WO2014206279A1

WO2014206279A1 - Lactic acid bacteria and preparation method and use of fermented grains or grain germ extracts

Info

Publication number: WO2014206279A1
Application number: PCT/CN2014/080665
Authority: WO
Inventors: 董英; 肖香; 张家艳; 徐斌; 崔恒林; 赵延胜; 周兴华
Original assignee: 江苏大学
Priority date: 2013-06-26
Filing date: 2014-06-24
Publication date: 2014-12-31

Abstract

Disclosed are a lactic acid bacteria, Lactobacillus plantarum, CGMCC No. 6016; the preparation method of fermented grains or grain germ extracts using the Lactobacillus plantarum; and the use of the Lactobacillus plantarum in fermented grains or grain germ extracts.

Description

Preparation method and use of lactic acid bacteria and fermented cereal or cereal germ extract

Technical field

The invention relates to the field of food biotechnology, and particularly relates to bio-transformation by fermentation of lactic acid bacteria by using cereal or grain germ (barley, wheat germ, etc.) as raw materials, thereby realizing the increase and enrichment of functional active components, and preparing to inhibit tumor cell proliferation. And a fermented extract of tumor growth, the inventive product can be used for the preparation of functional foods, food ingredients or pharmaceuticals. Background technique

According to the World Health Organization, the number of people worldwide who die from malignant tumors is more than 7 million a year, and the number is still rising. The treatment of cancer mainly includes surgery, chemotherapy, radiotherapy and targeted therapy (immunotherapy, gene therapy, angiogenesis inhibitors and targeted therapy), and its side effects still bring a lot of pain to patients. Therefore, the development of natural safe, non-toxic and anti-tumor active substances and their functional foods or drugs are the cravings of cancer patients.

Natural grain or grain germ is rich in a variety of functional substances, such as proteins, flavonoids, polyphenols, etc., with certain antioxidant and anti-tumor properties. However, due to the low content or activity of functional ingredients, it is difficult to achieve significant effects. Therefore, the use of biotransformation technology to improve the nutritional quality and functional properties of grain or grain germ has attracted the attention of researchers at home and abroad, and has achieved certain results.

The Hungarian patent (99/08694) discloses a method for detecting 2,6-dimethoxy-p-benzoquinone as an active substance and an antitumor product thereof (A _V emar). The method comprises the steps of: pulverizing the wheat germ, fermenting with commercial baker's yeast, freeze-drying or spray-drying the supernatant, and adding the dry powder to a certain concentration of the aqueous solution to be added to the tumor cell, thereby inhibiting metabolism and metastasis of the tumor cell; The 2,6-dimethoxy-p-benzoquinone was extracted with chloroform or ethanol as a marker, and was quantified by HPLC, but the other components of the extract were not analyzed. The US patent (2010/135580A2) is similar to the Hungarian patent, except that it uses ethanol extraction and obtains the polypeptide from Avemar by molecular sieves and electrophoresis, with a relative molecular mass between 5 and 100 KD, and through cells and animals. Model tests have demonstrated that this range of polypeptides inhibits the growth of tumor cells.

Japanese Patent (200510069392.8) discloses a preparation method of an antitumor preparation. The method comprises mixing wheat germ, soybean germ and rice germ in a certain ratio, heating the mixture with 110 ° C hot steam, and heating the steam heating product at 30 ° C for 48 h with a koji (Aspergillus oryzae). The obtained fermentation extract has an antitumor effect with an inhibition rate of 5% to 15%, and the patent does not analyze the antitumor component in the fermentation extract.

Kouta Funamoto, et al. of Chongcheng University in Japan found that the distillation residue of barley shochu has anti-tumor and immune activity in 2008, which can significantly inhibit the growth of human lung cancer cells, induce tumor cell apoptosis, and induce energy in vivo. The production of serum interferon (IFN-γ) in normal mice increases the number of natural killer cells (NK) and is injected through the tail vein without acute toxicity. Masa iro Ohgidani, et al. in 2012, after the injection of HepG2 hepatoma cells into SCID mice, the daily distillation of barley shochu distillation residue 2250 mg / kg body weight for 21 consecutive days, can significantly reduce tumor cells, induce tumor cell apoptosis , prolong the life of SCID mice, and no side effects. ¥ and the literature report on anti-tumor research on fermentation products, mainly, and its inhibitory effect on tumors has not yet reached a satisfactory level.

