KR101164876B1 - Broccoli production fermented by lactic acid bacteria, which is anti-inflammatory and anti-Helicobacter pylori and method for preparation thereof - Google Patents

Abstract

The present invention relates to a functional fermentation material having an effect of anti-inflammatory and anti-helicobacter fermented broccoli with lactic acid bacteria, and a method for preparing the same, more specifically, Lactobacillus plantarum MG207; Lactobacillus paracasei MG310; Lactobacillus casei MG311; Lactobacillus acidophilus MG501; Streptococcus thermophilus MG510; And Bifidobacterium longum MG723 relates to a functional fermentation material having the effect of anti-inflammatory and anti-helicobacter fermented broccoli with lactic acid bacteria, characterized in that any one or more selected from the group consisting of.
In addition, to minimize the destruction of sulforaphane and at the same time to effectively kill the general bacteria, adding 5 ~ 10wt% broccoli to distilled water; 25-35 minutes sterilization at 75-85; 2 hours to 3 hours at room temperature; It relates to a method for producing a functional fermentation material having an effect of anti-inflammatory and anti-Helicobacter fermented broccoli, characterized in that it comprises a step of sterilizing 25 to 35 minutes at 75 to 85 again.
On the other hand, broccoli is fermented with lactic acid bacteria to suppress the proliferation of Helicobacter pylori bacteria as well as to increase the cytokine material that inhibits inflammation that can be caused by Helicobacter bacteria and to reduce the cytokine that activates inflammation. It is about.
Furthermore, the present invention relates to a method of treating a basic cryoprotectant to increase the survival rate of bacteria of a functional fermentation substance having an effect of anti-inflammatory and anti-helicobacter fermented broccoli with lactic acid bacteria.
Anti-inflammatory and anti-Helicobacter effective fermentation broccoli of the present invention with lactic acid bacteria through the functional fermentation and its manufacturing method to prevent various side effects caused by the abuse of antibiotics, and of course, can be taken naturally as food Minimize the destruction of sulforaphane and at the same time can effectively kill ordinary bacteria.

Description

Broccoli production fermented by lactic acid bacteria, which is anti-inflammatory and anti-Helicobacter pylori and method for preparation according to Broccoli fermented with lactic acid bacteria

The present invention relates to a functional fermentation material having an effect of anti-inflammatory and anti-helicobacter fermented broccoli with lactic acid bacteria and a method for producing the same.

More specifically, the lactic acid bacteria is Lactobacillus plantarum MG207; Lactobacillus paracasei MG310; Lactobacillus casei MG311; Lactobacillus acidophilus MG501; Streptococcus thermophilus MG510; And Bifidobacterium longum MG723 relates to a functional fermentation material having the effect of anti-inflammatory and anti-helicobacter fermented broccoli with lactic acid bacteria, characterized in that any one or more selected from the group consisting of.

In addition, to minimize the destruction of sulforaphane and at the same time to effectively kill the general bacteria, adding 5 ~ 10wt% broccoli to distilled water; 25-35 minutes sterilization at 75-85; 2 hours to 3 hours at room temperature; It relates to a method for producing a functional fermentation material having an effect of anti-inflammatory and anti-Helicobacter fermented broccoli, characterized in that it comprises a step of sterilizing 25 to 35 minutes at 75 to 85 again.

Furthermore, the present invention is a fermentation broccoli with lactic acid bacteria to inhibit the proliferation of Helicobacter pylori bacteria as well as to increase the cytokine material that inhibits the inflammation that can be caused by Helicobacter bacteria and to increase the cytokine activating inflammation of the functional fermentation material having the effect of reducing It provides a production method and a method of treating a basic cryoprotectant to increase the survival rate of lactic acid bacteria contained in a functional fermentation substance having an effect of anti-inflammatory and anti-helicobacter fermentation broccoli with lactic acid bacteria.

Helicobacter pylori has been identified as a major causative agent of chronic gastritis and peptic ulcers since it was first isolated from human gastric mucosa by marshall and warren in Australia in 1982.In 1994, the World Cancer Institute International Cancer Research Institute It has been identified as a carcinogen. The fungus is a Gram-negative bacillus with several flagella, which grows in the surface layer or mucus of the gastric mucosa. The most characteristic microbiological features are strong motility and urease enzyme activity. Strong motility is to move freely and live in the mucus of the stomach, the main role is the flagella. In addition, urease activity can effectively break down urea present in the gastric mucosa, and it is estimated that ammonia produced thereby counteracts the attack by gastric acid by neutralizing the surrounding environment of bacteria. Helicobacter pylori, which has these characteristics, is the causative agent of chronic gastritis, stomach, duodenal ulcer, and gastric cancer. About 60-70% of the adult population in Korea is infected. .

Helicobacter pylori is a motile spiral Gram-negative bacillus that lives in the mucous membrane of the gastric mucosa and has an enzyme called urease, which is ammonia produced by decomposing urea. It survives by neutralizing the adult environment. Helicobacter pylori is associated with chronic gastritis and gastric ulcers, in particular 70% of gastric ulcers, 90% of gastritis, and 90% of duodenal ulcers. It has been shown to be closely related to gasric mucosa associated lymphoma, and interest in anti-Helicobacter pylori has been steadily emerging.

Currently, antibiotics are generally used as a therapy for anti-Helicobacter pylori. However, the main side effects of antibiotic abuse include shock, the formation of resistant bacteria, alternating strains, gene effects, blood disorders, liver disorders, gastrointestinal bleeding, and hearing impairments. Some new antibiotics can cause serious discomfort when taken with alcohol. Among the risks of antibiotics, the composition of resistant bacteria, their alternations, and their effects on genes are serious side effects unique to antibiotics. Antibiotics can also be a major cause of kidney disease. A study by Fanos, a professor of pediatrics at the University of Verona, Italy, warned of the effects on the kidneys of antibiotics or a combination of antibiotics and other drugs. In particular, antibiotics commonly administered to underweight babies may exacerbate the kidneys (Fanos & Cataldi, 1999). In other words, complete eradication is difficult and 10 to 20% of patients are not properly sanitized, causing re-infection and treating antibiotics, causing a vicious cycle that adversely affects national health due to the severe side effects of the abuse and the increase of antibiotic resistance strains. .

Therefore, there is a need to develop a product that can prevent the Helicobacter pylori bacteria, and further exert a therapeutic effect by naturally ingesting like food without side effects.

