TWM587175U - Fermenter of the yogurt bio beverage - Google Patents

Abstract

The application discloses a fermenter of a yogurt bio beverage, wherein the fermenter includes a sealed top cover and a tank, the tank is covered by the sealed top cover, and thus an inner space is formed. The inner space produces the yogurt bio beverage had excellent sensory evaluation and effects of free radical clearance, anti-oxidation, high reduction capability, and the inhibition of cancer cell growth.

Description

Yogurt biotech beverage fermentation machine

This creation is about a fermenter, especially about a fermenter for yoge biotech drinks with anti-oxidation and inhibition of the growth of digestive tract cancer cells.

The intestinal-brain two-way information network is called the gut-brain axis. Later, it was discovered that the intestinal symbiotic flora contributed to this intestinal-brain axis. The three affected each other, so they became symbiotic bacteria. -Intestinal-brain axis (microbiota-gut-brain axis). The National Institute of Mental Health (NIMH) launched a special project in 2013 to explore the gut microbiota-brain communication mechanism, with the goal of developing new anti-psychotic drugs and non-invasive treatments. Since then, related research on the symbiotic bacteria-gut-brain axis has sprung up and has become the focus of neuroscience research. The main axis is to explore the interaction between the gut microbiota and the brain. The gut microbiota has important effects on the brain through neural networks, neuroendocrine systems, and the immune system, and disturbances in human behavior also change the composition of the gut microbiota. The literature has reported a symbiotic-intestinal-brain axis balance and has been linked to a variety of diseases, such as: inflammatory bowel disease (IBD), gastrointestinal cancer, gallstones, behavioral disorders, anxiety, Depression, chronic fatigue syndrome (CFS), hepatic encephalopathy, allergy, obesity, diabetes, atherosclerosis, etc. In cancer chemotherapy and post-operative care, human clinical studies have reported that by regulating the composition of the gut microbiota, it can improve the cardiopulmonary health of breast cancer patients and reduce the fear of cancer recurrence.

At present, western and traditional 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 Ayurveda, another medical system, uses a large amount of turmeric to improve gastrointestinal discomfort, neuroprotection, and prevention of Alzheimer's, its diversity The efficacy is also related to the gut-brain axis. However, due to different eating habits, the use of turmeric has regional restrictions, mainly in India.

In the western medical system, the use of symbiotic-intestinal-brain axis research for drug development is called micro-ecological drugs, which refer to pharmaceutical preparations made of normal microorganisms or substances that regulate the normal growth of microorganisms. Can be used for indications such as infection, diabetes, tumors, inflammatory diseases, and immune-related diseases. Microecological medicines include: live biotherapeutic product (LBP), small molecule microbiome modulator (SMMM), fecal microbiota transplant (FMT). Among them, LBP refers to the explicit control of the type and number of bacterial cells through the identification, screening and combination of human microorganisms, and the use of different types, numbers and combinations of bacterial cells for different indications to ensure the safety and effectiveness of medication. However, the U.S. Food and Drug Administration (FDA) has not yet approved any LBP to market, and only issued guidelines for early clinical trials of LBP in 2016, which makes the development of LBP a clear standard. SMMM refers to a substance that can selectively promote the growth and reproduction of one or more beneficial bacteria in the intestine of the host, and achieve therapeutic purposes by affecting the growth and reproduction of bacteria. Currently known SMMM drug development is only in clinical phase II. FMT refers to transplanting the functional flora of healthy human feces into the gastrointestinal tract of a patient and reconstructing the intestinal flora of the patient for the treatment of intestinal and extra-intestinal diseases. Although the United States included FMT in the guidelines for the treatment of recurrent Clostridium difficile infection in 2013, the policies of different countries vary widely, and FMT currently lacks unified and effective supervision. In general, the clinical trials of micro-ecological drugs have not progressed as expected, and there are currently no micro-ecological drugs approved for marketing. Another problem is that the number of clinical trials of micro-ecological drugs is relatively small, and human verification of the efficacy of the drugs is still not as expected.

The traditional Chinese medicine system does not use symbiotic-intestinal-brain axis research to develop drugs, but studies the interaction between traditional Chinese medicine and intestinal microbiota. Regulating effects of traditional Chinese medicine on intestinal microflora, for example: supplementary Chinese medicines containing polysaccharides have beneficial effects on beneficial bacteria and pathogenic bacteria, but on beneficial bacteria The planting effect is significantly better than that of pathogenic bacteria, and metabolites produced by well-growing beneficial bacteria indirectly inhibit the growth of pathogenic bacteria. For example, Codonopsis pilosula polysaccharide can promote the growth of Bifidobacterium in vitro, thereby increasing the metabolism of acetic acid, and enhancing the colonization resistance of Bifidobacterium. Intestinal microbiota's metabolic effect on traditional Chinese medicine, for example: It has been confirmed that many traditional Chinese medicine components can only produce medicinal active ingredients and achieve therapeutic effect only after being metabolized by intestinal microbiota. For example, baicalin contained in Scutellaria Baicalensis is difficult to be directly absorbed in the intestinal tract. Only baicalein hydrolyzed by the intestinal microbiota can be absorbed into the blood to function. Human clinical trials have confirmed that Gegen Qinlian Decoction can treat type 2 diabetes by altering the gut microbiota. Overall, the study of commensal bacteria-gut-brain axis in traditional Chinese medicine provided an acceptable explanation of the mechanism of action in western medicine, but it still failed to improve the compliance and drug compliance of traditional Chinese medicines due to high doses and long treatment times. The old problem of low medication adherence.

The research and development of the symbiotic bacteria-gut-brain axis by the aforementioned western medicine, traditional Chinese medicine, and Ayurveda medical systems focus on diseases, drugs, and treatments. On the whole, these developments are still in their infancy, and have not yet had industrial success stories or large-scale mass production. Therefore, it is still necessary to develop new products and technologies that can be applied to the symbiotic bacteria-gut-brain axis in order to achieve the efficacy of biomedicine.

In view of the shortcomings in the conventional technology, the applicant of this case has conscientiously experimented and researched, and devoted himself to this case. He conceived this case and can overcome the shortcomings of the prior art. The following is a brief description of this case.

The purpose of this creation is to provide a Yogurt drink, including: animal milk products and multiple bacteria powders, animal milk products including water and animal milk, multiple bacteria powders and animal milk products are mixed, in which multiple bacteria powders come from the corresponding multiple species Strains, multiple strains include: Bifidobacterium longum , Lactobacillus acidophilus , Lactobacillus paracasei , and Lactobacillus rhamnosus .