Lactic acid bacterium (LAB) is safe, non-toxic, non-pathogenic, and has unique probiotic properties. It is a globally recognized "GRAS" ( generally regarded as safe) grade food microorganism, commonly used in sour milk products, kimchi, Fermented meat products, probiotic drinks and other fermented foods. However, research and product development on the anti-tumor properties of lactic acid bacteria fermentation products have not been reported in patents or other literature. Based on this, screening lactic acid bacteria capable of fermenting grain and grain germ and obtaining high bioactive products, and confirming the anti-tumor function of the fermentation product, thereby preparing a highly effective anti-tumor function food, food ingredient or medicine, is a broad development. The field of research for the future.

The invention selects excellent starting strains from self-separated lactic acid bacteria or commercial lactic acid bacteria, optimizes fermentation conditions, establishes a fermentation process with high yield of fermentation extract and strong anti-tumor activity, and uses the extract to carry out various human tumor cells and Its tumor-bearing animal test confirmed that its extract has significant inhibition of tumor cell proliferation.

Summary of the invention

The invention firstly provides a lactic acid bacteria which is derived from natural fermented kimchi by screening, separation, enrichment and physical and chemical analysis, and then obtains excellent lactic acid bacteria by using grain or grain germ (barley, wheat germ, etc.) as raw materials. The direct-injection fermentation of the lactic acid bacteria obtains a fermentation extract having an activity of inhibiting tumor cell proliferation, and the extract is separated, purified and analyzed.

MTT, apoptosis and cell cycle method were used to study the anti-tumor function of its components in vitro; the antitumor activity of the extract was studied by using a tumor-bearing nude mouse model (human tumor cells); comprehensive evaluation of the fermentation extract of the present invention Anti-tumor function and explore its action pathway.

The invention is achieved by the following technical solutions:

1. Lactobacillus Dy-1, its proposed taxonomic name is Lactobacillus plantarum; the strain was sent to the Chinese Academy of Sciences for microbiology research on April 18, 2012 at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China. The deposit of the General Microbiology Center of the China Microbial Culture Collection Management Committee is CGMCC No.6016.

2. Preparation method of lactic acid bacteria fermented grain or cereal germ extract

(1) Screening and preparation of strains: The kimchi samples were separately diluted by appropriate gradients, coated on MRS double-layer plates, and cultured in an incubator at 30 ° C for 36 h. After the colonies grew, observe the growth and select The colonies are suitable (30~300), the plate is obviously dissolved and the colony is easy to pick up, the colony characteristics are recorded, and the colonies are selected and picked. Screening of a strain of lactic acid bacteria from kimchi to identify its functional properties, its suggested classification name

Plontorum ; The strain was deposited on April 18, 2012 at the General Microbiology Center of the China Microbial Culture Collection Management Committee of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China. The deposit number is CGMCC. No.6016; or choose Commercially available lactic acid bacteria such as one or more of Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus acidophilus, and Lactobacillus case are selected.

(2) Preparation of lactic acid bacteria fermented grain or grain germ and its fermented extract: Weigh the grain or grain germ after sieving and removing, smash it through a 30-140 mesh sieve, and place it in a conical flask according to the mass to volume ratio. 1:6~1: 15 ratio, preferably 1:7 ratio is added to distilled water, then lactic acid bacteria are added to the grain or grain germ, wherein the lactic acid bacteria account for 2~8% of the quality of fresh grain or grain germ powder, and the mixture is even; The bottle is sealed with gauze, placed in a constant temperature incubator, and fermented at 20~40 °C for 12~72h, wherein the fermentation temperature is preferably 30~35 °C, after fermentation for 24~48 h, the fermentation is 3000~12000g at room temperature. /min Centrifuge for 10~40min, preferably 5,000~6500g/min for 20~30min; collect the supernatant and heat dry, spray dry or freeze dry, preferably freeze-dry, to obtain fermented grain or grain germ fermentation Things.

(3) Separation and purification of lactic acid bacteria fermented grain or grain germ extract: using ammonium sulfate precipitation, high-performance liquid phase (mobile phase is methanol to water ratio of 20%~15%, flow rate is lm! Jmin, column temperature is 25°) C, detection wavelength is 288 nm.), gel filtration chromatography (Sephadex G-50 and P Surpedex S-200), SDS-PAGE (5% concentrated gel, 10% ~ 12% separation gel) on fermented grain or grain germ The extract is isolated and purified to obtain a protein or polypeptide in the lactic acid bacteria fermented cereal or cereal germ extract.

