TWI835820B - 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 biotech drink with antioxidant properties and inhibiting growth of digestive tract cancer cells and preparation method thereof

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

The bidirectional information network between the gut and the brain is called the gut-brain axis. Later, it was discovered that the symbiotic microbiota in the gut contributes to this gut-brain axis. The three interact with each other, thus becoming the microbiota-gut-brain axis. In 2013, the National Institute of Mental Health (NIMH) launched a special project to explore the gut microbiota-brain communication mechanism, with the aim of developing new drugs and non-invasive treatments for mental illness. Since then, research related to the microbiota-gut-brain axis has sprung up like mushrooms after rain, becoming the focus of neuroscience research, with the main axis being to explore the interaction between the gut microbiota and the brain. The gut microbiota has an important impact on the brain through the neural network, neuroendocrine system, and immune system, and disturbances in human behavior will also change the composition of the gut microbiota. The literature has reported the mutual balance of the commensal bacteria-gut-brain axis and has been proven to be associated with a variety of diseases, such as inflammatory bowel disease (IBD), gastrointestinal cancer, cholelithiasis, behavioral disorders, anxiety, depression, chronic fatigue syndrome (CFS), hepatic encephalopathy, allergies, obesity, diabetes, atherosclerosis, etc. In the care of cancer chemotherapy and post-surgery, 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 traditional Chinese medicine systems are actively developing or integrating the symbiotic bacteria-gut-brain axis Research on drug development, disease treatment and its mechanism of action; while another medical system - Ayurveda (Ayurveda) uses a large amount of turmeric to improve digestive tract discomfort, neuroprotection, and prevent Alzheimer's disease, and its diversity The efficacy is also related to the gut-brain axis. However, due to different dietary habits, the use of turmeric has regional restrictions, mainly in India.

In the Western medicine system, the development of drugs using the symbiotic bacteria-gut-brain axis research is called microbial drugs, which refers to drug preparations made from normal microorganisms or substances that regulate the normal growth of microorganisms. It can be applied to indications such as infection, diabetes, tumors, inflammatory diseases, and immune-related diseases. Microbial drugs include: live biotherapeutic products (LBP), small molecule microbiome modulators (SMMM), and fecal microbiota transplants (FMT). Among them, LBP refers to the identification, screening, and combination of human microorganisms to clearly control the types and quantities of bacteria, and use different types, quantities, and combinations of bacteria for different indications to ensure the safety and effectiveness of medication. However, the U.S. Food and Drug Administration (FDA) has not approved any LBP for marketing so far. It only issued the early clinical trial guidelines for LBP in 2016, which made the development of LBP have clear standards. SMMM refers to substances that can selectively promote the growth and reproduction of one or more beneficial bacteria in the host intestine, and achieve therapeutic purposes by affecting the growth and reproduction of bacteria. Currently, the development of known SMMM drugs has only reached the second phase of clinical trials. FMT refers to the transplantation of functional flora from healthy human feces into the patient's gastrointestinal tract to rebuild the patient's intestinal flora for the treatment of intestinal and extraintestinal diseases. Although the United States has included FMT in the treatment guidelines for recurrent Clostridium difficile infection in 2013, the policies of different countries vary greatly, and FMT currently lacks unified and effective supervision. Overall, the clinical trials of microbial drugs have not progressed as expected, and no microbial drugs have been approved for marketing. Another problem is that the number of participants in clinical trials of microbial drugs is relatively small, and human verification of drug efficacy is still not as good as expected.

The traditional Chinese medicine system does not use the commensal bacteria-gut-brain axis to research and develop drugs, but studies the interaction between traditional Chinese medicine and intestinal microbiota. The regulatory effect of traditional Chinese medicine on intestinal microbiota. For example: tonic traditional Chinese medicine containing polysaccharides has a supporting effect on beneficial bacteria and pathogenic bacteria, but it has a negative effect on beneficial bacteria. The supporting effect is obviously better than that of pathogenic bacteria, and the metabolites produced by good-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 acetate metabolism and enhancing the colonization resistance of bifidobacteria. The metabolic effect of intestinal microbiota on traditional Chinese medicine, for example: It has been confirmed that many traditional Chinese medicine ingredients can only produce medicinal ingredients and achieve therapeutic effects after being metabolized by intestinal microbiota. For example, baicalin (baicalin) contained in scutellaria baicalensis is difficult to be directly absorbed in the intestines. Only when it is hydrolyzed by intestinal microbiota into baicalein (baicalein) can it be absorbed into the blood and play its role. Human clinical trials have confirmed that Gegen Qinlian Decoction can treat type 2 diabetes by changing the intestinal microbiota. Overall, research on Chinese medicine on the commensal bacteria-gut-brain axis provides an explanation of the mechanism of action that is acceptable to Western medicine, but it still cannot improve drug compliance and problems caused by Chinese medicine due to too high dosage and too long treatment time. Low medication adherence is an old problem.

The above-mentioned research and development of the symbiotic bacteria-gut-brain axis in Western medicine, Chinese medicine and Ayurvedic medical systems focus on diseases, drugs and treatments. Overall, these developments are still in their infancy, and there are no industrial success stories or large-scale mass production yet. Therefore, it is still necessary to develop novel products and technologies that can be applied to the commensal bacteria-gut-brain axis to achieve biomedical effects.

In view of the deficiencies in the prior art, the applicant in this case finally conceived this case after careful experimentation and research, and with a spirit of perseverance, which was able to overcome the deficiencies of the prior art. The following is a brief description of this case.

The object of the present invention is to provide a yogurt drink, comprising: an animal milk product and a complex bacterial powder, wherein the animal milk product comprises water and animal milk, and the complex bacterial powder is mixed with the animal milk product, wherein the complex bacterial powder comes from a corresponding plurality of strains, and the plurality of strains comprises: Bifidobacterium longum , Lactobacillus acidophilus , Lactobacillus paracasei and Lactobacillus rhamnosus .

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

Another object of the present invention is to provide a separation method for 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, wherein the main component of the n-hexane extract is a short-chain fatty acid with a double bond and a hydroxyl group; extracting the first residue with ethyl acetate to obtain an ethyl acetate extract and a second residue, wherein the main component of the ethyl acetate extract is glycolipid; extracting the second residue with ethanol to obtain an ethanol extract and a third residue, wherein the main components of the ethanol extract include disaccharides and oligosaccharides; and extracting the third residue with water to obtain a water extract and a fourth residue, wherein the main components of the water extract include polysaccharides, glycoproteins and proteins.

Another object of the present invention is to provide a method for using the aforementioned yogurt drink to inhibit 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 a combination thereof.

The present invention discloses a fermentation machine for preparing yogurt drinks, comprising: a tank body and a sealed top cover. The sealed top cover is assembled with the tank body so that a space enclosed by the sealed top cover and the tank body is sealed. A 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 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 to stir the water, milk powder and bacterial powder. The resistance temperature increaser is arranged outside the tank body and coupled to the motor controller to sterilize water and milk powder, and ferment the bacterial powder in the mixture formed by water and milk powder at 37°C to 43°C to prepare the yogurt drink.