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

Another purpose of this creation is to provide a method for separating the aforementioned Yogurt drink, comprising: freeze-drying the Yogurt drink to obtain a powdery product; extracting the powdery product with n-hexane to obtain a n-hexane extract and a first residue, The main component of the n-hexane extract is a short-chain fatty acid having a double bond and a hydroxyl group; 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 of Glycolipids; 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, 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 the group consisting of blood cancer cells, brain cancer cells, gastric cancer cells, colorectal cancer cells, and / or combinations thereof.

This creation discloses a fermentation machine for preparing yogurt drinks, which includes a tank body and a sealed top cover. The sealing top cover is assembled with the tank body, so that a space surrounded by the sealing top cover and the tank body is sealed. The feeding opening is arranged on the sealed top cover, and water, milk powder and bacterial powder are sent into the space through the feeding opening. The motor controller is disposed outside the tank, the stirring motor is coupled to the motor controller, and the stirring arm is disposed in the space and connected to the stirring motor for stirring water, milk powder and bacterial powder. The resistance warmer is arranged outside the tank and is coupled to the motor controller, for disinfecting water and milk powder, and fermenting the bacteria powder in a mixture of water and milk powder at 37 ° C to 43 ° C to prepare the Yogurt drinks.

In this creation, the fermentation machine also includes a discharge port at the bottom of the tank, and Yogurt drinks are sent out from the discharge port. The temperature-controlling interlayer outside the tank is connected with the resistance warmer, and the temperature-controlling interlayer is controlled by the resistance-heater to adjust or maintain the temperature of the space. The sealing top cover further includes a cleaning port, the side of the tank body includes a heat conduction flow inlet, and the bottom of the tank body includes a heat conduction flow outlet.

In this creation, the fermentation machine also includes a temperature detector coupled to the motor controller. The motor controller also includes a screen, and the temperature detected by the temperature detector is displayed on the screen. The sealed top cover also includes an observation window for the user to visually observe the conditions in the tank.

The term "animal milk products" herein generally includes water and animal milk. Animal milk can be in liquid form or dried into powder form by pasteurization. The source of animal milk can be milk, cow's milk or goat's milk. Wait.

The various bacterial strains and cancer cell lines in this article can be obtained through commercial means, and there is no need to deposit biological materials. The Yogurt drink in this article is liquid under refrigerated and room temperature environments. In this paper, the strains in Yogurt beverages showed different 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‧‧‧Youge Drink Stock Solution

14‧‧‧ powder 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‧‧‧ Fourth residue

100‧‧‧ Fermenter

101‧‧‧ tank

102‧‧‧Temperature control interlayer

103‧‧‧ resistance warmer

104‧‧‧mixing arm

105‧‧‧Sealed top cover

106‧‧‧washing mouth

107‧‧‧Temperature detector

108‧‧‧Motor controller

109‧‧‧Screen

110‧‧‧mixing motor

M‧‧‧feeding port

N4‧‧‧ discharge port

N5‧‧‧Heat conduction inlet

N6‧‧‧ Thermal Outlet

S1‧‧‧Viewing window

Figure 1 shows the 16S rDNA sequencing result and NCBI comparison result of strain code LR-36, which is Lactobacillus rhamnosus .

Figure 2 shows the 16S rDNA sequencing result and NCBI comparison result 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. It does not have enzymes, oxidases, and motility, and does not produce endospores. It will grow in both aerobic and anaerobic environments.

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

Figure 4 (A) is a photomicrograph of the strain code UY-76. Gram-positive bacilli Enzymes, oxidases, and motility do not produce endospores, and they grow in both aerobic and anaerobic environments.

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

FIG. 5 is a flowchart of the separation method 3.

FIG. 6 (A) is a ¹ H-NMR spectrum of 11 n-hexane extract (11-H) of Yogurt Beverage.

Figure 6 (B) is a ¹ H-NMR spectrum of 11 ethyl acetate extract (11-E) of Yogurt Beverage.

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

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

Fig. 7 (A) is a ¹ H-NMR spectrum of the 12 n-hexane extract (12-H) of Yogurt Beverage.

Figure 7 (B) is a ¹ H-NMR spectrum of the 12 ethyl acetate extract (12-E) of Yogurt Beverage.

Figure 7 (C) is the ¹ H-NMR spectrum of the 12 ethanol extract (12-A) of Yogurt Beverage.

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

FIG. 8 (A) is a ¹ H-NMR spectrum of 13 n-hexane extract (13-H) of Yogurt Beverage.

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

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

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

FIG. 9 (A) is a ¹ H-NMR spectrum of the 14 n-hexane extract (14-H) of Yogurt Beverage.

Figure 9 (B) is a ¹ H-NMR spectrum of the 14 ethyl acetate extract (14-E) of Yogurt Beverage.

FIG. 9 (C) is a ¹ H-NMR spectrum of Yogurt 14 ethanol extract (14-A).

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

Figure 10 is a histogram of the total polyphenol content of each extract obtained by separation method 3 of Yogurt drinks 11 to 14.

Figure 11 is a histogram of the total flavanone flavonoid content of each extract obtained by separation method 3 of Yogurt drinks 11 to 14.

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

FIG. 13 is an HPLC spectrum of each extract at 203 nm obtained by separation method 3 of Yogurt Drink 11.

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

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

FIG. 16 is an HPLC spectrum of each extract at 203 nm obtained by separation method 3 of Yogurt Beverage 14.

Figure 17 is a histogram of DPPH free radical scavenging rate of each extract obtained by separation method 3 of Yogurt beverages 11 to 14.

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

FIG. 19 is a histogram of the reducing power of each extract obtained by separation method 3 of Yogurt drinks 11 to 14.

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

FIG. 21 is a histogram of the survival inhibition rate of brain cancer cells of each extract obtained by separation method 3 of Yogurt Drinks 11 to 14.

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

FIG. 23 is a histogram of the survival inhibition rate of colorectal cancer cells of each extract obtained by separation method 3 of Yogurt drinks 11 to 14.

FIG. 24 is a histogram of the survival inhibition rate of prostate cancer cells of each extract obtained by separation method 3 of Yogurt Drinks 11 to 14.

Figure 25 is a flowchart of Yogurt beverage production system.

Picture 26: Yogurt Beverage Fermentor with Antioxidant and Inhibition of Digestive Tract Cancer Cell Growth The schematic.