The lactic acid bacteria selected in the step (1) are activated by MRS liquid medium and cultured at a high density, and a protective agent (20% skim milk, 10% trehalose, 14% sodium glutamate, 6% sorbitol, and the like) are added. 6%Vc, or 22% inulin, 12% sodium glutamate, 5% sorbitol), then prepare the directly used lactic acid bacteria Dy-1 starter by vacuum freeze-drying, and seal it in a sterile vacuum packaging bag. Deposit for use.

The grain described therein is preferably barley or glutinous rice or buckwheat. The grain germ described therein is preferably a wheat germ or a soybean germ.

The use of lactic acid bacteria to ferment cereals or grain germ extracts can be used to prepare anti-tumor functional foods or drug development.

The preparation method of the food having anti-tumor function is carried out according to the following steps:

The fermented cereal or cereal germ extract of the present invention is added to fermented dairy products and other acidic beverages in a certain ratio to prepare a functional beverage; it can also be added as food ingredients to foods such as bread, noodles, sausages and biscuits. The isolated and purified active fraction is directly used as a functional food or for anti-tumor drug development.

There is no particular weight requirement for adding the fermented cereal or cereal germ extract of the present invention to a foodstuff, and the amount is usually from 1% to 15%, preferably from 4% to 10% by mass; the extract may also be directly or After separation and purification, it is directly used as food or medicine.

Advantages of the invention:

(1) The present invention utilizes autonomously isolated lactic acid bacteria to ferment cereal or grain germ to prepare a fermentation extract having anti-tumor function Take the object, with independent intellectual property rights.

(2) The invention adopts simple pretreatment (non-high temperature heating) on grain or grain germ, has low production cost, adopts direct-injection fermentation, is convenient, fast, safe and stable, and is convenient for quality control.

(3) The invention uses cereal or grain germ as raw material, has wide source and large reserves, and bio-transforms it to significantly improve the biological function and nutritional quality of grain or grain germ, and guides enterprises to apply modern biotechnology to produce healthy and high. Valued products.

(4) The present invention studies the anti-tumor activity active ingredients in the extract, and clarifies that the protein or polypeptide with a relative molecular mass below 40KD has significant anti-tumor effect, indicating that the moderate degradation of protein macromolecules during the fermentation of lactic acid bacteria is resistant. An important pathway for tumor activity, and also provides a theoretical basis for the development of isolated foods or drugs. DRAWINGS

Figure 1 Growth inhibition rate of LFWGE on colon cancer HT-29 cells

Figure 2 Apoptosis map

Figure 3 Effect of LFWGE on cell cycle of colon cancer HT-29

Figure 4 Growth inhibition rate of FBE on colon cancer HT-29 cells

Figure 5 LFWGE water soluble protein SDS-PAGE map

Figure 6 gel filtration chromatography peptide map

Figure 7: Inhibition of HT-29 cell proliferation by crude extract of crude protein and crude polyphenol extraction in FBE

Figure 8 Effect of LFWGE on tumor volume of tumor-bearing nude mice

Figure 9 Inhibition rate of LFWGE on tumor-bearing nude mice

Figure 10 Effect of LFWGE on the content of tumor necrosis factor-α in serum of tumor-bearing nude mice

Figure 11 Effect of LFWGE on the content of interferon-γ in serum of tumor-bearing nude mice

Figure 12 Effect of LFWGE on serum interleukin-4 content in tumor-bearing nude mice

Figure 13 Effect of LFWGE on the expression of CyclinDl, Bax and Bcl-2 proteins in HT-29 cells

Figure 14 Effect of LFWGE on the expression of Caspase-3 protein in HT-29 cells

Figure 15 Effect of FBE on tumor volume of tumor-bearing nude mice

Figure 16 Inhibition rate of FBE on tumor-bearing nude mice

Figure 17 Effect of FBE on the content of malondialdehyde in the liver of nude mice

Figure 18 Effect of FBE on the activity of total superoxide dismutase in the liver of tumor-bearing mice

Figure 19 Effect of FBE on the activity of glutathione peroxidase in the liver of tumor-bearing mice

Figure 20 Effect of FBE on the content of interferon-γ in serum of tumor-bearing nude mice

Figure 21 Effect of FBE on serum interleukin-4 levels in tumor-bearing nude mice Figure 22 Effect of FBE on the content of tumor necrosis factor-α in serum of tumor-bearing nude mice

detailed description

The present invention will be further described below in conjunction with the embodiments.

