, having high reducing power and inhibiting the growth of cancer cells.

Description

Yoghourt biotechnological beverage with functions of resisting oxidation and inhibiting growth of digestive tract cancer cells and preparation method thereof

Technical Field

The invention relates to a yoghourt biotechnological beverage and a preparation method thereof.

Background

The two-way communication network between intestine and brain is called intestine-brain axis (gut-brain axis), and later, the symbiotic flora in the intestinal tract is found to contribute to the gut-brain axis, and the three influence each other, thus becoming symbiotic bacteria-gut-brain axis (microbe-gut-brain axis). The National Institute of Mental Health (NIMH) initiated a special program in 2013 exploring the gut microbiota-brain communication mechanism with the aim of developing new drugs against mental diseases and non-invasive treatment methods. Since then, the research on symbiotic bacteria, intestine and brain axis, has been developed like bamboo shoots in the spring after rain, and has been the focus of neuroscience research, mainly for discussing the interaction between intestinal microbiota and brain. The intestinal microbiota has important influence on the brain through a neural network, a neuroendocrine system and an immune system, and the composition of the intestinal microbiota is changed by the disturbance of human behavior. The literature has reported symbiont-gut-brain axis interrelationships and demonstrated to be associated with a variety of diseases, such as: inflammatory Bowel Disease (IBD), cancer of the gastrointestinal tract, cholelithiasis, behavioral disorders, anxiety, depression, Chronic Fatigue Syndrome (CFS), hepatic encephalopathy (hepaciphalopathy), allergy, obesity, diabetes, atherosclerosis, and the like. In cancer chemotherapy and post-operative care, human clinical studies have reported improvement of cardiopulmonary health and reduction of fear of cancer recurrence in breast cancer patients by modulating the composition of the gut microbiota.

At present, western medicine and traditional Chinese medicine systems are actively developing or combining the research of symbiotic bacteria-intestine-brain axis to research drug development, disease treatment and action mechanism conversion; while another medical system, Ayurveda, uses a large amount of turmeric for improving gut discomfort, neuroprotection, and prevention of alzheimer's disease, its diverse efficacy is also related to the gut-brain axis. However, the use of turmeric is restricted regionally due to different dietary habits, mainly in India.

In the western medicine system, the drug development by utilizing the symbiotic bacteria-intestine-brain axis research is called as a micro-ecological drug, which refers to a drug preparation prepared by utilizing normal microorganisms or substances for regulating the normal growth of the microorganisms. Can be used for treating infection, diabetes, tumor, inflammation diseases, immune related diseases, etc. The micro-ecological medicine comprises: a Live Biological Product (LBP), a small molecule micro-ecological modulator (SMMM), a fecal bacteria transplant (FMT). LBP means that the type and quantity of the bacteria are definitely controlled through identification, screening and combination of human microorganisms, and the safety and effectiveness of medication are ensured by adopting different types, quantities and combination of the bacteria for different indications. However, the U.S. Food and Drug Administration (FDA) has not yet approved any LBP for marketing, and only in 2016 early clinical trial guidelines for LBP were published, so that LBP development has clear criteria. SMMM refers to a substance that selectively promotes the growth and reproduction of one or more beneficial bacteria in the intestinal tract of a host, and achieves therapeutic goals by affecting the growth and reproduction of the bacteria. Currently known SMMM drugs are only developed to the second clinical stage. FMT refers to the treatment of intestinal and parenteral diseases by transplanting functional flora of healthy human feces to the gastrointestinal tract of a patient and reconstructing the intestinal flora of the patient. Although FMT has been listed in the united states as a treatment guideline for recurrent Clostridium difficile infection (Clostridium difficile infection) in 2013, the policies in different countries vary greatly and FMT currently lacks uniform and effective regulation. Overall, the clinical trials for probiotics have not progressed as expected and there is currently no approved commercial probiotics. Another problem is that the number of clinical trials of probiotics is relatively small and the human validation of the efficacy of the drug is not as expected.

The traditional Chinese medicine system does not utilize symbiotic bacteria-intestine-brain axis to research and develop medicines, but researches the interaction between traditional Chinese medicines and intestinal microbiota. The regulation effect of traditional Chinese medicines on intestinal microbiota is as follows: the tonifying traditional Chinese medicine containing polysaccharide has the function of supporting beneficial bacteria and pathogenic bacteria, but the supporting effect on the beneficial bacteria is obviously better than that of the pathogenic bacteria, and metabolites generated by the beneficial bacteria with good growth indirectly inhibit the growth of the pathogenic bacteria. For example, codonopsis pilosula polysaccharides can promote the growth of bifidobacteria in vitro, thereby increasing the metabolism of acetic acid and enhancing the colonization resistance (colonization resistance) of bifidobacteria. The metabolic effects of intestinal microbiota on traditional Chinese medicine are for example: at present, it is proved that many traditional Chinese medicine components can generate drug effect components only through the metabolism of intestinal microbiota to achieve the treatment effect. For example, baicalin (baicailin) contained in scutellaria baicalensis is difficult to be directly absorbed in the intestinal tract, and is hydrolyzed into baicalein (baicailein) by intestinal microbiota, and can be absorbed into the blood to act. Clinical tests of human bodies prove that the pueraria root, scutellaria and coptis decoction can treat the second-type diabetes by changing the intestinal microbiota. Overall, the traditional Chinese medicine research of symbiotic bacteria-intestine-brain axis provides the western medically acceptable action mechanism explanation, but still cannot solve the old problem that the traditional Chinese medicine has low medicine taking compliance (drug compliance) and medicine taking compliance (medicine compliance) due to too high dosage and too long treatment time.

The research and development of the medical system of western medicine, traditional Chinese medicine and ayurvedic rodgersia on symbiotic bacteria-intestine-brain axis focuses on diseases, medicines and treatment. Overall, these developments are still in their infancy and have not been successful in industry or in large-scale production. Therefore, there is still a need to develop novel products and techniques applicable to symbiotic bacteria-gut-brain axis for biomedical efficacy.

In view of the deficiencies in the prior art, the applicant has devised the present invention through careful experiments and studies and with a clear spirit, and the following is a brief description of the present invention to overcome the deficiencies in the prior art.

Disclosure of Invention

The invention aims to provide a yoghourt beverage, which comprises the following components: animal milk product and plural bacterial powder, animal milk product includes water and animal milk, plural bacterial powder is mixed with animal milk product, plural bacterial powder comes from corresponding plural bacterial strains, plural bacterial strains include: bifidobacterium longum (Bifidobacterium longum), Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus paracasei (Lactobacillus paracasei) and Lactobacillus rhamnosus (Lactobacillus rhamnosus).

The yogurt drink further comprises a group consisting of the following strains: bifidus (Bifidobacterium bifidum), Bifidobacterium breve (Bifidobacterium breve), Bifidobacterium infantis (Bifidobacterium infantis), Bifidobacterium longum (Bifidobacterium lactis), Enterococcus faecalis (Enterococcus faecium), Lactobacillus casei (Lactobacillus casei), Lactobacillus bulgaricus (Lactobacillus subsp. bulgaricus), Lactobacillus fermentum (Lactobacillus fermentum), Lactobacillus helveticus (Lactobacillus helveticus), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus plantarum (Lactobacillus salivarius), Lactobacillus salivarius (Lactobacillus thermophilus) and Streptococcus thermophilus (Streptococcus thermophilus).

Another object of the present invention is to provide a method for separating the aforesaid yogurt drink, comprising: freeze-drying the yoghurt drink to obtain a powdery product; extracting the powdery product by using n-hexane to obtain an n-hexane extract and a first residue, wherein the main component of the n-hexane extract is a short-chain fatty acid with double bonds and hydroxyl groups; extracting the first residue with ethyl acetate to obtain an ethyl acetate extract and a second residue, wherein the major component of the ethyl acetate extract is glycolipid; extracting the second residue with ethanol to obtain ethanol extract and third residue, wherein the ethanol extract comprises major components including disaccharide and oligosaccharide; and extracting the third residue with water to obtain an aqueous extract and a fourth residue, wherein the major components of the aqueous extract comprise polysaccharides, glycoproteins, and proteins.

Another object of the present invention is to provide a use of the yogurt drink for inhibiting the growth of cancer cells, wherein the cancer cells are selected from the group consisting of blood cancer cells, brain cancer cells, stomach cancer cells, colorectal cancer cells and/or combinations thereof.

The invention discloses a fermentation machine for preparing yoghourt drinks, which comprises: a tank body and a sealing top cover. The sealing top cover is assembled with the tank body, so that a space enclosed by the sealing top cover and the tank body is closed. The feed opening is arranged on the sealing top cover, and water, milk powder and bacterial powder are fed into the space through the feed opening. The motor controller is arranged outside the tank body, the stirring motor is coupled to the motor controller, and the stirring arm is arranged in the space and connected to the stirring motor for stirring water, milk powder and bacterial powder. The resistance heater is arranged outside the tank body and coupled with the motor controller, and is used for sterilizing water and milk powder, and fermenting the bacterial powder in a mixture formed by the water and the milk powder at 37-43 ℃ to prepare the yoghourt beverage.

In the invention, the fermentation machine also comprises a discharge hole at the bottom of the tank body, and the yoghourt drink is discharged from the discharge hole. The temperature control interlayer outside the tank body is connected with the resistance temperature increasing device, and the temperature control interlayer is controlled by the resistance temperature increasing device to adjust or maintain the temperature of the space. Still include the washing mouth on the sealed top cap, the cell body side includes the heat conduction inflow mouth, and the cell body bottom includes the heat conduction egress mouth.

In the invention, the fermentation machine also comprises a temperature-sensing detector coupled with the motor controller, the motor controller also comprises a screen, and the temperature sensed by the temperature-sensing detector is displayed on the screen. The seal cap also includes a viewing window for a user to visually observe conditions within the tank.

The term "animal milk product" as used herein generally includes water and animal milk, which may be in liquid form or dried to powder form by pasteurization, and which may be derived from human, bovine or sheep milk, etc.