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

In this creation, the active metabolites of Yogurt are separated by active guidance, and the active ingredients are determined and prepared into beverages or medicaments suitable for oral administration, while still maintaining the efficacy of the ingredients.

Experiment 1. Isolation and identification of strains:

Take 1ml of raw cow's milk as a sample, use Lactobacilli MRS Broth as a dilution solution to make a 10-fold serial dilution (concentration multiples of 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 ), respectively 200 μl of the dilution was dropped on sterile MRS agar culture medium, and then plated. The petri dish was cultured in an anaerobic incubator at 37 ° C for 24 hours. After that, single colonies of different types, colors, and sizes were selected aseptically and continued to be cultured, and colonies were repeatedly selected and cultured to isolate strains, and it was determined that the colonies on the Petri dish were all of the same shape, color, and size. Finally, the isolated single strain was stored and identified. The identification of Eugenic species was based on the observation of a single colony on the petri dish with the naked eye, and then two identical colonies were selected for Gram staining to determine that they were Gram-positive or negative. It is preferred to observe the colonies at 4 and 40 times multiples with a phase difference microscope. A single colony was selected for DNA extraction and 16S PCR test. The sequences were compared with the NCBI database, and the results were identified.

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

After isolation and identification, a total of 20 strains of strains were obtained.

Experiment 2: Fermentation of Yogurt:

The design model of the fermentation preparation experiment of the present invention is based on the strain species type and ratio, fermentation temperature, and fermentation time as manipulation variables. After fermentation, viscosity and pH value are used as quality control standards. The experimental group with Yogurt viscosity below 100 centipoise (cP) and pH value above 5.0 was judged as unfermented or incompletely fermented, and the fermentation time was too long (more than 12 hours) due to poor production efficiency. The preparation method of Experiment 2 is: 250ml of 75 ° C hot water is added to 36g milk powder for high temperature sterilization, and becomes Animal milk products. Allow it to cool to 35-43 ° C and add 0.3 g of bacteria powder. Then put them in different temperature (37 ° C, 40 ° C, 43 ° C) incubator for different time (8 hours, 10 hours, 12 hours), then measure the viscosity (Brookfield DVE RV viscometer) and pH value (Milwaukee pH600 pH meter) ). Table 4 shows the strains contained in the 14 successfully fermented strain combinations.

It was found that the effect of different milk powders on the success of fermentation was not significant; the weight ratio between species of bacteria had little effect on the fermentation results. Therefore, the weight ratio of (bacterial powder) between the strains of each experimental group is 1: 1, and the more strains, the more likely it is to successfully ferment, that is, the more strains, the more successful the fermentation. The higher the experimental reproducibility. Next, 3 replicate experiments were performed with 4 groups with the highest reproducibility of successful fermentation (Table 4 strain combinations 11 to 14) to confirm the optimal fermentation conditions for different strain combinations (that is, the highest viscosity and The lowest pH value), when the Yogurt viscosity is higher than 100 cp and the pH value is lower than 5.0, it is judged that the fermentation is complete. The experimental results are shown in Table 5.

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

Bacterial combination 11: The fermentation temperature is fixed at 37 ° C, and the fermentation time needs 12 hours to complete the fermentation; the fermentation temperature is fixed at 40 ° C, the fermentation time is 10 hours to complete the fermentation, and the fermentation time 8 or 12 hours cannot be completely fermented; the fermentation temperature is fixed to Fermentation can be completed at 43 ° C for 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 cannot be completed at 37, 40, and 43 ° C; the fermentation time is fixed at 10 hours, and the fermentation temperature can be completed at 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. The fermentation temperature is 37 and 43 ° C.

Bacterial combination 12: The fermentation temperature is fixed at 37 ° C, and the fermentation time can be completed in 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 ° C, and the fermentation time is 8, 10, 12 All fermentation can be completed in hours, the longer the fermentation time, the higher the viscosity, the lower the pH value; the fermentation temperature is fixed at 43 ° C, and the fermentation time can be completed in 8, 10, 12 hours. The longer the fermentation time, the lower the viscosity, the higher the pH The lower the value. The fermentation time is fixed at 8 hours, and the fermentation temperature is 40, 43 ° C. It can be completely fermented. 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 is 37, 40, 43 ° C. The higher the fermentation temperature, the higher the viscosity, the lower the pH value, and then the higher value. The fermentation time is fixed at 12 hours. The fermentation temperature can be 37, 40, and 43 ° C. The higher the fermentation temperature, the higher the viscosity and then the lower the pH value. Low after high.

Bacterial combination 13: The fermentation temperature is fixed at 37 ° C, and the fermentation time can be completely completed 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 ° C and the fermentation time is 8 and 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 ° C. The fermentation time can be completed in 8, 10, and 12 hours. The longer the fermentation time, the lower the viscosity and then the higher the viscosity. , The lower the pH value. The fermentation time is fixed at 8 hours. 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 value. Set to 10 hours, fermentation temperature can be complete at 37, 40, and 43 ° C. The higher the fermentation temperature, the higher the viscosity, the lower the pH value, and the lower the pH value. The fermentation time is fixed at 12 hours, and the fermentation temperature is 37, 40, and 43 ° C. All fermentation can be completed, the higher the fermentation temperature, the lower the viscosity, then the higher, the lower the pH value and then the same.

Bacterial combination 14: The fermentation temperature is fixed at 37 ° C, and the fermentation time can be completely completed 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 ° C and the fermentation time is 8 and 10 It can complete fermentation 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. The fermentation time can be completed in 8, 10, and 12 hours. The longer the fermentation time, the higher the viscosity and 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 ° C. The higher the fermentation temperature, the lower the viscosity and then the higher the pH value; the fermentation time is fixed at 10 hours and the fermentation temperature is 37, 40, 43 ° C. All fermentation can be completed, the higher the fermentation temperature, the higher the viscosity, the lower the pH value, and then the same; the fermentation time is fixed at 12 hours, and the fermentation temperature is 37, 40, and 43 ° C. After low, the pH value is low and then high.

Experiment 3. Sensory Evaluation of Yogurt Drinks:

Yogurt has a variety of effects on maintaining good 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 taking Yogurt containing Lactobacillus acidophilus; Consuming 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 sufficient amount of yogurt can achieve the effect of maintaining physical health.

