TW202102121A - Yogurt bio beverage for anti-oxidation and anti-proliferation against human digestive tract cancer cell lines, and its preparation method - Google Patents

Abstract

The present invention discloses a yogurt bio beverage for anti-oxidation and anti-proliferation against human digestive tract cancer cell lines and its preparation method. The yogurt beverage includes eight to tweleve species of bacteria, which fermentate at 37℃ to 43℃ for 8 hours to 12 hours using the animal milk product. The yogurt beverage has excellent sensory evaluation, and could further contain sauces and solid (fruit) ingredients to enhance the whole sensation for eating. The yogurt beverage also is subjected to the solvents with the gradually-enhanced polarities to identify the main components therein. It is proved that the present yogurt bevenage has effects of free radical clearance, anti-oxidation, high reduction capability, and the inhibition of cancer cell growth.

Description

Yogurt biotechnology drink with anti-oxidation and inhibiting growth of digestive tract cancer cells and preparation method thereof

The invention relates to a yogurt drink and a preparation method thereof.

The two-way information network between the gut and the brain is called the gut-brain axis. Later, it was discovered that the symbiotic flora of the intestine contributes to this gut-brain axis, and the three influence each other, so they become symbiotic bacteria. -Microbiota-gut-brain axis. The National Institutes of Mental Health (NIMH) launched a special project to explore the mechanism of intestinal microbiota-brain communication in 2013, with the purpose of developing new anti-psychiatric drugs and non-invasive treatments. Since then, related studies on the symbiotic bacteria-gut-brain axis have sprung up and become the focus of neuroscience research, with the main focus being to explore the interaction between the gut microbiota and the brain. The gut microbiota exerts an important influence on the brain through the neural network, neuroendocrine system and immune system, and the disturbance of human behavior will also change the composition of the gut microbiota. The literature has reported the mutual balance relationship between symbiotic bacteria-gut-brain axis, and confirmed that it is related to a variety of diseases, such as: inflammatory bowel disease (IBD), gastrointestinal cancer, cholelithiasis, behavior disorder, anxiety, Depression, chronic fatigue syndrome (CFS), hepatic encephalopathy (hepatic encephalopathy), allergies, obesity, diabetes, atherosclerosis, etc. In cancer chemotherapy and post-surgery care, human clinical studies have reported that regulating the composition of the intestinal microbiota can improve the cardiopulmonary health of breast cancer patients and reduce the fear of cancer recurrence.

At present, Western medicine and Chinese medicine systems are actively developing, or combining symbiotic bacteria-gut-brain axis Research on drug development, disease treatment and its mechanism of action; and another medical system, Ayurveda, uses a large amount of turmeric to improve digestive tract discomfort, neuroprotection, and prevent Alzheimer’s disease. Its diversity The efficacy of the intestine-brain axis is also related. However, due to different dietary habits, the use of turmeric has regional restrictions, mainly in India.

In the Western medical system, the use of symbiotic bacteria-gut-brain axis research for drug development is called microecological drugs, which refers to pharmaceutical preparations made from normal microorganisms or substances that regulate the normal growth of microorganisms. It can be applied to indications such as infection, diabetes, tumor, inflammatory disease, immune-related disease, etc. Microecological drugs include: live biotherapeutic product (LBP), small molecule microbiome modulator (SMMM), and fecal microbiota transplant (FMT). Among them, LBP refers to the clear control of the types and numbers of bacteria through the identification, screening and combination of human microorganisms, and the use of different types, numbers and combinations of bacteria for different indications to ensure the safety and effectiveness of the medication. However, the U.S. Food and Drug Administration (FDA) has not yet approved any LBP listing, and only released early clinical trial guidelines for LBP in 2016, making the development of LBP have clear standards. SMMM refers to a substance that can selectively promote the growth and reproduction of one or more beneficial bacteria in the host's intestines, and achieve the purpose of treatment by affecting the growth and reproduction of bacteria. The current known SMMM drug development has only reached the second clinical stage. FMT refers to transplanting the functional flora of healthy human feces to the patient's gastrointestinal tract to rebuild the patient's intestinal flora for the treatment of intestinal and extra-intestinal diseases. Although the United States has included FMT in the treatment guidelines for recurrent Clostridium difficile infection in 2013, the policies of different countries are very different, and FMT currently lacks unified and effective supervision. On the whole, the progress of clinical trials of microecological drugs has not been as expected, and there are currently no microecological drugs approved for marketing. Another problem is that the number of clinical trials of microecological drugs is relatively small, and the human verification of drug efficacy is still not as expected.

The Chinese medicine system does not use symbiotic bacteria-gut-brain axis to research and develop drugs, but to study the interaction between Chinese medicine and gut microbiota. The regulating effect of traditional Chinese medicine on the intestinal microbiota. For example, tonic traditional Chinese medicine containing polysaccharides can support beneficial bacteria and pathogenic bacteria, but for beneficial bacteria The planting effect is significantly better than that of pathogenic bacteria, and the metabolites produced by well-growing beneficial bacteria indirectly inhibit the growth of pathogenic bacteria. For example, Codonopsis pilosula polysaccharide can promote the growth of bifidobacteria in vitro, thereby increasing acetic acid metabolism and enhancing the colonization resistance of bifidobacteria. The metabolic effects of the gut microbiota on traditional Chinese medicine. For example, it has been confirmed that many traditional Chinese medicine components can only produce medicinal ingredients through the metabolism of the gut microbiota to achieve therapeutic effects. For example, baicalin contained in Scutellaria baicalensis is difficult to be directly absorbed in the intestinal tract. Only when it is hydrolyzed by the intestinal microbiota into baicalein can it be absorbed into the blood and play a role. Human clinical trials have confirmed that Gegen Qinlian Decoction can treat type 2 diabetes by changing the intestinal microbiota. On the whole, the study of symbiotic bacteria-gut-brain axis of traditional Chinese medicine provides an explanation of the mechanism acceptable to western medicine, but it still fails to improve the drug compliance and drug compliance caused by the high dose and long treatment time of traditional Chinese medicine. The old problem of low medication adherence.

The research and development of the above-mentioned western medicine, traditional Chinese medicine and Ayurvedic medical system for the symbiotic bacteria-gut-brain axis focuses on diseases, medicines and treatments. On the whole, these developments are still in their infancy, and there are no successful industrial cases or large-scale mass production. Therefore, it is still necessary to develop novel products and technologies that can be applied to the symbiotic bacteria-gut-brain axis to achieve biomedical effects.

In view of the shortcomings of the prior art, the applicant in this case, after careful experimentation and research, and with a spirit of perseverance, finally conceived this case, which can overcome the shortcomings of the previous technology. The following is a brief description of the case.

The purpose of the present invention is to provide a yogurt drink, including: animal milk products and plural bacteria powder, animal milk products including water and animal milk, plural bacteria powder and animal milk products are mixed, wherein the plural bacteria powder comes from the corresponding plural species Strains, plural strains include: Bifidobacterium longum (Bifidobacterium longum), Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus paracasei (Lactobacillus paracasei) and Rhamnosus ( Lactobacillus rhamnosus ).

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

Another object of the present invention is to provide a method for separating the aforementioned yogurt drink, comprising: freeze-drying the yogurt drink to obtain a powdered product; extracting the powdered product with n-hexane to obtain a n-hexane extract and a first residue, The main component of the n-hexane extract is short-chain fatty acids with double bonds and hydroxyl groups; the first residue is extracted with ethyl acetate to obtain an ethyl acetate extract and a second residue, wherein the ethyl acetate extract The main component is glycolipid; the second residue is extracted with ethanol to obtain an ethanol extract and a third residue, where the main components of the ethanol extract include disaccharides and oligosaccharides; and the third residue is extracted with water, A water extract and a fourth residue are obtained, wherein the main components of the water extract include polysaccharides, glycoproteins and proteins.

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

The invention discloses a fermentation machine for preparing yogurt 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 sealed. The feeding port is arranged on the sealed top cover, and water, milk powder and bacterial powder are fed into the space through the feeding port. The motor controller is arranged outside the tank, 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. Resistance warmer It is arranged outside the tank and coupled to the motor controller for disinfecting water and milk powder, and fermenting the bacterial powder in the mixture formed by the water and milk powder at 37°C to 43°C to prepare the yogurt drink.

In the present invention, the fermentation machine further includes a discharge port at the bottom of the tank, and the yogurt drink is sent out from the discharge port. The temperature control interlayer outside the tank is connected with the resistance warmer, and the temperature control interlayer is controlled by the resistance warmer to adjust or maintain the temperature of the space. The sealing top cover also includes a cleaning port, the side of the tank body includes a heat conduction inflow port, and the bottom of the tank body includes a heat conduction outflow port.