Antibiotics, commonly used as a therapy for Helicobacter pylori, can be a side effect of abuse, resulting in shock, composition of resistant bacteria, balance, gene effects, blood disorders, liver disorders, gastrointestinal bleeding, and hearing impairments. have. Furthermore, among the risks of antibiotics, the composition of resistant bacteria, myeloid alteration, and the effects on genes are serious side effects unique to antibiotics and may be a major cause of kidney disease. In addition, even if antibiotics are used to fight Helicobacter pylori, complete decontamination is difficult, and 10 to 20% of patients are not properly eradicated and are prone to reinfection. Therefore, it would be necessary to develop a product that can be naturally ingested like food without side effects to prevent Helicobacter pylori, and further exert a therapeutic effect.

Broccoli has used green shoots of cabbage from the Roman era, and its main ingredients are glucoraphanin and sulforaphane, which are antibacterial activity against Helicobacter pylori, a cause of gastric cancer and gastric ulcer. It has been reported to improve the cardiovascular health and to reduce the risk of cancer by strengthening the system's antioxidant defenses and reduce the inflammatory response. Broccoli also contains twice as much vitamin C as lemon and is rich in vitamin B1, vitamin B2, vitamin E, potassium and calcium, making it a well-being food for busy modern people today.

Intestinal bacteria present in our intestine can be divided into beneficial bacteria and harmful bacteria, and their types are about 2000 species and the number of bacteria is about 100 trillion. They continue to multiply and keep the growth of different bacteria at all times, so they usually live in the gut at a constant rate, unless there is a rapid change in the gut environment. Harmful and beneficial bacteria are in a competitive relationship. Therefore, which one is superior determines whether the intestines are healthy or frequently causing problems. When the balance of intestinal bacteria is broken and the harmful bacteria are increased or the beneficial bacteria are too small, the intestinal function decreases, and chronic symptoms such as chronic diarrhea, abdominal pain, bloating, smell, malnutrition, sepsis, and shock appear. Harmful bacteria mainly break down proteins, producing harmful or toxic substances such as ammonia, hydrogen sulfide, amines, phenols, and indole, which are harmful to the human body. These harmful substances can penetrate the bloodstream through the urethra and spread throughout the body, causing serious problems with the immune system. It also causes inflammatory diseases to prevail and buy harm. Lactobacillus, known as enteric beneficial bacteria, inhibits the growth of harmful intestinal bacteria and rot bacteria, prevents the production of intestinal rot products such as ammonia, indole and hydrogen sulfide, which are the causes of various intestinal diseases, and creates antibiotics that act in the intestine, thus making the intestinal flora total. . In addition, it promotes the activation of immune mechanisms (macrophages, lymphocytes) and the production of antibodies in the blood to improve immunity, and also inhibits the growth of harmful bacteria that produce free radicals and carcinogens in the intestine and induces death by anti-inflammatory action. It has been reported that vitamin K and the water-soluble vitamin B group (B ₁ , B ₂ , B ₆ , B ₁₂ ) are synthesized to improve the nutrition of the intestines. In addition, it exhibits antimicrobial activity against Helicobacter pylori, improves stressful constipation and diarrhea, inhibits cholesterol synthesis, and is effective in improving allergy and skin care.

Therefore, the contents of the present invention is to develop a product fermented with lactic acid bacteria that can settle the powdered organic brokerage well in the intestine and work, and do not have to worry about side effects like antibiotics by always ingesting, and inhibits Helicobacter pylori at low cost Therefore, the purpose is to prevent and treat all the diseases caused by the bacteria in advance.

The technical objects to be achieved by the present invention are not limited to the above-mentioned technical problems, and other technical subjects which are not mentioned can be clearly understood by those skilled in the art from the description of the present invention .

In order to solve the problems of the prior art, the present invention provides a functional fermentation material having an effect of anti-inflammatory and anti-helicobacter fermented broccoli with lactic acid bacteria and a method for producing the same.

Preferably, the lactic acid bacteria is Lactobacillus plantarum MG207; Lactobacillus paracasei MG310; Lactobacillus casei MG311; Lactobacillus acidophilus MG501; Streptococcus thermophilus MG510; And Bifidobacterium longumMG723 may be any one or more selected from the group consisting of.

In addition, the production method comprises the steps of adding 5 ~ 10wt% broccoli powder to distilled water to minimize the destruction of sulforaphane from the fermentation broccoli powder and at the same time effectively kill the general bacteria; 25-35 minutes sterilization at 75-85; 2 hours to 3 hours at room temperature; Again, 75 to 85 may include 25 to 35 minutes sterilization step.

On the other hand, broccoli is fermented with lactic acid bacteria to inhibit the proliferation of Helicobacter pylori bacteria as well as to increase the cytokine material that inhibits inflammation that can be caused by Helicobacter bacteria and to reduce the cytokine activating inflammation to reduce functional cytokines production method to provide.

The present invention also provides a method of treating a basic cryoprotectant to increase the survival rate of lactic acid bacteria contained in a functional fermentation material having an effect of anti-inflammatory and anti-helicobacter fermented broccoli with lactic acid bacteria.

Furthermore, the basic cryoprotectant may be skim milk, whole milk, whey, casein.

Preferably, the skimmed milk is added to the broccoli fermentation 10 ~ 15wt%, sucrose (Sucrose) 4 ~ 6wt%, maltose (Maltose) 4 ~ 6wt%, Trehalose (Trehalose) 4 ~ 6wt% More may be added.

More preferably, the skim milk may be added to 10-15 wt% of broccoli fermentation, 5 wt% of sucrose, 5 wt% of maltose, and 5 wt% of trehalose. have.

The present invention is an anti-inflammatory and anti-Helicobacter effective fermentation broccoli fermented with lactic acid bacteria, and through the preparation method thereof, antibiotics commonly used as a therapy for Helicobacter pylori bacteria shock, composition of resistant bacteria, At the same time to prevent the balance, gene effects, blood disorders, liver disorders, gastrointestinal bleeding, deafness, etc., there is an advantageous effect that can be taken naturally as food.