The various bacterial and cancer cell lines described herein are commercially available and do not require biological material storage. The yogurt drink herein is liquid under refrigerated and room temperature environments. The strains in the yogurt drink herein exhibit unequal fermentation results at temperatures of 37 ℃ to 43 ℃ and fermentation times of 8 hours to 12 hours.

Drawings

FIG. 1 shows the results of sequencing of 16S rDNA of the strain No. LR-36 and comparison of NCBI, which shows rhamnosus (Lactobacillus rhamnosus).

FIG. 2 shows the result of sequencing the 16S rDNA of strain LPL-68 and the result of comparison with NCBI, which shows Lactobacillus plantarum (Lactobacillus plantarum).

FIG. 3(A) is a photomicrograph of strain number UY-58. Gram-positive bacilli have no catalase, oxidase and motility, do not produce endospores, and grow in aerobic and anaerobic environments.

FIG. 3(B) shows the 16S rDNA sequencing result of the strain number UY-58. Closest to Lactobacillus fermentum, the similarity is 99.9%.

FIG. 4(A) is a photomicrograph of the strain number UY-76. Gram-positive bacilli have no catalase, oxidase and motility, do not produce endospores, and grow in aerobic and anaerobic environments.

FIG. 4(B) shows the 16S rDNA sequencing result of the strain number UY-76. Closest to Lactobacillus helveticus (Lactobacillus helveticus), the similarity is 100%.

Fig. 5 is a flow chart of the separation method 3.

FIG. 6(A) shows 11-hexane extract (11-H) of yogurt drink¹H-NMR spectrum.

FIG. 6B shows 11 ethyl acetate extract (11-E) of yogurt drink¹H-NMR spectrum.

FIG. 6(C) shows the 11-ethanol extract (11-A) of the yogurt drink¹H-NMR spectrum.

FIG. 6(D) shows the 11-W aqueous extract of yogurt drink¹H-NMR spectrum.

FIG. 7(A) shows the 12-hexane extract (12-H) of yogurt drink¹H-NMR spectrum.

FIG. 7B shows the 12-ethyl acetate extract (12-E) of the yogurt drink¹H-NMR spectrum.

FIG. 7(C) shows the 12-ethanol extract (12-A) of the yogurt drink¹H-NMR spectrum.

FIG. 7(D) shows the water extract (12-W) of the yogurt drink 12¹H-NMR spectrum.

FIG. 8(A) shows the hexane extract 13-H from yogurt drink 13¹H-NMR spectrum.

FIG. 8B shows 13 ethyl acetate extracts (13-E) of yogurt drink¹H-NMR spectrum.

FIG. 8(C) shows the alcohol extract 13-A of yogurt drink 13¹H-NMR spectrum.

FIG. 8(D) is a drawing of a 13 water extract (13-W) of yogurt drink 13¹H-NMR spectrum.

FIG. 9(A) shows the 14-hexane extract (14-H) of yogurt drink¹H-NMR spectrum.

FIG. 9B shows the 14-ethyl acetate extract (14-E) of the yogurt drink¹H-NMR spectrum.

FIG. 9(C) shows the 14-ethanol extract (14-A) of the yogurt drink¹H-NMR spectrum.

FIG. 9(D) shows the 14-W aqueous extract of yogurt drink¹H-NMR spectrum.

Fig. 10 is a bar graph of the total polyphenol content analysis of each extract obtained from the yogurt drinks 11 to 14 by the separation method 3.

Fig. 11 is a bar graph of the total flavanone flavonoid content of each extract obtained from the yogurt drinks 11 to 14 by the separation method 3.

Fig. 12 is a histogram of the total polysaccharide content of each extract obtained from the yogurt drinks 11 to 14 by the separation method 3.

FIG. 13 is an HPLC spectrum of 203nm wavelength of each extract obtained from the yogurt drink 11 by the separation method 3.

FIG. 14 is an HPLC spectrum at 203nm of each extract obtained by the separation method 3 of the yogurt drink 12.

FIG. 15 is an HPLC spectrum at 203nm of each extract obtained by separation method 3 of yogurt drink 13.

FIG. 16 is an HPLC spectrum at 203nm of each extract obtained from the yogurt drink 14 by the separation method 3.

Fig. 17 is a bar graph of DPPH free radical clearance of each extract obtained from the yogurt drinks 11 to 14 by the separation method 3.

FIG. 18 is a bar graph of the total antioxidant capacity-ABTS free radical clearance of each extract obtained from yogurt drinks 11-14 by separation method 3.

Fig. 19 is a bar graph of the reduction force measurements of the extracts obtained by the separation method 3 of the yogurt drinks 11 to 14.

FIG. 20 is a bar graph showing the inhibition rate of blood cancer cell survival of each extract obtained by the separation method 3 of yogurt drinks 11 to 14.

Fig. 21 is a bar graph of the survival inhibition rate of brain cancer cells of each extract obtained by the separation method 3 of the yogurt drinks 11 to 14.

Fig. 22 is a bar graph of the gastric cancer cell survival inhibition rate of each extract obtained by the separation method 3 of the yogurt drinks 11 to 14.

Fig. 23 is a histogram showing the inhibition rate of survival of colon cancer cells of each extract obtained by the separation method 3 of yogurt drinks 11 to 14.

Fig. 24 is a bar graph of the prostate cancer cell survival inhibition rate of each extract obtained by the separation method 3 of the yogurt drinks 11 to 14.

FIG. 25 is a flow diagram of a yogurt drink production system.

FIG. 26 is a diagram of a yogurt drink fermentation machine.

Reference numerals:

the method comprises the following steps of 3, yogurt drink stock solution 12, powdery product 14, N-hexane extract 16, first residue 18, ethyl acetate extract 20, second residue 22, ethanol extract 24, third residue 26, water extract 28, fourth residue 30, fermentation machine 100, tank body 101, temperature control interlayer 102, resistance warmer 103, stirring arm 104, sealing top cover 105, cleaning opening 106, temperature sensing detector 107, motor controller 108, screen 109, stirring motor 110, feeding opening M, discharging opening N4, heat conduction flow inlet N5, heat conduction flow outlet N6 and observation window S1.

Detailed Description

The above objects and advantages of the present invention will become more readily apparent to those of ordinary skill in the art after reviewing the following detailed description and accompanying drawings.

The invention leads and separates the activity of the active metabolite of the yoghourt, determines the active ingredients and prepares the active ingredients into drinks or medicaments suitable for oral administration, and still maintains the efficacy of the ingredients.

Experiment 1, separation and identification of strains:

taking 1ml of raw milk as a sample, and taking lactobacillus MRS culture medium (Lactobacillus MRS Broth) as diluent to carry out 10-fold serial dilution (the concentration fold is 10 respectively^-1、10^-2、10^-3、10^-4) 200 mul of the diluted material was dropped on sterile MRS agar medium and then coated. The plates were placed in an anaerobic incubator at 37 ℃ for 24 hours. And then, continuously culturing the single colonies with different shapes, colors and sizes in an aseptic manner, repeatedly selecting the colonies and culturing to separate strains, and determining that the colonies on the culture dish are all the same in shape, color and size. And finally, performing strain preservation and identification on the separated single strain. The identification of the yoghourt strain is to observe a single colony on a culture dish by naked eyes, select two identical colonies for gram staining and determine the colony as gram positive or negative bacteria. The colonies are preferably observed under a phase-contrast microscope at a magnification of 4 and 40 times. Single colonies were selected for DNA extraction and 16S PCR experiments, and the sequences were aligned with the NCBI database and the results used to complete the identification.

As shown in FIG. 1 and FIG. 2, the strain numbers LR-36 and LPL-68 are Lactobacillus rhamnosus (Lactobacillus rhamnosus) and Lactobacillus plantarum (Lactobacillus plantarum) respectively after 16S rDNA sequencing and NCBI comparison. Furthermore, as shown in FIG. 3(A), FIG. 3(B) and Table 1, the strain number UY-58 was confirmed to be a gram-positive bacterium by microscopic observation and identification, DNA extraction, 16S rDNA sequencing (SEQ ID NO:1), API 50CHL identification, and was closest to Lactobacillus fermentum (similarity of 99.9%). FIGS. 4(A) and 4(B) show the strain number UY-76 identified by microscopic observation, DNA extraction, 16S rDNA sequencing (SEQ ID NO:2), and API 50CHL as gram-positive bacteria, and most closely related to Lactobacillus helveticus (Lactobacillus helveticus, similarity 100%).

TABLE 1 API 50CHL identification system analysis results of strain number UY-58 (Lactobacillus fermentum)

TABLE 2 API 50CHL identification system analysis results of strain number UY-76 (Lactobacillus helveticus)

After separation and identification, 20 strains are obtained in total, and are arranged in the sequence of Latin names as shown in Table 3.

TABLE 3 Chinese and Latin names of 20 strains

Experiment 2, fermentation preparation of yogurt:

the design model of the fermentation preparation experiment of the invention takes the strain species and proportion, the fermentation temperature and the fermentation time as the operation factors, and takes the viscosity and the pH value (pH) as the quality control standard after fermentation. The viscosity of the yoghourt is below 100 centipoise (cP) and the pH value is above 5.0, the yoghourt is judged to be not fermented or not completely fermented, and experimental groups with overlong fermentation time (more than 12 hours) are eliminated due to poor production efficiency. The preparation method of experiment 2 was: 250ml of hot water with the temperature of 75 ℃ is added into 36g of milk powder for high-temperature sterilization to obtain the animal milk product. When the mixture is cooled to 35-43 ℃, 0.3g of bacterial powder is added. The resulting mixture was then placed in incubators at different temperatures (37 ℃, 40 ℃, 43 ℃) for different periods of time (8 hours, 10 hours, 12 hours), after which the viscosity (Brookfield DVE RV viscometer) and the pH (Milwaukee pH600 pH meter) were measured. Table 4 shows the strains contained in the combination of 14 constituent successfully fermented strains.