In the present invention, the fermentation machine further includes a temperature sensor coupled to the motor controller, and the motor controller further includes a screen, and the temperature sensed by the temperature sensor is displayed on the screen. The sealing top cover also includes an observation window for the user to visually observe the condition in the tank.

The term "animal milk products" as used herein generally includes water and animal milk. Animal milk can exist in liquid form, or pasteurized and dried into powder form, and the source of animal milk can be human milk, cow milk or goat milk Wait.

The various bacterial strains and cancer cell strains in this article can be obtained through commercial methods, and there is no need to deposit biological materials. The yogurt drink in this article is liquid under refrigeration and room temperature. The strains in the yogurt drink in this article showed varying fermentation results at a temperature of 37°C to 43°C and a fermentation time of 8 hours to 12 hours.

3‧‧‧Separation method

12‧‧‧ Yogurt drink stock solution

14‧‧‧Powdered product

16‧‧‧N-hexane extract

18‧‧‧First residue

20‧‧‧Ethyl acetate extract

22‧‧‧Second residue

24‧‧‧Ethanol extract

26‧‧‧Third residue

28‧‧‧Water extract

30‧‧‧The fourth residue

100‧‧‧Fermenting machine

101‧‧‧Slot body

102‧‧‧Temperature control sandwich

103‧‧‧Resistance Warmer

104‧‧‧Stirring arm

105‧‧‧Sealed top cover

106‧‧‧Cleaning port

107‧‧‧Temperature detector

108‧‧‧Motor Controller

109‧‧‧Screen

110‧‧‧Stirring motor

M‧‧‧Feeding port

N4‧‧‧Discharge port

N5‧‧‧Heat conduction inlet

N6‧‧‧Heat conduction outlet

S1‧‧‧Observation window

Figure 1 shows the 16S rDNA sequencing results of the strain code LR-36 and the NCBI comparison results. It is Lactobacillus rhamnosus (Lactobacillus rhamnosus).

Figure 2 shows the 16S rDNA sequencing results and NCBI comparison results of the strain code LPL-68, which is Lactobacillus plantarum .

Figure 3 (A) is a photomicrograph of the strain code UY-58. It is a gram-positive bacillus, has no catalase, oxidase and motility, does not produce endospores, and can grow in both aerobic and anaerobic environments.

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

Figure 4 (A) is a photomicrograph of the strain code UY-76. It is a gram-positive bacillus, has no catalase, oxidase and motility, does not produce endospores, and can grow in both aerobic and anaerobic environments.

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

Figure 5 is a flowchart of separation method 3.

^{Figure 6 (A) shows the 1} H-NMR spectrum of the 11-hexane extract (11-H) of Yogurt Drink.

^{Figure 6 (B) shows the 1} H-NMR spectrum of the 11 ethyl acetate extract (11-E) of Yogurt Drink.

^{Figure 6 (C) shows the 1} H-NMR spectrum of the 11 ethanol extract (11-A) of Yogurt Drink.

^{Figure 6 (D) is the 1} H-NMR spectrum of the 11 water extract (11-W) of Yogurt Drink.

^{Figure 7 (A) shows the 1} H-NMR spectrum of the 12-hexane extract (12-H) of Yogurt Drink.

^{Figure 7 (B) is the 1} H-NMR spectrum of the 12 ethyl acetate extract (12-E) of Yogurt Drink.

^{Figure 7 (C) shows the 1} H-NMR spectrum of the 12 ethanol extract (12-A) of Yogurt Drink.

^{Figure 7 (D) shows the 1} H-NMR spectrum of the 12-water extract (12-W) of Yogurt Drink.

^{Figure 8 (A) shows the 1} H-NMR spectrum of the 13-hexane extract (13-H) of Yogurt Drink.

^{Figure 8 (B) shows the 1} H-NMR spectrum of the 13 ethyl acetate extract (13-E) of Yogurt Drink.

^{Figure 8 (C) shows the 1} H-NMR spectrum of the 13 ethanol extract (13-A) of Yogurt Drink.

^{Figure 8 (D) is the 1} H-NMR spectrum of the 13 water extract (13-W) of Yogurt Drink.

^{Figure 9 (A) is the 1} H-NMR spectrum of the 14-H hexane extract (14-H) of Yogurt Drink.

^{Figure 9 (B) shows the 1} H-NMR spectrum of the 14 ethyl acetate extract (14-E) of Yogurt Drink.

^{Figure 9 (C) shows the 1} H-NMR spectrum of the ethanol extract of Yogurt Drink 14 (14-A).

^{Figure 9 (D) shows the 1} H-NMR spectrum of the 14 water extract (14-W) of Yogurt Drink.

Figure 10 is a bar graph of the total polyphenol content analysis of each extract obtained by separation method 3 of Yogurt Drinks 11-14.

Figure 11 shows the total of each extract obtained by separation method 3 of Yogurt Drinks 11-14 Flavanone flavonoid content analysis histogram.

Figure 12 is a histogram of total polysaccharide content analysis of each extract obtained by separation method 3 of Yogurt Drinks 11-14.

Figure 13 shows the HPLC spectrum of each extract obtained by separation method 3 of Yogurt Drink 11 at a wavelength of 203 nm.

Figure 14 shows the HPLC spectrum of each extract obtained by separation method 3 of Yogurt Drink 12 at a wavelength of 203 nm.

Figure 15 shows the HPLC spectrum of each extract obtained by separation method 3 of Yogurt Drink 13 at a wavelength of 203 nm.

Figure 16 shows the HPLC spectrum of each extract obtained by separation method 3 of Yogurt Drink 14 at a wavelength of 203 nm.

Figure 17 is a bar graph of the DPPH free radical scavenging rate of each extract obtained from Yogurt Drinks 11 to 14 through separation method 3.

Figure 18 is a histogram of the total antioxidant capacity-ABTS free radical scavenging rate of each extract obtained by the separation method 3 of Yogurt Drinks 11-14.

Figure 19 is a bar graph of the reducing power of each extract obtained by separation method 3 of yogurt drinks 11-14.

Figure 20 is a histogram of the survival inhibition rate of blood cancer cells of each extract obtained from Yogurt Drinks 11 to 14 by separation method 3.

Figure 21 is a histogram of the survival inhibition rate of brain cancer cells of each extract obtained from Yogurt Drink 11 to 14 through separation method 3.

Figure 22 is a histogram of the survival inhibition rate of gastric cancer cells of each extract obtained from Yogurt Drinks 11 to 14 through separation method 3.

Figure 23 is a histogram of the survival inhibition rate of colorectal cancer cells of each extract obtained from Yogurt Drink 11 to 14 through separation method 3.

Figure 24 shows the prostate cancer of each extract obtained by separation method 3 of Yogurt Drinks 11 to 14 Histogram of cell survival inhibition rate.

Figure 25 is the flow chart of the Yogurt beverage production system.

Picture 26 shows the yogurt beverage fermentation machine.

The above-mentioned objects and advantages of the present invention will become more immediately apparent to those with ordinary knowledge in the technical field after referring to the following detailed description and drawings.

In the present invention, the active metabolites of yogurt are separated by active guidance, the active ingredients are determined and prepared into drinks or medicaments suitable for oral administration, and the efficacy of the ingredients is still maintained.

Experiment 1. Isolation and identification of strains:

Take 1ml of raw cow's milk as a sample, and use Lactobacilli MRS Broth as a diluent to perform 10-fold serial dilutions (concentration multiples of 10 ^-1 , 10 ^-2 , 10 ^-3 , and 10 ^-4 ), and take them separately 200μl of the dilution was dropped into sterile MRS agar culture medium, and then plated. Place the petri dish in an anaerobic incubator at 37°C for 24 hours. After that, single colonies of different types, colors, and sizes are aseptically selected and continued to be cultured, and the colonies are selected and cultured repeatedly to isolate the bacteria, and it is determined that the colonies on the petri dish have the same shape, color, and size. Finally, the isolated single strain is preserved and identified. The identification of the yogurt strain is to observe the single colony on the petri dish with the naked eye, and then select two identical colonies for Gram staining to determine the Gram-positive or negative bacteria. Preferably, the colony is observed by a phase-contrast microscope at 4 and 40 times magnification. Select a single colony for DNA extraction and 16S PCR test, compare the sequence with the NCBI database, and complete the identification based on the result.