1. Screening process of lactic acid bacteria

(1) Strain screening

Firstly, the collected kimchi samples were appropriately diluted by gradient and applied to the MRS double-layer plate method. The cells were cultured at 30 ° C for 36 h in an incubator. After the colonies grew, observe the growth of the plates and select the colonies in the plates. Appropriate (30~300) and calcium lysate are obvious for easy picking of colonies, record colony characteristics, select colonies, and pick colonies.

The isolated strain was subjected to multiple scribing or coating separation to obtain a pure strain. Pick a small amount of bacteria from the storage of the sputum, carry out Gram staining, record the staining results by microscopic observation, check whether the strain is a pure strain, and observe the shape of the strain to determine the size of the bacteria. Initial identification. The impure bacteria are again enriched and separated, the method is the same as above.

(2) strain screening

The acid-producing ability of the obtained strains was compared by the acid-producing gas-producing test to obtain a strain with high acid yield; and the potential facultative anaerobic lactic acid bacteria were obtained by observing the strains which simultaneously produced acid and gas.

According to the recorded colony conditions, pure rod-shaped or chain-like Gram-positive bacteria were selected, and the acid-producing gas production test was carried out in the sugar-fermented acid-producing medium, and the temperature was incubated at 25 ° C for 48 hours in the incubator after inoculation. The pH was measured.

2. Identification of strains

(1) Morphological observation

Three strains with the highest acid yield were isolated from naturally fermented cabbage kimchi, showing white colony bulge, round, tidy edges and smooth surface on solid medium; facultative anaerobic in liquid medium , growth to a certain amount of turbidity; Gram stain observation (see Table 1) are positive, the bacterial body rounded straight rod shape, width 0.8~1.2μηι, length 1~2μηι single or in pairs, not formed Endogenous spores.

(2) Physiological and biochemical tests

Physiological and biochemical tests on the above three strains (see Table 2) showed that the three strains of arginine ammonia production test, hydrogen sulfide production test, catalase test, gelatin liquefaction test, nitrate reduction test and 吲哚The test was negative; lactic acid qualitative test showed that lactic acid was produced; in the sugar fermentation test (see Table 3), no fermented rhamnose, partially weakly fermented arabinose and pine triose, the other sugar alcohols could be fermented, neither Produce gas. Physiological and biochemical tests identified the three strains as Lactobacillus Plantarum. However, it is still necessary to select Dy-1 with better fermentation performance for 16s rDNA amplification for further identification. (3) 16s rDNA PCR amplification product sequence

Pick the appropriate amount of Dy-1 bacteria, add it to MRS medium, incubate at 37 °C overnight, take 1mL of bacterial solution, centrifuge at 5000xg for 5min, discard the supernatant, add 300μί cell lysate and 50μί protease, and lyse at 90 °C in water bath. After lOmin, the supernatant was taken at 12000 xg for 10 min, and the PCR reaction was carried out with the v3 universal primer.

The PCR product was purified and sequenced by Shanghai Biotech. The sequence is as follows:

CAAGAGCCTGTCAGC According to the results of Blast analysis in Genbank, the 16S rRNA gene sequence of Dy-1 is only 3 bases different from the 16S rRN A gene sequence of Lactobacillus plantar um strain L3, and its similarity is more than 99%, which should belong to the same species. That is Lactobacillus plantarum. The strain was deposited on April 18, 2012 at the General Microbiology Center of the China Microbial Culture Collection Management Committee of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China. The deposit number is CGMCC No. 6016, its suggested classification name is Lactobacillus plantarum, Lactobacillus plantar called it lactic acid bacteria Dy-1

3. Anti-tumor function test

In vitro anti-tumor function: The fermented cereal or cereal germ extract of the present invention is added to human HT-29 colon cancer cells at a concentration of 0.125 4 mg/mL, and cultured for a period of time (for example, 48 hours), and the anti-tumor effect is observed and evaluated. . The method is as follows -

1 Cell proliferation assay: MTT method was used.

2 Apoptosis and cell cycle assay: Flow cytometry was used for analysis.

Compared with the control without the fermented cereal or cereal germ extract of the present invention, the number of living cells of the tumor can be reduced to 50% or less, and the number of living cells can be optimally reduced to 10% or less; Up to 50% or more, up to 90% or more.