Tables 4 and 14 strains contained in the combination of successfully fermented strains

The results show that different milk powders have little influence on whether the fermentation can be successfully carried out; the weight ratio between the species of the strain has little influence on the fermentation result. Therefore, the ratio of the (powder) to the (powder) by weight of the strains in each experimental group was 1:1, and the higher the number of the strains, the easier the successful fermentation, i.e., the higher the experimental reproducibility of the successful fermentation. Then, 3 repeated experiments were performed with 4 groups (strain combinations 11 to 14 in table 4) having the highest reproducibility of successful fermentation, and it was determined that the fermentation was complete when the optimal fermentation conditions (i.e., the highest viscosity and the lowest pH could be achieved within the shortest fermentation time) for the different strain combinations were confirmed, and the viscosity of the yogurt was higher than 100cp and the pH was lower than 5.0. The results of the experiment are shown in Table 5.

TABLE 5 fermentation test of Strain combinations 11 to 14

Results of 3 replicates of strain combinations 11 to 14 fermentatively prepared show that:

and (3) strain combination 11: the fermentation temperature is fixed to be 37 ℃, and the fermentation time needs 12 hours to be complete; the fermentation temperature is fixed to be 40 ℃, the fermentation time is 10 hours, the fermentation can be completed, and the fermentation time is 8 hours or 12 hours, so that the fermentation can not be completed; the fermentation temperature is fixed at 43 ℃, the fermentation time is 10-12 hours, the fermentation can be completed, and the viscosity is higher and the pH value is lower as the fermentation time is longer. The fermentation time is fixed to be 8 hours, and the fermentation temperature is 37 ℃, 40 ℃ and 43 ℃ which cannot be completely fermented; the fermentation time is fixed to be 10 hours, the fermentation temperature is 40 ℃ and 43 ℃ and can be completely fermented, the higher the fermentation temperature is, the lower the viscosity is, and the higher the pH value is; the fermentation time is fixed to 12 hours, and the fermentation temperature is 37 ℃ and 43 ℃ to complete the fermentation.

And (3) strain combination 12: the fermentation temperature is fixed at 37 ℃, the fermentation time can be completely fermented within 10 and 12 hours, and the viscosity is higher and the pH value is lower as the fermentation time is longer; the fermentation temperature is fixed at 40 ℃, the fermentation time can be completely fermented within 8, 10 and 12 hours, and the viscosity is higher and the pH value is lower as the fermentation time is longer; the fermentation temperature is fixed at 43 ℃, the fermentation time can be completely fermented within 8, 10 and 12 hours, the viscosity is firstly low and then high, and the pH value is lower as the fermentation time is longer. The fermentation time is fixed to be 8 hours, the fermentation temperature is 40 ℃ and 43 ℃ and can be completely fermented, the higher the fermentation temperature is, the lower the viscosity is, and the higher the pH value is; the fermentation time is fixed to be 10 hours, the fermentation temperature is 37 ℃, 40 ℃ and 43 ℃ and can be completely fermented, the higher the fermentation temperature is, the higher the viscosity is, the lower the viscosity is, and the lower the pH value is, the higher the pH value is; the fermentation time is fixed to be 12 hours, the fermentation temperature is 37 ℃, 40 ℃ and 43 ℃ to be completely fermented, the higher the fermentation temperature is, the higher the viscosity is, the lower the viscosity is, and the lower the pH value is, the higher the pH value is.

And (3) strain combination 13: the fermentation temperature is fixed at 37 ℃, the fermentation time can be completely fermented within 8, 10 and 12 hours, and the viscosity is higher and the pH value is lower as the fermentation time is longer; the fermentation temperature is fixed at 40 ℃, the fermentation time can be completely fermented within 8, 10 and 12 hours, and the viscosity is higher and the pH value is lower as the fermentation time is longer; the fermentation temperature is fixed at 43 ℃, the fermentation time can be completely fermented within 8, 10 and 12 hours, the viscosity is firstly low and then high, and the pH value is lower as the fermentation time is longer. The fermentation time is fixed to be 8 hours, the fermentation temperature can be completely fermented at 37 ℃, 40 and 43 ℃, and the higher the fermentation temperature is, the higher the viscosity is, and the lower the pH value is; the fermentation time is fixed to be 10 hours, the fermentation temperature is 37 ℃, 40 ℃ and 43 ℃ and can be completely fermented, the higher the fermentation temperature is, the higher the viscosity is, the lower the viscosity is, and the lower the pH value is, the higher the pH value is; the fermentation time is fixed to be 12 hours, the fermentation temperature is 37 ℃, 40 ℃ and 43 ℃ can be completely fermented, the higher the fermentation temperature is, the viscosity is firstly low and then high, and the pH value is firstly low and then unchanged.

Strain combination 14: the fermentation temperature is fixed at 37 ℃, the fermentation time can be completely fermented within 8, 10 and 12 hours, and the viscosity is higher and the pH value is lower as the fermentation time is longer; the fermentation temperature is fixed at 40 ℃, the fermentation time can be completely fermented within 8, 10 and 12 hours, and the viscosity is higher and the pH value is lower as the fermentation time is longer; the fermentation temperature is fixed at 43 ℃, the fermentation time can be completely fermented within 8, 10 and 12 hours, and the viscosity is higher and the pH value is unchanged as the fermentation time is longer. The fermentation time is fixed to be 8 hours, the fermentation temperature is 37 ℃, 40 ℃ and 43 ℃ and can be completely fermented, the higher the fermentation temperature is, the higher the viscosity is, the lower the viscosity is, the higher the viscosity is, and the lower the pH value is; the fermentation time is fixed to be 10 hours, the fermentation temperature is 37 ℃, 40 ℃ and 43 ℃ and can be completely fermented, the higher the fermentation temperature is, the higher the viscosity is, the lower the viscosity is, and the lower the pH value is, the lower the viscosity is, the lower the pH value is; the fermentation time is fixed to be 12 hours, the fermentation temperature is 37 ℃, 40 ℃ and 43 ℃ to be completely fermented, the higher the fermentation temperature is, the higher the viscosity is, the lower the viscosity is, and the lower the pH value is, the higher the pH value is.

Experiment 3, sensory evaluation of yogurt drink:

yogurt has diverse effects on maintaining health. For example, experiments prove that the yoghourt can effectively reduce the total cholesterol and the ratio of the total cholesterol to the high-density lipoprotein cholesterol after being eaten for a long time; the yogurt containing Lactobacillus acidophilus can reduce serum cholesterol for hypercholesterolemic patients; the high density cholesterol can be increased by eating yogurt containing Lactobacillus acidophilus and Bifidobacterium longum for hypercholesterolemic patients; the risk of suffering from diabetes can be effectively reduced by taking the yoghourt for a long time; the edible yogurt can also prevent cardiovascular diseases. Therefore, the effect of maintaining the health can be achieved by eating a sufficient amount of the yoghourt for a long time.

In order to achieve the aim, the strain combinations 1 to 14 are fermented and prepared into the yoghurt drinks 1 to 14, and the yoghurt drinks are analyzed and screened by sensory evaluation. Sensory evaluation is a standard defined and developed by the american society for food and technology, and measures the characteristics of yogurt drinks using three senses, namely sight, smell, and taste. The experimental design for sensory evaluation included: general methods of organoleptic analysis (ISO 6658:2005), sensory evaluation glossary (ISO 5492:2008), sensory color test (visual colorimetry: ISO 11037:1999), sensory texture test (texture profile: ISO 11036:1994), sensory flavor test (flavor profile: ISO 6564:1985), descriptive analysis (qualitative description of organoleptic properties: ISO 11035:1994, evaluation of the intensity of organoleptic properties: ISO 4121: 2003). The experimental method adopts descriptive analysis, ranking method and preference method for evaluation.

1. Evaluation of yogurt drinks 1 to 14 with descriptive analysis:

the method calls 11 high-sensory-acuity assessors who have practical experience of fermented product development, fermented milk product development, yogurt product development and hand-operated beverage shops to perform experiments, wherein 5 males and 6 females are assessed in a conference discussion mode, differences in appearance, texture and flavor among yogurt drinks are judged, and finally the differences are described by vision (color and luster, cleanliness), smell (milk fragrance, aroma and odor uniqueness), taste (acidity, sweetness, smoothness and alcohol thickness) and overall taste (flavor expression and aftertaste), and the taste is evaluated by a five-point evaluation method (1: very dislike, 2: dislike, 3: dislike and dislike, 4: like and 5: very like). The results of the experiment are shown in Table 6.

TABLE 6 organoleptic evaluation results of yogurt drinks 1 to 14 by descriptive analysis

Total score of 55 points, score of 11-26 is low preference level, score of 26-41 is medium preference level, score of above 41 is high preference level

2. Evaluation of the yogurt drinks 1 to 14 was performed by a rank-order evaluation method:

the number and gender of the appraisers are as described above, the appraisers divide the yogurt drinks 1 to 14 into 3 grades (high, medium and low preference grades) according to the preference degrees of the appraisers, and each grade is arranged from left to right according to the highest preference degree to the lowest preference degree. The results are shown in Table 7.

Table 7, sensory evaluation results of yogurt drinks 1 to 14 by the rank-order scoring method:

preference level	Yogurt drink group (highest to lowest preference from left to right)
		Height of	14、12、13、11
In	7、9、6、5、10、8、4
		Is low in	3、2、1

3. Evaluation of yogurt drinks 11 to 14 by a preference evaluation method:

and (3) determining whether the yogurt drinks 11 to 14 with high preference grade by a sequential grading method are significantly different or not by a preference grading method, knowing the relationship between the preference degree and the product characteristics, and grading by a consumer questionnaire method by adopting a preference five-grade grading method (1: very dislike, 2: dislike, 3: dislike and dislike, 4: like, 5: very like). Also for the aroma, sweetness and sourness, five points (1 point: too light, 2 points: light, 3 points: moderate, 4 points: strong, 5 points: too strong) were recorded, with better scores being closer to 3 points.