As shown in Figures 1 and 2, after 16S rDNA sequencing and NCBI alignment, the strain codes LR-36 and LPL-68 are Lactobacillus rhamnosus and Lactobacillus plantarum , respectively. In addition, as shown in Figure 3 (A), Figure 3 (B) and Table 1, the strain code UY-58 was identified by microscope observation, DNA extraction, 16S rDNA sequencing (SEQ ID NO:1), API 50 CHL identification confirmed that it is a gram-positive bacteria, and the closest to Lactobacillus fermentum (Lactobacillus fermentum, similarity is 99.9%). Figure 4 (A) and Figure 4 (B) show that the strain code UY-76 was identified by microscope observation, DNA extraction, 16S rDNA sequencing (SEQ ID NO: 2), API 50 CHL identification, and confirmed to be Gram Positive bacilli, and closest to Lactobacillus helveticus (Lactobacillus helveticus, 100% similarity).

After isolation and identification, a total of 20 strains were obtained, which are arranged in the order of Latin names as shown in Table 3.

Experiment 2. Fermentation preparation of yogurt:

The design model of the fermentation preparation experiment of the present invention is based on the type and ratio of strains, fermentation temperature, and fermentation time as operating variables, and viscosity and pH value (pH) are used as quality control standards after fermentation. Yogurt with a viscosity below 100 centipoise (cP) and a pH value above 5.0 is judged as unfermented or incompletely fermented In general, the experimental group with too long fermentation time (more than 12 hours) was also eliminated due to poor production efficiency. The preparation method of experiment 2 is: adding 250 ml of hot water at 75° C. into 36 g of milk powder for high-temperature sterilization to become an animal milk product. When it is cooled to 35~43℃, add 0.3g of bacterial powder. Then put in different temperature (37℃, 40℃, 43℃) incubator and react for different time (8 hours, 10 hours, 12 hours), and then measure the viscosity (Brookfield DVE RV viscometer) and pH value (Milwaukee pH600 pH meter) ). Table 4 shows the strains contained in the 14 groups of successfully fermented strain combinations.

It was found that different milk powders had little effect on the success of fermentation; the weight ratio between strains had little effect on the fermentation results. Therefore, the weight ratio of bacterial species in each experimental group is 1:1, and the more bacterial species, the easier it is to successfully ferment, that is, the more bacterial species, the more successful the fermentation is. The higher the reproducibility of the experiment. Next, start with The 4 groups with the highest reproducibility of successful fermentation (strain combinations 11 to 14 in Table 4) were subjected to 3 repeated experiments to confirm the best fermentation conditions for different combinations of strains (that is, to achieve the highest viscosity and lowest pH in the shortest fermentation time) ), when the yogurt viscosity is higher than 100cp and the pH value is lower than 5.0, the fermentation is judged to be complete. The experimental results are shown in Table 5.

The results of 3 repeated experiments prepared by fermentation of the combination of strains 11 to 14 showed that:

Strain combination 11: The fermentation temperature is fixed at 37°C, and the fermentation time takes 12 hours to complete the fermentation; the fermentation temperature is fixed at 40°C, and the fermentation time is 10 hours to complete the fermentation, and the fermentation time is 8 or 12 hours to complete the fermentation; the fermentation temperature is fixed to Fermentation can be completed at 43°C and fermentation time of 10 or 12 hours. The longer the fermentation time, the higher the viscosity and the lower the pH. The fermentation time is fixed at 8 hours, and the fermentation temperature is 37, 40, and 43℃. The fermentation time is fixed at 10 hours. The fermentation temperature is 40 and 43℃, and the fermentation can be completed. The higher the fermentation temperature, the lower the viscosity and the higher the pH value; The fermentation time is fixed at 12 hours, and the fermentation temperature is 37 and 43°C to complete the fermentation.

Strain combination 12: The fermentation temperature is fixed at 37℃, and the fermentation time is 10 or 12 hours. The fermentation time is 10 or 12 hours. The longer the fermentation time, the higher the viscosity and the lower the pH value. The fermentation temperature is fixed at 40℃, and the fermentation time is 8, 10, 12 Fermentation can be completed within hours, the longer the fermentation time, the higher the viscosity, and the lower the pH; the fermentation temperature is fixed at 43℃, and the fermentation time is 8, 10, and 12 hours. The lower the value. The fermentation time is fixed at 8 hours, the fermentation temperature is 40, 43℃, and the fermentation can be completed. The higher the fermentation temperature, the lower the viscosity and the higher the pH; the fermentation time is fixed at 10 hours, and the fermentation temperature can be completed at 37, 40, and 43℃. The higher the fermentation temperature, the higher the viscosity first, then the lower the pH value; the fermentation time is fixed at 12 hours, and the fermentation temperature is 37, 40, and 43 ℃, and the fermentation can be completed. The higher the fermentation temperature, the higher the viscosity first and then the lower pH value. High after low.

Strain combination 13: The fermentation temperature is fixed at 37℃, and the fermentation time is 8, 10, and 12 hours. The fermentation time is 8, 10, and 12 hours. The longer the fermentation time, the higher the viscosity and the lower the pH; the fermentation temperature is fixed at 40℃, and the fermentation time is 8, 10 , The fermentation can be completed in 12 hours, the longer the fermentation time, the higher the viscosity and the lower the pH value; the fermentation temperature is fixed at 43℃, and the fermentation time can be completed in 8, 10, and 12 hours. The longer the fermentation time, the lower the viscosity and the higher the pH, and the lower the pH. The fermentation time is fixed at 8 hours, and the fermentation temperature can be completed at 37, 40, and 43°C. The higher the fermentation temperature, the higher the viscosity and the lower the pH; the fermentation time is fixed at 10 hours, and the fermentation temperature can be at 37, 40, and 43°C. Fermentation is complete. The higher the fermentation temperature, the higher the viscosity first, then the lower the pH value. The fermentation time is fixed at 12 hours, and the fermentation temperature is 37, 40, and 43 ℃. The fermentation can be completed. The higher the fermentation temperature, the viscosity first becomes lower and then higher. The pH value is low first and then unchanged.

Strain combination 14: The fermentation temperature is fixed at 37°C, and the fermentation time is 8, 10, and 12 hours. The fermentation time is 8, 10, and 12 hours. The longer the fermentation time, the higher the viscosity and the lower the pH; the fermentation temperature is fixed at 40°C, and the fermentation time is 8, 10 , The fermentation can be completed in 12 hours, the longer the fermentation time, the higher the viscosity, and the lower the pH; the fermentation temperature is fixed at 43℃, and the fermentation time can be completed in 8, 10, and 12 hours. The longer the fermentation time, the higher the viscosity and the pH. The value does not change. The fermentation time is fixed at 8 hours, and the fermentation temperature can be completed at 37, 40, and 43℃. The higher the fermentation temperature, the higher the viscosity and the lower the pH value. The fermentation time is fixed at 10 hours, and the fermentation temperature is 37, 40, and 43℃. Fermentation can be completed. The higher the fermentation temperature, the higher the viscosity first, then the lower the pH value. The fermentation time is fixed at 12 hours, and the fermentation temperature is 37, 40, and 43 ℃. The fermentation can be completed. The higher the fermentation temperature, the higher the viscosity. The latter is low, and the pH value is first low and then high.

Experiment 3. Sensory evaluation of yogurt drink:

Yogurt has a variety of effects for maintaining good health. For example, experiments have confirmed that long-term consumption of yogurt can effectively reduce total cholesterol and the ratio of total cholesterol to high-density lipoprotein cholesterol; patients with hypercholesterolemia eating yogurt containing Lactobacillus acidophilus can reduce serum cholesterol; patients with hypercholesterolemia Eating yogurt containing Lactobacillus acidophilus and Bifidobacterium longum can increase high-density cholesterol; long-term consumption of yogurt can effectively reduce the risk of diabetes; eating yogurt can also prevent cardiovascular disease. Therefore, long-term consumption of adequate amounts of yogurt can achieve the effect of maintaining good health.

In order to achieve the above-mentioned goal, the present invention fermented strain combinations 1-14 to prepare yogurt drinks 1-14, and performed sensory evaluation analysis and screening. The sensory product is rated as the evaluation standard defined and developed by the American Food Science and Technology Association. It uses the three senses of sight, smell and taste to measure the characteristics of yogurt drinks. The experimental design of sensory evaluation includes: sensory analysis method General Law (ISO 6658:2005), sensory evaluation vocabulary (ISO 5492:2008), color sensory inspection (visual colorimetry: ISO 11037:1999), texture sensory inspection (texture profile: ISO 11036:1994), flavor sensory inspection (Flavour profile: ISO 6564: 1985), description analysis (qualitative description of sensory characteristics: ISO 11035: 1994, evaluation of the intensity of sensory characteristics: ISO 4121: 2003). The experimental methods are evaluated by descriptive analysis, ranking method and hedonic scale.