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

In the present invention, the fermentation machine further includes a temperature detector coupled to the motor controller. The motor controller further includes a screen, and the temperature sensed by the temperature detector is displayed on the screen. The sealed top cover also includes an observation window for users to visually observe the conditions inside the tank.

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

Various bacterial strains and cancer cell lines described in this article are commercially available and do not require storage of biological materials. The yogurt drink in this article is liquid in refrigerated and room temperature environments. The strains in the yogurt drink in this article showed varying fermentation results at temperatures from 37°C to 43°C and fermentation times from 8 hours to 12 hours.

3‧‧‧Separation method

12‧‧‧Yogurt drink concentrate

14‧‧‧Powdered products

16‧‧‧n-Hexane extract

18‧‧‧First residue

20‧‧‧Ethyl acetate extract

22‧‧‧Second residue

24‧‧‧Ethanol Extract

26‧‧‧Third residue

28‧‧‧Aqueous extract

30‧‧‧Fourth residue

100‧‧‧Fermentation machine

101‧‧‧Trough

102‧‧‧Temperature control interlayer

103‧‧‧Resistance Heater

104‧‧‧Stirring arm

105‧‧‧Sealing 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 and NCBI alignment results of strain code LR-36, which is Lactobacillus rhamnosus .

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

Figure 3 (A) is a photomicrograph of strain code UY-58. It is a Gram-positive bacillus that does not have catalase, oxidase and motility. It does not produce endospores and can grow in both aerobic and anaerobic environments.

Figure 3 (B) shows the 16S rDNA sequencing results of strain code UY-58. It is closest to Lactobacillus fermentum , with a similarity of 99.9%.

Figure 4 (A) is a photomicrograph of strain code UY-76. It is a Gram-positive bacillus that does not have catalase, oxidase and motility. It does not produce endospores and can grow in both aerobic and anaerobic environments.

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

Figure 5 is a flow chart of separation method 3.

Figure 6 (A) is the ¹ H-NMR spectrum of the 11 n-hexane extract (11-H) of yogurt drink.

Figure 6 (B) is the ¹ H-NMR spectrum of the yogurt drink 11 ethyl acetate extract (11-E).

Figure 6 (C) is the ¹ H-NMR spectrum of the ethanol extract of yogurt drink 11 (11-A).

Figure 6 (D) is the ¹ H-NMR spectrum of yogurt drink 11 water extract (11-W).

Figure 7 (A) is the ¹ H-NMR spectrum of the 12-n-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) is the ¹ H-NMR spectrum of yogurt drink 12 ethanol extract (12-A).

Figure 7 (D) is the ¹ H-NMR spectrum of yogurt drink 12 water extract (12-W).

Figure 8 (A) is the ¹ H-NMR spectrum of the n-hexane extract of yogurt drink 13 (13-H).

Figure 8 (B) is the ¹ H-NMR spectrum of yogurt drink 13 ethyl acetate extract (13-E).

Figure 8 (C) is the ¹ H-NMR spectrum of yogurt drink 13 ethanol extract (13-A).

Figure 8 (D) is the ¹ H-NMR spectrum of yogurt drink 13 water extract (13-W).

Figure 9 (A) is the ¹ H-NMR spectrum of the 14 n-hexane extract (14-H) of yogurt drink.

Figure 9 (B) is the ¹ H-NMR spectrum of yogurt drink 14 ethyl acetate extract (14-E).

Figure 9 (C) is the ¹ H-NMR spectrum of the ethanol extract of yogurt drink 14 (14-A).

Figure 9 (D) is the ¹ H-NMR spectrum of the water extract of yogurt drink 14 (14-W).

Figure 10 is a bar chart showing the total polyphenol content of each extract obtained from yogurt drinks 11 to 14 using separation method 3.

Figure 11 shows the total amount of each extract obtained by separation method 3 of yogurt drinks 11 to 14. Flavanone flavonoid content analysis histogram.

Figure 12 is a bar graph showing the analysis of the total polysaccharide content of each extract of yogurt drinks 11 to 14 obtained by separation method 3.

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

Figure 14 is 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 is the HPLC spectrum of each extract obtained from yogurt drink 14 by separation method 3 at a wavelength of 203 nm.

Figure 17 is a histogram of the DPPH radical scavenging rates of each extract obtained from yogurt drinks 11 to 14 through separation method 3.

Figure 18 is a bar graph showing the total antioxidant capacity of each extract obtained from yogurt drinks 11 to 14 using separation method 3 - ABTS free radical scavenging rate.

Figure 19 is a histogram showing the reducing power measurement of each extract obtained from Yoghurt Drinks 11 to 14 through separation method 3.

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

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

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

Figure 23 is a bar graph showing the inhibition rate of colon cancer cell survival of each extract obtained from yogurt drinks 11 to 14 by separation method 3.

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

Figure 25 is a flow chart of the yogurt drink production system.

Picture 26 is a diagram of the yogurt fermentation machine.

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

This invention carries out activity-guided separation of active metabolites of yogurt, determines the active ingredients and prepares them into beverages or medicines suitable for oral administration, while still maintaining the efficacy of the ingredients.

Experiment 1. Isolation and identification of bacterial strains:

Take 1ml of raw milk as a sample, and dilute it 10 times with Lactobacilli MRS Broth as a diluent (concentration multiples are ^10-1 , ^10-2 , ^10-3 , ^10-4 respectively). Take 200μl of the dilution and drop it on the sterile MRS agar medium, and then apply it to the plate. Place the culture plate in a 37℃ anaerobic incubator for 24 hours. Then, aseptically select single colonies with different shapes, colors, and sizes for further culture, and repeatedly select colonies and culture to separate the strains, and make sure that the colonies on the culture plate are of the same shape, color, and size. Finally, the separated single strains are preserved and identified. The identification of yogurt strains is to first observe the single colony on the culture dish with the naked eye, then select two identical colonies for Gram staining to determine whether they are Gram-positive or Gram-negative bacteria. It is best to observe the colonies under 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 results.

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

After isolation and identification, a total of 20 bacterial strains were obtained, which are listed in Table 3 in order of their Latin names.

Experiment 2: Fermentation preparation of yogurt:

The design model of the fermentation preparation experiment of the present invention uses the strain type and ratio, fermentation temperature, and fermentation time as the manipulated variables, and the viscosity and pH value (pH) after fermentation as the quality control standards. Yogurt with a viscosity below 100 centipoise (cP) and a pH value above 5.0 is judged as unfermented or incompletely fermented. The experimental group with a fermentation time of too long (more than 12 hours) is also eliminated due to poor production efficiency. The preparation method of Experiment 2 is: add 250ml of 75℃ hot water to 36g milk powder for high temperature sterilization to become an animal milk product. After cooling to 35~43℃, add 0.3g bacterial powder. Then they were placed in incubators at different temperatures (37℃, 40℃, 43℃) for different reaction times (8 hours, 10 hours, 12 hours), and then the viscosity (Brookfield DVE RV viscometer) and pH value (Milwaukee pH600 pH meter) were measured. Table 4 shows the strains contained in the 14 successful fermentation combinations.