The 1 st consumer questionnaire was conducted at department store streets without interference from the external environment and without mutual influence. The evaluation subjects were 67 untrained consumers, of which 24 were male (35.8%), 43 were female (64.2%). The age distribution was: 25 people under 20 years old (37.3%), 21 people under 20-29 years old (31.3%), 13 people under 30-39 years old (19.4%), 3 people under 40-49 years old (4.5%), 4 people under 50-59 years old (6%), 1 person over 60 years old (1.5%). The first three major occupational distributions are: 30 students (44.8%), 17 service (25.4%), 5 military classmates (7.4%), and 15 others (22.4%).

The 2 nd consumer questionnaire was also conducted in the university school district without interference from the external environment and without mutual influence. The evaluation subjects were 69 untrained consumers, among which 31 men (44.9%), 38 women (55.1%). The age distribution was: 50 people 18-22 years old (72.5%), 15 people 23-30 years old (21.8%), 4 people less than 18 years old (5.8%). The career distribution was all students (69, 100%).

The total number of participating persons is 136 by combining the 1 st and 2 nd consumer questionnaire results. Referring to table 8, the overall preference degree is best for the yogurt drink 14, with scores of preference greater than 4; secondly, a yoghourt drink 12; the yoghourt drinks 11 and 13 have similar scores and are disliked and not unpleasant when the scores are both more than 3. In addition, the number of

yogurt drinks

11, 12, 13, and 14 rated as the most preferred (first order) yogurt drink by all 136 consumers was 32, 38, 31, and 35, respectively.

TABLE 8 sensory evaluation results of the preference scoring methods (Consumer questionnaire) for yogurt drinks 11 to 14

Yogurt drink group	11	12	13	14
					Score of	3.238±0.865	3.643±0.807	3.273±0.857	4.206±0.759

Under the process of pure nature and without adding any flavoring, the fragrance of the yoghourt drinks 11 to 14 is not greatly different and is slightly weak; the sweet tastes of the yoghourt drinks 11 to 14 are moderate; the sourness of the yogurt drinks 11 to 14 was not very different, and was slightly less (as shown in the results of table 9).

Table 9, five-score sensory evaluation results of flavor, sweetness and sourness of yogurt drinks 11 to 14

Yogurt drink group	Fragrance	Sweet taste	Sour taste
				11	2.60±1.015	3.06±0.918	2.54±0.953
12	2.66±1.000	3.14±0.873	2.65±0.972
				13	2.59±0.815	3.09±0.896	2.60±0.961
14	2.69±0.886	2.96±0.858	2.39±0.939

Experiment 4, sensory evaluation of yoghurt drink ingredients:

in order to increase the eating diversity of the yogurt drink, improve the daily yogurt intake of the user (for example, 200g can be consumed within 30 minutes), and ensure that the user does not feel greasy after drinking for a long time, experiment 4 adds a formula design of a hand-shaking beverage based on the yogurt drink 14, and evaluates the formula by a descriptive analysis method. Experiment 4 high sensory acuity panelists 5 with experience in fermented product development, fermented dairy product development, yogurt product development, hand operated beverage shop practice were summoned to conduct experiments, of which 3 men and 2 women were evaluated in a conference discussion.

1. The formula design of the salty and sweet yoghourt drink comprises the following steps:

adding sesame paste as sauce: the sesame paste is added with the yoghourt to be smashed and ground together after being boiled, the sesame paste is aromatic in flavor and contains oil, the flavor and the oil are compatible, the whole flavor can generate salty taste, but the flavor is too specific, and the acceptability is not high. Adding cucumber as an ingredient: the small cucumber is diced and added with the yoghourt to be smashed and ground together, the flavor of the small cucumber covers the flavor of the yoghourt, the whole vegetable taste is obvious, and the unique flavor of the yoghourt is lacked. Adding corn as an ingredient: corn kernels of the canned non-flavored corn are added into the yoghourt to be smashed and ground together, the corn has slight sweet taste, the overall sweet taste can be improved, but the corn flavor and the yoghourt flavor are combined into a special flavor, and the acceptance is very low. Sensory evaluation results: the formula design of the yoghurt drink is not suitable for salty and sweet taste.

2. The formula design of the yoghourt beverage sauce comprises the following steps:

the yoghourt drink is purely natural in preparation process, does not contain any seasoning, is moderate in overall flavor sweetness and slightly weak in fragrance and sourness, and belongs to a elegant and not greasy taste type. Different sauce formulations bring different sensory feelings. Adding brown sugar to obtain sauce: the brown sugar decocted into syrup is added into the yoghourt to be smashed and ground together, and the brown sugar is rich and special in flavor and is just compatible with the sour and sweet flavor of the yoghourt. Adding matcha as sauce: the Japanese imported matcha powder is added into the yoghourt to be smashed and ground together, and the matcha is slightly bitter and is not compatible with the sour and sweet flavor of the yoghourt. Adding honey to obtain a juice: the honey is directly added into the yoghourt to be smashed and ground together, and the special faint scent of the honey can highlight the sour and sweet flavor of the yoghourt.

3. The formula design of the solid ingredients of the yoghourt drink comprises the following steps:

the yogurt drink belongs to non-Newtonian fluid (non-Newtonian fluid, which means that the viscosity of fluid is an indeterminate value under a certain temperature and pressure), the viscosity of the yogurt drink changes due to the pressure or speed, and the viscosity increases when the pressure is higher, even becomes a temporary solid. If solid ingredients are added into the yoghourt drink, the drinking and the sucking are difficult. In order to overcome the problem, 200g of the yoghourt beverage is contained in a cylindrical cup (the upper circle has a diameter of 9 cm, the lower circle has a diameter of 5.6 cm and the cup is 13.5 cm high), so that a user can easily suck the solid ingredients in the yoghourt beverage by using a suction pipe with a diameter of 1-1.5 cm and a length of 17.5-20 cm, and the phenomenon that the solid ingredients are remained after the beverage is drunk up can not occur. In a preferred embodiment, at room temperature, the temperature rise of the yogurt drink affects the viscosity and thus the ease with which the solid ingredients are sucked through the straw. Therefore, the crushed ice layer is added on the upper layer of the yoghourt drink for cold preservation (the size of the crushed ice is controlled to be 0.2-0.4 cubic centimeter, water can be melted out too fast when the size of the crushed ice is less than 0.2 cubic centimeter, the crushed ice is sucked into the upper layer of the yoghourt drink when the size of the crushed ice is more than 0.4 cubic centimeter, and the taste of the crushed ice is not good), the volume ratio of the crushed ice layer to the yoghourt drink is 1: 3-1: 5, the yoghourt drink is maintained at 8-12 ℃ within 30 minutes, and sensory evaluation results of solid ingredients are reproducible. The evaluation of the sensory evaluation of the solid ingredients is mainly as follows: smoothness of taste and overall balance; further comprises visual stimulation, and promotes the desire of the user to drink the yoghourt drink (promotes appetite and enhances active intake). The appearance of the yoghourt drink can present the effects of layering, gradual layering and special grains. The added solid ingredients include, but are not limited to, pearl, coconut, coffee jelly, vermicelli, taro, pudding, xiancao jelly, tofu pudding, mung bean, red bean, coix seed, purple rice, oat, and the sensory evaluation results are shown in table 10. In addition, fruits including but not limited to watermelon, mango, strawberry, lychee, dragon fruit, passion fruit, avocado, banana, papaya, kiwi, orange, grapefruit, pineapple can also be used as solid ingredients, and the sensory evaluation results are shown in table 11.

TABLE 10 sensory evaluation results of solid ingredients for yogurt drinks

TABLE 11 sensory evaluation results of solid fruit ingredients for yogurt drinks

Experiment 5, preparation of yogurt drink extract:

the types and contents of the active metabolites of the yoghurt drink are different depending on strains, formulas and fermentation environments. In order to determine the active ingredients of the yoghourt drink, the yoghourt drinks 11 to 14 with high sensory evaluation preference grade are selected for extraction and distribution extraction, and the obtained divided layers are subjected to ingredient analysis and activity test.

1. The separation method 1:

heating the stock solution of the yoghourt drink, decompressing, concentrating and drying to obtain paste. And extracting the paste with ethanol for 3 times to obtain ethanol extract. The ethanol extract was filtered under vacuum, heated, and concentrated under reduced pressure to obtain a crude extract. And then adding ethyl acetate: water (1:1(v/v)) was used to partition the crude extract. However, the crude extract has too much emulsion layer, which results in poor separation effect and failure of subsequent separation experiment. If dichloromethane is used instead: water (1:1(v/v)) is used for partition extraction of the crude extract, and the problem of too much emulsion layer still exists.

2. The separation method 2:

the original liquid of the yoghourt drink is not dried, but is diluted by adding water (1:1(v/v)), and then ethyl acetate (1:1(v/v)) is added for partition extraction, so that a large amount of emulsifying layers are still generated, and the separation effect is poor. If dichloromethane (1:1(v/v)) or n-butanol (1:1(v/v)) is added for partition extraction, there is still the problem of too much emulsion layer.

3. The separation method 3:

freeze-drying the stock solution of the yoghourt drink to obtain a powdery product, and sequentially extracting the powdery product by using a solvent with gradually increased polarity. The extraction rate of dichloromethane is too low, the extraction effect of n-hexane is not obviously different, solvent residue is generated after n-butanol extraction, and subsequent solvent extraction is difficult, so that dichloromethane and n-butanol are not suitable to be used as extraction solvents of the separation method.

As shown in fig. 5, the separation method 3 is performed by a solvent permutation and combination extraction test, and finally, n-hexane, ethyl acetate, ethanol and water are sequentially used for extraction. That is, the yogurt drink stock solution 12 is freeze-dried to obtain a powdery product 14, and then n-hexane is used to extract the powdery product 14 to obtain n-hexane extract 16 (sample nos. 11-H, 12-H, 13-H, 14-H) and a first residue 18. Next, the first residue 18 was extracted with ethyl acetate to obtain ethyl acetate extracts 20 (sample Nos. 11-E, 12-E, 13-E, 14-E) and a second residue 22. Thereafter, the second residue 22 was extracted with ethanol to obtain ethanol extracts 24 (sample Nos. 11-A, 12-A, 13-A, 14-A) and a third residue 26. Finally, the third residue 26 is extracted with water to obtain an aqueous extract 28 (sample Nos. 11-W, 12-W, 13-W, 14-W) and a fourth residue 30. The extract and the residue were each concentrated under reduced pressure with heating. The results of the experiments with the various extracts of yogurt drinks 11 to 14 are shown in table 12. Table 12 shows: the separation method can overcome the problem of an emulsion layer generated by liquid-liquid distribution extraction, separate, concentrate and collect various active ingredients of the yoghourt drink, and obviously confirm the difference of various extracts in appearance and flavor by vision and smell.