1 is a graph showing the Effect of pre-heating on sulforaphane. A is control, B is 60 pre-heating, C is 80 pre-heating, D is 100 pre-heating, E is 121 pre-heating.
Figure 2 is a graph showing the Comparison of control and microwave treatment in sulforaphane from Broccoli seed meal. The graph on the left is control, and the graph on the right shows 60 cases.
3 is a graph showing the Effect of broccoli culture broth fermented by lactic acid bacteria on IL-10 production in the LPS (lipopolysaccharide) stimulated RAW 264.7 cells. The white bar on the left shows the supernatant treatment, the middle gray bar shows the lactic acid bacteria cell treatment, and the right black bar shows the supernatant and lactic acid bacteria cell mixed treatment. Also, '0' is fermented broccoli, '1' is fermented with Lactobacillus plantarum MG207, '2' is fermented with Lactobacillus paracasei MG310, '3' is fermented with Lactobacillus casei MG311, '4' is fermented with Lactobacillus acidophilus MG501, and '5' is Bifidobacterium. The longum MG723 fermentation broth, '6', represents the Streptococcus thermophilus MG510 fermentation broth.
Figure 4 shows the Effect of broccoli culture broth fermented by lactic acid bacteria on TNF-a production in the LPS (lipopolysaccharide) stimulated RAW 264.7 cells. The white bar on the left shows the supernatant treatment, the middle gray bar shows the lactic acid bacteria cell treatment, and the right black bar shows the supernatant and lactic acid bacteria cell mixed treatment. Also, '0' is fermented broccoli, '1' is fermented with Lactobacillus plantarum MG207, '2' is fermented with Lactobacillus paracasei MG310, '3' is fermented with Lactobacillus casei MG311, '4' is fermented with Lactobacillus acidophilus MG501, and '5' is Bifidobacterium. The longum MG723 fermentation broth, '6', represents the Streptococcus thermophilus MG510 fermentation broth.

The present invention relates to a functional fermentation material having an effect of anti-inflammatory and anti-helicobacter fermented broccoli with lactic acid bacteria and a method for producing the same.

The lactic acid bacteria is Lactobacillus plantarum MG207; Lactobacillus paracasei MG310; Lactobacillus casei MG311; Lactobacillus acidophilus MG501; Streptococcus thermophilus MG510; And Bifidobacterium longumMG723 may be any one or more selected from the group consisting of.

The production method includes the steps of adding 5 ~ 10wt% broccoli powder to distilled water in order to minimize the destruction of sulforaphane from the fermentation broccoli powder and at the same time effectively kill general bacteria; 25-35 minutes sterilization at 75-85; 2 hours to 3 hours at room temperature; Again, 75 to 85 may include 25 to 35 minutes sterilization step.

The present invention also provides a method of producing a functional fermentation material having the effect of anti-inflammatory and anti-helicobacter by fermenting broccoli with lactic acid bacteria.

Furthermore, the present invention relates to a method of treating a basic cryoprotectant to increase the survival rate of lactic acid bacteria contained in a functional fermentation material having an effect of anti-inflammatory and anti-helicobacter fermented broccoli with lactic acid bacteria.

Skim milk is added to the broccoli fermentation 10 ~ 15wt%, sucrose (Sucrose) 4-6wt%, maltose (Maltose) 4-6wt%, trehalose (4-6wt%) Can be.

More preferably, the skim milk may be added to 10-15 wt% of broccoli fermentation, 5 wt% of sucrose, 5 wt% of maltose, and 5 wt% of trehalose. have.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

Example 1 Broccoli Sample Heat Treatment and General Bacterial Count

8 g of broccoli powder was added to 100 ml of distilled water (8%, w / v), followed by heat treatment at 60 to 30 minutes, heat treatment at 80 to 30 minutes, heat treatment at 100 to 30 minutes, and autoclaving at 121 to 20 minutes. However, in the case of 60 and 80, the sample prepared by heat treatment for 30 minutes at 60 and 80 for the second time after being left at 2 hours 37 after the first heat treatment was diluted by 10-fold serial dilution with 0.9% sterile saline solution and then nutrient agar (nutrient agar). ), The colonies generated after 37 to 48 hours of incubation were measured, and the number of viable cells per ml of culture medium was calculated by multiplying the average colony by the dilution factor.

Example 2 Sulforaphane Analysis of Broccoli Samples

Acetonitrile (B & J, USA), ethyl acetate (B & J, USA), ethanol (B & J, USA) were used as HPLC solvents for extraction and analysis of broccoli samples. Daejung, Korea) used reagent grade. Sulforapane standards were purchased from Sigma-Aldrich (St. Louis, Mo., USA). Sulforaphane extraction and purification from the sample was mixed with 150 g of ultra pure distilled water and 5 g of broccoli powder for 25 hours for 2 hours for autolysis of glucosinolates by myrosinase. To extract the crude organic solvent, add the mixed solution together with 150 mL ethyl acetate in a separatory funnel and shake it vigorously for 10 minutes using a funnel shaker (Funnel shaker, Eyela, Tokyo, Japan) and filter the crude extract with filter paper (Advantec No.131, USA). After filtration, the supernatant was separated from the separatory funnel. The solvent in the crude extract was evaporated using a rotary vacuum condenser and dissolved in 150 mL of hexane-ethyl acetate (hexane: ethyl acetate, 8: 2, v / v), followed by medium pressure liquid chromatography (Isco, USA). Solid-Phase Extraction (SPE) was performed. A normal phase column (230-400 mesh, silica, 40 g) was installed in the instrument to adjust 3 mL per minute under vacuum at 100 mL hexane, then the crude extract was loaded and the flow rate was adjusted to 10 mL per minute. Washed with 500 mL ethyl acetate. After adjusting the flow rate again to 3 mL per minute, the extract eluted with 50 mL of ethanol was dried with a rotary vacuum condenser and dissolved in 10 mL of acetonitrile.

Example 3 Helicobacter pylori Culture

Helicobacter pylori (KCTC 5335, ATCC 700392) is a plated medium prepared by adding 3% of Lake Horse Blood (Oxoid) to sterilized Brucella broth (BBL) and spreading it on a flat plate. 37, 5% CO Colonies cultured for 2 days under ₂ conditions were collected and used after diluting in 0.85% sterile saline.