The results showed that different milk powders have little impact on whether fermentation can be successful; the weight ratio between bacterial species also has little impact on the fermentation results. Therefore, the weight ratio of (bacteria powder) between the strains in each experimental group is 1:1, and the more strains, the easier it is for successful fermentation. That is, the more strains, the more likely it is for successful fermentation. The higher the experimental reproducibility. Then, with The 4 groups with the highest reproducibility of successful fermentation (strain combinations 11 to 14 in Table 4) were subjected to three repeated experiments to confirm the optimal fermentation conditions of different bacterial species combinations (that is, the highest viscosity and lowest pH value can be reached 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 complete. The experimental results are shown in Table 5.

The results of three repeated experiments prepared by fermentation of strain combinations 11 to 14 show:

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 At 43°C, fermentation can be completed within 10 or 12 hours. The longer the fermentation time, the higher the viscosity and the lower the pH value. The fermentation time is fixed at 8 hours, and fermentation cannot be completed at fermentation temperatures of 37, 40, and 43°C. The fermentation time is fixed at 10 hours, and fermentation can be completed at fermentation temperatures of 40 and 43°C. 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 or 43°C to complete the fermentation.

Strain combination 12: When the fermentation temperature is fixed at 37°C, the fermentation can be completed within 10 or 12 hours. The longer the fermentation time, the higher the viscosity and the lower the pH value. When the fermentation temperature is fixed at 40°C, the fermentation can be completed within 8, 10 or 12 hours. The longer the fermentation time, the higher the viscosity and the lower the pH value. When the fermentation temperature is fixed at 43°C, the fermentation can be completed within 8, 10 or 12 hours. The longer the fermentation time, the lower the viscosity first and then the higher the pH value. The fermentation time is fixed at 8 hours, and the fermentation temperature can be completely fermented at 40 or 43℃. The higher the fermentation temperature, the lower the viscosity and the higher the pH value. The fermentation time is fixed at 10 hours, and the fermentation temperature can be completely fermented at 37, 40, or 43℃. The higher the fermentation temperature, the higher the viscosity first and then the lower the pH value, and the lower the pH value first and then the higher the pH value. The fermentation time is fixed at 12 hours, and the fermentation temperature can be completely fermented at 37, 40, or 43℃. The higher the fermentation temperature, the higher the viscosity first and then the lower the pH value, and the lower the pH value first and then the higher the pH value.

Strain combination 13: The fermentation temperature is fixed at 37℃, and the fermentation time can be completely fermented at 8, 10, and 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 can be completely fermented at 8, 10, and 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 completely fermented at 8, 10, and 12 hours. The longer the fermentation time, the lower the viscosity first and then the higher the pH value. The fermentation time is fixed at 8 hours, and the fermentation temperature can be completely fermented 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 can be completely fermented at 37, 40, and 43℃. The higher the fermentation temperature, the higher the viscosity first and then lower, and the pH value first and then higher. The fermentation time is fixed at 12 hours, and the fermentation temperature can be completely fermented at 37, 40, and 43℃. The higher the fermentation temperature, the lower the viscosity first and then higher, and the pH value first and then remains unchanged.

Strain combination 14: The fermentation temperature is fixed at 37°C, and the fermentation time is 8, 10, and 12 hours, and the fermentation can be completed. The longer the fermentation time, the higher the viscosity and the lower the pH value; the fermentation temperature is fixed at 40°C, and the fermentation time is 8, 10 , can be completely fermented 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°C, and the fermentation time can be complete in 8, 10, or 12 hours. The longer the fermentation time, the higher the viscosity and the lower the pH value. The value remains unchanged. The fermentation time is fixed at 8 hours, and the fermentation temperature is 37, 40, and 43°C. The fermentation can be completed. The higher the fermentation temperature, the viscosity is first lower and then higher, and the pH value is lower. The fermentation time is fixed at 10 hours, and the fermentation temperature is 37, 40, and 43°C. All can be completely fermented. The higher the fermentation temperature, the viscosity will first increase and then decrease, and the pH value will first decrease and then remain unchanged. The fermentation time is fixed at 12 hours. The fermentation temperature can be complete at 37, 40, and 43°C. The higher the fermentation temperature, the higher the viscosity will be. then low, pH value first low and then high.

Experiment 3: Sensory evaluation of yogurt drinks:

Yogurt has a variety of effects on maintaining physical health. For example, experiments have shown 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 can reduce serum cholesterol by eating yogurt containing Lactobacillus acidophilus; patients with hypercholesterolemia can increase high-density cholesterol by eating yogurt containing Lactobacillus acidophilus and Bifidobacterium longum; long-term consumption of yogurt can effectively reduce the risk of diabetes; yogurt consumption can also prevent cardiovascular disease. Therefore, long-term consumption of sufficient yogurt can achieve the effect of maintaining physical health.

In order to achieve the above goal, the present invention fermentes the bacterial strain combinations 1 to 14 to prepare yogurt drinks 1 to 14, and conducts sensory evaluation analysis and screening. The sensory evaluation is a criterion defined and developed by the American Society of Food Technologists, which 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 Theory of Laws (ISO 6658:2005), Sensory Evaluation Vocabulary (ISO 5492:2008), Color Sensory Testing (Visual Colorimetry: ISO 11037:1999), Texture Sensory Testing (Texture Profile: ISO 11036:1994), Flavor Sensory Testing (Flavor profile: ISO 6564:1985), descriptive analysis (qualitative description of sensory properties: ISO 11035:1994, assessment of intensity of sensory properties: ISO 4121:2003). The experimental methods were evaluated using descriptive analysis, ranking method and hedonic scale.

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

This experiment was conducted by recruiting 11 highly sensitive sensory tasters with experience in fermented product development, fermented milk product development, yogurt product development, and hand-shaken beverage store practice. Among them, 5 men and 6 women conducted a discussion in a meeting to judge the differences in appearance, texture, and flavor between yogurt drinks. Finally, the differences were described by vision (color, cleanliness), smell (milky aroma, fragrance, unique flavor), taste (acidity, sweetness, smoothness, mellowness), and overall preference (flavor expression, aftertaste). The evaluation was conducted using a five-point preference rating method (1 point: very dislike, 2 points: dislike, 3 points: neither like nor hate, 4 points: like, 5 points: very like). The experimental results are shown in Table 6.