In order to achieve the above-mentioned objectives, the present invention fermentes the strain combinations 1 to 14 to prepare Yogurt drinks 1 to 14 and performs sensory evaluation analysis and screening. Sensory products are evaluated and developed by the American Institute of Food Science and Technology. They use the three senses of sight, smell and taste to measure the characteristics of Yogurt beverages. The experimental design of sensory evaluation includes: general analysis of sensory analysis methods (ISO 6658: 2005), word vocabulary of sensory evaluation (ISO 5492: 2008), color sensory test (visual colorimetry: ISO 11037: 1999), texture sensory test (texture profile: ISO 11036: 1994), flavor sensory test (flavor profile: ISO 6564: 1985), descriptive analysis (qualitative description of sensory characteristics (Description: ISO 11035: 1994, Evaluation of sensory characteristic strength: ISO 4121: 2003). The experimental method uses descriptive analysis, ranking method and hedonic scale for evaluation.

1. Evaluation of Yogurt beverages 1 to 14 by descriptive analysis:

This is a gathering of 11 highly sensory and sensitive judges with fermented product development, fermented dairy product development, yogurt product development, and hands-on beverage store practical experience, including 5 males and 6 females. Appraisal, judging the differences in appearance, texture and flavor among Yogurt beverages, and finally based on visual (color, cleanliness), smell (milk, aroma, unique smell), taste (acidity, sweetness, smoothness, Alcohol thickness), and overall preferences (flavor performance, aftertaste) describe the different traits, using a five-point scale of preference (1 point: very dislike, 2 points: dislike, 3 points: dislike and hate, 4 points : Like, 5 points: like very much). The experimental results are described in Table 6.

2. Evaluation of Yogurt Drinks 1 to 14 using the scoring method:

The number and gender of the reviewers are as described above. The reviewers divide Yogurt beverages 1 to 14 into 3 levels according to their preferences (high, medium, and low preferences), and each level is based on the highest and lowest preferences from left to left. To the right. The results are shown in Table 7.

3. Evaluation of Yogurt Drinks 11 to 14 by preference score method:

The scoring method is used to rank 11 and 14 of Yogurt beverages with high preference level, and then confirm whether there is a significant difference with the preference scoring method, and understand the relationship between the degree of preference and product characteristics. 1 point: very dislike, 2 points: dislike, 3 points: dislike and hate, 4 points: like, 5 points: very like). It also records the aroma, sweetness, and sourness on a five-point scale (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 more good.

The first consumer questionnaire survey was held in a department store shopping street. This was conducted without being disturbed by the external environment and without mutual influence. The evaluation target was 67 untrained consumers, including 24 males (35.8%) and 43 females (64.2%). The age distribution is: 25 people (37.3%) under 20 years old, 21 people (31.3%) 20-30 years old, 13 people (19.4%) 30-39 years old, 3 people (4.5%) 40-49 years old, 50- Four were 59 years old (6%), and one was over 60 years old (1.5%). The top three occupations are: 30 students (44.8%), 17 in the service industry (25.4%), 5 in military education (7.4%), and the remaining 15 (22.4%).

The second consumer questionnaire survey was held in the university school district, and this was also conducted without interference from the external environment and without mutual influence. The evaluation target is a total of 69 untrained consumers. There were 31 males (44.9%) and 38 females (55.1%). The age distribution was: 50 people (72.5%) between 18-22 years old, 15 people (21.8%) between 23-30 years old, and 4 people (5.8%) under 18 years old. The occupation distribution is all students (69, 100%).

Based on the results of the first and second consumer questionnaires, the total number of participants was 136. Please refer to Table 8. The overall preference is Yogurt Drink 14 with a score of more than 4 likes, followed by Yogurt Drink 12; Yogurt Drinks 11 and 13 have similar scores, and those who have scores greater than 3 also dislike. it's OK. In addition, Yogurt Drinks 11, 12, 13 and 14 were rated by all 136 consumers as their favorite (first place). The number of Yogurt Drinks was 32, 38, 31 and 35 respectively.

In the pure natural process without any seasoning, the aroma difference of Yogurt beverages 11 to 14 is not much, and they are slightly lighter; the sweetness of Yogurt beverages 11 to 14 is moderate; the sourness of Yogurt beverages 11 to 14 The difference is not large, and all are slightly faint (as shown in the results in Table 9).

Experiment 4. Sensory evaluation of ingredients for yogurt

In order to increase the variety of Yogurt beverages, increase the daily Yogurt intake of the user (for example, 200g in 30 minutes), and not greasy after long-term drinking, Experiment 4 added Yogurt 14 as the base. Shake the beverage formula and evaluate with descriptive analysis. Experiment 4 convened 5 high-sensory and sensitive judges with fermented product development, fermented dairy product development, Youge product development, and hand-operated beverage store practical experience, including 3 males and 2 females. Discuss the way to discuss and evaluate.

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

Add sesame sauce as the sauce: After the sesame sauce is boiled, add Yogurt to break and grind. The sesame sauce is rich in flavor and contains oil. After the two are compatible, the flavor contains oily taste, which makes the overall flavor produce a salty taste. not tall. Gherkin is added as an ingredient: diced cucumber and yoghurt are crushed and ground together. The flavor of the cucumber masks the yoghurt flavor. The overall vegetable flavor is obvious and lacks the unique flavor of yoghurt. Add corn as an ingredient: Add the unflavored canned corn kernels to Yogurt and crush and grind them together. The corn is slightly sweet and can improve the overall sweetness, but the flavor of corn and Yogurt are combined into a special flavor with low acceptance. . Sensory evaluation results: The formula design of Yogurt drinks is not suitable for salty and sweet flavors.

2. The formula design of Yogurt sauce:

The Yogurt beverage of the present invention has a pure and natural manufacturing process without any seasoning. The overall flavor is moderately sweet, and the aroma and sour taste are slightly lighter, which belongs to an elegant and not greasy mouth shape. Different sauce recipes bring different sensory sensations. Add brown sugar as sauce: add brown sugar boiled into syrup to Yogurt and crush and grind. The brown sugar has a strong and special flavor, which is compatible with the sweet and sour flavor of Yogurt. Add matcha tea as a sauce: Add matcha powder imported from Japan to Yogurt and crush and grind it. The matcha tea is slightly bitter, which is not compatible with Yogurt's sweet and sour flavor. Add honey to the juice: Add honey directly to Yogurt and crush and grind it. The unique fragrance of honey can highlight the sweet and sour flavor of Yogurt.