TABLE 12 extracts from yogurt drinks 11 to 14 obtained by separation method 3

Experiment 6, analysis of the ingredients of the yogurt drink extract:

1. nuclear magnetic resonance spectroscopy (NMR) analysis:

yogurt drinks 11 to 14 were isolated by method 3 to obtain 16 extracts, which were subjected to NMR analysis using isotopic (deuterium) solvents of hydrogen: CDCl₃、acetone-d6、MeOH-d4、C₅D₅N, DMSO-D6 and D₂O solubility of the extract was measured, and as a result, C was found₅D₅N, DMSO-d6 is preferred for 16 extractsThe solubility of (a). In that¹C was found in H-NMR preliminary experiments₅D₅The solvent signal for N masks the signal of the extract, and DMSO-d6 was finally selected as the solvent for NMR analysis, with the concentrations of the extract being 25mg/ml, 50mg/ml, 75mg/ml and 100 mg/ml. Found when the n-hexane extract (11-H, 12-H, 13-H, 14-H), the ethyl acetate extract (11-E, 12-E, 13-E, 14-E), and the ethanol extract (sample nos. 11-A, 12-A, 13-A, 14-A) were at 50mg/ml¹The H-NMR spectrum showed the optimum magnetic field and characteristic signal, with the optimum sample concentration of the aqueous extracts (11-W, 12-W, 13-W, 14-W) being 25 mg/ml. After the n-hexane extracts (11-H, 12-H, 13-H and 14-H) and the ethyl acetate extracts (11-E, 12-E, 13-E and 14-E) are added with DMSO-d6 and before testing, the sample solubility can be improved by heating to 37-40 ℃, and further, the optimization can be carried out¹Characteristic signals of H-NMR spectra. The experiment was carried out with a nuclear magnetic resonance spectrometer JEOL ECS 400MHz FT-NMR with a resolution of 400MHz and chemical shifts in ppm (expressed as coupling constants) in Hertz (J) of 16 extracts as shown in FIGS. 6(A) to 9(D)¹The H-NMR spectrum and the NMR signal analysis and the major component determination of 16 extracts in table 13 show that the NMR characteristic signals and major components of the n-hexane extract, ethyl acetate extract, ethanol extract, and water extract are different, and that the NMR signals of yogurt drinks 11 to 14 are also different.

TABLE 13 NMR Signal analysis and principal component determination of extracts obtained by separation method 3 for yogurt drinks 11 to 14

2. Analysis of total polyphenol (total polyphenol) content:

16 extracts are obtained from 11 to 14 yoghourt drinks through a separation method 3, the extracts are analyzed by a Folin-Ciocalteu colorimetric method, the principle is that phosphotungstic acid (phosphotungstic acid) and phosphomolybdic acid (phosphomolybdybdic acid) in a Folin-Ciocalteu reagent are oxidized and colored by polyphenol molecules, and gallic acid (gallic acid) is used as a standard substance. The experimental method comprises the following steps: dissolving gallic acid (5mg/mL) in methanol to obtain 50, 100, 250 and 500 μ g/mL standards, adding 20 μ L of each standard to 1.6mL with secondary water, adding 100 μ L of 2N Folin-Ciocalteu reagent, and adding 300 μ L of 20% Na₂CO₃Then mixed, reacted at 40 ℃ for 40 minutes, and the absorbance was measured at 725 nm. In addition, secondary water is used to replace the standard substance for zeroing. A standard curve is drawn by the relationship between concentration and absorbance. mu.L of the sample to be tested (1mg/mL) was treated in the same manner and the total polyphenol content per gram of extract was calculated according to the standard curve. As shown in table 14 and fig. 10, the total polyphenol content in the extract of the yogurt drink 11 was the highest at 11-E and the lowest at 11-W; in the extract of the yoghourt drink 12, the content of 12-A is the highest, and the content of 12-W is the lowest; in the extract of the yoghourt drink 13, the content of 13-H is the highest, and the content of 13-W is the lowest; the extract of the yogurt drink 14 has the highest 14-E content and the lowest 14-W content. The total polyphenol content of 14-E was the highest and 11-W was the lowest for 16 extracts. The total polyphenol content in 4 n-hexane extracts (11-H to 14-H) is 18.2-24.0 mg_{Gallic acid}(ii) in terms of/g. The total polyphenol content in the 4 ethyl acetate extracts (11-E to 14-E) is 9.4-40.6 mg_{Gallic acid}(ii) in terms of/g. The total polyphenol content of 4 ethanol extracts (11-A to 14-A) is 11.3-28.3 mg_{Gallic acid}(ii) in terms of/g. The total polyphenol content in 4 water extracts (11-W to 14-W) is 2.6-7.1 mg_{Gallic acid}/g。

TABLE 14 analysis of the content of Total polyphenols, Total flavanone flavonoids and Total polysaccharides in the extracts obtained by separation of yogurt drinks 11-14 according to method 3

3. Analysis of total flavanone flavonoid content:

the principle is that 2,4-Dinitrophenylhydrazine (DNP) can be used for derivatizing aldehyde groups or ketone groups on a flavanone structure, so that the flavanone structure has light absorption characteristics at a specific wavelength, and hesperetin (hesperetin) is used as a standard product. The experimental method comprises the following steps: 1mL of a sample to be tested was added to 2mL of DNP solution and 2mL of methanol, and the mixture was heated in a water bath at 50 ℃ for 50 minutes. After cooling, 5mL of a KOH/methanol solution was added thereto, the mixture was allowed to stand at room temperature for 2 minutes, and 1mL of the mixture was put into a centrifuge bottle containing 5mL of methanol, shaken, and centrifuged at 1108 Xg for 10 minutes. The filtered supernatant was quantified to 25mL with methanol and the absorbance was measured at 494 nm. The measured absorbance is compared with a standard curve of a standard substance for quantification, and the content of flavanone flavonoid in each gram of extract is converted. As shown in Table 14 and FIG. 11, the total flavanone flavonoids measured in the extract of yogurt drink 11 showed the highest 11-A content and the lowest 11-E content; in the extract of the yoghourt drink 12, the content of 12-A is the highest, and the content of 12-H is the lowest; in the extract of the yoghourt drink 13, the content of 13-A is the highest, and the content of 13-H is the lowest; the extract of the yogurt drink 14 has the highest 14-A content and the lowest 14-H content. The total of 16 extracts was found to have the highest total flavanone flavonoid content of 12-A and the lowest total flavanone flavonoid content of 12-H. The total flavanone flavonoid content in the 4 n-hexane extracts (11-H to 14-H) is 15.0-23.1 mg_Hesperetin(ii) in terms of/g. The total flavanone flavonoid content in the 4 ethyl acetate extracts (11-E to 14-E) is 16.0-26.0 mg_Hesperetin(ii) in terms of/g. The total flavanone flavonoid content in the 4 ethanol extracts (11-A to 14-A) is 62.5-91.3 mg_Hesperetin(ii) in terms of/g. The total flavanone flavonoid content in the 4 aqueous extracts (11-W to 14-W) is in the range of 28.2-49.5 mg_Hesperetin/g。

4. Analysis of total polysaccharide content:

the analysis is carried out by a phenol-sulfuric acid method (phenol-sulfuric acid assay) and the principle is that pentose and hexose are acidicUnder high temperature, the furfural is decomposed into furfural (furfurfural) due to dehydration, and the furfural further reacts with phenol to generate an orange product, the total polysaccharide content is calculated according to the change of absorbance, and glucose with the concentration of 0, 10, 20, 30, 40 and 50 mu g/ml is used as a standard substance. The experimental method comprises the following steps: 0.5mL of 5% phenol solution is added into 0.5mL of 10mg/mL of sample to be tested, then 2.5mL of concentrated sulfuric acid is added and mixed evenly, the mixture is reacted in a water bath at 100 ℃ for 20 minutes, and the light absorption value is measured by 490nm wavelength after the temperature is cooled. As shown in Table 14 and FIG. 12, the total polysaccharide content in the extract of yogurt drink 11 was the highest at 11-W and the lowest at 11-H; in the extract of the yoghourt drink 12, the content of 12-W is the highest, and the content of 12-E is the lowest; in the extract of the yoghourt drink 13, the content of 13-W is the highest, and the content of 13-H is the lowest; the extract of the yoghurt drink 14 has the highest content of 14-W and the lowest content of 14-H. The total of 16 extracts was highest in 14-W total polysaccharide content and lowest in 14-H. The total polysaccharide content of 4 n-hexane extracts (11-H to 14-H) is 7.6-11.2 mg_Glucose(ii) in terms of/g. The total polysaccharide content of the 4 ethyl acetate extracts (11-E to 14-E) is 9.7-18.4 mg_Glucose(ii) in terms of/g. The total polysaccharide content of the 4 ethanol extracts (11-A to 14-A) is 223.2 to 249.4mg_Glucose(ii) in terms of/g. The total polysaccharide content of the 4 water extracts (11-W to 14-W) is 414.4-494.1 mg_Glucose/g。

5. Thin Layer Chromatography (TLC) analysis:

the solvent system was tested in combination with an ionic solvent system (n-hexane, dichloromethane, chloroform, methanol) or a non-ionic solvent system (n-hexane, toluene, ethyl acetate, acetone, methanol), and with an acid (glacial acetic acid (GAA), Formic Acid (FA)) or a base (10% ammonia, 25% ammonia, diethylamine) according to normal phase thin layer chromatography commonly used in the pharmacopoeia of taiwan, second edition. As shown in Table 15, the n-hexane extract and the ethyl acetate extracts (11-H, 11-E, 12-H, 12-E, 13-H, 13-E, 14-H, and 14-E) have similar polarities, and the TLC main spots have good analysis effect in the same solvent system. Neither the ethanol extract (11-A to 14-A) nor the water extract (11-W to 14-W) had an ideal analytical effect in normal phase thin layer chromatography, and the TLC main spot was not present in R_f 0.3-0.8。