In vivo anti-tumor function: The abdominal cancer of 46-week-old BALB/c nude mice was subcutaneously inoculated into human colon cancer HT-29 cells (inoculation amount: 1 10 ⁶ 2 10 ⁷ cells/nude mice), and nude mice were visually observed after 2 15 days. At the inoculation site, the tumor was started and the stomach was started. The tumor-bearing nude mice were randomly divided into 5 groups, namely, the negative control group, the 5-FU positive control group, the high dose group, the low dose group, and the high dose group +5-FU group. 10 12 nude mice in each group were continuously intragastrically administered for 14 50 days; the tumor volume was measured daily, the tumor growth curve was drawn, and the tumor inhibition rate was calculated according to the tumor weight; the interferon (IFN- _{Y) in the} serum of the tumor-bearing nude mice was determined at the end of the experiment. ), the concentration of interleukin-4 (IL-4) and tumor necrosis factor (TNF-a). The results showed that the inhibition rate of the fermented cereal or grain extract group was 30% 60% compared with the negative control group.

Example 1

Weigh 20.0g of pulverized fresh wheat germ (hereinafter referred to as wheat germ) powder in a 500mL Erlenmeyer flask, add 100ml of distilled water to it, add 0.4g of lactic acid bacteria Dy-1 starter, mix evenly; The gauze was sealed and placed in a constant temperature incubator for 24 hours at 30 ° C. After centrifugation at 6500 g / min for 20 min, the supernatant was collected and lyophilized.

Weigh 0.5g of lyophilized powder (LFWGE) dissolved in 50ml of deionized water, pass 0.22um filter, and the filtrate is 0.125 mg/mL 0.25mg/mL 0.5 mg/mL 1.0 mg/mL 2.0 mg/mK 4.0 Mg/ml was added to HT-29 colon cancer cells, cultured for 24 h, 48 h, 72 h, and the inhibition rate of LFWGE on colon cancer HT-29 cells was determined by MTT assay (Fig. 1). The results showed that the concentration of extract was positively correlated with the inhibition rate of tumor cell proliferation, and the maximum inhibition rate reached 94.2%. 1.0 mg/mL of LFWGE was added to colon cancer cells, and after 48 hours of cell culture, cells were collected, and apoptosis was detected by flow cytometry (Fig. 2). The results showed that early apoptosis was 66.5% (Fig. 2, lower right corner) and late apoptosis was 27.6% (Fig. 2, upper right corner). LFWGE of 0.2 mg/mL, 0.4 mg/mL, and 0.8 mg/mL was added to colon cancer HT-29 cells, and cells were cultured for 48 hours, and the cells were collected, and the cell cycle was detected by flow cytometry (Fig. 3). The results showed that with the increase of concentration, the 0 ₁ phase also prolonged, and the S phase shortened, indicating that LFWGE can effectively block the cell phase.

Example 2

The filtrate in Example 1 was also added to SGC-7901 gastric cancer cells at 0.125 mg/mL, 0.25 mg/ml, 0.5 mg/mK 1.0 mg/mK 2.0 mg/mK 4.0 mg/ml, respectively. At 24h, 48h, 72h, the inhibition rate of LFWGE on gastric cancer SGC-7901 cells was determined by MTT assay. The results showed that the concentration of extract was positively correlated with the inhibition rate of tumor cell proliferation, and the maximum inhibition rate reached 89.6%.

The lmg/ml LFWGE was added to the SGC-7901 gastric cancer cells, and the cells were cultured for 48 hours, and the cells were collected, and the apoptosis of the cells was detected by flow cytometry. Early apoptosis was 48.5% and late apoptosis was 22.4%. LFWGE of 0.2 mg/ml, 0.4 mg/ml, and 0.8 mg/ml was added to gastric cancer cells, and the cells were cultured for 48 hours, and the cells were collected, and the cell cycle was detected by flow cytometry. The results showed that with the increase of concentration, the phase ₁ was also prolonged, and the S phase was shortened, indicating that LFWGE can effectively block the phase ₁ of cells.

Example 3

20.0 g of barley flour crushed through a 60 mesh sieve was weighed into a 500 mL Erlenmeyer flask, and a 1:6 ratio of water was added thereto to be uniformly mixed, and the mixture was hydrolyzed with 0.015% β-glucanase at 40 ° C for 30 min or not. Fermentation was carried out at 30 °C for 48 h by adding 4% lactic acid bacteria Dy-1 starter. After the fermentation was completed, the fermentation product was centrifuged at 5000 g/min for 20 min at room temperature, and the supernatant was collected and freeze-dried for 24 h to obtain fermented barley extract. (FBE).