1. Use descriptive analysis to evaluate yogurt drinks 1 to 14:

This is to convene 11 high sensory acuity reviewers with practical experience in fermented product development, fermented dairy product development, yogurt product development, and hand-crank beverage shops to conduct the experiment, including 5 males and 6 females, which will be discussed in a meeting. Tasting, judging the differences in appearance, texture, and flavor between yogurt drinks, and finally looking at (color, cleanliness), smell (milk aroma, aroma, unique odor), taste (acidity, sweetness, smoothness, Body weight), and overall preferences (flavor performance, after rhyme) to describe the different traits, using a five-point scoring method of preference (1 point: very dislike, 2 points: dislike, 3 points: dislike or dislike, 4 points : I like it, 5 points: I like it very much) for evaluation. The experimental results are shown in Table 6.

好等級高

Good grade high

2. Evaluation of yogurt drinks from 1 to 14 based on the ranking method:

The number and gender of the tasters are as described above. The tasters classify yogurt drinks from 1 to 14 into 3 levels (high, medium, and low) according to their preferences, and each level is based on the degree of preference from the highest to the lowest. Arrange to the right. The results are shown in Table 7.

3. Evaluate yogurt drinks from 11 to 14 based on the preference score method:

Use the preference scoring method to confirm whether there are significant differences between the yogurt drinks with high preference grades from 11 to 14 and to understand the relationship between the preference degree and product characteristics, and conduct a consumer questionnaire survey, using the preference five-point scoring method ( 1 point: dislike very much, 2 points: dislike, 3 points: dislike or dislike, 4 points: like, 5 points: like very much) for rating. The aroma, sweetness, and sourness are also recorded on a five-point system (1 point: too light, 2 points: light, 3 points: moderate, 4 points: strong, 5 points: too strong). The closer the score is to 3, the better good.

The first consumer questionnaire survey was held in the department store shopping street, which was conducted without interference from the external environment and mutual influence. The evaluation targets were 67 untrained consumers, including 24 men (35.8%) and 43 women (64.2%). Age distribution: 25 people (37.3%) under 20 years old, 21 people (31.3%) 20-29 years old, 13 people (19.4%) 30-39 years old, 3 people 40-49 years old (4.5%), 50- 4 persons (6%) were 59 years old and 1 person (1.5%) was 60 years old and above. The distribution of the top three occupations is: 30 students (44.8%), 17 (25.4%) in the service industry, 5 (7.4%) in the military, public education, and 15 (22.4%).

The second consumer questionnaire survey was held in the university school district. Environment interference and without mutual influence. The evaluation targets were 69 untrained consumers, including 31 males (44.9%) and 38 females (55.1%). The age distribution is: 50 people (72.5%) 18-22 years old, 15 people 23-30 years old (21.8%), 4 people under 18 years old (5.8%). Occupation distribution are all students (69 people, 100%).

Based on the results of the first and second consumer questionnaire surveys, the total number of participants was 136. Please refer to Table 8. The overall preference is yogurt drink 14 best, with a score greater than 4, followed by yogurt drink 12. The scores of yogurt drink 11 and 13 are similar, and those with a score greater than 3 do not like it. it's OK. In addition, the number of yogurt drinks 11, 12, 13 and 14 rated by all 136 consumers as the most favorite (first order) yogurt drinks were 32, 38, 31 and 35, respectively.

Under the natural process without any seasoning, the aroma of yogurt drinks 11-14 is not much different, and they are all slightly light; the sweetness of yogurt drinks 11-14 is moderate; the sour taste of yogurt drinks 11-14 The difference is not big, all are slightly lighter (as shown in the results in Table 9).

Experiment 4. Sensory evaluation of yogurt drink ingredients:

In order to increase the diversification of yogurt drink consumption, increase the user's daily yogurt intake (for example, can drink 200g in 30 minutes) and drink it for a long time without being greasy, experiment 4 uses yogurt drink 14 as a base to add The formulation of the shake drink is designed and evaluated by the descriptive analysis method. real Test 4 convened 5 high sensory appraisers with practical experience in fermented product development, fermented dairy product development, yogurt product development, and hand-cranked beverage shops to conduct the experiment, including 3 men and 2 women, and discuss them in a meeting. Conduct a tasting.

1. Recipe design of yogurt drink with salty and sweet taste:

Add sesame paste as the sauce: After the sesame paste is boiled, add yogurt to crush and grind together. The sesame paste has a strong fragrance and contains oil. When the two are compatible, the flavor contains oily taste, which makes the overall flavor taste salty, but the flavor is too specific and acceptable. not tall. Add cucumber as an ingredient: diced cucumber and add yogurt to crush and grind together. Cucumber flavor masks yogurt flavor. The overall vegetable taste is obvious and lacks the unique flavor of yogurt. Add corn as an ingredient: add corn kernels from canned unseasoned corn to yogurt and crush and grind together. The corn is slightly sweet, which can enhance the overall sweetness, but the corn flavor and yogurt flavor merge into a special flavor, which is very low. . Sensory evaluation result: The formula design of yogurt drink is not suitable for salty and sweet taste.

2. The recipe design of yogurt drink sauce:

The yogurt beverage of the present invention has a pure natural process without any seasoning, and the overall flavor is moderately sweet, aroma and acidity are slightly weak, and it is elegant and not greasy. Different sauce recipes bring different sensory feelings. Add brown sugar as sauce: add the brown sugar boiled into syrup to yogurt and crush and grind together. The brown sugar has a strong and special flavor, which is just compatible with the sweet and sour flavor of yogurt. Add matcha to the sauce: add Japanese imported matcha powder to yogurt to crush and grind together. Matcha is slightly bitter and incompatible with the sweet and sour flavor of yogurt. Add honey as syrup: Add honey directly to yogurt and grind together. The unique fragrance of honey can highlight the sweet and sour flavor of yogurt.

3. Formula design of solid ingredients of yogurt beverage:

Yogurt drinks belong to non-Newtonian fluid (non-Newtonian fluid, which means that the viscosity of the fluid at a certain temperature and pressure is indeterminate), and its viscosity will change due to the pressure or speed. The higher the pressure, the higher the viscosity. Increase, and even become a temporary solid. Adding solid ingredients to yogurt beverages will cause difficulty in drinking and smoking. To overcome this problem, put 200g of yogurt drink in a cylindrical cup (upper circle diameter 9 cm, lower circle diameter 5.6 cm, cup height 13.5 cm), let use People can use a straw with a diameter of 1 to 1.5 cm and a length of 17.5 to 20 cm to easily suck the solid ingredients in the yogurt drink, and the solid ingredients will not be left after the drink has been drunk. In a preferred embodiment, at room temperature, the increase in the temperature of the yogurt drink will affect the viscosity and thus the difficulty of sucking the solid ingredients through the straw. Therefore, add a layer of crushed ice to the upper layer of the yogurt to keep cold (the size of the crushed ice should be controlled between 0.2 and 0.4 cubic centimeters, because less than 0.2 cubic centimeters will melt water too quickly, and more than 0.4 cubic centimeters will suck the crushed ice cubes. Good), the volume ratio of the crushed ice layer and the yogurt drink is 1:3~1:5, so that the yogurt drink can be maintained at 8~12℃ within 30 minutes, so that the sensory evaluation results of the solid ingredients are reproducible. The key points of the sensory evaluation of solid ingredients are: smoothness of taste and overall balance; further including visual stimulation, which enhances the user's desire to drink yogurt beverages (promoting appetite and enhancing active intake). The appearance of yogurt drinks can show the effects of layering, gradation, and special textures. The added solid ingredients include, but are not limited to, pearl, coconut, coffee jelly, vermicelli, taro balls, pudding, grass jelly, bean curd, mung bean, red bean, coix seed, purple rice, oats, and the sensory evaluation results are shown in Table 10. Narrated. In addition, fruits can also be used as solid ingredients, including but not limited to watermelon, mango, strawberry, lychee, dragon fruit, passion fruit, avocado, banana, papaya, kiwi, orange, grapefruit, and pineapple. The results are shown in Table 11.

Experiment 5. Preparation of Yogurt Drink Extract:

The types and content of active metabolites of yogurt drinks vary by strain, formula, and fermentation environment. In order to determine the active ingredients of the yogurt drink of the present invention, yogurt drinks 11 to 14 with high sensory preference ratings are selected for extraction and distribution extraction, and the obtained divided layers are then subjected to component analysis and activity testing.