Example 4 Preparation of Broccoli Fermentation for Anti-Helicobacter Pylori

Fermentation strains include Bacillus coagulans MG520; Bifidobacterium bifidum MG731; Bifidobacterium breve MG729; Bifidobacterium longum MG723; Bifidobacterium thermophilum MG748; Enterococcus faecium MG89; Lactobacillus acidophilus MG501; L. brevis MG18; Lactobacillus casei sub. casei MG311; Lactobacillus casei sub. casei MG727; Lactobacillus casei sub. rhamnosus MG316; Lactobacillus casei sub. rhamnosus MG515; L. delbrueckii sub. bulgaricus MG515; L. fermentum MG590; Lactobacillus paracasei MG310; Lactobacillus plantarum MG207; L. reuteri MG505Y; L. salivarius MG595; Lactococcus lactis MG530; Leuconostoc mesenteroides MG865; treptococcus faecalis MG511; Streptococcus thermophilus MG510; 22 kinds of lactic acid strains were selected and used. The strain is a strain directly isolated from kimchi, dairy products, feces in the laboratory of the applicant Mediogen Co., Ltd. of the present invention, it can be purchased from the company. Each of these species was enriched in MRS medium, followed by medium culture in a minimum medium (1.5% peptone, 1.5% glucose, 0.1% yeast extract), followed by adding 8% organic broccoli powder to distilled water and pH7. Broccoli solution was prepared by sterilization twice at 80 for 30 minutes. 22 lactic acid bacteria were inoculated separately in 1% each of the prepared 8% broccoli solution, followed by anaerobic incubation for 24 to 72 hours at 37, followed by centrifugation at 6,000 rpm for 20 minutes (Supra 22K, Hanil science Co. Ltd, Korea). Was taken. The supernatant was calibrated with pH 4.0 and pH 7.0 with 0.9% lactic acid to pH 5.5 and pH 7.0, filtered through a sterile 0.2 μm syringe filter, Cellulose Acetate, Advantec, and then filtered. It was concentrated to the fold and used as a sample.

Example 5 Acidity Measurement of Fermented Product

The pH was measured using a pH meter (pH meter, Istec720P, Korea), the acidity was measured according to the food method. In other words, 10 ml of herbal fermentation broth is added with the same amount of distilled water, 0.5 ml of 1% phenophthalein alcohol is added, and titrated with 0.1 N sodium hydroxide (NaOH) solution to measure the consumption when light red color is maintained for 30 seconds. The total acid was titrated by the following equation, and the appropriate value was converted into lactic acid content (%) and expressed. The following f represents the factor of 0.1N sodium hydroxide.

Example 6 Measurement of Lactic Acid Bacteria

The viable cell count was diluted 10 times with 0.9% sterile physiological saline, and then inoculated with Lactobacillus, Lactococcus, Enterococcus, Streptococcus genus in BCP plate count agar, and Bifidobacterium genus inoculated with BL agar for 37, 48 hours anaerobic jar. The number of colonies generated after incubation at was measured and the average number of colonies was multiplied by the dilution factor to calculate the number of viable cells per ml of culture.

Example 7 Helicobacter Pylori Inhibition Measurement

Brucella broth (including 3% of Lakee horse blood) calibrated to pH4.0, pH5.5 and pH7.0, respectively, and 2.5%, 1.0%, 0.5% and 0.1% broccoli fermentation filtration concentrate calibrated to the same pH as Brucella broth, respectively. After the addition of%, H. pylori inoculated with 0.85% sterile saline was inoculated 100μm each to make a mixed broth. After reacting the mixed broth at 37, 5% CO ₂ for 24 hours, smear 100μl onto Brucella agar plate (including 3% of Horse Horse Blood) and incubate for 4 days under conditions of 37, 5% CO _{2 to} colonize H. pylori. It measured and confirmed.

Example 8 Measurement of Urease Inhibition

In order to determine the inhibitory activity of Helicobacter pylori urease on broccoli supernatant fermented with lactic acid bacteria, H. pylori colonies incubated for 72 hours in Brucella agar (including 3% of Lake horse blood) were collected and washed with 0.85% sterile saline. It was diluted to / 20 and suspended at 0.89 at absorbance of 600 nm. Broccoli fermentation broths prepared at pH 4.0, pH 5.5 and pH 7.0 prepared in advance were added to fresh Brucella broth (including 3% of Lake horse blood) in 1%, 5% and H suspended in 0.85% sterile saline. 10% (v / v) of pylori was inoculated and incubated at 37 and 5% CO ₂ for 1 hour. In order to measure the activity of urease, the sample was mixed with 30% (v / v) of urea brtoh (Merck, Germany) and reacted at 30 for 1 hour, and then absorbance was measured at 560 nm.

Example 9 RAW 264.7 Cell Culture and Sample Treatment

The mouse macrophage cell line RAW 264.7 from Korea Cell Line Bank was distributed and stored in a nitrogen tank. The cells used for the experiment were 10% fetal bovine serum, streptomycin (100 µg / ml), and penicillin ( 100U / ml) was incubated in Dulbecco`s modified Eagle's medium (DMEM, GIBCO, USA) at 37, 5% CO ₂ conditions. Raw cells (p = 30) to be used for the experiment were added to 2x10 ⁵ cells / ml in a 24-well culture plate and cultured for 18 hours, and then the samples were treated. Broccoli culture samples to be used in the experiment, one was divided into the whole culture (whole), the other was centrifuged to the culture medium (medium) filtered through a membrane filter.

Pure lactic acid bacteria grown in lactic acid bacteria culture medium was used diluted to 5 x 10 ⁷ CFU / ml for each strain. 50 μl of the sample was diluted in 550 μl of DMEM medium (1:12), and 600 μl of the sample was treated in wells in which RAW 264.7 cells were cultured. After 16 hours of incubation, the sample was removed, and 500 μl of DMEM medium containing 100 ng / ml of LPS was added thereto for 8 hours, followed by the recovery of the culture solution, and centrifuged supernatant was used for cytokine measurement. As a negative control sample, 10% broccoli unfermented filtrate, which was not cultured with bacteria, was diluted in the same ratio. As a positive control, lipopolysaccharide (Sigma, USA) of Pseudomonas aerogunosa was added to DMEM medium at a concentration of 100 ng / ml.

Example 10 Cytokine Measurement

Quantification of IL-10 and TNF-a present in the sampled supernatant was performed using an ELISA kit (KOMA, Korea). 50 μl of the supernatant of the cell was added to the well with the anti-mouse IL-10 and anti-mouse TNF-a attached with 100 μl of the assay diluent, and then the biotinylated detection antibody was added. After four washings with PBS buffer, streptavidin-horseradish peroxidase conjugate was added and reacted at room temperature for 30 minutes. After four more washings with PBS, 100μl of tetramethylbenzidine and hydrogen peroxide were added to detect attached peroxidase conjugate, and then 100μl of 1M H ₂ SO ₄ solution was added to terminate the reaction and absorbed at 450 nm in the microplate reader. Was measured. Cytokine concentration was quantified by preparing a linear dose-response standard curve.

<Example 11> Lyophilization of broccoli fermented products and production of raw materials

After adding the lyophilized protective agent at a concentration of 5-30% to the fermentation broth, it was homogenized and then pre-frozen at -70 for 2 hours and lyophilized for 48 hours. The temperature of the chamber inside the dryer was adjusted to start at -10, rise by 10 in 2 hours and fix at 30. After lyophilization was finished, the powder was ground, viable cell count was measured, and the survival rate was calculated by comparing with the initial viable cell number. The measured samples were diluted 10 times serially with 0.9% sterile saline solution, and then Lactobacillus, Lactococcus, Enterococcus and Streptococcus were inoculated in BCP plate count agar and Bifidobacterium was inoculated in BL agar and incubated in anaerobic jars for 37 and 48 hours. Thereafter, the number of colonies generated was measured, and the average number of colonies was multiplied by the dilution factor to calculate the number of viable cells per g of culture medium.