TABLE 15 Normal phase thin layer chromatography analysis of extracts from yogurt drinks 11 to 14 obtained by separation method 3

6. High Performance Liquid Chromatography (HPLC) analysis:

the analysis was performed with reference to the reverse phase high performance liquid chromatography method commonly used in the pharmacopoeia in taiwan (second edition), under the following conditions: the HPLC is Agilent 1100 series, the detector is G1315B photodiode array detector, the autosampler is G1329A autosampler, the chromatography column is Intersil ODS-3V 250x 4.6mm (5 μm), the solvent A of mobile phase is acetonitrile, and the solvent B is 0.1% H₃PO₄The flow rate was 1ml/min, the column temperature was 30 ℃ and the detection wavelengths were 203, 210, 254, 280 and 365 nm. Vehicle system conditions were as follows: the mobile phase comprises solvents A and B, the linear gradient is 0-20 minutes 0% A-10% A, 20-40 minutes 10% A-40% A, 40-60 minutes 40% A-100% A, 60-70 minutes 100% A, and the flow rate and the column temperature are as described above. As shown in table 16 and fig. 13 to 16, no significant chromatographic peak was observed in both the n-hexane extract (11-H to 14-H) and the ethyl acetate extract (11-E to 14-E) at 5 detection wavelengths, and the ethanol extract (11-a to 14-a) and the water extract (11-W to 14-W) exhibited a preferable chromatographic peak at only 203 nm.

TABLE 16 area and height of the fractions of ethanol extract and water extract subjected to HPLC at 203nm

Extracts of plants	Peak residence time (minutes)	Percentage of area (%)	Percent height (%)
				11-A	16.854	3.56	2.04
12-A	16.860	1.74	3.17
				13-A	16.846	5.01	3.65
14-A	16.971	5.65	3.40
				11-W	40.837	30.59	29.17
12-W	40.437	73.38	100
				13-W	40.230	78.08	100
14-W	40.306	83.43	100

Experiment 7, antioxidant activity test of yogurt drink extract:

1. determination of the ability to scavenge 1, 1-diphenyl-2-trinitrophenylhydrazine (2, 2-diphenyl-1-piperidinylhydrazyl, DPPH) free radical:

lipid rancidity is caused by the generation of free radicals in the process of lipid autooxidation, and antioxidants scavenge lipid peroxide free radicals by supplying hydrogen, thereby inhibiting oxidation chain reactions. DPPH is commonly used in antioxidant studies to evaluate the hydrogen donating ability of antioxidants. The DPPH methanol solution has strong light absorption at 517nm, and the light absorption value is reduced when the DPPH methanol solution is reduced by an antioxidant. Therefore, a lower absorbance at 517nm indicates a higher hydrogen-donating ability of the antioxidant. The experimental method comprises the following steps: a4 mL sample of the extract (50mg of the extract dissolved in 15mL of 50% ethanol) was added to 1mL of a freshly prepared 10mM DPPH methanol solution, mixed with shaking and allowed to stand at room temperature for 30 minutes, and the absorbance at 517nm was measured. The results are expressed as percent scavenging, with higher percent scavenging indicating better hydrogen donating ability of the test sample. As shown in Table 17 and FIG. 17, extracts 11-A and 14-A showed better DPPH radical scavenging ability among 16 extracts.

TABLE 17 determination of DPPH radical scavenging, Total antioxidant, reducing powers of various extracts obtained by separation method 3 of yogurt drinks 11 to 14

2. Total antioxidant capacity (TEAC) determination:

2,2' -Azinobis- (3-ethylbenzthiazoline-6-sulfonate) (ABTS) is catalyzed by hydrogen peroxide and peroxidase to generate oxidation reaction to form stable blue-green water-soluble ABTS ·⁺A cationic radical. A standard curve was prepared using an ultraviolet-visible glossmeter with a strong absorbance at 620nm against a standard water-soluble vitamin E analog (trolox). When ABTS ·⁺When the cation free radical and the antioxidant are reduced, the color of the solution is lightened, and the light absorption value is reduced, thereby converting and eliminating ABTS⁺The ability of cationic radicals. The better scavenging capacity indicates a greater resistance to the oxide hydrogen donor. The experimental method comprises the following steps: placing the prepared ABTS in a refrigerator for 1 hour to generate stable blue-green ABTS⁺An aqueous radical solution. Add 80. mu.L of extract sample (50mg of extract in 15mL of 50% ethanol solution) to 96-well plate and 120. mu.L of ABTS⁺Free radical aqueous solution, total volume 200. mu.L. Shaking the mixture at room temperature in dark for 10 minutes, and measuring the light absorption value at 620 nm. Lower absorbance indicates elimination of ABTS⁺The better the radical effect, the clearance was calculated as a percentage. As shown in table 17 and fig. 18, the total antioxidant capacity results for the extract of yogurt drink 11 were the best 11-a and the weakest 11-E; in the extract of the yogurt drink 12, the 12-A capacity is the best, and the 12-H capacity is the weakest; in the extract of the yogurt drink 13, 13-A has the best ability, and 13-H has the weakest ability; of the extracts of yogurt drink 14, 14-A was the best and 14-H was the weakest. Of the 16 extracts, 12-A had the best overall antioxidant capacity and 12-H was the weakest.

3. And (3) reduction force determination:

the antioxidant can convert red blood salt (K)₃Fe(CN)₆) Reducing into yellow blood salt (K)₄Fe(CN)₆) The salt of yellow blood is then mixed with Fe³⁺The Prussian blue is generated by the action, the light absorption value is measured at the wavelength of 620nm to detect the generation amount of the Prussian blue, and the higher the light absorption value is, the stronger the reducing power of the antioxidant is. The experimental method comprises: mu.L of extract sample (50mg of extract dissolved in 15mL of 50% ethanol solution) was added to a microcentrifuge tube, followed by 200. mu.L of 1% hematite in a total volume of 400. mu.L. After mixing, the mixture was placed in a 50 ℃ water bath for 20 minutes, after completion of the reaction, the mixture was cooled on ice for 10 minutes, mixed with 200. mu.L of 10% trichloroacetic acid (TCA), and centrifuged at 3,000rpm for 10 minutes. Add 75. mu.L of supernatant to a 96-well plate, add 75. mu.L of ultrapure water and 30. mu.L of 0.1% FeCl₃.6H₂O, and measuring the absorbance at 620nm after mixing. Higher absorbance indicates higher reducing power. As shown in Table 17 and FIG. 19, the extract of the yogurt drink 11 exhibited the best reducing power of 11-A, and the equal and weakest reducing powers of 11-H and 11-E; in the extract of the yoghurt drink 12, the 12-A reducing power is the best, and the 12-W is the weakest; in the extract of the yoghourt drink 13, the 13-A reducing power is the best, and the 13-H is the weakest; in the extract of the yogurt drink 14, the 14-A reducing power is the best, and the 14-H reducing power is the weakest. Of the 16 extracts, 12-A reduction was the best and 13-H reduction was the weakest.

Experiment 8, test of cancer cell growth inhibition activity of yogurt drink extract:

for organs related to the symbiotic bacteria, intestine and brain axes, active metabolites were tested for cancer cell growth inhibitory activity against 16 extracts obtained by isolation method 3 in yogurt drinks 11 to 14, which were selected from oral cancer (Ca9-22, Cal-27 cell line), leukemia (K562, Molt 4 cell line), brain cancer (GBM8401, U87MG cell line), stomach cancer (KATO III, SNU-1 cell line), colon cancer (DLD-1 cell line), and prostate cancer (LN-cap, PC-3 cell line) from the beginning of oral intake, via the digestive tract, blood, circulatory system, and urinary system (excretory system). The experimental method was a cell viability assay (MTT assay) in which various cancer cells were implanted in 96-well plates at a cell density of 7X 10 per well³cells/100. mu.L, and additional samples (in 100. mu.g/mL) at different concentrations (DMSO as control) were placed at 37 ℃ in 5% CO₂The cell incubator was left standing for 72 hours. Then, 50. mu.L of MTT was added thereto and cultured for 1 hour. After centrifugation at 2000rpm for 3 minutes, 200. mu.L DMSO was added to the removed supernatant, and the mixture was shaken on a shaker until the purple crystals were completely dissolved, and absorbance values of 570mm and 620mm were read. The results are presented after treatment of the different samples at a concentration of 100. mu.g/mL, most preferablyThe percentage of cancer cell growth is inhibited well. The human clinical treatment of oral cancer is mainly surgery and radiotherapy (electrotherapy), and 16 extracts in the experiment do not show the effect of inhibiting the growth of oral cancer cells. The results of the experiments with other cancer cells are shown in table 18 and fig. 20 to 24.

TABLE 18 cancer cell survival inhibition ratio (%)

1. Inhibition of growth activity of leukemia cells (K562, Molt 4 cell line):

as shown in fig. 20, in the extract of the yogurt drink 11, the inhibitory activity of 11-a was the best and 11-W was the weakest in inhibiting the growth of the leukemia cells K562; in the extract of the yoghurt drink 12, the inhibitory activity of 12-A is the best, and 12-W is the weakest; in the extract of the yogurt drink 13, the inhibitory activity of 13-W is the best, and 13-H is the weakest; of the extracts from yogurt drink 14, 14-A had the best inhibitory activity and 14-W was the weakest. Of the 16 extracts combined, 12-A had the best activity for inhibiting the growth of cancer cells. As shown in FIG. 20, in the extract of the yogurt drink 11, the inhibitory activity of 11-H was the best, and neither of 11-E and 11-W had the inhibitory activity, in terms of inhibiting the growth of blood cancer cells Molt 4; in the extract of the yoghourt drink 12, the inhibition activity of 12-A is the best, and 12-H and 12-W have no inhibition activity; in the extract of the yogurt drink 13, the inhibitory activity of 13-A is the best, and 13-E is the weakest; in the extract of the yogurt drink 14, the inhibitory activity of 14-E was the best, and neither 14-A nor 14-W had inhibitory activity. Of the 16 extracts combined, 14-E had the best inhibitory activity.