Weigh 0.5g of FBE dissolved in 50ml of deionized water, pass 0.22um filter, and the filtrate is 0.125mg/mL, 0.25mg/mL, 0.5mg/mL, 1.0mg/mL, 2.0mg/ml, 4.0 Mg/ml was added to HT-29 tumor cells, cultured for 24h, 48h, 72h, and the inhibition rate of FBE on colon cancer HT-29 cells was determined by MTT assay. The results showed that the extract concentration was positively correlated with the inhibition rate of tumor cell proliferation, and the maximum inhibition rate reached 95.6% (Fig. 4).

Example 4

100 g of pulverized fresh wheat germ was mixed with 1 L of distilled water and 4 g of lactic acid bacteria Dy-1 starter, and the mixture was fermented at 30 ° C for 48 h at 250 rpm. The obtained fermentation product was centrifuged at 6000 g for 15 min, and the supernatant was collected and freeze-dried to obtain a LFWGE lyophilized powder. The distribution of water-soluble proteins in LFWGE was detected by SDS-PAGE (Fig. 5). In order to separate the protein components in LFWGE, the protein in the LFWGE aqueous solution was precipitated by (NH4) ₂ S0 ₄ precipitation method, the precipitate was dissolved in PBS, and the resulting solution was subjected to gel filtration chromatography on Sephadex G-50 and Surpedex S-200. Peaks 1 and 2 were collected separately (Figure 6) and lyophilized separately. Peak 1 and 1 were determined by MTT method The inhibition rate of the peak 2 component on HT-29 cells. The results showed that peak 1 had a significant inhibition rate on HT-29 cells, with a maximum of 88.6% (Table 3).

Example 5

100 g of pulverized fresh barley was mixed with 1 L of distilled water and 4 g of lactic acid bacteria Dy-1 starter, and the mixture was fermented at 250 rpm for 24 h at 30 °C. The obtained fermentation product was centrifuged at 5000 g for 15 min, and the supernatant was collected and freeze-dried to obtain a fermented barley extract (FBE) lyophilized powder. The FBE was dissolved, and the protein was precipitated with 90% saturation of (NH ₄ ) ₂ S0 ₄ , centrifuged at 6000 g/min for 15 min, and the precipitate was dissolved in distilled water and dialyzed 3 times with a dialysis bag having a molecular weight of 8000-14000 Da. After lyophilization, the protein was coarsely extracted. Things. Weigh lg FBE, using 70% ethanol solution ratio 1:60, 60 ° C reflux leaching lh, extract 2 times, centrifuge at 4500g / min for 15min to obtain the filtrate, rotary evaporation to obtain a concentrated solution, freeze-dried, much phenol crude Things. 500 ug/mL crude protein extract and crude polyphenol extract were added to HT-29 tumor cells, and the cells were cultured for 24 hours, and the crude protein extract and polyphenol crude extract were determined by MTT method for colon cancer HT-29. Cell inhibition rate. The results showed that crude protein extract and crude polyphenol extract were positively correlated with tumor cell proliferation inhibition rate (Fig. 7).

Example ό

The LFWGE prepared above was dissolved in physiological saline to prepare an aqueous solution having a concentration of 0.2 g/ml and 0.1 g/ml, and was treated by a sterile membrane for use. Human HT-29 colon cancer cells were inoculated subcutaneously into 4-6 weeks old BALB/c nude mice, and 2 days later, 2g/kg/d, lg/kg/d, 2g/kg/d+5-FU (25 mg) /kg/d, ip, 7 days) The dose of LFWGE was administered to the tumor-bearing nude mice for 30 days of continuous gavage, and the same dose of saline group and intraperitoneal injection of 5-FU group (25 mg/kg/). d, ip, 7 days) Comparison was performed to determine the antitumor effect of LFWGE by measuring changes in tumor volume and final tumor inhibition rate (Figures 8 and 9). The results showed that the tumor inhibition rates of 2g/kg/d, lg/kg/d, 2g/kg/d+5-FU LFWGE group reached 49.37%, 40.7%, and 47.8%, respectively.