1. Separation method 1:

The yogurt drink stock solution is heated under reduced pressure, concentrated and dried to obtain a paste. Then the paste was continuously extracted 3 times with ethanol to obtain an ethanol extract. The ethanol extract was vacuum filtered, heated and concentrated under reduced pressure to obtain a crude extract. The crude extract was partitioned and extracted with ethyl acetate: water (1:1 (v/v)). However, there are too many emulsified layers of the crude extract, resulting in poor separation effect, and subsequent separation experiments cannot be carried out. If it is changed to dichloromethane: water (1:1 (v/v)) to partition and extract the crude extract, there is still a problem of too much emulsified layer.

2. Separation method 2:

The yogurt drink stock solution is not dried, but diluted with water (1:1(v/v)), and then ethyl acetate (1:1(v/v)) is added for distribution extraction, which still produces a large amount of emulsified layer, resulting in The separation effect is not good. If dichloromethane (1:1(v/v)) or n-butanol (1:1(v/v)) is added instead for partition extraction, there is still a problem of too much emulsified layer.

3. Separation method 3:

The yogurt drink stock solution is freeze-dried to obtain a powdered product, and then the powdered product is sequentially extracted with a solvent with increasing polarity. It is found that the extraction rate of dichloromethane is too low, and there is no significant difference in the extraction effect of n-hexane. After extraction with n-butanol, there is a problem of residual solvent and it is difficult to continue the subsequent solvent extraction. Therefore, dichloromethane and n-butanol are not suitable for use. The extraction solvent for this separation method.

As shown in the separation method 3 shown in Figure 5, after the solvent permutation and combination extraction test, the final extraction is carried out in order with n-hexane, ethyl acetate, ethanol and water. That is, the yogurt drink stock solution 12 is freeze-dried to obtain a powdered product 14, and then the powdered product 14 is extracted with n-hexane to obtain a n-hexane extract 16 (sample code: 11-H, 12-H, 13-H, 14-H) and the first residue 18. Then, the first residue 18 is extracted with ethyl acetate to obtain the ethyl acetate extract 20 (sample code: 11-E, 12-E, 13-E, 14-E) and the second residue 22. After that, the second residue 22 is extracted with ethanol to obtain an ethanol extract 24 (sample code: 11-A, 12-A, 13-A, 14-A) and a third residue 26. Finally, the third residue 26 is extracted with water to obtain the water extract 28 (sample code: 11-W, 12-W, 13-W, 14-W) and the fourth residue 30. The extract and residue must be heated and concentrated under reduced pressure. The experimental results of various extracts of yogurt drinks from 11 to 14 are shown in Table 12. Show. Table 12 shows that this separation method can overcome the problem of liquid-liquid distribution and extraction to produce an emulsified layer. It separates, concentrates and collects various active ingredients of yogurt beverages, and visually and olfactorily confirms the appearance and flavor of various extracts. The difference.

Experiment 6. Component analysis of yogurt drink extract:

1. Nuclear magnetic resonance spectroscopy (NMR) analysis:

The yogurt drinks 11 to 14 obtained 16 extracts by separation method 3. These extracts were analyzed by NMR, and the hydrogen isotopes (deuterium) solvents: CDCl ₃ , acetone-d6, MeOH-d4, C ₅ D ₅ N, The solubility of the extracts was tested by DMSO-d6 and D _{2 O, and it was found that C 5} D ₅ N and DMSO-d6 had better solubility for the 16 extracts. In the ¹ H-NMR preliminary experiment, it was found that _{the solvent signal of C 5} D ₅ N would obscure the signal of the extract. Finally, DMSO-d6 was selected as the solvent for NMR analysis. The concentration of the extract was 25mg/ml, 50mg/ml, 75mg/ ml and 100mg/ml. Found n-hexane extract (11-H, 12-H, 13-H, 14-H), ethyl acetate extract (11-E, 12-E, 13-E, 14-E), ethanol extract ( Sample code: 11-A, 12-A, 13-A, 14-A) ¹ H-NMR spectrum at 50mg/ml shows the best magnetic field and characteristic signal, water extract (11-W, 12-W , 13-W, 14-W) the best sample concentration is 25mg/ml. Hexane extract (11-H, 12-H, 13-H, 14-H) and ethyl acetate extract (11-E, 12-E, 13-E, 14-E) after adding DMSO-d6 、Before the test, heating to 37~40℃ can further improve the solubility of the sample, and then optimize the characteristic signal of the ^{1 H-NMR spectrum.} The experiment was carried out with a JEOL ECS 400MHz FT-NMR with a resolution of 400MHz, chemical shifts were expressed in ppm (δ), and coupling constants were expressed in Hertz ( J ). ^{As shown in the 1} H-NMR spectra of the 16 extracts in Figure 6 (A) to Figure 9 (D) and the NMR signal analysis and main component determination of the 16 extracts in Table 13, the n-hexane extract, The NMR characteristic signals and main components of ethyl acetate extract, ethanol extract, and water extract are all different, and the NMR signals of yogurt drinks 11 to 14 are also different.

2. Analysis of total polyphenol content:

The yogurt drinks 11 to 14 were separated by method 3 to obtain 16 extracts. These extracts were analyzed by the Folin-Ciocalteu colorimetric method. The principle is the phosphotungstic acid and phosphomolybdic acid in the Folin-Ciocalteu reagent. (phosphomolybdic acid) is oxidized by polyphenol molecules to show color, and gallic acid is used as the standard product. The experimental method is: after dissolving gallic acid (5mg/mL) in methanol, make 50, 100, 250 and 500μg/mL standard products, take 20μL of each standard product and supplement the volume to 1.6mL with secondary water, and then add 100μL 2N Folin-Ciocalteu reagent, and then add 300μL of 20% Na ₂ CO ₃ and mix, react at 40°C for 40 minutes, and measure the absorbance at 725nm. In addition, the standard product was replaced with secondary water for zeroing. Draw a standard curve based on the relationship between concentration and absorbance. 20μL of the test sample (1mg/mL) was processed in the same way, and the total polyphenol content in each gram of extract was calculated according to the standard curve. As shown in Table 14 and Figure 10, the extract of Yogurt Drink 11 has the highest content of 11-E and the lowest 11-W; the Extract of Yogurt Drink 12 has the highest content of 12-A. 12-W is the lowest; in the extract of Yogurt Drink 13, the content of 13-H is the highest and 13-W is the lowest; in the extract of Yogurt Drink 14, the content of 14-E is the highest and 14-W is the lowest. Taking 16 extracts together, 14-E has the highest total polyphenol content and 11-W the lowest. In the 4 n-hexane extracts (11-H to 14-H), the total polyphenol content ranged from 18.2~24.0mg _{Gallic acid} /g. In the 4 ethyl acetate extracts (11-E to 14-E), the total polyphenol content ranged from 9.4~40.6mg _{Gallic acid} /g. In the 4 ethanol extracts (11-A to 14-A), the total polyphenol content ranged from 11.3~28.3mg _{Gallic acid} /g. In the 4 water extracts (11-W to 14-W), the total polyphenol content ranges from 2.6 to 7.1 mg _{Gallic acid} /g.

3. Analysis of total flavanone flavonoid content:

The principle is that 2,4-dinitrophenylhydrazine (2,4-dinitrophenylhydrazine, DNP) will derivatize the aldehyde or ketone group on the flavanone structure to make it absorb light at a specific wavelength. And hesperetin (hesperetin) as the standard product. The experimental method is as follows: add 1 mL of the sample to be tested into 2 mL of DNP solution and 2 mL of methanol, and place it in a 50°C water bath to heat for 50 minutes. After cooling, add 5 mL of KOH methanol solution and mix, let stand at room temperature for 2 minutes, then put 1 mL of the mixed solution into a centrifuge bottle containing 5 mL of methanol, shake and centrifuge at 1108×g for 10 minutes. The filtered supernatant was quantified to 25 mL with methanol, and the absorbance was measured at a wavelength of 494 nm. The measured absorbance value is compared with the standard curve of the standard product and converted into the content of flavanone flavonoids per gram of extract. As shown in Table 14 and Figure 11, the total flavanone flavonoid determination results show that in the extract of Yogurt Drink 11, the content of 11-A is the highest and 11-E is the lowest; in the extract of Yogurt Drink 12, the content of 12-A is The highest, 12-H is the lowest; in the extract of Yogurt Drink 13, the content of 13-A is the highest, and the content of 13-H is the lowest; in the extract of Yogurt Drink 14, the content of 14-A is the highest and 14-H is the lowest. Combining 16 extracts, 12-A has the highest total flavanone flavonoid content, and 12-H has the lowest content. In the 4 n-hexane extracts (11-H to 14-H), the total flavanone flavonoid content ranged from _{15.0-23.1mg Hesperetin} /g. In the 4 ethyl acetate extracts (11-E to 14-E), the total flavanone flavonoid content ranged from 16.0~26.0mg _Hesperetin /g. In the 4 ethanol extracts (11-A to 14-A), the total flavanone flavonoid content ranged from 62.5 to 91.3 _{mg Hesperetin} /g. In the 4 water extracts (11-W to 14-W), the total flavanone flavonoid content ranged from 28.2 to _{49.5 mg Hesperetin} /g.