Example 12 Statistical Processing

In order to secure the objective data indicating the superiority of the effects, the significance test between groups was performed. More specifically, Student's t-test was used, and when the significance level was P> 0.05, it was determined that the result was significant.

Experimental Example 1 microwave treatment of lyophilized powder of organic broccoli

After freeze-drying the organic brokerage, microwave treatment was performed as a pretreatment to reduce general bacteria. As a result, the average bacterial count in the untreated group was 3.0X10 ⁵ cfu / ml and 5.0X10 ³ cfu / ml in the treated group. As a result of HPLC analysis of sulforaphane content as shown in FIG. 1, the microwave treated group contained about 7.0 ug per gram of powder and the untreated group contained about 2.6 ug. It was about 2.7 times higher. This is shown in [Table 1].

Changes in General Bacterial Counts and Sulforaphane Contents by Pretreatment of Broccoli Ingredients Way General bacteria count (cfu / ml) Sulforaphane content per 1g broccoli powder (㎍) Microwave Untreated 3.0 × 10 ⁵ 2.6 Microwave Treatment 5.0 × 0 ³ 7.0

Experimental Example 2 Measurement of General Bacteria in Freeze-Dried Powder of Organic Broccoli

Microwave-treated organic broccoli lyophilized powder was added to distilled water to 8% content, followed by heat treatment at 60, 80, and 100 minutes for 30 minutes, and autoclaving at 121 for 20 minutes, as shown in [Table 2]. About 2.8 × 10 ³ cfu / ml of normal bacteria were detected, 80 × ¹ 1.8 × 10 ¹ cfu / ml was detected, and 100 and 121 autoclaves were not detected. However, in the heat treatment of 100 and 121, the loss of sulfolapane, which is the main component of broccoli, was feared, and two heat treatments were performed at 60 and 80 for about 3 hours, resulting in 2.7 X 10 ² cfu / ml of normal bacteria at 60, Since it was not detected, the heat treatment temperature for the future fermentation test was selected after 80 to 30 minutes of heat treatment, and then left to stand for 3 hours, followed by 80 to 30 minutes of heat treatment.

Changes in General Bacterial Count According to Heat Treatment Temperature and Method Heat treatment temperature General bacteria count (cfu / ml) Before heat treatment Primary heat treatment Secondary heat treatment 60 ° C
5.0 × 0 ³ 2.8 × 0 ³ 2.7 × 0 ² 80 ℃ 2.8 × 0 ² ND * 100 ℃ ND - 121 ℃ ND -

* ND: not detected

<Experiment 3> Changes according to the heat treatment temperature of sulforaphane, the main ingredient of broccoli

As shown in FIG. 2, in order to ferment the lyophilized powder of organic brokery, the killing of the general bacteria remaining after the microwave treatment is essential. For this, the number of general bacteria is reduced as much as possible and the decomposition of sulforaphane, the main ingredient of broccoli, is As a result of HPLC analysis of the optimum heat treatment temperature that could be minimized, the sulforaphane content was maintained at about 97% for 60 minutes for 30 minutes, and at 84% for 80, 45% for 100, and high pressure for 120 minutes for 120 minutes. The sulforaphane was all destroyed. Therefore, the proper temperature at which sulforaphane is not destroyed is 60 and 80. However, when sterilization is performed at 60, the killing temperature of the normal bacteria contained in broccoli powder is low. After sterilization twice, the sulforaphane content was reduced to about 66% (see Table 3).

Content of sulforaphane depending on the heat treatment temperature Heat sulforaphane (%) No heat 60 ° C 80 ℃ 100 ℃ 121 ℃ 1 time 100 96.88 83.50 44.92 0.0 Episode 2 - - 66.18 13.68 -

Experimental Example 4 Broccoli Fermentation and Strain Selection

As a result of measuring growth time and pH and acidity change of 22 lactic acid bacteria in broccoli in [Table 4], as shown in [Table 4] and [Table 5], pH and acidity were large after 24 hours incubation. The number of viable cells in culture was better in 24 hours than in 48 hours, Bifidobacterium longum MG723 showed the best growth rate of 5.3 × 10 ⁸ cfu / ml, and in Lactobacillus genus Lactobacillus acidophilusMG501, Lactobacillus plantarum MG207, Lactobacillus paracasei MG310 , Lactobacillus casei sub. The growth of rhamnosus MG311 was 5.5 × 10 ⁸ cfu / ml, 35.5 × 10 ⁸ cfu / ml, 54.5 × 10 ⁸ cfu / ml, and 23.5 × 10 ⁸ cfu / ml, respectively. Streptococcus thermophilus MG510 was 5.7 × 10 ^{8 in} Streptococcus genus. Excellent at cfu / ml. In addition, Enterococcus faecium MG89, Lactobacillus casei sub. Rhamnosus MG515 and Streptococcus faecalis MG511 were also excellent, but according to the redundancy and consumer preference, Bifidobacterium longum MG723, Lactobacillus acidophilusMG501, Lactobacillus plantarum MG207, Lactobacillus paracasei MG310, Lactobacillus casei sub. Six species such as rhamnosus MG311 and Streptococcus thermophilus MG510 were selected and prepared by fermentation for 24 hours using the six strains mentioned above for the preparation of broccoli fermentation samples.