好等級高

Good, high level

2. Use the ranking method to evaluate yogurt drinks from 1 to 14:

The number and gender of the tasters are as described above. The tasters divided yogurt drinks 1 to 14 into three levels (high, medium, and low) according to their preference, and each level was arranged from left to right from the highest to the lowest preference. The results are shown in Table 7.

3. Evaluate yogurt drinks 11 to 14 using the preference rating method:

Yogurts 11 to 14 with high preference levels in the ranking method were further evaluated by the preference rating method to confirm whether there were significant differences and to understand the relationship between preference levels and product characteristics. The survey was conducted using a consumer questionnaire and a five-point preference rating system (1 point: very dislike, 2 points: dislike, 3 points: neither like nor hate, 4 points: like, 5 points: very like) was used for scoring. The aroma, sweetness, and sourness were also recorded using 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 points, the better.

The first consumer questionnaire survey was held in a department store shopping street, where there was no interference from the external environment and no mutual influence. A total of 67 untrained consumers were evaluated, including 24 males (35.8%) and 43 females (64.2%). The age distribution is: 25 people (37.3%) are under 20 years old, 21 people are 20-29 years old (31.3%), 13 people are 30-39 years old (19.4%), 3 people are 40-49 years old (4.5%), 50- 4 people are 59 years old (6%), and 1 person is over 60 years old (1.5%). The top three occupations are: 30 students (44.8%), 17 people in the service industry (25.4%), 5 people in the military, public education, and education, and the remaining 15 people (22.4%).

The second consumer questionnaire survey was conducted in the university district, without any external interference or mutual influence. The evaluation subjects were 69 untrained consumers, including 31 males (44.9%) and 38 females (55.1%). The age distribution was: 50 people aged 18-22 (72.5%), 15 people aged 23-30 (21.8%), and 4 people under 18 (5.8%). The occupation distribution was all students (69 people, 100%).

Based on the results of the first and second consumer questionnaires, the total number of participants was 136. Please refer to Table 8. In terms of overall preference, Yogurt Drink 14 is the best, with a score greater than 4 points for liking it; followed by Yogurt Drink 12; Yogurt Drink 11 and 13 have similar scores, with scores greater than 3 points being disliked. Not annoying. In addition, the number of yogurt drinks 11, 12, 13 and 14 rated as the most preferred (first-ranked) yogurt drinks by all 136 consumers were 32, 38, 31 and 35 respectively.

Under the purely natural process without adding any seasonings, the aroma of Yogurt Beverages 11 to 14 is not much different, and they are all slightly lighter; the sweetness of Yogurt Beverages 11 to 14 is moderate; the sour taste of Yogurt Beverages 11 to 14 The difference is not big, both are slightly lighter (as shown in the results in Table 9).

Experiment 4: Sensory evaluation of yogurt drink ingredients:

In order to increase the diversity of yogurt drinks, improve the daily yogurt intake of users (for example, being able to drink 200g within 30 minutes) and drink for a long time without getting tired of it, Experiment 4 used Yogurt Drink 14 as the base to add the recipe design of hand-shaken drinks, and evaluated it using descriptive analysis. Experiment 4 recruited 5 highly sensitive sensory tasters with practical experience in fermented product development, fermented milk product development, yogurt product development, and hand-shaken drink stores to conduct the experiment, including 3 men and 2 women, and conducted the evaluation in the form of meeting discussion.

1. Salty and sweet yogurt drink recipe design:

Adding sesame paste as sauce: After boiling sesame paste, add yogurt and grind together. Sesame paste has a strong aroma and contains oil. After the two are compatible, the flavor contains oil, which makes the overall flavor have a salty taste, but the flavor is too special and not well accepted. Adding cucumber as ingredient: Dice cucumber and add yogurt to grind together. The cucumber flavor covers the yogurt flavor, and the overall vegetable flavor is obvious, lacking the unique flavor of yogurt. Adding corn as ingredient: Add corn kernels from unseasoned canned corn to yogurt and grind together. Corn has a slightly sweet taste, which can enhance the overall sweetness, but the corn flavor and yogurt flavor are integrated into a special taste, which is very low in acceptance. Sensory evaluation results: The formula design of yogurt drink is not suitable for salty and sweet taste.

2. Formula design of yogurt drink sauce:

The yogurt drink of the present invention is made in a purely natural process without any seasoning. The overall taste is moderately sweet, with a slightly lighter aroma and sourness, and is light and elegant. Different sauce recipes bring different sensory experiences. Adding brown sugar as sauce: Add brown sugar boiled into syrup to yogurt and grind together. The brown sugar has a rich and special flavor, which is compatible with the sweet and sour flavor of yogurt. Adding matcha as sauce: Add Japanese imported matcha powder to yogurt and grind together. Matcha is slightly bitter and is incompatible with the sweet and sour flavor of yogurt. Adding 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 for yogurt drinks:

Yogurt is a non-Newtonian fluid (a fluid whose viscosity is not constant at a certain temperature and pressure). Its viscosity changes with the pressure or speed. The greater the pressure, the greater the viscosity, and it may even become a temporary solid. If solid ingredients are added to yogurt, it will be difficult to drink and suck. To overcome this problem, 200g of yogurt is placed in a cylindrical cup (upper circle diameter 9 cm, lower circle diameter 5.6 cm, cup height 13.5 cm), so that users can easily suck the solid ingredients in the yogurt with a straw with a diameter of 1~1.5 cm and a length of 17.5~20 cm, and there will be no phenomenon of solid ingredients remaining after the drink is finished. 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 with a straw. Therefore, a crushed ice layer is added to the upper layer of the yogurt drink to keep it cold (the size of the crushed ice is controlled to be between 0.2 and 0.4 cubic centimeters, because less than 0.2 cubic centimeters will melt and release water too quickly, and more than 0.4 cubic centimeters will suck the crushed ice cubes and the taste is not good). The volume ratio of the crushed ice layer to the yogurt drink is 1:3 to 1:5, so that the yogurt drink is maintained between 8 and 12°C within 30 minutes, so that the sensory evaluation results of the solid ingredients are reproducible. The evaluation focus of the sensory evaluation of solid ingredients is: the smoothness of the taste, the overall balance; further including the visual stimulation, enhancing the user's desire to drink yogurt after watching (promoting appetite, enhancing active intake). The appearance of yogurt can present the effects of layering, gradient, and special textures. The added solid ingredients include but are not limited to pearls, coconut, coffee jelly, vermicelli, taro balls, pudding, grass jelly, tofu pudding, mung bean, red bean, coix seed, purple rice, and oatmeal. The results of the sensory evaluation are shown in Table 10. 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 of the sensory evaluation are shown in Table 11.

Experiment 5, Preparation of Yogurt Drink Extract:

The types and contents of active metabolites in yogurt drinks vary depending on the strain, formula, and fermentation environment. In order to determine the active ingredients of the yogurt drinks of the present invention, yogurt drinks 11 to 14 with high sensory tasting preference levels were selected for extraction and distribution extraction, and the obtained fractions were further subjected to component analysis and activity testing.