4. Analysis of total polysaccharide content:

This analysis is carried out by the phenol-sulfuric acid assay. The principle is that the five-carbon and six-carbon sugars will be decomposed into furfural due to dehydration under acidic and high-temperature conditions. The furfural is further combined with phenol. The reaction produces an orange product. The total polysaccharide content is calculated by the change in absorbance, and 0, 10, 20, 30, 40, 50 μg/ml glucose is used as the standard product. The experimental method is as follows: add 0.5mL of 5% phenol solution to 0.5mL of 10mg/mL test product, then add 2.5mL of concentrated sulfuric acid to mix uniformly, react in a water bath at 100℃ for 20 minutes, and measure the absorbance at a wavelength of 490nm after cooling the temperature. As shown in Table 14 and Figure 12, the total polysaccharide determination results show that in the extract of Yogurt Drink 11, 11-W has the highest content and 11-H is the lowest; in the extract of Yogurt Drink 12, 12-W has the highest content. 12-E is the lowest; in the extract of Yogurt Drink 13, the content of 13-W is the highest and 13-H is the lowest; in the extract of Yogurt Drink 14, the content of 14-W is the highest and 14-H is the lowest. Combining 16 extracts, 14-W has the highest total polysaccharide content and 14-H the lowest. The total polysaccharide content of the 4 n-hexane extracts (11-H to 14-H) ranges from 7.6 to 11.2 mg _Glucose /g. The total polysaccharide content of the 4 ethyl acetate extracts (11-E to 14-E) ranges from 9.7 to 18.4 mg _Glucose /g. The total polysaccharide content of the 4 ethanol extracts (11-A to 14-A) ranges from 223.2~249.4mg _Glucose /g. The total polysaccharide content of the 4 water extracts (11-W to 14-W) ranges from 414.4 to 494.1 mg _Glucose /g.

5. Thin layer chromatography (TLC) analysis:

Refer to the normal phase thin layer chromatography commonly used in the Taiwan Chinese Pharmacopoeia (Second Edition) for analysis. The solvent system is an ionic solvent system (n-hexane, dichloromethane, chloroform, methanol) or a non-ionic solvent system ( N-hexane, toluene, ethyl acetate, acetone, methanol), and adding acid (glacial acetic acid (GAA), formic acid (FA)) or alkali (10% ammonia, 25% ammonia, diethylamine) for testing. As shown in Table 15, the n-hexane extract and ethyl acetate extract (11-H, 11-E, 12-H, 12-E, 13-H, 13-E, 14-H, 14-E) The polarities are relatively similar, and the main spots of TLC can have a good analysis effect in the same solvent system. Ethanol extracts (11-A to 14-A) and water extracts (11-W to 14-W) have no ideal analytical results in normal phase thin layer chromatography, and the main spots of TLC cannot be found in R _f 0.3- 0.8.

6. High performance liquid chromatography (HPLC) analysis:

Analyze with reference to the reverse phase high performance liquid chromatography method commonly used in Taiwan Chinese Pharmacopoeia (Second Edition). The conditions are as follows: the high performance liquid chromatograph is Agilent 1100 series, and the detector is G1315B photodiode array detector. The autosampler is G1329A autosampler, the chromatography column is Intersil ODS-3V 250 x 4.6mm (5μm), the mobile phase solvent A is acetonitrile, solvent B is 0.1% H ₃ PO ₄ , and the flow rate is 1ml/min. The column temperature is 30°C, and the detection wavelengths are 203, 210, 254, 280 and 365nm. The conditions of the solvent system are as follows: the mobile phase includes 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, 100% A for 60~70 minutes, the flow rate and column temperature are as described above. As shown in Table 16 and Figures 13 to 16, among the 5 detection wavelengths, the n-hexane extract (11-H to 14-H) and the ethyl acetate extract (11-E to 14-E) are both No significant chromatographic peak was observed. The ethanol extract (11-A to 14-A) and the water extract (11-W to 14-W) only showed a better chromatographic peak at 203nm.

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-pricrylhydrazyl, DPPH) free radicals:

Lipids generate free radicals in the process of self-oxidation to cause lipid rancidity. Antioxidants provide hydrogen to scavenge lipid peroxide free radicals, thereby inhibiting the chain reaction of oxidation. DPPH is often used in antioxidant research to evaluate the hydrogen supply capacity of antioxidants. DPPH methanol solution has a strong absorbance at 517nm, and the absorbance decreases when it is reduced by antioxidants. Therefore, the lower the absorbance value at 517 nm, the stronger the hydrogen supply capacity of the antioxidant. The experimental method is: add 4 mL of extract sample (50 mg of extract dissolved in 15 mL of 50% ethanol solution) to 1 mL of freshly prepared 10 mM DPPH methanol solution, shake and mix, place at room temperature for 30 minutes, and detect the absorbance at 517nm wavelength. The result is expressed as a removal percentage, and the higher the removal percentage, the better the hydrogen supply capacity of the test sample. As shown in Table 17 and Figure 17, the extracts 11-A and 14-A have better DPPH free radical scavenging ability among the 16 extracts.

2. Determination of total antioxidant capacity (trolox equivalent antioxidant capacity, TEAC):

2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) produces an oxidation reaction catalyzed by hydrogen peroxide and peroxidase to form a stable blue-green water-soluble ABTS ⁺ cationic radical. Use an ultraviolet-visible photometer, with a strong absorbance value at 620nm, and make a standard curve with a standard water-soluble vitamin E analogue (trolox). When ABTS˙ ⁺ cationic free radicals are reduced with antioxidants, the color of the solution will become lighter and the light absorption value will decrease, which can be converted into ^{the ability to remove ABTS˙ +} cationic free radicals. The better the scavenging ability, the stronger the antioxidant hydrogen supply ability. The experimental method is: put the prepared ABTS in the refrigerator for 1 hour to produce a stable blue-green ABTS ⁺ free radical aqueous solution. Add 80 μL of the extract sample (50 mg of the extract dissolved in 15 mL of 50% ethanol solution) into a 96-well plate, and then add 120 μL of ABTS˙ ⁺ free radical aqueous solution, with a total volume of 200 μL. Shake for 10 minutes in the dark at room temperature, and measure the absorbance at 620 nm. The lower the absorbance value, the better the removal of ABTS˙ ⁺ free radicals. The removal rate is calculated as a percentage. As shown in the results of total antioxidant capacity shown in Table 17 and Figure 18, among the extracts of Yogurt Drink 11, 11-A has the best ability and 11-E is the weakest; among the extracts of Yogurt Drink 12, 12-A The best ability, 12-H is the weakest; among the extracts of yogurt drink 13, 13-A has the best ability and 13-H is the weakest; among the extracts of yogurt drink 14, 14-A has the best ability, 14- H is the weakest. Among the 16 extracts, 12-A has the best total antioxidant capacity and 12-H the weakest.

3. Determination of reducing power:

Antioxidants will reduce the red blood salt (K ₃ Fe(CN) ₆ ) to yellow blood salt (K ₄ Fe(CN) ₆ ), and the yellow blood salt will react with Fe ^{3+ to} produce Prussian blue. The absorbance value is measured at 620nm. , To detect the amount of Prussian blue produced, the higher the absorbance value, the stronger the reducing power of the antioxidant. The experimental method is: add 200 μL of extract sample (50 mg of extract dissolved in 15 mL of 50% ethanol solution) into a microcentrifuge tube, and then add 200 μL of 1% red blood salt, the total volume is 400 μL. After mixing, place it in a 50°C water bath for 20 minutes. After the reaction is completed, place it on ice to cool for 10 minutes, then add 200 μL of 10% trichloroacetic acid (TCA) to mix, and centrifuge at 3,000 rpm for 10 minutes. Add 75μL of supernatant to a 96-well plate, then add 75μL of ultrapure water and 30μL of 0.1% FeCl ₃ . 6H ₂ O, after mixing, measure the absorbance at 620 nm. The higher the absorbance value, the stronger the reducing power. As shown in Table 17 and Figure 19, among the extracts of Yogurt Drink 11, 11-A has the best reducing power, while 11-H and 11-E have the same reducing power and the weakest; among the extracts of Yogurt Drink 12 , 12-A has the best reducing power and 12-W is the weakest; among the extracts of yogurt drink 13, 13-A has the best reducing power and 13-H is the weakest; among the extracts of yogurt drink 14, 14-A The reducing power is the best, and the 14-H reducing power is the weakest. Among the 16 extracts, 12-A has the best reducing power and 13-H has the weakest reducing power.