Broccoli fermentation results after 24 hours incubation No Strain After fermentation
Culture pH Acidity
(%) Viable cell count
(× 10 ⁸ cfu / ml) 0 control - 0.18 - One Bacillus coagulans MG520 4.4 1.02 0.86 2 Bifidobacterium bifidum MG731 5.5 0.20 <10 ⁵ 3 Bifidobacterium breve MG729 4.3 1.14 0.25 4 Bifidobacterium longum MG723 4.1 1.32 5.3 5 Bifidobacterium thermophilum MG748 4.1 1.32 0.35 6 Enterococcus faecium MG89 4.3 1.14 6.7 7 L. acidophilus MG501 3.8 1.60 5.5 8 L. brevis MG18 5.0 0.35 1.5 9 L. casei sub. casei MG311 4.1 1.32 23.5 10 L. casei sub. casei MG727 4.1 1.32 4.7 11 L. casei sub. rhamnosus MG316 4.0 1.43 16 12 L. casei sub. rhamnosus MG515 4.2 1.25 9.5 13 L. delbrueckii sub. bulgaricus MG515 4.2 1.25 0.39 14 L. fermentum MG590 4.4 1.02 1.2 15 L. paracasei MG310 4.1 1.32 54.5 16 L. plantarum MG207 3.9 1.50 35.5 17 L. reuteri MG505Y 4.6 0.82 1.1 18 L. salivarius MG595 4.1 1.32 1.5 19 Lactococcus lactis MG530 4.5 0.96 0.20 20 Leuconostoc mesenteroides MG865 4.5 0.96 0.48 21 Streptococcus faecalis MG511 4.5 0.96 5.5 22 Streptococcus thermophilus MG510 4.4 1.02 5.7

Broccoli fermentation results after 48 hours of incubation No Strain After fermentation
Culture pH Acidity
(%) Viable cell count
(× 10 ⁸ cfu / ml) 0 control - 0.18 - One Bacillus coagulans MG520 4.4 1.02 0.09 2 Bifidobacterium bifidum MG731 5.5 0.20 <105 3 Bifidobacterium breve MG729 4.3 1.14 0.15 4 Bifidobacterium longum MG723 4.1 1.32 3.3 5 Bifidobacterium thermophilum MG748 4.1 1.32 0.15 6 Enterococcus faecium MG89 4.3 1.14 3.7 7 L. acidophilus MG501 3.8 1.60 2.5 8 L. brevis MG18 5.0 0.35 0.8 9 L. casei sub. casei MG311 4.1 1.32 0.26 10 L. casei sub. casei MG727 4.1 1.32 2.7 11 L. casei sub. rhamnosus MG316 4.0 1.43 8.0 12 L. casei sub. rhamnosus MG515 4.2 1.25 4.8 13 L. delbrueckii sub. bulgaricus MG515 4.2 1.25 0.2 14 L. fermentum MG590 4.4 1.02 0.7 15 L. paracasei MG310 4.1 1.32 6.2 16 L. plantarum MG207 3.9 1.50 3.3 17 L. reuteri MG505Y 4.6 0.82 0.3 18 L. salivarius MG595 4.1 1.32 0.8 19 Lactococcus lactis MG530 4.5 0.96 0.1 20 Leuconostoc mesenteroides MG865 4.5 0.96 0.2 21 Streptococcus faecalis MG511 4.5 0.96 2.3 22 Streptococcus thermophilus MG510 4.4 1.02 2.1

Experimental Example 5 Inhibition of Helicobacter Pylori of Broccoli Fermentation

Inhibitory activity against Helicobacter pylori was found to be more effective at lower pH. In control (-), lactic acid itself showed lower inhibitory effect in Brucella medium calibrated to pH 4.0 and 5.5 with lactic acid. It was shown that H. pylori growth was inhibited and that the pH adjusted to 5.5 showed that the growth of H. pylori was inhibited at 1.0 ~ 2.5% concentration. It turns out that synergistic effect is suppressed. In order to rule out the effects of lactic acid, the samples neutralized to pH 7.0 showed a viable cell count of 1.2X10 ⁶ cfu / ml in pure Brucella medium, which was control (-), but in medium containing broccoli unfermented solution, which was added to control (+). Compared with the (-) group, the number of viable bacteria was reduced from 1/10 to 1/100, and the effect of brokerage extract was shown. The growth of H. pylori was completely inhibited at the concentration of 2.5% of the filtrate fermented with, and the number of viable cells was reduced to 1/10 to 1/100 even at the concentration of 0.1 to 0.5%. In the concentration of 1.0 ~ 2.5% of fermented product filtrate, the number of viable cells decreased from 1/10 to 1/100 compared to the control (+). It can be seen that the pylori inhibitor is produced. However, at the concentration of 0.1-0.5%, it showed no inhibitory effect except the samples fermented with L. acidophilus and S. thermophilus strains.

Inhibition of Helicobacter pylori Growth by Broccoli Fermentation Concentration and pH Supernatant pH
sample H. pylori viable count (cfu / ml) pH 4.0 pH 5.5 pH 7.0 Control (-) ^* ND ^*** 1.1 x 10 ⁴ 1.2 × 10 ⁶ Control (+) ^** 2.5% ND ND 3.1 × 10 ⁴ 1.0% ND 2.1 × 10 ² 1.4 × 10 ⁵ 0.5% ND 1.1 x 10 ³ 1.6 × 10 ⁵ 0.1% ND 1.7 × 10 ³ 1.7 × 10 ⁵ L. plantarum MG207
Fermented products 2.5% ND ND 2.0 x 10 ³ 1.0% ND ND 2.1 × 10 ⁴ 0.5% ND 5.0 × 10 ¹ 3.7 × 10 ⁵ 0.1% ND 3.0 × 10 ² 5.8 × 10 ⁵ L. paracasei MG310
Fermented products 2.5% ND ND 5.0 × 10 ³ 1.0% ND ND  2.6 × 10 ⁴ 0.5% ND 2.4 × 10 ² 2.9 × 10 ⁵ 0.1% ND 2.7 × 10 ² 3.1 × 10 ⁵ Lc asei MG311
Fermented products 2.5% ND ND 4.0 × 10 ³ 1.0% ND ND 1.3 × 10 ⁴ 0.5% ND 3.3 × 10 ² 2.4 × 10 ⁵ 0.1% ND 5.4 × 10 ² 3.9 × 10 ⁵ L. acidophilus MG501
Fermented products 2.5% ND ND ND 1.0% ND ND 2.3 × 10 ³ 0.5% ND 2.0 × 10 ¹ 3.4 × 10 ⁴ 0.1% ND 4.3 × 10 ² 5.7 × 10 ⁵ S. thermophilus MG510
Fermented products 2.5% ND ND ND 1.0% ND ND 2.7 × 10 ² 0.5% ND ND 1.3 × 10 ³ 0.1% ND 5.5 × 10 ² 3.3 × 10 ⁴ Bi. longum MG723
Fermented products 2.5% ND ND 2.0 x 10 ⁴ 1.0% ND 4.0 × 10 ¹ 2.2 × 10 ⁵ 0.5% ND 2.7 × 10 ² 3.6 × 10 ⁵ 0.1% ND 7.0 × 10 ² 4.4 × 10 ⁵

* Control (-): Brucella broth + H.pylori

** Control (+): Brucella broth + H.pylori + Broccoli Unfermented Supernatant

*** ND: Not detected

Experimental Example 6 Measurement of Urease Inhibition

As a result of testing the inhibitory effect on the urease activity of Helicobacter pylori using broccoli fermentation, there was no significant difference in the inhibition of urease activity according to the pH of the sample, and there were differences in the fermentation supernatants of Streptococcus thermophilus and Bifidobacterium longum depending on the concentration. No significant difference was seen in the remaining strains. 5% addition of unfermented solution showed about 29% inhibition, and Lactobacillus paracasei showed about 56% inhibition. L. acidophilus showed a significant inhibitory effect of 55-67% depending on the concentration. Especially, fermentation supernatants of Streptococcus thermophilus and Bifidobacterium longum showed high inhibition rate of 60-80 depending on the concentration, inhibiting the growth of H. pylori. This may be indirectly effective (see Table 7).