1. Separation method 1:

The yogurt solution was dried by heating and decompressing the solution to obtain a paste. The paste was then extracted with ethanol three times to obtain an ethanol extract. The ethanol extract was vacuum filtered, heated, decompressed and concentrated to obtain a crude extract. The crude extract was then partitioned and extracted with ethyl acetate: water (1:1 (v/v)). However, the crude extract had too many emulsion layers, resulting in poor separation results and inability to perform subsequent separation experiments. If the crude extract was partitioned and extracted with dichloromethane: water (1:1 (v/v)), there was still a problem of too many emulsion layers.

2.Separation method 2:

The yogurt solution was not dried but diluted with water (1:1 (v/v)) and then ethyl acetate (1:1 (v/v)) was added for distribution extraction, which still produced a large amount of emulsion layers, resulting in poor separation effect. If dichloromethane (1:1 (v/v)) or n-butanol (1:1 (v/v)) was added for distribution extraction, there was still a problem of too many emulsion layers.

3.Separation method 3:

The original liquid of the yogurt drink is freeze-dried to obtain a powdery product, which is then extracted sequentially with solvents of increasing polarity. It was found that the extraction rate of methylene chloride is too low, and there is no significant difference in the extraction effect between n-butanol and n-butanol. There is a problem of solvent residue after n-butanol extraction, and it is difficult to continue the subsequent solvent extraction. Therefore, methylene chloride and n-butanol are not suitable as Extraction solvent for this separation method.

As shown in the separation method 3 of FIG. 5 , after the solvent arrangement and combination extraction test, the extraction is finally performed in sequence with n-hexane, ethyl acetate, ethanol and water. That is, the yogurt drink stock solution 12 is freeze-dried to obtain a powder product 14, and then the powder product 14 is extracted with n-hexane to obtain 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 ethyl acetate extract 20 (sample code: 11-E, 12-E, 13-E, 14-E) and the second residue 22. Afterwards, the second residue 22 was extracted with ethanol to obtain ethanol extract 24 (sample code: 11-A, 12-A, 13-A, 14-A) and the third residue 26. Finally, the third residue 26 was extracted with water to obtain water extract 28 (sample code: 11-W, 12-W, 13-W, 14-W) and the fourth residue 30. The extracts and residues were heated and compressed and concentrated. The experimental results of various extracts of yogurt drinks 11 to 14 are shown in Table 12. Table 12 shows that this separation method can overcome the problem of emulsion layer produced by liquid-liquid partition extraction, separate, concentrate and collect various active ingredients in yogurt drinks, and clearly confirm the differences in appearance, properties and flavor of various extracts by visual and olfactory detection.

Experiment 6: Analysis of ingredients of yogurt extract:

1. Nuclear Magnetic Resonance Spectroscopy (NMR) Analysis:

Yogurt drinks 11 to 14 were separated by method 3 to obtain 16 extracts. These extracts were subjected to NMR analysis. The solubility of the extracts was tested with hydrogen isotope (deuterium) solvents: CDCl ₃ , acetone-d6, MeOH-d4, C ₅ D ₅ N, DMSO-d6 and D ₂ O. The results showed that C ₅ D ₅ N and DMSO-d6 had better solubility for all 16 extracts. In the ¹ H-NMR preliminary experiment, it was found that the solvent signal of C ₅ D ₅ N would mask the signal of the extract. Finally, DMSO-d6 was selected as the solvent for NMR analysis. The concentrations of the extracts were 25 mg/ml, 50 mg/ml, 75 mg/ml and 100 mg/ml. It was found that the 1 H-NMR spectra of n-hexane extracts (11-H, 12-H, 13-H, 14-H), ethyl acetate extracts (11-E, 12-E, 13-E, 14-E), and ethanol extracts (sample code: 11-A, 12-A, 13-A, 14-A) at ⁵⁰ mg/ml showed the best magnetic field and characteristic signals, and the best sample concentration of water extracts (11-W, 12-W, 13-W, 14-W) was 25 mg/ml. After adding DMSO-d6 and before testing, heating the n-hexane extracts (11-H, 12-H, 13-H, 14-H) and ethyl acetate extracts (11-E, 12-E, 13-E, 14-E) to 37~40℃ can further improve the sample solubility and optimize the characteristic signals of the ¹ H-NMR spectra. The experiment was conducted using a JEOL ECS 400MHz FT-NMR nuclear magnetic resonance spectrometer with a resolution of 400MHz, chemical shifts expressed in ppm (δ), and coupling constants expressed in Hertz ( J ). As shown in the ^1H -NMR spectra of the 16 extracts in Figures 6 (A) to 9 (D) and the NMR signal analysis and major component determination of the 16 extracts in Table 13, the NMR characteristic signals and major components of the n-hexane extract, ethyl acetate extract, ethanol extract, and water extract are all different, and the NMR signals between yogurt drinks 11 to 14 are also different.

2. Analysis of total polyphenol content:

Yogurt drinks 11 to 14 were separated by separation method 3 to obtain 16 extracts, which were analyzed by Folin-Ciocalteu colorimetric method. The principle is that phosphotungstic acid and phosphomolybdic acid in the Folin-Ciocalteu reagent are oxidized by polyphenol molecules to produce color, and gallic acid is used as a standard. The experimental method is as follows: After dissolving gallic acid (5 mg/mL) in methanol, 50, 100, 250 and 500 μg/mL standards are prepared. 20 μL of each standard is taken and the volume is supplemented to 1.6 mL with secondary water. Then 100 μL of 2N Folin-Ciocalteu reagent and 300 μL of 20% Na ₂ CO ₃ are added and mixed. The mixture is reacted at 40°C for 40 minutes and the absorbance is measured at 725 nm. In addition, secondary water is used to replace the standard and the value is zeroed. A standard curve is drawn based on the relationship between concentration and absorbance. 20 μL of the sample to be tested (1 mg/mL) is treated in the same way, and the total polyphenol content per gram of extract is calculated according to the standard curve. As shown in Table 14 and Figure 10, the total polyphenol content of the extract of yogurt drink 11 was the highest in 11-E and the lowest in 11-W; the extract of yogurt drink 12 was the highest in 12-A and the lowest in 12-W; the extract of yogurt drink 13 was the highest in 13-H and the lowest in 13-W; the extract of yogurt drink 14 was the highest in 14-E and the lowest in 14-W. In the 16 extracts, 14-E had the highest total polyphenol content and 11-W had the lowest. The total polyphenol content of the 4 n-hexane extracts (11-H to 14-H) ranged from 18.2 to 24.0 mg _{Gallic acid} /g. The total polyphenol content of the 4 ethyl acetate extracts (11-E to 14-E) ranged from 9.4 to 40.6 mg _{Gallic acid} /g. The total polyphenol content of the four ethanol extracts (11-A to 14-A) ranged from 11.3 to 28.3 mg _{Gallic acid} /g. The total polyphenol content of the four water extracts (11-W to 14-W) ranged from 2.6 to 7.1 mg _{Gallic acid} /g.