3. Formula design of solid ingredients for yogurt

Yogurt drinks are non-Newtonian fluids (non-Newtonian fluids are fluids with a non-constant value at a certain temperature and pressure). Their viscosity will change due to the pressure or speed. The greater the pressure, the higher the viscosity. Increase or even become temporary solid. Adding solid ingredients to Yogurt beverages will cause difficulties in drinking and smoking. In order to overcome this problem, 200g Yogurt beverages are accommodated in cylindrical cups (diameter of the upper circle is 9 cm, diameter of the lower circle is 5.6 cm, and cup height is 13.5 cm). The straws can easily suck the solid ingredients in Yogurt beverages, and the phenomenon of solid ingredients remaining after drinking the beverage will not occur. In the preferred embodiment, At room temperature, the temperature increase of Yogurt beverages will affect the viscosity and thus the difficulty of sucking solid ingredients with a straw. Therefore, add crushed ice to the upper layer of yogurt to keep cold. Good), the volume ratio of crushed ice layer and Yogurt drink is 1: 3 ~ 1: 5, so that Yogurt drink is maintained between 8 ~ 12 ℃ within 30 minutes, and the sensory evaluation results of solid ingredients are reproducible. The evaluation focus of the solid ingredient sensory evaluation is: smoothness of the taste and overall balance; further including visual stimulus, which enhances the user's desire to watch and drink Yogurt beverages (promote appetite, enhance active intake). The appearance of Yogurt drinks can show the effects of layering, gradation and special texture. The added solid ingredients include, but are not limited to, pearl, coconut fruit, coffee jelly, vermicelli, taro, pudding, frozen grass jelly, bean curd, mung bean, red bean, barley kernel, purple rice, oatmeal. The results of sensory evaluation are shown in Table 10. Described. 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 Beverage Extract:

The types and contents of active metabolites in Yogurt beverages vary depending on the strain, formula, and fermentation environment. In order to determine the active ingredients of the Yogurt beverage of the present invention, Yogurt beverages 11 to 14 with high sensory appraisal preferences are selected for extraction and partition extraction, and the obtained divided layers are further subjected to component analysis and activity testing.

1. Separation method 1:

The yogurt beverage stock solution was concentrated under heating and reduced pressure to dry, to obtain a paste. again The paste was continuously extracted three times with ethanol to obtain an ethanol extract. The ethanol extract was filtered under vacuum, 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 performed. If dichloromethane: water (1: 1 (v / v)) is used for partition extraction of the crude extract, there is still a problem of too many emulsified layers.

2. Separation method 2:

The yogurt liquid was not dried, but diluted with water (1: 1 (v / v)), and then added with ethyl acetate (1: 1 (v / v)) for partition extraction. A large amount of emulsified layer was still produced, 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 many emulsified layers.

3. Separation method 3:

The yogurt liquid is freeze-dried to obtain a powdery product, and the powdery product is sequentially extracted with a solvent of increasing polarity. It is found that the extraction rate of dichloromethane is too low, and there is no significant difference between the extraction effect of n-hexane. The solvent residue problem after n-butanol extraction is difficult to continue the subsequent solvent extraction. Therefore, neither dichloromethane nor n-butanol is suitable as Extraction solvent for this separation method.

As shown in the separation method 3 shown in FIG. 5, after a solvent array combination extraction test, finally extraction is performed sequentially with n-hexane, ethyl acetate, ethanol, and water. That is, the Yogurt drink stock solution 12 is freeze-dried to obtain a powdery product 14, and the powdery 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. Next, the first residue 18 was extracted with ethyl acetate to obtain an ethyl acetate extract 20 (sample code: 11-E, 12-E, 13-E, 14-E) and a second residue 22. Thereafter, 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 a water extract 28 (sample code: 11-W, 12-W, 13-W, 14-W) and a fourth residue 30. The extract and residue were each concentrated by heating under reduced pressure. The experimental results of various extracts of Yogurt beverages 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. Various active ingredients of Yogurt were separated, concentrated and collected, and the differences in appearance properties and flavor of various extracts were clearly confirmed by sight and smell.

Experiment 6. Composition analysis of Yogurt beverage extract:

1. Nuclear magnetic resonance spectroscopy (NMR) analysis:

Yogurt beverages 11 to 14 are obtained by separation method 3 to obtain 16 extracts. These extracts are subjected to NMR analysis. The hydrogen isotopes (deuterium) solvents are: CDCl ₃ , acetone-d6, MeOH-d4, C ₅ D ₅ N, DMSO-d6 and D ₂ O tested the solubility of the extracts. As a result, it was found that C ₅ D ₅ N and DMSO-d6 had better solubility for 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 concentration of the extract was 25 mg / ml, 50 mg / ml, 75 mg / ml and 100mg / ml. 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) The ¹ H-NMR spectrum at 50 mg / ml showed the best magnetic field and characteristic signals. Water extract (11-W, 12-W , 13-W, 14-W) The optimal sample concentration is 25 mg / ml. N-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 testing, 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 performed with a nuclear magnetic resonance spectrometer JEOL ECS 400MHz FT-NMR. The resolution is 400MHz, the chemical shift is expressed in ppm (δ), and the coupling constants are expressed in Hertz ( J ). As shown in the ¹ H-NMR spectra of the 16 extracts in FIGS. 6 (A) to 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 beverages 11 to 14 are also different.

2. Analysis of total polyphenol content:

Yogurt beverages 11 to 14 are separated into 16 extracts by separation method 3. These extracts are analyzed by the Folin-Ciocalteu colorimetric method. The principle is the phosphotungstic acid and phosphomolybdic acid in the Folin-Ciocalteu reagent (phosphomolybdic acid) is colored by oxidation of polyphenol molecules, and gallic acid is used as a standard. The experimental method is: After dissolving gallic acid (5mg / mL) in methanol, make 50, 100, 250, and 500 μg / mL standards, take 20 μL of each standard to replenish the volume to 1.6 mL with secondary water, and then add 100 μL 2N Folin-Ciocalteu reagent, 300 μL of 20% Na ₂ CO ₃ was added and mixed, and the mixture was reacted at 40 ° C. for 40 minutes, and the absorbance was measured at 725 nm. In addition, the standard product was replaced by secondary water and zeroed. Draw a standard curve based on the relationship between concentration and absorbance. 20 μL of the test sample (1 mg / mL) was treated 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 content of 11-E was highest in the extract of Yogurt Drink 11 and the lowest in 11-W; the content of 12-A was highest in the extract of Yogurt Drink 12, 12-W is the lowest; 13-H content is highest in the extract of Yogurt Drink 13, 13-W is the lowest; 14-E content is highest in the Yogurt Drink 14 extract and 14-W is the lowest. Based on 16 extracts, the total polyphenol content of 14-E was the highest and 11-W was the lowest. In the 4 n-hexane extracts (11-H to 14-H), the total polyphenol content ranged from 18.2 to 24.0 mg _{Gallic acid} / g. In the four ethyl acetate extracts (11-E to 14-E), the total polyphenol content ranged from 9.4 to 40.6 mg _{Gallic acid} / g. In the four ethanol extracts (11-A to 14-A), the total polyphenol content ranged from 11.3 to 28.3 mg _{Gallic acic} / g. In the four water extracts (11-W to 14-W), the total polyphenol content ranged from 2.6 to 7.1 mg of _{Gallic acid} / g.