2. Inhibiting the growth activity of brain cancer cells (GBM8401 and U87MG cell lines):

as shown in FIG. 21, in the extract of yogurt drink 11, 11-A had the best inhibitory activity and 11-E had the weakest inhibitory activity against the growth of brain cancer cells GBM 8401; in the extract of the yoghurt drink 12, the inhibitory activity of 12-A is the best, and 12-W is the weakest; in the extract of the yogurt drink 13, the inhibitory activity of 13-H is the best, and 13-W is the weakest; of the extracts from yogurt drink 14, 14-A had the best inhibitory activity and 14-H was the weakest. Of the 16 extracts combined, 11-A had the best inhibitory activity. As shown in fig. 21, in the extract of the yogurt drink 11, the inhibitory activity of 11-a was the best and 11-W was the weakest in terms of inhibiting the growth of the cells U87 MG; in the extract of the yoghurt drink 12, the inhibitory activity of 12-H is the best, and 12-W is the weakest; in the extract of the yoghourt drink 13, the inhibition activity of 13-H is the best, and 13-E and 13-W have no inhibition activity; of the extracts from yogurt drink 14, 14-W had the best inhibitory activity, and 14-E was the weakest. Of the 16 extracts combined, 14-W was the most potent inhibitor.

3. Inhibiting the growth activity of gastric cancer cells (KATO III, SNU-1 cell line):

as shown in FIG. 22, in the extract of yogurt drink 11, 11-H had the best inhibitory activity and 11-W was the weakest in inhibiting the growth of gastric cancer cells KATO III; in the extract of the yoghourt drink 12, the inhibition activity of 12-E is the best, and 12-H and 12-W have no inhibition activity; only 13-E in the extract of yogurt drink 13 had inhibitory activity; of the extracts from yogurt drink 14, 14-W had the best inhibitory activity, and 14-A was the weakest. Of the 16 extracts combined, 14-W was the most potent inhibitor. As shown in FIG. 22, in the extract of yogurt drink 11, 11-H had the best inhibitory activity and 11-A had the weakest inhibitory activity against the growth of gastric cancer cells SNU-1; in the extract of the yoghurt drink 12, the inhibitory activity of 12-E is the best, and 12-W is the weakest; in the extract of the yogurt drink 13, the inhibitory activity of 13-H is the best, and 13-A is the weakest; of the extracts from yogurt drink 14, 14-A had the best inhibitory activity and 14-E was the weakest. Of the 16 extracts combined, 12-E had the best inhibitory activity.

4. Inhibiting the growth activity of colorectal cancer cells (DLD-1 cell line):

as shown in FIG. 23, in the extract of the yogurt drink 11, 11-H inhibitory activity was the best and 11-W was the weakest in the inhibition of the growth of DLD-1 cells in colorectal cancer; in the extract of the yoghurt drink 12, the inhibitory activity of 12-H is the best, and 12-E is the weakest; in the extract of the yogurt drink 13, the inhibitory activity of 13-H is the best, and 13-W is the weakest; of the extracts from yogurt drink 14, 14-A had the best inhibitory activity and 14-E was the weakest. Of the 16 extracts combined, 11-A had the best inhibitory activity.

5. Inhibiting the growth activity of prostate cancer cells (LN-cap, PC-3 cell line):

as shown in FIG. 24, 11-A had the best inhibitory activity and 11-E the weakest in the extract from yogurt drink 11 with respect to the inhibition of the growth of prostate cancer cells LN-cap; in the extract of the yoghurt drink 12, the inhibitory activity of 12-H is the best, and 12-E is the weakest; in the extract of the yoghourt drink 13, the inhibition activity of 13-A is the best, and 13-H and 13-E have no inhibition activity; of the extracts of yogurt drink 14, 14-W had the best inhibitory activity, and 14-H was the weakest. Of the 16 extracts combined, 11-A had the best inhibitory activity. As shown in FIG. 24, in the extract of yogurt drink 11, 11-E had the best inhibitory activity and 11-H the weakest, in terms of inhibiting the growth of prostate cancer cells PC-3; in the extract of the yoghurt drink 12, the inhibitory activity of 12-H is the best, and 12-W is the weakest; in the extract of the yoghourt drink 13, the inhibition activity of 13-E is the best, and 13-H and 13-W have no inhibition activity; of the extracts from yogurt drink 14, 14-E had the best inhibitory activity, and 14-A was the weakest. Of the 16 extracts combined, 13-E had the best inhibitory activity.

Experiment 9, yogurt drink production system:

the past liquid yogurt drinks are divided into two forms in production: home-made small-volume production and large-volume production in tonnage plants. The household small-scale production has the problems of unstable quality, subsequent modulation and inconvenient operation. The mass production of ton-scale factories faces the problem that 2 to 3 days or more have passed since the consumers purchased and drunk the drinks, the freshly fermented yogurt drinks cannot be drunk the same day, and the drinks cannot be drunk up within the shelf life or are not preserved properly and deteriorate. Therefore, the invention provides a production system of liquid yoghourt drink, which can simultaneously solve the problems of home-made small-quantity production and ton-grade factory large-quantity production of yoghourt drink, can produce kilogram-grade liquid yoghourt drink by a single fermentation machine, can be fermented and manufactured in a transparent process on site, and can be finished in 18 hours, so that the yoghourt drink drunk by consumers every day is fresh and fermented on the same day, and the terminal product is eaten in a hand-operated drinking mode.

As shown in fig. 25 and the structure of the fermentation machine 100 shown in fig. 26, the liquid yogurt drink production system of the present invention sequentially includes: high-pressure cleaning, high-temperature sterilization, milk-making emulsification, secondary sterilization, inoculation fermentation, finished product sampling, finished product collection, cooling and refrigeration, homogenizing and blending, ingredient addition, filling and packaging and product serving.

The machine 100 for preparing yogurt drinks comprises: the tank comprises a tank body 101, a sealing top cover 105 assembled with the tank body 101, a feeding port M arranged on the sealing top cover 105, a motor controller 108 arranged outside the tank body 101, a stirring motor 110 coupled to the motor controller 108, a stirring arm 104 arranged in the space of the tank body 101 and connected to the stirring motor 110, and a resistance heater 103 arranged outside the tank body 101 and coupled to the motor controller 108. Wherein, the space enclosed by the sealing top cover 105 and the tank body 101 is in a closed state; water, milk powder and fungus powder are fed into the space through a feeding port M; the resistance heater 103 is used for sterilizing water and milk powder, and the bacteria powder is stirred by the stirring arm 104 in the mixture formed by the water and the milk powder at 37-43 ℃ to prepare the yoghourt drink.

Further, the fermentation machine 100 further includes a discharge port N4 disposed at the bottom of the tank 101 for discharging the yogurt drink or the waste liquid (or the waste water). Alternatively, the fermentation apparatus 100 further includes a temperature control interlayer 102 disposed outside the tank 101 and connected to the resistance heater 103, wherein the temperature control interlayer 102 is controlled by the resistance heater 103 to adjust or maintain the temperature in the space. Alternatively, the sealing top cover 105 further comprises a cleaning opening 106, and the fermentation machine 100 further comprises a heat conduction inflow opening N5 arranged on the side edge of the tank body 101 and a heat conduction outflow opening N6 arranged at the bottom. Alternatively, the fermentation machine 100 further comprises a temperature-sensitive detector 107 coupled to the motor controller 108, and the temperature sensed by the temperature-sensitive detector 107 is displayed on a screen 109 of the motor controller 108. Alternatively, a viewing window S1 in the seal cap 105 may allow the user to visualize the conditions within the tank 101.

The process of fig. 25 is as follows:

(1) high-pressure cleaning and high-temperature sterilization: opening a feeding port M → cleaning the inner wall of the tank body 101 by using a high-pressure spray gun → confirming that the water level is at a proper position by an observation window S1 → opening a discharge port N4 for discharging → the tank body 101 is filled with water again → the temperature detected by the temperature detector 107 is displayed on a screen 109 of the motor controller 108 → is displayed and heated to above 72 deg.C → is reached to a sterilization temperature after → standing for 30 minutes → opening the discharge port N4 for discharging to a wastewater tank (not shown).

(2) Milk making and emulsifying: opening the feeding port M → injecting clean water → feeding milk powder → turning on the stirring motor 110 → emulsifying → closing the feeding port M → confirming the emulsification through the observation window S1.

(3) Secondary sterilization: open warming → view screen 109 shows heating above 72 deg.C → after reaching sterilization temperature → close warming → rest for 30 minutes.

(4) Inoculating bacteria and fermenting: injecting cooling water from the heat conduction inlet N5 → turning on a circulating pump (not shown) → performing heat exchange temperature control with the cooling water → the observation screen 109 displaying gradual cooling to 43 ℃ → turning off the cooling water source and the circulating pump → the feed port M is disinfected with 75% alcohol → rapidly opening the feed port M for feeding bacteria and turning off the screw → turning on the stirring motor 110 to stir the stirring arm 104 for 20 minutes → turning off the stirring motor 110 → standing for fermentation.

(5) Sampling finished products: spraying purified water on a discharge port N4, spraying 75% alcohol for disinfection → taking out a 300 ml sample → carrying out viscosity and pH value detection → reaching the standard of the tank collection → carrying out the tank collection.

(6) And (3) finished product collection: spraying purified water to a discharge port N4 → spraying 75% alcohol for disinfection → using 304 food-grade stainless steel containers for receiving in batches.

(7) Cooling and refrigerating: and (4) preparing the yoghourt drink → refrigerating at 4 ℃.