3. Analysis of total flavanone flavonoid content:

The principle is that 2,4-dinitrophenylhydrazine (DNP) will derivatize the aldehyde or ketone group on the flavanone structure to make it have light absorption properties at a specific wavelength. And hesperetin (hesperetin) was used as the standard. The experimental method is as follows: add 1 mL of the sample to be tested to 2 mL of DNP solution and 2 mL of methanol, and place it in a 50°C water bath for heating for 50 minutes. After cooling, add 5 mL of KOH methanol solution and mix, let it 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 value was measured at a wavelength of 494 nm. The measured absorbance value is compared with the standard curve of the standard substance and converted into the flavanone content per gram of extract. As shown in Table 14 and the total flavanone flavonoid measurement results shown in Figure 11, in the extract of Yogurt Drink 11, 11-A has the highest content and 11-E has the lowest; in the extract of Yogurt Drink 12, the 12-A content The highest, 12-H is the lowest; among the extracts of Yogurt Drink 13, the 13-A content is the highest and 13-H is the lowest; among the extracts of Yogurt Drink 14, the 14-A content is the highest and 14-H is the lowest. Comprehensive of the 16 extracts, 12-A has the highest total flavanone flavonoid content, and 12-H has the lowest. Among the four n-hexane extracts (11-H to 14-H), the total flavanone flavonoid content ranged from 15.0 to 23.1 mg _Hesperetin /g. Among the four ethyl acetate extracts (11-E to 14-E), the total flavanone content ranged from 16.0 to 26.0 mg _Hesperetin /g. Among the four ethanol extracts (11-A to 14-A), the total flavanone content ranged from 62.5 to 91.3 mg _Hesperetin /g. Among the four water extracts (11-W to 14-W), the total flavanone 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 five-carbon sugars and six-carbon sugars will decompose into furfural due to dehydration under acidic and high-temperature conditions. Furfural is further combined with phenol. The reaction produces an orange product, and the total polysaccharide content is calculated based on the change in absorbance value, and 0, 10, 20, 30, 40, and 50 μg/ml glucose are used as standards. The experimental method is: add 0.5 mL of 5% phenol solution to 0.5 mL of 10 mg/mL product to be tested, then add 2.5 mL of concentrated sulfuric acid, mix evenly, react in a 100°C water bath for 20 minutes, and measure the absorbance value at a wavelength of 490 nm after cooling. As shown in Table 14 and Figure 12, the total polysaccharide measurement results show that the extract of Yogurt Drink 11 has the highest 11-W content and the lowest 11-H content; the extract of Yogurt Drink 12 has the highest 12-W content. 12-E is the lowest; among the extracts of Yogurt Drink 13, 13-W has the highest content and 13-H is the lowest; among the extracts of Yogurt Drink 14, 14-W has the highest content and 14-H has the lowest. Comprehensive of the 16 extracts, 14-W has the highest total polysaccharide content and 14-H has the lowest. The total polysaccharide content of the four n-hexane extracts (11-H to 14-H) ranged from 7.6 to 11.2 mg _Glucose /g. The total polysaccharide content of the four ethyl acetate extracts (11-E to 14-E) ranged from 9.7 to 18.4 mg _Glucose /g. The total polysaccharide content of the four ethanol extracts (11-A to 14-A) ranged from 223.2 to 249.4 mg _Glucose /g. The total polysaccharide content of the four water extracts (11-W to 14-W) ranged from 414.4 to 494.1 mg _Glucose /g.

5. Thin layer chromatography (TLC) analysis:

Refer to the normal phase thin layer chromatography method 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 adding 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 good analytical results in the same solvent system. Neither the ethanol extract (11-A to 14-A) nor the water extract (11-W to 14-W) had ideal analytical results in normal phase thin layer chromatography, and the main TLC spots did not exist at R _f 0.3- 0.8.

6. High Performance Liquid Chromatography (HPLC) Analysis:

The analysis was performed with reference to the reversed-phase HPLC method commonly used in the Taiwan Pharmacopoeia (Second Edition) under the following conditions: the HPLC instrument was an Agilent 1100 series, the detector was a G1315B photodiode array detector, the autosampler was a G1329A autosampler, the chromatography column was an Intersil ODS-3V 250 x 4.6 mm (5 μm), the mobile phase solvent A was acetonitrile, the solvent B was 0.1% H ₃ PO ₄ , the flow rate was 1 ml/min, the column temperature was 30°C, and the detection wavelengths were 203, 210, 254, 280, and 365 nm. The solvent system conditions are as follows: the mobile phase includes solvents A and B, the linear gradient is 0% A-10% A from 0 to 20 minutes, 10% A-40% A from 20 to 40 minutes, 40% A-100% A from 40 to 60 minutes, and 100% A from 60 to 70 minutes, and the flow rate and column temperature are as described above. As shown in Table 16 and Figures 13 to 16, among the five detection wavelengths, no significant chromatographic peaks were observed for the n-hexane extracts (11-H to 14-H) and the ethyl acetate extracts (11-E to 14-E), and only 203 nm for the ethanol extracts (11-A to 14-A) and the water extracts (11-W to 14-W) showed a better chromatographic peak.

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:

Free radicals are generated during the self-oxidation of lipids, causing lipid rancidity. Antioxidants scavenge lipid peroxide free radicals by providing hydrogen, thereby inhibiting the oxidation chain reaction. DPPH is often used in antioxidant research to evaluate the hydrogen-donating ability of antioxidants. DPPH methanol solution has strong absorption at 517nm, and the absorbance value decreases when reduced by antioxidants. Therefore, the lower the absorbance value at 517nm, the stronger the hydrogen-donating ability 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) into 1 mL of freshly prepared 10 mM DPPH methanol solution, shake and mix, then place at room temperature for 30 minutes, and detect the absorbance value at 517nm wavelength. The results are expressed in terms of clearance percentage. The higher the clearance percentage, the better the hydrogen supply capacity of the test sample. As shown in Table 17 and Figure 17, 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 stable blue-green water-soluble ABTS˙ ⁺ cationic free radicals. Use a UV-visible light photometer, which has a strong absorbance value at 620nm, and compare it with the standard water-soluble vitamin E analog (trolox) to prepare a standard curve. When ABTS˙ ⁺ cationic free radicals and antioxidants are reduced, the color of the solution will become lighter and the absorbance value will decrease, thus converting the ability to scavenge ABTS˙ ⁺ cationic free radicals. The better the scavenging ability, the stronger the antioxidant's hydrogen-donating ability. The experimental method is: place the prepared ABTS in the refrigerator for 1 hour to produce a stable blue-green ABTS˙ ⁺ free radical aqueous solution. Add 80 μL of extract sample (50 mg of 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, making the total volume 200 μL. Shake at room temperature for 10 minutes in the dark, and measure the absorbance value at 620 nm. The lower the absorbance value, the better the effect of scavenging ABTS˙ ⁺ free radicals, and the scavenging rate is calculated as a percentage. As shown in the total antioxidant capacity results in Table 17 and Figure 18, among the extracts of Yogurt Drink 11, 11-A has the best ability and 11-E has the weakest ability; among the extracts of Yogurt Drink 12, 12-A 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 and 14- H is the weakest. Among the 16 extracts, 12-A had the best total antioxidant capacity, and 12-H had the weakest.