3. Analysis of total flavanone flavonoid content:

The principle is that 2,4-dinitrophenylhydrazine (DNP) will derivatize the aldehyde group or keto group on the flavanone structure to make it have light absorption properties at a specific wavelength. Hesperetin is used as a standard. The experimental method is as follows: 1 mL of the sample to be tested is added to 2 mL of DNP solution and 2 mL of methanol, and the solution is heated in a 50 ° C water bath for 50 minutes. After cooling, 5 mL of KOH methanol solution was added and mixed, and the mixture was allowed to stand at room temperature for 2 minutes. Then, 1 mL of the mixed solution was charged into a centrifuge bottle containing 5 mL of methanol, and centrifuged 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 was compared with the standard curve of the standard and quantified, and converted into the flavanone flavonoid content per gram of extract. As shown in Table 14 and Figure 11, the total flavanone flavonoid measurement results showed that the content of 11-A was highest in the extract of Yogurt Drink 11, and the content of 11-E was lowest; the content of 12-A in the extract of Yogurt Drink 12 The highest, 12-H; the extract of Yogurt 13, the highest content of 13-A, the lowest of 13-H; the extract of Yogurt 14, the highest content of 14-A, the lowest of 14-H. Based on 16 extracts, the total flavanone flavonoid content of 12-A was the highest, and 12-H was the lowest. In the 4 n-hexane extracts (11-H to 14-H), the total flavanone flavonoid content ranged from 15.0 to 23.1 mg _Hesperetin / g. The content of total flavanone flavonoids in the 4 ethyl acetate extracts (11-E to 14-E) ranged from 16.0 to 26.0 _{mg Hesperetin} / g. In the four ethanol extracts (11-A to 14-A), the total flavanone flavonoid content ranged from 62.5 to 91.3 _{mg Hesperetin} / g. In the four water extracts (11-W to 14-W), the total flavanone flavonoid content ranged from 28.2 to _{49.5 mg Hesperetin} / g.

4- Total polysaccharide content analysis:

This analysis is performed by the phenol-sulfuric acid assay. The principle is that five-carbon sugars and six-carbon sugars are decomposed into furfural due to dehydration under acidic and high temperature conditions. Furfural is further reacted with phenol An orange-yellow product is formed by the reaction, and the total polysaccharide content is converted by changing the absorbance value, and 0, 10, 20, 30, 40, 50 μg / ml glucose is used as a standard. The experimental method is as follows: 0.5 mL of a 5% phenol solution is added to 0.5 mL of a 10 mg / mL test substance, and then 2.5 mL of concentrated sulfuric acid is added, and the mixture is uniformly reacted in a water bath at 100 ° C for 20 minutes. After the temperature is cooled, the absorbance is measured at a wavelength of 490 nm. As shown in Table 14 and Figure 12, the total polysaccharides in the extract of Yogurt 11 have the highest 11-W content and the lowest of 11-H; the extract of Yogurt 12 has the highest 12-W content. 12-E is the lowest; 13-W content is highest in the extract of Yogurt Drink 13, 13-H is the lowest; 14-W content is highest in the extract of Yogurt Drink 14, and 14-H is the lowest. Comprehensive 16 extracts, the total polysaccharide content of 14-W is the highest, and 14-H is 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 ethanol 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 commonly used in Taiwan Chinese Pharmacopoeia (Second Edition) for analysis. The solvent system is ionic solvent system (n-hexane, dichloromethane, chloroform, methanol) or non-ionic solvent system ( Tests were performed with n-hexane, toluene, ethyl acetate, acetone, methanol), and addition of acid (glacial acetic acid (GAA), formic acid (FA)) or addition of base (10% ammonia, 25% ammonia, diethylamine). 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 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) did not have satisfactory results in normal-phase thin-layer chromatography. The main spots of TLC did not exist at R _f 0.3- 0.8.

Table 15.Normal Phase Thin Layer Chromatographic Analysis of Extracts from Yogurt Drinks 11 to 14 via Separation Method 3

6. High performance liquid chromatography (HPLC) analysis:

The analysis was performed with reference to the reversed-phase high-performance liquid chromatography commonly used in Taiwan ’s Chinese Pharmacopoeia (Second Edition). The autosampler is G1329A autosampler, the chromatography column is Intersil ODS-3V 250 x 4.6mm (5μm), the solvent A of the mobile phase is acetonitrile, the 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% to 20 minutes, 0% A to 10% A, 20 to 40 minutes, 10% A to 40% A, and 40 to 60 minutes, 40% A to 100%. A, 100% for 60 to 70 minutes, flow rate and column temperature are as described above. As shown in Table 16 and Figures 13 to 16, among the five detection wavelengths, n-hexane extract (11-H to 14-H) and ethyl acetate extract (11-E to 14-E) are all No significant chromatographic peaks were observed. The ethanol extracts (11-A to 14-A) and water extracts (11-W to 14-W) exhibited better chromatographic peaks only at 203 nm.

Experiment 7. Antioxidant activity test of Yogurt beverage extract:

1. Determination of 1,1-diphenyl-2-trinitrophenylhydrazine (2,2-diphenyl-1-pricrylhydrazyl, DPPH) free radical capacity:

Lipids produce free radicals during the process of self-oxidation and cause rancidity of lipids. Antioxidants scavenge lipid peroxide free radicals by supplying hydrogen, thereby inhibiting the oxidative chain reaction. DPPH is often used in antioxidant research to evaluate the hydrogen supply capacity of antioxidants. DPPH methanol solution has strong light absorption at 517nm, and the absorption value decreases when it is reduced by the antioxidant. Therefore, the lower the absorbance at 517 nm, the stronger the hydrogen supply ability of the antioxidant. The experimental method is as follows: 4 mL of extract sample (50 mg of extract in 15 mL of 50% ethanol solution) is added to 1 mL of freshly prepared 10 mM DPPH methanol solution, shaken and mixed, and then placed at room temperature for 30 minutes, and the absorbance at 517 nm is measured. . The result is expressed as a percentage of removal, and the higher the percentage of removal, 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 ability to scavenge DPPH free radicals in 16 extracts.