(8) Homogenizing and blending, and adding ingredients: taking yogurt drink → adding ice blocks → homogenizing with a homogenizer → blending with solid ingredients or solid fruit ingredients.

(9) Filling and packaging, and taking out the product: finally, checking whether the beverage or the ingredients are seeped → finished.

The invention belongs to a difficult innovation, has high industrial value and is applied by law. Furthermore, the invention may be modified in any way by a person skilled in the art, without thereby departing from the scope of protection as claimed in the appended claims.

Sequence listing

<110> Yuyang biomedical products Ltd

<120> yoghourt raw beverage with functions of oxidation resistance and inhibition of growth of cancer cells in digestive tract and preparation method thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1478

<212> DNA

<213> Lactobacillus fermentum

<400> 1

tggctcagga tgaacgccgg cggtgtgcct aatacatgca agtcgaacgc gttgrcccaa 60

ttgattgatg gtgcttgcac ctgattgatt ttggtcgcca acgagtggcg gacgggtgag 120

taacacgtag gtaacctgcc cagaagcggg ggacaacatt tggaaacaga tgctaatacc 180

gcataacaac gttgttcgca tgaacaacgc ttaaaagatg gcttctcgct atcacttctg 240

gatggacctg cggtgcatta gcttgttggt ggggtaaygg cctaccaagg cgatgatgca 300

tagccgagtt gagagactga tcggccacaa tgggactgag acacggccca tactcctacg 360

ggaggcagca gtagggaatc ttccacaatg ggcgcaagcc tgatggagca acaccgcgtg 420

agtgaagaag ggtttcggct cgtaaagctc tgttgttaaa gaagaacacg tatgagagta 480

actgttcata cgttgacggt atttaaccag aaagtcacgg ctaactacgt gccagcagcc 540

gcggtaatac gtaggtggca agcgttatcc ggatttattg ggcgtaaaga gagtgcaggc 600

ggttttctaa gtctgatgtg aaagccttcg gcttaaccgg agaagtgcat cggaaactgg 660

ataacttgag tgcagaagag ggtagtggaa ctccatgtgt agcggtggaa tgcgtagata 720

tatggaagaa caccagtggc gaaggcggct acctggtctg caactgacgc tgagactcga 780

aagcatgggt agcgaacagg attagatacc ctggtagtcc atgccgtaaa cgatgagtgc 840

taggtgttgg agggtttccg cccttcagtg ccggagctaa cgcattaagc actccgcctg 900

gggagtacga ccgcaaggtt gaaactcaaa ggaattgacg ggggcccgca caagcggtgg 960

agcatgtggt ttaattcgaa gctacgcgaa gaaccttacc aggtcttgac atcttgcgcc 1020

aaccctagag atagggcgtt tccttcggga acgcaatgac aggtggtgca tggtcgtcgt 1080

cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tgttactagt 1140

tgccagcatt aagttgggca ctctagtgag actgccggtg acaaaccgga ggaaggtggg 1200

gacgacgtca gatcatcatg ccccttatga cctgggctac acacgtgcta caatggacgg 1260

tacaacgagt cgcgaactcg cgagggcaag caaatctctt aaaaccgttc tcagttcgga 1320

ctgcagtgcc gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccatgag 1380

agtttgtaac acccaaagtc ggtggggtaa ccttttagga gccagccgcc taaggtggga 1440

cagatgatta gggtgaagtc gtaacaaggg taaaccga 1478

<210> 2

<211> 1515

<212> DNA

<213> Lactobacillus helveticus (Lactobacillus helveticus)

<400> 2

gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc gagcagaacc agcagattta 60

cttcggtaat gacgctgggg acgcgagcgg cggatgggtg agtaacacgt ggggaacctg 120

ccccatagtc tgggatacca cttggaaaca ggtgctaata ccggataaga aagcagatcg 180

catgatcagc ttataaaagg cggcgtaagc tgtcgctatg ggatggcccc gcggtgcatt 240

agctagttgg taaggtaacg gcttaccaag gcaatgatgc atagccgagt tgagagactg 300

atcggccaca ttgggactga gacacggccc aaactcctac gggaggcagc agtagggaat 360

cttccacaat ggacgcaagt ctgatggagc aacgccgcgt gagtgaagaa ggttttcgga 420

tcgtaaagct ctgttgttgg tgaagaagga tagaggtagt aactggcctt tatttgacgg 480

taatcaacca gaaagtcacg gctaactacg tgccagcagc cgcggtaata cgtaggtggc 540

aagcgttgtc cggatttatt gggcgtaaag cgagcgcagg cggaagaata agtctgatgt 600

gaaagccctc ggcttaaccg aggaactgca tcggaaactg tttttcttga gtgcagaaga 660

ggagagtgga attccatgtg tagcggtgga atgcgtagat atatggaaga acaccagtgg 720

cgaaggcgac tctctggtct gcaactgacg ctgaggctcg aaagcatggg tagcgaacag 780

gattagatac cctggtagtc catgccgtaa acgatgagtg ctaagtgttg ggaggtttcc 840

gcctctcagt gctgcagcta acgcattaag cactccgcct ggggagtacg accgcaaggt 900

tgaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga 960

agcaacgcga agaaccttac caggtcttga catctagtgc catcctaaga gattaggagt 1020

tcccttcggg gacgctaaga caggtggtgc atggctgtcg tcagctcgtg tcgtgagatg 1080

ttgggttaag tcccgcaacg agcgcaaccc ttgttattag ttgccagcat taagttgggc 1140

actctaatga gactgccggt gataaaccgg aggaaggtgg ggatgacgtc aagtcatcat 1200

gccccttatg acctgggcta cacacgtgct acaatggaca gtacaacgag aagcgagcct 1260

gcgaaggcaa gcgaatctct gaaagctgtt ctcagttcgg actgcagtct gcaactcgac 1320

tgcacgaagc tggaatcgct agtaatcgcg gatcagaacg ccgcggtgaa tacgttcccg 1380

ggccttgtac acaccgcccg tcacaccatg gaagtctgca atgcccaaag ccggtggcct 1440

aaccttcggg aaggagccgt ctaaggcagg gcagatgact ggggtgaagt cgtaacaagg 1500

tagccgtagg agaac 1515

Claims

1. A yogurt drink comprising:

animal milk products, including water and animal milk;

a plurality of bacterial powders mixed with the animal milk product, wherein the plurality of bacterial powders are from a corresponding plurality of strains comprising: bifidobacterium longum (Bifidobacterium longum), Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus paracasei (Lactobacillus paracasei) and Lactobacillus rhamnosus (Lactobacillus rhamnosus).

2. The yogurt drink of claim 1, wherein the plurality of bacterial strains further comprises: bifidus (Bifidobacterium bifidum), Bifidobacterium breve (Bifidobacterium breve), Bifidobacterium rett (Bifidobacterium lactis), Lactobacillus casei (Lactobacillus casei), Lactobacillus bulgaricus (Lactobacillus delbrueckii subsp. bulgaricus), Lactobacillus helveticus (Lactobacillus helveticus), Lactobacillus plantarum (Lactobacillus plantarum) and Streptococcus thermophilus (Streptococcus thermophilus).

3. The yogurt drink of claim 1, wherein the plurality of bacterial strains further comprises: bifidus (bifidus), Bifidobacterium referans (Bifidobacterium lactis), Enterococcus faecalis (Enterococcus faecium), Lactobacillus casei (Lactobacillus casei), Lactobacillus fermentum (Lactobacillus fermentum), Lactobacillus plantarum (Lactobacillus plantarum), and Streptococcus thermophilus (Streptococcus thermophilus).

4. The yogurt drink of claim 1, wherein the plurality of bacterial strains further comprises: lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus salivarius and Streptococcus thermophilus.

5. Yoghurt drink according to any one of claims 1 to 4, characterized in that the bacterial powder weight of each of said plurality of bacterial strains is the same.

6. The yogurt drink of any one of claims 1 to 4, further comprising a sauce ingredient selected from one of the group consisting of brown sugar, honey, and combinations thereof.

7. The yogurt drink of any one of claims 1 to 4, further comprising a solid ingredient selected from one of the group consisting of pearls, coconut, coffee jelly, taro, pudding, red beans, purple rice, oats, and combinations thereof.

8. The yogurt drink of any one of claims 1 to 4, further comprising a solid fruit ingredient selected from one of the group consisting of mango, strawberry, dragon fruit, passion fruit, avocado, banana, kiwi, orange, grapefruit, and combinations thereof.

9. Yoghurt drink according to any one of claims 1 to 4, wherein the plurality of bacterial strains are fermented in the animal milk product at 37 to 43 ℃ for 8 to 12 hours.

10. A method of isolating the yoghurt drink of any one of claims 1 to 4, comprising:

freeze-drying the yoghurt drink to obtain a powdery product;

extracting the powdery product by using normal hexane to obtain a normal hexane extract and a first residue, wherein the main component of the normal hexane extract is a short-chain fatty acid with double bonds and hydroxyl groups;

extracting the first residue with ethyl acetate to obtain an ethyl acetate extract and a second residue, wherein the major amount of the glycolipid in the ethyl acetate extract;

extracting the second residue with ethanol to obtain an ethanol extract and a third residue, wherein the ethanol extract comprises major components including disaccharide and oligosaccharide; and

and extracting the third residue with water to obtain an aqueous extract and a fourth residue, wherein the major components of the aqueous extract comprise polysaccharide, glycoprotein and protein.

11. The separation method according to claim 10, wherein the n-hexane extract and the ethyl acetate extract are subjected to component analysis by normal phase thin layer chromatography, and a solvent system of the normal phase thin layer chromatography is toluene: ethyl acetate: formic acid 5: 4:1 (v/v/v).

12. The separation method according to claim 10, wherein the ethanol extract and the aqueous extract are subjected to a composition analysis by reverse phase high performance liquid chromatography.

13. Use of the yoghurt drink according to any one of claims 1 to 4 for the preparation of a food product for inhibiting the growth of cancer cells selected from the group consisting of blood cancer cells, brain cancer cells, stomach cancer cells, colon cancer cells and combinations thereof.