Experiment 8. Test of inhibiting cancer cell growth activity of Yogurt drink extract:

For the related organs of the symbiotic bacteria-gut-brain axis, the active metabolites will be ingested from the oral cavity and will pass through the digestive tract, blood, circulatory system, and urinary system (excretion system). Oral cancer (Ca9-22, Cal- 27 cell line), blood cancer (K562, Molt 4 cell line), brain cancer (GBM8401, U87MG cell line), gastric cancer (KATO III, SNU-1 cell line), colorectal cancer (DLD-1 cell line), prostate cancer ( LN-cap, PC-3 cell strains) were tested for the cancer cell growth inhibitory activity of 16 extracts from Yogurt Drink 11 to 14 obtained by separation method 3. The experimental method is cell viability analysis (MTT assay), in which various cancer cells are implanted into 96-well plates with a cell density of 7×10 ³ cells/100μL per well, and then samples of different concentrations (within 100μg/mL) are added ( Take DMSO as the control group) and place them in a 37°C, 5% CO ₂ cell incubator for 72 hours. Then add 50μL MTT and incubate for 1 hour. Centrifuge at 2000 rpm for 3 minutes, remove the supernatant and add 200 μL of DMSO, place on a shaker and shake until the purple crystals are completely dissolved, and read the absorbance values at 570 mm and 620 mm. Results are presented as the different samples processed in the 100 μ g / mL concentration, the best percentage of growth inhibition of cancer cells. The human clinical treatment of oral cancer is mainly surgery and radiotherapy (electrotherapy). The 16 extracts in this experiment did not show the effect of inhibiting the growth of oral cancer cells. The experimental results of other cancer cells are shown in Table 18 and Figures 20 to 24.

1. Inhibit the growth activity of blood cancer cells (K562, Molt 4 cell lines):

As shown in Figure 20, in terms of inhibiting the growth of blood cancer cells K562, among the extracts of Yogurt Drink 11, 11-A has the best inhibitory activity and 11-W is the weakest; among the extracts of Yogurt Drink 12, 12- A has the best inhibitory activity and 12-W is the weakest; among the extracts of yogurt drink 13, 13-W has the best inhibitory activity and 13-H is the weakest; among the extracts of yogurt drink 14, 14-A The inhibitory activity is the best, and 14-W is the weakest. Among the 16 extracts, 12-A has the best cancer cell growth inhibitory activity. As shown in Figure 20, in terms of inhibiting the growth of blood cancer cells Molt 4, among the extracts of Yogurt Drink 11, 11-H has the best inhibitory activity, and 11-E and 11-W have no inhibitory activity; Yogurt Drink 12 has the best inhibitory activity. Among the extracts, 12-A has the best inhibitory activity, 12-H and 12-W have no inhibitory activity; among the extracts of Yogurt Drink 13, 13-A has the best inhibitory activity and 13-E is the weakest; Yogurt Among the extracts of Drink 14, 14-E has the best inhibitory activity, and both 14-A and 14-W have no inhibitory activity. Among the 16 extracts, 14-E has the best inhibitory activity.

2. Inhibit the growth activity of brain cancer cells (GBM8401, U87MG cell line):

As shown in Figure 21, in terms of inhibiting the growth of brain cancer cells GBM8401, among the extracts of Yogurt Drink 11, 11-A has the best inhibitory activity and 11-E is the weakest. Among the extracts of Yogurt Drink 12, 12 -A has the best inhibitory activity and 12-W is the weakest; among the extracts of yogurt drink 13, 13-H has the best inhibitory activity and 13-W is the weakest; among the extracts of yogurt drink 14, 14-A The inhibitory activity is the best, and 14-H is the weakest. Among the 16 extracts, 11-A has the best inhibitory activity. As shown in Figure 21, in terms of inhibiting the growth of U87MG cells, among the extracts of Yogurt Drink 11, 11-A has the best inhibitory activity and 11-W is the weakest; among the extracts of Yogurt Drink 12, 12-H The inhibitory activity of yogurt is the best, and 12-W is the weakest. Among the extracts of yogurt drink 13, 13-H has the best inhibitory activity. Neither 13-E nor 13-W has inhibitory activity; in the extract of yogurt drink 14, there is no inhibitory activity. 14-W has the best inhibitory activity, 14-E The weakest. Among the 16 extracts, 14-W has the best inhibitory activity.

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

As shown in Figure 22, in terms of inhibiting the growth of gastric cancer cells KATO III, among the extracts of Yogurt Drink 11, 11-H has the best inhibitory activity and 11-W is the weakest. Among the extracts of Yogurt Drink 12, 12 -E has the best inhibitory activity, 12-H and 12-W have no inhibitory activity; in the extract of Yogurt Drink 13, only 13-E has inhibitory activity; in the extract of Yogurt Drink 14, 14-W is inhibited The activity is the best, and 14-A is the weakest. Among the 16 extracts, 14-W has the best inhibitory activity. As shown in Figure 22, in terms of inhibiting the growth of gastric cancer cells SNU-1, among the extracts of Yogurt Drink 11, 11-H has the best inhibitory activity and 11-A is the weakest. Among the extracts of Yogurt Drink 12, The inhibitory activity of 12-E is the best, and 12-W is the weakest. Among the extracts of Yogurt Drink 13, 13-H has the best inhibitory activity and 13-A is the weakest. Among the extracts of Yogurt Drink 14, 14- A has the best inhibitory activity and 14-E has the weakest. Among the 16 extracts, 12-E has the best inhibitory activity.

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

As shown in Figure 23, in terms of inhibiting the growth of colorectal cancer cells DLD-1, among the extracts of Yogurt Drink 11, 11-H has the best inhibitory activity and 11-W is the weakest; among the extracts of Yogurt Drink 12 , 12-H has the best inhibitory activity and 12-E is the weakest; among the extracts of yogurt drink 13, 13-H has the best inhibitory activity and 13-W is the weakest; among the extracts of yogurt drink 14, 14 -A has the best inhibitory activity and 14-E has the weakest. Among the 16 extracts, 11-A has the best inhibitory activity.

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

As shown in Figure 24, in terms of inhibiting the growth of prostate cancer cells LN-cap, among the extracts of Yogurt Drink 11, 11-A has the best inhibitory activity and 11-E is the weakest; among the extracts of Yogurt Drink 12 , 12-H has the best inhibitory activity, and 12-E is the weakest; among the extracts of yogurt drink 13, 13-A has the best inhibitory activity, 13-H and 13-E have no inhibitory activity; yogurt drink 14 has the best inhibitory activity. Among the extracts, 14-W has the best inhibitory activity and 14-H has the weakest. Among the 16 extracts, 11-A has the best inhibitory activity. As shown in Figure 24, in terms of inhibiting the growth of prostate cancer cell PC-3, among the extracts of Yogurt Drink 11, 11-E has the best inhibitory activity and 11-H is the weakest; Yogurt Drink 12 is the extract Among them, 12-H has the best inhibitory activity, and 12-W is the weakest; among the extracts of Yogurt Drink 13, 13-E has the best inhibitory activity, and both 13-H and 13-W have no inhibitory activity; Yogurt Drinks Among the 14 extracts, 14-E has the best inhibitory activity and 14-A has the weakest. Among the 16 extracts, 13-E has the best inhibitory activity.

Experiment 9. Yogurt Beverage Production System:

In the past, liquid yogurt beverages were produced in two forms: home-made small-scale production and mass production in ton-level factories. Household small-scale production has the problems of unstable quality, need for subsequent modulation, and inconvenient operation. Mass production in a ton-level factory will face 2 to 3 days or more from the time consumers buy and drink the drink, the freshly fermented yogurt drink cannot be consumed on the same day, and often it cannot be consumed within the shelf life, or it is improperly preserved. The problem. Therefore, the present invention provides a liquid yogurt beverage production system, which can simultaneously solve the above-mentioned home-made small-volume production and large-scale production of yogurt beverages in ton-level factories. A single fermentation machine can produce kilogram-level liquid yogurt beverages. It can be fermented on-site with a transparent process and completed in 18 hours. The yogurt drinks that consumers drink every day are freshly fermented that day, and the end products are served by hand shake.

As shown in the manufacturing process shown in Figure 25 and the structure of the fermentation machine 100 shown in Figure 26, the liquid yogurt beverage production system of the present invention sequentially includes: high pressure cleaning, high temperature sterilization, milk emulsification, secondary sterilization, inoculation Fermentation, finished product sampling, finished product collection, cooling and refrigeration, homogenizing preparation, adding ingredients, filling and packaging, product serving.