Inhibition of Urease Activity of Broccoli Fermentation Sample and Concentration % inhibition of urease activity pH 4.0 pH 5.5 pH 7.0 Control One 21.7 23.3 23.3 5 27.1 29.5 29.5 L. plantarum MG207 One 32.6 34.1 34.2 5 37.2 38.6 38.8 L. paracasei MG310 One 56.3 55.3 55.2 5 59.7 56.2 56.2 L. casei MG311 One 20.0 21.1 21.0 5 21.2 22.4 22.4 L. acidophilus MG501 One 53.3 55.2 55.5 5 67.4 65.2 60.5 Str. thermophilus MG510 One 59.7 67.7 40.3 5 79.8 73.9 42.4 Bi. longum MG723 One 59.7 65.1 64.3 5 83.7 68.8 66.4

Experimental Example 7 Cytokine Production by Broccoli Fermentation

When broccoli fermentation broth was added to Raw 264.7 macrophage, the amount of IL-10 known as anti-inflammatory cytokine was measured. Samples obtained by mixing the fermentation broth of each strain 1:12 and measuring the amount of IL-10 present in the Raw 264.7 cell culture medium were mixed with the lactic acid bacteria in the fermentation supernatant, the pure lactic acid cell used for fermentation, and the fermentation supernatant. Was carried out. Experimental results showed that 640 pg / ml of the control LPS and 880 pg / ml of the broccoli unfermented supernatant were the highest in the Lactobacillus acidophilus MG501 supernatant (1800 pg / ml), and Bifidobacterium longum MG723 and Lactobacillus plantarum MG207 1700 pg / ml, Lactobacillus aracasei MG310 and Lactobacillus casei MG311 produced 1200 ~ 1300 pg / ml, producing about 1.5 ~ 2 times more IL-10 than broccoli unfermented solution.

Lactobacillus acidophilus MG501 cells showed the highest yield at 2600 pg / ml, and Lactobacillus plantarum MG207 and Bifidobacterium longum MG723 had 2200 ~ 2300 pg / ml, Streptococcus thermophilus MG510 had 1800 pg / ml and LactobaMGus paracasei case. The production of MG311 was about 1300 pg / ml, which was higher than that of the supernatant, and the samples containing both broccoli fermentation supernatant and lactic acid bacteria were analyzed. Lactobacillus acidophilus MG501 and Bifidobacterium longum MG723 samples were about 3000 pg / ml. Lactobacillus plantarum MG207 and Streptococcus thermophilus MG510 samples produced 2500-1029 pg / ml of IL-10, 1.3-1.4 times higher than supernatant alone and 1.3-1.4 times higher than cells alone. Anti-inflammatory effect is expected by showing The internal production of TNF-a produced 1074 pg / ml for the control LPS, 976 pg / ml for the broccoli unfermented supernatant, and the lowest yield was approximately 829 pg / ml for the Streptococcus thermophilus MG510 supernatant. It is similar to or less than the control group. Lactobacillus acidophilus MG501 cells showed the lowest value of 995 pg / ml when only Lactobacillus cells were treated. TNF-a levels were similar or slightly higher than those of the control group. Bifidobacterium longum MG723 produced the least amount of 768 pg / ml of Bifidobacterium longum MG723 after processing the fermented supernatant and cell mixture sample. The remaining samples were about 850 ~ 990pg / ml, which was lower than the control group. Feed (see [FIG. 3] and [FIG. 4]).

 Experimental Example 8 Lyophilization of Broccoli Fermentation and Preparation of Raw Materials

 After cell broth fermentation was performed by lyophilization of broccoli to increase the viability of the cells, the cell survival rate was tested. The best survival rate was shown, followed by whole milk, whey, and casein. The results are shown in [Table 8].

 Therefore, in future tests, skim milk 10% was added based on the cost of mass production and other components were added to each concentration to check the survival rate. 5% and 10% concentrations of broccoli fermentation products based on 10% of Skim milk and broccoli fermented with 9 types of protective agents such as glucose, sucrose, maltose, lactose, trehalose, solbitol, xylitol, mannitol and modified starch for 24 hours. As a result, L. plantrum MG207 fermented products showed better survival rate than other protectants with 53 ~ 58% survival rate of trehalose, sucrose and maltose. The survival rate was about 48%, and L.casei MG311 fermentation was about 50%.

 L. acidophilus MG501 fermentation showed a survival rate of about 45% in sucrose and trehalose samples. The fermented thermophilus MG510 showed a high survival rate of 60-68% in glucose, sucrose, maltose and trehalose samples, and the viability of Bifidobacterium longum MG723 fermentation showed about 42% survival in 10% sucrose and trehalose samples. ] Reference).

Therefore, the final protective agent composition consisted of skim milk 10%, sucrose 5%, maltose 5%, trehalose 5%, and the survival rate after freeze-drying of the fermentation products of each strain by incubation time using these protective ingredients is shown in Table 10. In case of fermentation with L. plantrum MG207, the survival rate of lyophilized samples was the highest as 92% after 12 hours of cultivation. The survival rates of the samples were 86.5%, 87.8%, 80.6%, and 66.4%, respectively. In case of thermophilus MG510, the survival rate of lyophilized sample after 36 hours of cultivation was the best (97%). Therefore, L. plantrum MG207 was cultured for 12 hours, and Lactobacillus paracasei MG310, L.casei MG311, Lactobacillus acidophilusMG501, and Bi.longum MG723 were cultured for 24 hours. thermophilus MG510 was carried out by taking a fermentation product incubated for 36 hours.