The levels of tumor necrosis factor (TNF-a), interferon (IFN-γ) and interleukin-4 (IL-4) in the serum of tumor-bearing nude mice were determined by enzyme-linked immunosorbent assay kit. The results showed that LFWGE significantly increased the levels of IFN, IL-4 and TNF-α in the serum of tumor-bearing nude mice (Fig. 10, Fig. 11 and Fig. 12), which was related to the inhibition of HT-29 colon cancer cells.

The expression of related factors in tumor tissues of nude mice was determined by immunoblotting. It was found that LFWGE down-regulated the mRNA and protein expression of Bcl-2 and CyclinDl; up-regulated the mRNA and protein expression of Bax and Caspase-3 (Fig. 13, 14). Induction of apoptosis in tumor cells of nude mice bearing tumors.

Example 7

The FBE prepared above was dissolved in physiological saline to a concentration of 0.2 g/ml and 0.1 g/ml, and was treated by a sterile membrane for use. Human HT-29 colon cancer cells were inoculated subcutaneously into 4-6 week old BALB/c nude mice, and 2 days later, 2g/kg/d, lg/kg/d, 2g/kg/d+5-FU ( 25 mg/kg/d, ip, 7 days) The dose of FBE was administered to the tumor-bearing nude mice for 30 days, and the same dose of saline group and intraperitoneal injection of 5-FU group (25 mg/). Kg/d, 7 days) In contrast, the antitumor effect of FBE was confirmed by the volume of the transplanted tumor measured by the vernier caliper and the final tumor suppressing rate (Fig. 15, 16). The results showed that the inhibition rate of the 2g/kg/d+5-FU mixed dose group could reach 53.36%.

The contents of superoxide dismutase (SOD) (Fig. 17), glutathione-peroxidase (GSH-PX) (Fig. 18) and malondialdehyde (MDA) in the liver of tumor-bearing nude mice were determined by kit ( Figure 19). The results show that FBE can increase the enzyme activity of SOD and GSH-PX and decrease the content of MDA.

The concentration of interferon (IFN-γ) (Fig. 20), interleukin-4 (Fig. 21) and tumor necrosis factor (TNF-α) (Fig. 22) in the serum of tumor-bearing nude mice was determined by enzyme-linked immunosorbent assay kit. show, FBE simultaneously can be increased IFN- _Y, the content of interleukin-4 and TNF-α in.

Table 1 Screening to obtain the morphological characteristics of the strain with the highest acid yield

Code pH Titration Acidity (%) Characteristics Bacterial Size (μηι) Pure No

3.70 0.528 rod shape 0.8X1.1 pure

3.70 0.528 rod shape 0.8X1.2 pure

3.69 0.528 rod shape 0.8X1.0 pure

Table 2 Physiological and biochemical test results of isolated strains

Code arginine ammonia production test hydrogen sulfide hydrogen peroxide enzyme gelatin liquefaction nitrate reduction 吲哚 test

Test test test

DY-1

s ₃ Note: + means positive, one means negative

Table 3 Inhibition rate of two-part peptides in LFWGE against HT-29 cells Sample Concentration (g/mL) Absorbance (A490) Inhibition rate (%)

200 0.100±0.012 88.6

100 0.381 ±0.021 56.8

Pe kl 50 0.432±0.015 51.0

25 0.631 ±0.024 28.4 200 0.873 ± 0.052 0.9

100 0.861 + 0.073 23

50 0.892 ± 0.017 - 1.2

25 0.871 ±0.013 1.1

SEQUENCE LISTING

<110> Jiangsu University

<120> Preparation method and use of lactic acid bacteria and fermented cereal or cereal germ extract

<160> 1

<170> Patentln version 3.3

Please follow the "Precautions" to correctly fill in the fields that are not determined by the Patent Office in columns 2 and 4 of the columns of this table.

Title: Preparation of Lactic Acid Bacteria and Fermented Cereal or Grain Germ Extract 1 Application No.