2. Total antioxidant capacity (TEAC) determination:

2,2'-Azinobis- (3-ethylbenzthiazoline-6-sulphonate) (ABTS) produces an oxidation reaction under the catalysis of hydrogen peroxide and peroxidase to form a stable blue-green water-soluble ABTS˙ ⁺ cationic radical. A UV-Vis photometer was used, which had a strong absorbance at 620 nm, and a standard curve was prepared against a water-soluble vitamin E analog (trolox) of the standard. When ABTS˙ ⁺ cationic radicals and antioxidants are reduced, the solution color will become lighter and the absorbance value will be reduced, so that the ability to remove ABTS˙ ⁺ cationic radicals will be converted. The better the scavenging ability, the stronger the antioxidant hydrogen supplying ability. The experimental method is as follows: the prepared ABTS is placed in a refrigerator for 1 hour, so that it produces a stable blue-green ABTS˙ ⁺ radical aqueous solution. 80 μL of the extract sample (50 mg of the extract in 15 mL of a 50% ethanol solution) was added to a 96-well plate, and 120 μL of ABTS (R) ⁺ free radical aqueous solution was added, and the total volume was 200 μL. Shock at room temperature for 10 minutes at room temperature, and measure the absorbance at 620 nm. The lower the absorbance value, the better the effect of scavenging ABTS˙ ⁺ free radicals. The clearance rate is calculated as a percentage. As shown in Table 17 and Figure 18, the results of total antioxidant capacity showed that among the extracts of Yogurt 11, 11-A had the best ability and 11-E was the weakest; among the extracts of Yogurt 12, 12-A The best ability, 12-H is the weakest; among the extracts of Yogurt 13, 13-A has the best ability, 13-H is the weakest; among the extracts of Yogurt 14, 14-A has the best ability, 14- H is the weakest. Among the 16 extracts, 12-A had the best total antioxidant capacity, and 12-H was the weakest.

3. Determination of reducing power:

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

Experiment 8: Yogurt Beverage Extract Inhibits Cancer Cell Growth Activity Test:

For related organs of the commensal bacteria-gut-brain axis, the active metabolites start from oral intake and 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 line) were tested for the activity of inhibiting the growth of cancer cells in 16 extracts obtained from Yogurt Drinks 11 to 14 after separation method 3. The experimental method is cell survival analysis (MTT assay). Various cancer cells are implanted into 96-well disks, and the cell density of each well is 7 × 10 ³ cells / 100 μL. DMSO was used as the control group) and placed in a 37 ° C, 5% CO ₂ cell incubator for 72 hours. An additional 50 μL of MTT was added and incubated for 1 hour. Centrifuge at 2000 rpm for 3 minutes, remove the supernatant and add 200 μL DMSO, shake on a shaker until the purple crystals are completely dissolved, and read the absorbance 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 radiation therapy (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. Inhibiting 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, 11-A had the best inhibitory activity, and 11-W had the weakest extract. Among the extracts of Yogurt Drink 12, 12- A had the best inhibitory activity, 12-W was the weakest; among the extracts of Yogurt 13, 13-W had the best inhibitory activity, 13-H was the weakest; among the extracts of Yogurt 14, 14-A The inhibitory activity was the best, and 14-W was the weakest. Among 16 extracts, 12-A had the best inhibitory activity against cancer cell growth. 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 had the best inhibitory activity, and 11-E and 11-W had no inhibitory activity. Among the extracts, 12-A had the best inhibitory activity, and 12-H and 12-W had no inhibitory activity. Among the extracts of Yogurt 13, 13-A had the best inhibitory activity, and 13-E was the weakest; Yogurt Among the extracts of drink 14, 14-E had the best inhibitory activity, and 14-A and 14-W had no inhibitory activity. Among 16 extracts, 14-E had the best inhibitory activity.

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

As shown in Figure 21, in inhibiting the growth of brain cancer cell GBM8401, among the extracts of Yogurt 11, 11-A had the best inhibitory activity, and 11-E was the weakest. Among the extracts of Yogurt 12, 12 -A had the best inhibitory activity, 12-W was the weakest; among the extracts of Yogurt 13, 13-H had the best inhibitory activity, 13-W was the weakest; among the extracts of Yogurt 14, 14-A The best inhibitory activity was 14-H. Among the 16 extracts, 11-A had the best inhibitory activity. As shown in Figure 21, in terms of inhibiting the growth of cells U87MG, 11-A had the best inhibitory activity and 11-W was the weakest in the extract of Yogurt 11; 12-H in the extract of Yogurt 12 The best inhibitory activity is 12-W, and the weakest is 12-W. Among the extracts of Yogurt 13, 13-H has the best inhibitory activity, and neither 13-E, 13-W has inhibitory activity. 14-W had the best inhibitory activity, and 14-E had the weakest. Among 16 extracts, 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 suppressing the growth of gastric cancer cell KATO III, Among the extracts of Pin 11, 11-H had the best inhibitory activity, and 11-W was the weakest. Among the extracts of Yogurt 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 has inhibitory activity; among the extracts of Yogurt Drink 14, 14-W has the best inhibitory activity and 14-A has the weakest. Among 16 extracts, 14-W had the best inhibitory activity. As shown in Figure 22, in inhibiting the growth of gastric cancer cells SNU-1, the extract of Yogurt 11 has the best inhibitory activity, and 11-A is the weakest. Among the extracts of Yogurt 12, 12-E had the best inhibitory activity, 12-W was the weakest; among the extracts of Yogurt 13, 13-H had the best inhibitory activity, 13-A was the weakest; among the extracts of Yogurt 14, 14- A had the best inhibitory activity, and 14-E was the weakest. Among 16 extracts, 12-E had the best inhibitory activity.

4. Inhibition of colorectal cancer cell (DLD-1 cell line) growth activity:

As shown in Figure 23, in 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 was the weakest; among the extracts of Yogurt Drink 12 , 12-H has the best inhibitory activity, 12-E is the weakest; among the extracts of Yogurt 13, 13-H has the best inhibitory activity, 13-W is the weakest; -A had the best inhibitory activity, and 14-E had the weakest. Among the 16 extracts, 11-A had the best inhibitory activity.