Survival Results from Basic Cryoprotectant Treatment Protection
sample % Survival after lyophilization. L. plantarum MG207 L. paracasei MG310 L. casei MG311 L. acidophilus MG501 Str. thermophilus MG510 Bi. longum MG723 No additives 0.4 10.6 0.4 42.0 Skim milk 10% 48.1% 35.5% 32.5% 30.5% 54.8% 22.5% Skim milk 15% 42.5% 30.6% 28.7% 26.0% 52.1% 20.7% Whole milk 10% 31.0% 25.5% 22.0% 20.5% 40.5% 18.0% Whole milk 15% 36.7% 30.0% 27.0% 22.4% 35.5% 20.2% Whey 10% 36.0% 26.9% 23.5% 21.5% 40.0% 17.5% Whey 15% 38.5% 30.5% 28.9% 25.7% 42.2% 18.5% Casein 10% 21.5% 23.4% 21.2% 18.2% 32.5% 18.2% Casein 15% 20.0% 22.4% 20.5% 20.0% 29.2% 20.0%

Survival rate according to the concentration of other protectant components in 10% of skim milk Lyoprotectants and Concentrations % Survival after lyophilization. L. plantarum MG207 L. paracasei MG310 L. casei MG311 L. acidophilus MG501 Str. thermophilus MG510 Bi. longum MG723 Control
(skim milk 10%) 48.1% 35.5% 32.5% 30.5% 54.8% 22.5% Glucose 5% 42.0 38.9 36.8 35.6 60.0 26.4 10% 35.1 37.7 35.4 36.8 62.1 25.2 Sucrose 5% 55.5 45.5 48.5 40.5 66.2 37.5 10% 50.2 48.6 50.6 45.6 66.5 42.4 Maltose 5% 52.4 46.3 45.1 37.1 62.4 36.2 10% 53.6 47.6 48.5 38.5 63.6 39.6 Lactose 5% 22.9 34.4 36.5 30.5 42.8 22.5 10% 33.6 36.6 38.2 32.8 43.1 28.5 Trehalose 5% 58.1 44.1 46.8 40.7 68.6 37.7 10% 55.3 48.2 50.6 45.5 65.4 41.6 Sorbitol 5% 20.6 24.8 22.5 20.0 40.0 24.1 10% 30.0 29.1 26.4 22.8 42.0 28.1 Xylitol 5% 10.4 22.5 24.8 20.8 40.6 25.7 10% 15.4 29.5 24.5 24.5 45.4 28.5 Mannitol 5% 12.5 17.0 16.5 20.5 42.2 26.0 10% 11.2 11.3 14.4 24.4 41.3 24.0 Modified starch 5% 10.8 15.8 12.8 14.2 30.0 4.8 10% 11.1 10.4 12.4 14.2 31.2 2.4

Survival Rate after Lyophilization by Culture Time with Optimal Protective Agent Incubation time % Survival after lyophilization. L. plantarum MG207 L. paracasei MG310 L. casei MG311 L. acidophilus MG501 Str. thermophilus MG510 Bi. longum MG723 12 92.0 45.5 42.5 40.5 50.8 36.5 24 69.6 86.5 87.8 80.6 90.5 66.4 36 50.2 61.8 66.4 56.7 97.0 45.2 48 45.5 40.9 48.8 40.5 86.6 34.8

* Freeze protection: Sucrose 5%, Maltose 5%, Trehalose 5%, Skim milk 10%

On the basis of the embodiments with reference to the accompanying drawings it was described in detail. However, this is not intended to limit the scope of the present invention to this intended to illustrate the present invention, there are various equivalents that can replace them. In addition, the terms or words used in the specification and claims should not be construed as being limited to the common or dictionary meanings, and the inventors should properly define the concept of terms in order to best explain their invention in the best way. It should be interpreted as meaning and concept corresponding to the technical idea of the present invention based on the principle that it can.

Antibiotics are commonly used as a countermeasure against Helicobacter pylori, which is estimated to be infected by about 60% to 70% of the adult population in Korea, due to chronic gastritis, stomach, duodenal ulcer and gastric cancer. However, abuse of antibiotics can lead to various side effects, such as shock and the composition of resistant bacteria. Therefore, there is no side effect, such as food intake as naturally as possible to prevent the Helicobacter pylori bacteria, and further develop a product that can exhibit a therapeutic effect will be urgently needed.

In this respect, broccoli, which contains glucopanin and sulfolapan, has antibacterial activity against Helicobacter pylori, which enhances cardiovascular health by strengthening tissue antioxidant defenses and reducing inflammatory responses. It has the effect of reducing risk.

Accordingly, the present invention provides a functional fermentation substance having an anti-inflammatory and anti-helicobacter effect of fermenting broccoli with lactic acid bacteria and a method for preparing the same, while preventing various side effects caused by abuse of antibiotics, and can be naturally consumed as food. Of course, there are industrial applications in that it minimizes the destruction of sulforaphane and effectively kills general bacteria.

Furthermore, the present invention is a fermentation broccoli with lactic acid bacteria to inhibit the proliferation of Helicobacter pylori bacteria, as well as to increase the cytokine material that inhibits inflammation that can be caused by Helicobacter bacteria and to increase the cytokine activating inflammation of the functional fermentation material having the effect of reducing the It is highly industrially feasible in that it provides a method of producing and treating a basic cryoprotectant to increase the survival rate of lactic acid bacteria contained in a functional fermentation substance having an effect of anti-inflammatory and anti-helicobacter fermenting broccoli with lactic acid bacteria. Would be excellent.

Claims (10)

delete delete In the method of producing a functional fermentation material having the effect of anti-inflammatory and anti-helicobacter fermented broccoli with lactic acid bacteria,
(1) a first step of washing, grinding, and lyophilizing broccoli, followed by microwave treatment as a pretreatment process to reduce general bacteria;
(2) After the microwave treatment, after sterilization for 25 minutes to 35 minutes at 75 ~ 85 ℃, the temperature is left for 2 hours to 3 hours, the second step to sterilize 25 minutes to 35 minutes at 75 ~ 85 ℃ again ;
(3) Lactobacillus plantarum 35.5 × 10 ⁸ cfu / ml after the sterilization; Lactobacillus paracasei 54.5 × 10 ⁸ cfu / ml; And Bifidobacterium longum 5.3 × 10 ⁸ cfu / ml after inoculating any one of the lactic acid bacteria selected from the group consisting of the third step of fermentation for 24 hours.
(4) after the fermentation, the fourth step of treating any of the basic cryoprotectant selected from the group consisting of whole milk, whey and casein to increase the survival rate of lactic acid bacteria; And
(5) after the freeze-drying, the fifth step of powdering; manufacturing method of a functional fermentation material, characterized in that it comprises a.
Method for producing a functional fermentation material having the effect of anti-inflammatory and anti-helicobacter fermented broccoli with lactic acid bacteria. delete delete delete delete delete delete delete

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