Method and use

Square mode

2 Applicant Jiangsu University 3 Application Date

4 Name of genetic resources: Lactobacillus plantarum Lactobacillus plantarum

6 Access to genetic resources

I Genetic resources are taken from: □ animals □ plants microbes

II Acquisition method: □ Purchase □ Gift or exchange Port depository □ Seed bank (species bank) Oral gene library 図 Self-collection □ Entrusted collection □ Others

8Get the time February 2012

9 provider name

(name)

Non

10 providers are located

Country or region

7

Θ Provider contact

the way

China Jiangsu Province Zhenjiang City Jiangsu University

Home, province (city))

Θ Collector Name

(name)

Θ Collector contact

13505280069

the way

(name)

13505280069

System mode

(@Get the time February 2012

(Country, Province China Jiangsu Province Zhenjiang City Jiangsu University Campus Laboratory (City))

June 5, 2013

Claims

Claims WO 2014/206279 PCT/CN2014/080665

1. Lactobacillus Dy-1, its proposed classification name is Lactobacillus plantarum; the deposit number is CGMCC No.6016.

2. A method for preparing lactic acid bacteria fermented cereal or cereal germ extract, which is characterized by following the following steps:

(1) Selection of strains: Choose Lactobacillus plantarum·σ<:ί:ο/5σ(:/7/υ5 ρ/σηί:σ ·υΜ, CGMCC No.6016; or choose commercially available excellent lactic acid bacteria, such as Lactobacillus plantarum One or more of Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus acidophilus and Lactobacillus case is the starting strain;

(2) Preparation of grains or grain germs fermented by lactic acid bacteria and fermentation extracts: weigh the grains or grain germs after screening and impurity removal, crush them through a 30 to 140 mesh sieve, place them in an Erlenmeyer flask, and mix according to the mass to volume ratio. Add distilled water at a ratio of 1:6~1:15, preferably 1:7, and then add lactic acid bacteria Dy-1 starter to the grain or grain germ, where the lactic acid bacteria Dy-1 starter accounts for the mass of fresh grain or grain germ powder. 2~8%, mix evenly; seal the Erlenmeyer flask with gauze, place it in a constant temperature incubator, and ferment at 20~40°C for 12~72h, with the preferred fermentation temperature being 30~35°C and fermentation for 24~48 h. Afterwards, the fermentation product is centrifuged at room temperature at 3000~12000g/min for 10~40min, preferably at 5000~6500g/min for 20~30min; the supernatant is collected and subjected to hot air drying, spray drying or freeze drying, preferably freeze drying. , thereby obtaining an extract of fermented grains or grain germ;

(3) Separation and purification of lactic acid bacteria fermented grains or grain germ extracts: ammonium sulfate precipitation, high-performance liquid phase (the mobile phase is methanol and water in a ratio of 20% to 15%, the flow rate is lm!Jmin, and the column temperature is 25° C, the detection wavelength is 288nm.), gel filtration chromatography (SephadexG-50 and SurpedexS-200), SDS-PAGE (5% stacking gel, 10%~12% separation gel) for fermented cereals or cereal germs The extract is separated and purified to obtain the protein or peptide in the lactic acid bacteria fermented grain or grain germ extract.

3. The method for preparing lactic acid bacteria fermented cereals or cereal germ extract according to claim 1, characterized in that the lactic acid bacteria selected in step (1) are activated and cultured at high density using MRS liquid culture medium, and then a protective agent is added to The mass ratio is: 20% skim milk, 10% trehalose, 14% sodium glutamate, 6% sorbitol and 6% Vc, or the mass ratio is 22% inulin, 12% sodium glutamate and 5% sorbitol. After alcoholization, vacuum freeze-drying method is used to prepare lactic acid bacteria Dy-1 starter culture that can be used directly, and the fermentation agent is sealed and stored in sterile vacuum packaging bags for later use.

4. The method for preparing lactobacillus fermented cereals or cereal germ extracts according to claim 1, wherein the cereals are barley, barley, or buckwheat; and the cereal germs are wheat germs or soybean germs.

5. The use of lactic acid bacteria fermented cereals or cereal germ extracts can be used to prepare anti-tumor functional foods.

6. The use of lactic acid bacteria fermented cereals or cereal germ extracts according to claim 5, characterized in that the preparation method of foods with anti-tumor functions is carried out according to the following steps: fermenting cereals or cereal germ extracts in a certain proportion Add directly to fermented dairy products and other acidic beverages to make functional drinks; it can also be added as a food ingredient Claims

WO 2014/206279 PCT/CN2014/080665 In foods such as bread, noodles, sausages and biscuits; fermented cereals or cereal germ extracts can also be used directly as food; the isolated and purified active parts can be used as functional foods or for the development of anti-tumor drugs; There are no special weight requirements for adding fermented grain or grain germ extract to food. The amount added is generally 1% to 15% in terms of mass ratio, preferably 4% to 10%.