The fermentation machine 100 for preparing yogurt drinks includes: a tank body 101, a sealed top cover 105 assembled with the tank body 101, a feeding port M provided on the sealed top cover 105, and a motor controller 108 provided outside the tank body 101 , The stirring motor 110 coupled to the motor controller 108, the stirring arm 104 arranged in the space of the tank 101 and connected to the stirring motor 110, and the resistance warmer arranged outside the tank 101 and coupled to the motor controller 108 103. Among them, the space enclosed by the sealing top cover 105 and the tank body 101 is in a closed state; water, milk powder, and bacterial powder are fed into the space through the feeding port M; the resistance warmer 103 is used to sterilize the water and milk powder, and make The bacterial powder is stirred by the stirring arm 104 in the mixture formed by water and milk powder at 37°C to 43°C to prepare a yogurt drink.

Furthermore, the fermentation machine 100 further includes a discharge port N4 arranged at the bottom of the tank body 101 for discharging the yogurt drink or waste liquid (or waste water). Alternatively, the fermentation machine 100 further includes a temperature control interlayer 102 arranged outside the tank body 101 and connected to the resistance warmer 103. The temperature control interlayer 102 is used to adjust or maintain the temperature in the space under the control of the resistance warmer 103. temperature. Alternatively, the sealing top cover 105 further includes a cleaning port 106, and the fermentation machine 100 further includes a heat conduction inflow port N5 provided at the side of the tank body 101 and a heat conduction outflow port N6 at the bottom. Alternatively, the fermentation machine 100 further includes a temperature sensor 107 coupled to the motor controller 108, and the temperature sensed by the temperature sensor 107 is displayed on the screen 109 of the motor controller 108. Alternatively, the observation window S1 on the sealing top cover 105 allows the user to visually observe the condition in the tank body 101.

The manufacturing process in Figure 25 is as follows:

(1) High-pressure cleaning and high-temperature sterilization: open the feeding port M → use a high-pressure spray gun to clean the inner wall of the tank 101 → confirm the water level to the proper position through the observation window S1 → open the discharge port N4 to discharge → the tank 101 is filled up again Water → The temperature detected by the temperature sensor 107 is displayed on the screen 109 of the motor controller 108 → It shows that it is heated to above 72°C → After reaching the sterilization temperature → Let it stand for 30 minutes → Open the discharge port N4 and discharge to the waste water tank (not show).

(2) Milk emulsification: open the feeding port M → inject clean water → put in milk powder → turn on the stirring motor 110 → perform emulsification → close the feeding port M → confirm the emulsification status through the observation window S1.

(3) Secondary sterilization: turn on heating→observe that the screen 109 shows heating to above 72°C→after reaching the sterilization temperature→turn off heating→stand for 30 minutes.

(4) Inoculation and fermentation: inject cooling water from the heat conduction inlet N5 → turn on the circulating pump (not shown) → use cooling water for heat exchange and temperature control → observe the screen 109 shows that the temperature is gradually reduced to 43°C → turn off the cooling water source and circulation Pump → Sterilize the feeding port M with 75% alcohol → quickly open the feeding port M to inject bacteria and close the screw tightly → turn on the stirring motor 110 to make the stirring arm 104 operate and stir for 20 minutes → turn off the stirring motor 110 → stand for fermentation.

(5) Finished product sampling: spray the discharge port N4 with pure water and then spray 75% alcohol for disinfection → take out 300 ml sample → check the viscosity and pH value → the viscosity and pH value reach the sink standard → proceed to the sink.

(6) Finished product receiving tank: spray the discharge port N4 with pure water → spray 75% alcohol for disinfection → use 304 food grade stainless steel containers to receive in batches.

(7) Cooling and refrigeration: preparing yogurt drinks in large quantities → refrigerating at 4°C.

(8) Homogeneous preparation and addition of ingredients: take yogurt drink → add ice cubes → homogenize with a homogenizer → prepare solid ingredients or solid fruit ingredients for preparation.

(9) Filling the packaging and serving the product: Finally, check again whether the drink or ingredients ooze out → complete.

The present invention is really a difficult innovative invention and has deep industrial value, and it is necessary to file an application in accordance with the law. In addition, the present invention can be modified by persons with ordinary knowledge in the relevant technical field, but does not deviate from the scope of protection as claimed in the attached patent scope.

<110> Yuyang Biomedical Co., Ltd.

<120> Yogurt biotechnology drink with anti-oxidation and inhibiting the growth of digestive tract cancer cells and its preparation method

<160> 2

<210> 1

<211> 1478

<212> rDNA

<213> Lactobacillus fermentum

<220>

<223> 16S rDNA of strain code UY-58

<400> 1

<210> 2

<211> 1515

<212> rDNA

<213> Lactobacillus helveticus

<220>

<223> 16S rDNA of strain code UY-76

<400> 2

Claims (13)

A yogurt drink includes: an animal milk product, including water and animal milk; a plurality of bacterial powders mixed with the animal milk product, wherein the plural bacterial powders are derived from corresponding plural strains, and the plural strains include: long Bifidobacterium longum , Lactobacillus acidophilus , Lactobacillus paracasei , and Lactobacillus rhamnosus . The yogurt drink described in item 1 of the scope of patent application, wherein the plurality of strains further include: Bifidobacterium bifidum , Bifidobacterium breve , Bifidobacterium lactis ), Lactobacillus casei (Lactobacillus casei), Lactobacillus delbrueckii subsp. bulgaricus , Lactobacillus helveticus , Lactobacillus plantarum , and Streptococcus thermophilus . The yogurt drink described in item 1 of the scope of patent application, wherein the plurality of strains further include: Bifidobacterium bifidum , Bifidobacterium lactis , Enterococcus faecium , Lactobacillus casei , Lactobacillus fermentum , Lactobacillus plantarum , and Streptococcus thermophilus . The yogurt drink according to item 1 of the patent application, wherein the plurality of strains further include: Lactobacillus fermentum , Lactobacillus helveticus , Lactobacillus salivarius , and Streptococcus thermophilus ( Streptococcus thermophilus ). The yogurt drink according to any one of items 1 to 4 in the scope of the patent application, wherein the weight of the bacterial powder of each of the plurality of strains is the same. The yogurt drink according to any one of items 1 to 4 in the scope of the patent application, wherein the yogurt drink further includes a sauce ingredient selected from the group consisting of brown sugar, honey and combinations thereof One of them. The yogurt drink according to any one of items 1 to 4 in the scope of the patent application, wherein the yogurt drink further includes a solid ingredient selected from pearls, coconuts, coffee jelly, taro balls, pudding, and red beans One of the group consisting of, purple rice, oats and their combinations. The yogurt drink according to any one of items 1 to 4 in the scope of the patent application, wherein the yogurt drink further includes a solid fruit ingredient selected from the group consisting of mango, strawberry, dragon fruit, passion fruit, and avocado , Banana, kiwi, orange, grapefruit, and combinations thereof. The yogurt drink according to any one of items 1 to 4 in the scope of the patent application, wherein the plurality of strains are fermented in the animal milk product at 37°C to 43°C for 8 hours to 12 hours. A method for separating yogurt drink according to any one of items 1 to 4 in the scope of patent application, comprising: freeze-drying the yogurt drink to obtain a powdered product; extracting the powdered product with n-hexane to obtain a n-hexane extraction And a first residue, wherein the main components of the n-hexane extract are short-chain fatty acids with double bonds and hydroxyl groups; the first residue is extracted with ethyl acetate to obtain an ethyl acetate extract and A second residue, wherein the main component of the ethyl acetate extract is glycolipid; the second residue is extracted with ethanol to obtain an ethanol extract and a third residue, wherein the main component of the ethanol extract is The components include disaccharides and oligosaccharides; and the third residue is extracted with water to obtain a water extract and a fourth residue, wherein the main components of the water extract include polysaccharides, glycoproteins and proteins. The separation method described in item 10 of the scope of patent application, wherein the n-hexane extract and the ethyl acetate extract are subjected to component analysis by normal phase thin layer chromatography, and the solvent system of the normal phase thin layer chromatography is Toluene: ethyl acetate: formic acid = 5: 4: 1 (v/v/v). The separation method as described in item 10 of the scope of patent application, wherein the ethanol extract and the water extract are subjected to component analysis by reverse phase high performance liquid chromatography. A yogurt drink that uses any one of items 1 to 4 in the scope of patent application to inhibit the growth of cancer cells Wherein, the cancer cells are selected from one of the group consisting of blood cancer cells, brain cancer cells, gastric cancer cells, colorectal cancer cells and combinations thereof.

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