3.Measurement of reducing power:

Antioxidants will reduce hematite (K ₃ Fe(CN) ₆ ) to ferruginous hematite (K ₄ Fe(CN) ₆ ), which then reacts with Fe ³⁺ to generate Prussian blue. The absorbance is measured at 620nm to detect the amount of Prussian blue generated. The higher the absorbance, the stronger the reducing power of the antioxidant. The experimental method is: add 200μL of extract sample (50mg of extract dissolved in 15mL of 50% ethanol solution) to a microcentrifuge tube, and then add 200μL of 1% hematite, with a total volume of 400μL. After mixing, place in a 50℃ water bath for 20 minutes. After the reaction is complete, place on ice for 10 minutes, add 200μL of 10% trichloroacetic acid (TCA) and mix, centrifuge at 3,000rpm for 10 minutes. Add 75μL of supernatant to a 96-well plate, add 75μL of ultrapure water and 30μL of 0.1% FeCl _{3 ．} 6H ₂ O, mix, and measure the absorbance at 620nm. The higher the absorbance, 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 and weakest reducing power; among the extracts of yogurt drink 12, 12-A has the best reducing power, while 12-W has the weakest; among the extracts of yogurt drink 13, 13-A has the best reducing power, while 13-H has the weakest; among the extracts of yogurt drink 14, 14-A has the best reducing power, while 14-H has the weakest reducing power. Among the 16 extracts, 12-A has the best reducing power, while 13-H has the weakest reducing power.

Experiment 8: Test on the inhibitory activity of yogurt extract on cancer cell growth:

For organs related to the commensal bacteria-gut-brain axis, active metabolites start from oral intake and pass through the digestive tract, blood, circulatory system, and urinary system (excretory 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 lines) were used to test the cancer cell growth inhibitory activity of 16 extracts obtained from yogurt drinks 11 to 14 through separation method 3. The experimental method is cell viability analysis (MTT assay). Various cancer cells are implanted into a 96-well plate. The cell density in each well is 7×10 ³ cells/100 μL, and then samples of different concentrations (within 100 μg/mL) are added ( DMSO was used as the control group) and placed in a 37°C, 5% _CO2 cell culture incubator for 72 hours. Then add 50 μL MTT and incubate for 1 hour. Centrifuge at 2000 rpm for 3 minutes, remove the supernatant and add 200 μL DMSO, place on a oscillator and shake until the purple crystals are completely dissolved, and read the absorbance values at 570 mm and 620 mm. The results are presented as the best inhibition percentage of cancer cell growth after different samples were treated at a concentration of 100 μg /mL. Human clinical treatments for oral cancer are 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 has the weakest; among the extracts of yogurt drink 12, 12-A has the best inhibitory activity and 12-W has the weakest; among the extracts of yogurt drink 13, 13-W has the best inhibitory activity and 13-H has the weakest; among the extracts of yogurt drink 14, 14-A has the best inhibitory activity and 14-W has the weakest. Among the 16 extracts combined, 12-A has the best activity in inhibiting the growth of cancer cells. As shown in Figure 20, in terms of inhibiting the growth of blood cancer cell Molt 4, among the extracts of yogurt drink 11, 11-H has the best inhibitory activity, while 11-E and 11-W have no inhibitory activity; among the extracts of yogurt drink 12, 12-A has the best inhibitory activity, while 12-H and 12-W have no inhibitory activity; among the extracts of yogurt drink 13, 13-A has the best inhibitory activity, while 13-E has the weakest; among the extracts of yogurt drink 14, 14-E has the best inhibitory activity, while 14-A and 14-W have no inhibitory activity. Among the 16 extracts combined, 14-E has the best inhibitory activity.

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

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 had the best inhibitory activity, and 11-E had the weakest; among the extracts of yogurt drink 12, 12-A had the best inhibitory activity, and 12-W had the weakest; among the extracts of yogurt drink 13, 13-H had the best inhibitory activity, and 13-W had the weakest; among the extracts of yogurt drink 14, 14-A had the best inhibitory activity, and 14-H had the weakest. Among the 16 extracts combined, 11-A had 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 had the best inhibitory activity and 11-W had the weakest; among the extracts of yogurt drink 12, 12-H had the best inhibitory activity and 12-W had the weakest; among the extracts of yogurt drink 13, 13-H had the best inhibitory activity, and 13-E and 13-W had no inhibitory activity; among the extracts of yogurt drink 14, 14-W had the best inhibitory activity and 14-E had the weakest. Among the 16 extracts combined, 14-W had 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 had the best inhibitory activity, and 11-W had the weakest; among the extracts of yogurt drink 12, 12-E had the best inhibitory activity, and 12-H and 12-W had no inhibitory activity; among the extracts of yogurt drink 13, only 13-E had inhibitory activity; among the extracts of yogurt drink 14, 14-W had the best inhibitory activity, and 14-A had the weakest. Among the 16 extracts combined, 14-W had 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 had the best inhibitory activity and 11-A had the weakest; among the extracts of yogurt drink 12, 12-E had the best inhibitory activity and 12-W had the weakest; among the extracts of yogurt drink 13, 13-H had the best inhibitory activity and 13-A had the weakest; among the extracts of yogurt drink 14, 14-A had the best inhibitory activity and 14-E had the weakest. Among the 16 extracts combined, 12-E had 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 had the best inhibitory activity and 11-W had the weakest; among the extracts of yogurt drink 12, 12-H had the best inhibitory activity and 12-E had the weakest; among the extracts of yogurt drink 13, 13-H had the best inhibitory activity and 13-W had the weakest; among the extracts of yogurt drink 14, 14-A had the best inhibitory activity and 14-E had the weakest. Among the 16 extracts combined, 11-A had 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 had the best inhibitory activity and 11-E had the weakest; among the extracts of yogurt drink 12, 12-H had the best inhibitory activity and 12-E had the weakest; among the extracts of yogurt drink 13, 13-A had the best inhibitory activity, and 13-H and 13-E had no inhibitory activity; among the extracts of yogurt drink 14, 14-W had the best inhibitory activity and 14-H had the weakest. Among the 16 extracts combined, 11-A had the best inhibitory activity. As shown in Figure 24, in terms of inhibiting the growth of prostate cancer cells PC-3, among the extracts of yogurt drink 11, 11-E had the best inhibitory activity and 11-H had the weakest; among the extracts of yogurt drink 12, 12-H had the best inhibitory activity and 12-W had the weakest; among the extracts of yogurt drink 13, 13-E had the best inhibitory activity, and 13-H and 13-W had no inhibitory activity; among the extracts of yogurt drink 14, 14-E had the best inhibitory activity and 14-A had the weakest. Among the 16 extracts combined, 13-E had the best inhibitory activity.