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

As shown in Figure 24, in inhibiting the growth of prostate cancer cells LN-cap, among the extracts of Yogurt 11, 11-A had the best inhibitory activity, and 11-E was the weakest; among the extracts of Yogurt 12 , 12-H had the best inhibitory activity, and 12-E was 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. In the extract, 14-W had the best inhibitory activity, and 14-H had the weakest. Among the 16 extracts, 11-A had the best inhibitory activity. As shown in Figure 24, in inhibiting the growth of prostate cancer cells PC-3, 11-E had the best inhibitory activity and 11-H was the weakest among the extracts of Yogurt Drink 11; among the extracts of Yogurt Drink 12 , 12-H had the best inhibitory activity, and 12-W was 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, 14-E had the best inhibitory activity, and 14-A had the weakest. Among 16 extracts, 13-E had 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 tonnage factories. The family-type small-scale production has the problems of unstable quality, subsequent modulation and inconvenient operation. The mass production of ton-scale factories will face 2 to 3 days or more when consumers buy and drink beverages. Freshly fermented Yogurt beverages cannot be consumed on the same day, and often they cannot be consumed within the storage period, or are stored improperly. The problem. Therefore, the present invention provides a production system for liquid yogurt, which can simultaneously solve the above-mentioned problems of home-made small-scale production and mass production of yogurt in tonnage factories. A single fermentation machine can produce kilogram liquid yogurt. It can be fermented on-site in a transparent process and completed in 18 hours. The Yogurt drinks that consumers consume every day are freshly fermented on the day, and the end products are eaten by hand.

As shown in the process shown in FIG. 25 and the structure of the fermenter 100 shown in FIG. 26, the sequence of the liquid yogurt beverage production system of the present invention includes: high-pressure cleaning, high temperature sterilization, milk emulsification, secondary sterilization, and inoculation. Fermentation, finished product sampling, finished product collection, cooling and refrigerating, homogeneous preparation, adding ingredients, filling and packaging, and product meals.

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

Further, the fermenter 100 further includes a discharge port N4 provided at the bottom of the tank 101 and for discharging the Yogurt beverage or waste liquid (or waste water). Alternatively, the fermenter 100 further includes a temperature-controlling interlayer 102 disposed outside the tank 101 and connected to the resistance warmer 103, and the temperature-controlling interlayer 102 is used to adjust or maintain the space in the space under the control of the resistance warmer 103. temperature. Alternatively, seal the top cover 105 further includes a cleaning port 106, and the fermenter 100 further includes a heat conduction inlet N5 and a heat conduction outlet N6 at the bottom of the tank 101. Alternatively, the fermentation machine 100 further includes a temperature detector 107 coupled to the motor controller 108, and the temperature detected 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 condition in the groove body 101.

The process of 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 that the water level is in an appropriate position through the observation window S1 → open the discharge port N4 to discharge → the tank 101 is full again Water → The temperature detected by the temperature detector 107 is displayed on the screen 109 of the motor controller 108 → The display 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 Shows).

(2) Milk-making emulsification: Open the feeding port M → inject clean water → add milk powder → turn on the stirring motor 110 → perform emulsification → close the feeding port M → confirm the emulsification situation through the observation window S1.

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

(4) Bacteria fermentation: Inject cooling water from the heat conduction inlet N5 → turn on the circulation pump (not shown) → use the cooling water for heat exchange temperature control → observe the screen 109 to gradually reduce the temperature to 43 ° C → close the cooling water source and circulation Pump → Feeding port M is sterilized with 75% alcohol → Quickly open feeding port M to add bacteria and turn off and tighten → Turn on the stirring motor 110 to stir the stirring arm 104 for 20 minutes → Turn off the stirring motor 110 → Allow to stand for fermentation.

(5) Finished product sampling: spray the outlet N4 with pure water and spray with 75% alcohol to sterilize → take out a 300 ml sample → check the viscosity and pH value → the viscosity and pH value reach the tank collection standard → perform tank collection.

(6) Finishing tank: spray the outlet N4 with pure water → spray with 75% alcohol for disinfection → use 304 food grade stainless steel containers for batch receiving.

(7) Cooling and refrigerating: Youge drink volume preparation → 4 ℃ refrigerating.

(8) Homogenize and add ingredients: take Yogurt drink → add ice cube → use homogenizer for homogenization Quality → solid ingredients or solid fruit ingredients.

(9) Filling and packaging, product delivery: Finally, check whether the beverage or ingredients have leaked out again → complete.

This creation is really an incompetent creative creation, which is of great industrial value. In addition, the present creation can be modified in any way by those with ordinary knowledge in the technical field to which it belongs, without departing from the scope to be protected by the scope of the attached patent application.

[Sequence list]

<110> Yuyang Biomedical Co., Ltd.

<120> Fermenter for Yogurt Biotech Drinks

<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 UY-76

<400> 2

Claims (6)

A fermentation machine for preparing yogurt drinks, comprising: a tank body; a sealed top cover assembled with the tank body so that a space enclosed by the sealed top cover and the tank body is closed; a feeding port, Set on the sealed top cover, water, milk powder and bacterial powder are sent into the space through the feeding port; a motor controller is installed outside the tank; a stirring motor is coupled to the motor controller; a stirring An arm disposed in the space and connected to the stirring motor for stirring the water, the milk powder, and the fungus powder; and a resistance warmer provided outside the tank and coupled to the motor controller for The water and the milk powder are sterilized, and the bacteria powder is fermented in a mixture formed by the water and the milk powder at 37 ° C to 43 ° C to prepare the yogurt drink. The fermenter according to item 1 of the scope of patent application, wherein the fermenter further comprises a discharge port provided at the bottom of the tank for the Yogurt beverage to be sent out from the discharge port. The fermenter according to item 1 of the scope of patent application, wherein the fermenter further comprises a temperature-controlling interlayer disposed outside the tank, and the resistance warmer is connected to the temperature-controlling interlayer, and the temperature-controlling interlayer is used for receiving The resistance warmer is controlled to adjust or maintain the temperature of the space. The fermenter according to item 1 of the patent application scope, wherein the sealed top cover further includes a cleaning port, and the fermenter further includes a heat conduction flow inlet disposed at a side of the tank body and a heat conduction flow outlet at a bottom of the tank body. The fermenter according to item 1 of the patent application scope, wherein the fermenter further comprises a temperature detector coupled to the motor controller, and the motor controller further includes a screen, the temperature sensor detects the A temperature is displayed on the screen. The fermenter according to item 1 of the patent application scope, wherein the sealed top cover further includes an observation window for a user to visually observe the condition in the tank.