Experiment 9: Yogurt production system:

In the past, liquid yogurt drinks were produced in two forms: home-made small-scale production and ton-scale factory mass production. Home-based small-volume production has problems such as unstable quality, the need for subsequent preparation, and inconvenient operation. Large-scale production in ton-level factories will face the problem that 2 to 3 days or more have passed since consumers purchased and drank the drinks. Freshly fermented yogurt drinks cannot be consumed on the same day, and often cannot be consumed within the shelf life, or may deteriorate if stored improperly. problem. Therefore, the present invention provides a production system for liquid yogurt drinks, which can simultaneously solve the above-mentioned problems of home-made small-scale production and mass production of yogurt drinks in ton-level factories. A single fermentation machine can produce kilogram-level liquid yogurt drinks. It can be fermented and produced on-site using a transparent process, which can be completed in 18 hours. The yogurt drinks consumers drink every day are freshly fermented on the same day, and the final product can be served in a hand-shake manner.

As shown in the process shown in Figure 25 and the structure of the fermentation machine 100 shown in Figure 26, the sequence of the liquid yogurt production system of the present invention includes: high-pressure cleaning, high-temperature sterilization, milk emulsification, secondary sterilization, and sterilization. Fermentation, finished product sampling, finished product collection, cooling and refrigeration, homogenization, adding ingredients, filling and packaging, and product delivery.

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, the resistance heater arranged outside the tank 101 and coupled to the motor controller 108 103. Among them, the space surrounded by the sealed top cover 105 and the tank body 101 is in a sealed state; water, milk powder and bacterial powder are sent into the space through the feeding port M; the resistance temperature increaser 103 is used to sterilize the water and milk powder, and make The bacterial powder is stirred by the stirring arm 104 in a mixture of water and milk powder at a temperature between 37°C and 43°C to prepare a yogurt drink.

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

The manufacturing process in Figure 25 is as follows:

(1) High-pressure cleaning and high-temperature sterilization: Open the feed port M → Use a high-pressure spray gun to clean the inner wall of the tank 101 → Confirm the water level is at the appropriate position through the observation window S1 → Open the discharge port N4 to discharge → Fill the tank 101 with water again → The temperature detected by the temperature detector 107 is displayed on the screen 109 of the motor controller 108 → Display heating to above 72°C → After reaching the sterilization temperature → Stand for 30 minutes → Open the discharge port N4 to discharge to the wastewater tank (not shown).

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

(3) Secondary sterilization: Turn on heating → Observe the screen 109 to see if the temperature is heated to above 72°C → After reaching the sterilization temperature → Turn off heating → Leave it alone for 30 minutes.

(4) Inoculation fermentation: Inject cooling water from the heat conduction inlet N5 → turn on the circulation pump (not shown) → use the cooling water to perform heat exchange and temperature control → observe the screen 109 to gradually cool down to 43°C → close the cooling water source and circulation Pump → Disinfect the feeding port M with 75% alcohol → Quickly open the feeding port M to add bacteria and close it and tighten it → Turn on the stirring motor 110 and let the stirring arm 104 run and stir for 20 minutes → Close the stirring motor 110 → Leave to ferment.

(5) Sampling of finished products: Spray the discharge port N4 with pure water and then spray 75% alcohol for disinfection → Take out 300 ml of samples → Conduct viscosity and pH testing → The viscosity and pH value reach the tank collection standard → Carry out tank collection.

(6) Finished product collection: Spray the outlet N4 with pure water → Spray 75% alcohol for disinfection → Use 304 food grade stainless steel containers to collect in batches.

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

(8) Homogenize and prepare, add ingredients: take yogurt drink → add ice cubes → use homogenizer to homogenize → prepare solid ingredients or solid fruit ingredients.

(9) Filling, packaging, and serving: Finally, check again to see if there is any leakage of the beverage or ingredients → Completed.

This invention is truly an innovative invention with profound industrial value, and the application must be filed in accordance with the law. In addition, the present invention may be modified in any way by those with ordinary skill in the art without departing from the scope of protection as claimed in the appended patent application.

<110> Yuyang Biomedical Co., Ltd.

<120> Yogurt biotech drink with antioxidant properties 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 (10)

A yogurt drink comprises: an animal milk product, comprising water and animal milk; and a plurality of bacterial powders mixed with the animal milk product, wherein the plurality of bacterial powders are from a plurality of corresponding strains, and the plurality of strains are composed of Bifidobacterium longum , Lactobacillus acidophilus , Lactobacillus fermentum , Lactobacillus helveticus , Lactobacillus paracasei , Lactobacillus rhamnosus , Lactobacillus salivarius , and Streptococcus thermophilus . As described in item 1 of the patent application, the weight of the bacterial powder of each of the multiple strains is the same. The yogurt drink described in item 1 of the patent application, wherein the yogurt drink further includes a sauce ingredient, and the sauce ingredient is selected from one of the group consisting of brown sugar, honey and combinations thereof. The yogurt drink described in item 1 of the patent application, wherein the yogurt drink further includes a solid ingredient selected from the group consisting of pearls, coconut, coffee jelly, taro balls, pudding, red beans, purple rice, oats and One of the groups formed by its combination. The yogurt drink as described in item 1 of the patent application scope, wherein the yogurt drink also includes a solid fruit ingredient, and the solid fruit ingredient is selected from one of the group consisting of mango, strawberry, dragon fruit, passion fruit, avocado, banana, kiwi, orange, grapefruit and a combination thereof. For example, in the yogurt drink described in item 1 of the patent application, the plurality of bacterial 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 beverages in the first item of the patent application scope, comprising: freeze-drying the yogurt beverage to obtain a powder product; extracting the powder product with n-hexane to obtain a n-hexane extract and a first residue, wherein the main component of the n-hexane extract is a short-chain fatty acid with a double bond and a hydroxyl group; extracting the first residue with ethyl acetate to obtain an ethyl acetate extract and a The 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 components of the ethanol extract 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. As described in item 7 of the patent application, 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). For the separation method described in item 7 of the patent application, the ethanol extract and the water extract are analyzed for components by reverse phase high performance liquid chromatography. A use of an ethanol extract obtained by the separation method of claim 7 in the preparation of a drug for inhibiting the growth of cancer cells, wherein the cancer cells are selected from one of the groups consisting of blood cancer cells, brain cancer cells, gastric cancer cells, colorectal cancer cells and combinations thereof.

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